Turkiyersquode Mikrobiyota CalısmalarındaKullanılan Laboratuvar Yontemleri

Meltem Yalınay MD PhD

Gazi Uumlniversitesi Tıp Fakuumlltesi

Tıbbi Mikrobiyoloji AD

10122016

Mikrobiyota Analiz Youmlntemleri

Mikroskobi

Kuumlltuumlr

Kuumlltuumlre Dayalı Molekuumller Youmlntemler

Kuumlltuumlr Bağımsız Molekuumller Youmlntemler

Dizi Analizi

Yeni Nesil DizilemeBiyoinformatik

Analiz

Mikrobiyota Analiz Youmlntemleri-I

GaitaDNA

İzolasyonu

3 KLİMUD Kongresi Antalya 19-22 Kasım 2015

Mikrobiyota Analiz Youmlntemleri

Molekuler Yontemler

Kuumlltuumlre Dayalı Molekuumller Youmlntemler

Kuumlltuumlr Bağımsız Molekuumller Youmlntemler

Fenotipik Parmak İzi Analiz YoumlntemleriGenotipik Parmak Analizi Youmlntemleri

FISHKantitatif Dot BlotRAPDPZR-DGGE PZR-TGGET-RFLPAPMikroarray16S rRNArecA Gen AnaliziqPZRDNA dizilemeYeni nesil dizileme

Mikrobiyota Analiz YoumlntemleriYontem Avantaj Dezavantaj

qPZR

Filogenetik tanımlama Kantitatif Hızlı Yuumlksek duyarlılık

PZR bias Bilinmeyen tuumlrlere

uygulanamaz Tek hedef

DGGETGGE

bull Hızlı bull Yarı kantitatif bull Oumlrneklerin ileri testler iccedilin

kullanımına olanak sağlar

Filogenetiktanımlama yapamaz

PZR bias

T-RFLP Hızlı Yarı kantitatif Maliyet etkin

Filogenetiktanımlama yapamaz

PZR bias

16S rRNA Filogenetik tanımlama Kantitatif

PZR bias Zahmetli Pahalı Klonlama bias

Mikrobiyota Analiz YoumlntemleriYontem (Uumlretici firma) Avantaj Dezavantaj

Pirodizileme

(454 GS FLX+ Roche)

Okuma suumlresi uzundur Yuumlksek verimlilik Duyarlı Aynı zamanda birden fazla

oumlrneği analiz etme imkanı Koloni biası yoktur

Yuumlksek maliyet Homopolimerlerde

yuumlksek hata oranı Kısa sekans okuma Kapsamlı biyoinformatik

analize ihtiyaccedil duyar

Geri doumlnuumlşuumlmluuml terminatoumlr

dizileme

(HiSeq20002500 Illumina)

Etkili Okuma suumlrelerini iyileştiren

suumlreklilik Yuumlksek verimlilik Manuel iş azlığı

Uzun ccedilalışma suumlresi Kısa okuma uzunlukları Yuumlkseltme geliştirilme

Ligasyon

(5500xl SOLiD Life

Technologies)

Duumlşuumlk hata oranı Yuumlksek verimlilik

Ccedilok kısa uzunluklar Uzun ccedilalışma suumlresi

Gerccedilek zamanlı sekans (PacBio

RS Pacific Biosceince)

Oumlrnek hazırlaması kolay Reaktif maliyeti duumlşuumlk Ccedilok uzun okuma uzunluğu

Hata oranı yuumlksek Sistem maliyeti yuumlksek Sistem kurulumu zordur

Mikrobiyota Analiz Youmlntemleri

Bioresour Technol 2015 Sep192735-40 doi 101016jbiortech201505086 Epub 2015 Jun 9Use of PCR-DGGE based molecular methods to assessment of microbial diversity during anaerobic treatment of antibiotic combinationsAydin S1 Shahi A2 Ozbayram EG2 Ince B3 Ince O2Author information

AbstractAs it is currently often not know how anaerobic bioreactors eg for biogas production react if the substrate is contaminated by toxic compounds like antibiotics This study evaluated how anaerobic sequencing batch reactors were affected by amendments of different antibiotics and stepwise increasing concentrations The compositions of microbial community were determined in the seed sludge using 16S rRNA gene clone libraries and PCR-DGGE analyses were used for the detection of microbial community changes upon antibiotics additions According to PCR-DGGE results the syntrophic interaction of acetogens and methanogens is critical to the performance of the reactors Failure to maintain the stability of these microorganisms resulted in a decrease in the performance and stability of the anaerobic reactors Assessment of DGGE data is also useful for suggesting the potential to control ultimate microbial community structure especially derived from Gram-negative bacteria through bioaugmentation to successful for antibiotic biodegradationCopyright copy 2015 Elsevier Ltd All rights reserved

Mikrobiyota Analiz Youmlntemleri

Mikroskobi

Kuumlltuumlr

Kuumlltuumlre Dayalı Molekuumller Youmlntemler

Kuumlltuumlr Bağımsız Molekuumller Youmlntemler

Dizi Analizi

Yeni Nesil DizilemeBiyoinformatik

Analiz

Mikrobiyota Analiz Youmlntemleri-I

GaitaDNA

İzolasyonu

3 KLİMUD Kongresi Antalya 19-22 Kasım 2015

Mikrobiyota Analiz Youmlntemleri

Molekuler Yontemler

Kuumlltuumlre Dayalı Molekuumller Youmlntemler

Kuumlltuumlr Bağımsız Molekuumller Youmlntemler

Fenotipik Parmak İzi Analiz YoumlntemleriGenotipik Parmak Analizi Youmlntemleri

FISHKantitatif Dot BlotRAPDPZR-DGGE PZR-TGGET-RFLPAPMikroarray16S rRNArecA Gen AnaliziqPZRDNA dizilemeYeni nesil dizileme

Mikrobiyota Analiz YoumlntemleriYontem Avantaj Dezavantaj

qPZR

Filogenetik tanımlama Kantitatif Hızlı Yuumlksek duyarlılık

PZR bias Bilinmeyen tuumlrlere

uygulanamaz Tek hedef

DGGETGGE

bull Hızlı bull Yarı kantitatif bull Oumlrneklerin ileri testler iccedilin

kullanımına olanak sağlar

Filogenetiktanımlama yapamaz

PZR bias

T-RFLP Hızlı Yarı kantitatif Maliyet etkin

Filogenetiktanımlama yapamaz

PZR bias

16S rRNA Filogenetik tanımlama Kantitatif

PZR bias Zahmetli Pahalı Klonlama bias

Mikrobiyota Analiz YoumlntemleriYontem (Uumlretici firma) Avantaj Dezavantaj

Pirodizileme

(454 GS FLX+ Roche)

Okuma suumlresi uzundur Yuumlksek verimlilik Duyarlı Aynı zamanda birden fazla

oumlrneği analiz etme imkanı Koloni biası yoktur

Yuumlksek maliyet Homopolimerlerde

yuumlksek hata oranı Kısa sekans okuma Kapsamlı biyoinformatik

analize ihtiyaccedil duyar

Geri doumlnuumlşuumlmluuml terminatoumlr

dizileme

(HiSeq20002500 Illumina)

Etkili Okuma suumlrelerini iyileştiren

suumlreklilik Yuumlksek verimlilik Manuel iş azlığı

Uzun ccedilalışma suumlresi Kısa okuma uzunlukları Yuumlkseltme geliştirilme

Ligasyon

(5500xl SOLiD Life

Technologies)

Duumlşuumlk hata oranı Yuumlksek verimlilik

Ccedilok kısa uzunluklar Uzun ccedilalışma suumlresi

Gerccedilek zamanlı sekans (PacBio

RS Pacific Biosceince)

Oumlrnek hazırlaması kolay Reaktif maliyeti duumlşuumlk Ccedilok uzun okuma uzunluğu

Hata oranı yuumlksek Sistem maliyeti yuumlksek Sistem kurulumu zordur

Mikrobiyota Analiz Youmlntemleri

Bioresour Technol 2015 Sep192735-40 doi 101016jbiortech201505086 Epub 2015 Jun 9Use of PCR-DGGE based molecular methods to assessment of microbial diversity during anaerobic treatment of antibiotic combinationsAydin S1 Shahi A2 Ozbayram EG2 Ince B3 Ince O2Author information

AbstractAs it is currently often not know how anaerobic bioreactors eg for biogas production react if the substrate is contaminated by toxic compounds like antibiotics This study evaluated how anaerobic sequencing batch reactors were affected by amendments of different antibiotics and stepwise increasing concentrations The compositions of microbial community were determined in the seed sludge using 16S rRNA gene clone libraries and PCR-DGGE analyses were used for the detection of microbial community changes upon antibiotics additions According to PCR-DGGE results the syntrophic interaction of acetogens and methanogens is critical to the performance of the reactors Failure to maintain the stability of these microorganisms resulted in a decrease in the performance and stability of the anaerobic reactors Assessment of DGGE data is also useful for suggesting the potential to control ultimate microbial community structure especially derived from Gram-negative bacteria through bioaugmentation to successful for antibiotic biodegradationCopyright copy 2015 Elsevier Ltd All rights reserved

Mikrobiyota Analiz Youmlntemleri-I

GaitaDNA

İzolasyonu

3 KLİMUD Kongresi Antalya 19-22 Kasım 2015

Mikrobiyota Analiz Youmlntemleri

Molekuler Yontemler

Kuumlltuumlre Dayalı Molekuumller Youmlntemler

Kuumlltuumlr Bağımsız Molekuumller Youmlntemler

Fenotipik Parmak İzi Analiz YoumlntemleriGenotipik Parmak Analizi Youmlntemleri

FISHKantitatif Dot BlotRAPDPZR-DGGE PZR-TGGET-RFLPAPMikroarray16S rRNArecA Gen AnaliziqPZRDNA dizilemeYeni nesil dizileme

Mikrobiyota Analiz YoumlntemleriYontem Avantaj Dezavantaj

qPZR

Filogenetik tanımlama Kantitatif Hızlı Yuumlksek duyarlılık

PZR bias Bilinmeyen tuumlrlere

uygulanamaz Tek hedef

DGGETGGE

bull Hızlı bull Yarı kantitatif bull Oumlrneklerin ileri testler iccedilin

kullanımına olanak sağlar

Filogenetiktanımlama yapamaz

PZR bias

T-RFLP Hızlı Yarı kantitatif Maliyet etkin

Filogenetiktanımlama yapamaz

PZR bias

16S rRNA Filogenetik tanımlama Kantitatif

PZR bias Zahmetli Pahalı Klonlama bias

Mikrobiyota Analiz YoumlntemleriYontem (Uumlretici firma) Avantaj Dezavantaj

Pirodizileme

(454 GS FLX+ Roche)

Okuma suumlresi uzundur Yuumlksek verimlilik Duyarlı Aynı zamanda birden fazla

oumlrneği analiz etme imkanı Koloni biası yoktur

Yuumlksek maliyet Homopolimerlerde

yuumlksek hata oranı Kısa sekans okuma Kapsamlı biyoinformatik

analize ihtiyaccedil duyar

Geri doumlnuumlşuumlmluuml terminatoumlr

dizileme

(HiSeq20002500 Illumina)

Etkili Okuma suumlrelerini iyileştiren

suumlreklilik Yuumlksek verimlilik Manuel iş azlığı

Uzun ccedilalışma suumlresi Kısa okuma uzunlukları Yuumlkseltme geliştirilme

Ligasyon

(5500xl SOLiD Life

Technologies)

Duumlşuumlk hata oranı Yuumlksek verimlilik

Ccedilok kısa uzunluklar Uzun ccedilalışma suumlresi

Gerccedilek zamanlı sekans (PacBio

RS Pacific Biosceince)

Oumlrnek hazırlaması kolay Reaktif maliyeti duumlşuumlk Ccedilok uzun okuma uzunluğu

Hata oranı yuumlksek Sistem maliyeti yuumlksek Sistem kurulumu zordur

Mikrobiyota Analiz Youmlntemleri

Bioresour Technol 2015 Sep192735-40 doi 101016jbiortech201505086 Epub 2015 Jun 9Use of PCR-DGGE based molecular methods to assessment of microbial diversity during anaerobic treatment of antibiotic combinationsAydin S1 Shahi A2 Ozbayram EG2 Ince B3 Ince O2Author information

AbstractAs it is currently often not know how anaerobic bioreactors eg for biogas production react if the substrate is contaminated by toxic compounds like antibiotics This study evaluated how anaerobic sequencing batch reactors were affected by amendments of different antibiotics and stepwise increasing concentrations The compositions of microbial community were determined in the seed sludge using 16S rRNA gene clone libraries and PCR-DGGE analyses were used for the detection of microbial community changes upon antibiotics additions According to PCR-DGGE results the syntrophic interaction of acetogens and methanogens is critical to the performance of the reactors Failure to maintain the stability of these microorganisms resulted in a decrease in the performance and stability of the anaerobic reactors Assessment of DGGE data is also useful for suggesting the potential to control ultimate microbial community structure especially derived from Gram-negative bacteria through bioaugmentation to successful for antibiotic biodegradationCopyright copy 2015 Elsevier Ltd All rights reserved

Mikrobiyota Analiz Youmlntemleri

Molekuler Yontemler

Kuumlltuumlre Dayalı Molekuumller Youmlntemler

Kuumlltuumlr Bağımsız Molekuumller Youmlntemler

Fenotipik Parmak İzi Analiz YoumlntemleriGenotipik Parmak Analizi Youmlntemleri

FISHKantitatif Dot BlotRAPDPZR-DGGE PZR-TGGET-RFLPAPMikroarray16S rRNArecA Gen AnaliziqPZRDNA dizilemeYeni nesil dizileme

Mikrobiyota Analiz YoumlntemleriYontem Avantaj Dezavantaj

qPZR

Filogenetik tanımlama Kantitatif Hızlı Yuumlksek duyarlılık

PZR bias Bilinmeyen tuumlrlere

uygulanamaz Tek hedef

DGGETGGE

bull Hızlı bull Yarı kantitatif bull Oumlrneklerin ileri testler iccedilin

kullanımına olanak sağlar

Filogenetiktanımlama yapamaz

PZR bias

T-RFLP Hızlı Yarı kantitatif Maliyet etkin

Filogenetiktanımlama yapamaz

PZR bias

16S rRNA Filogenetik tanımlama Kantitatif

PZR bias Zahmetli Pahalı Klonlama bias

Mikrobiyota Analiz YoumlntemleriYontem (Uumlretici firma) Avantaj Dezavantaj

Pirodizileme

(454 GS FLX+ Roche)

Okuma suumlresi uzundur Yuumlksek verimlilik Duyarlı Aynı zamanda birden fazla

oumlrneği analiz etme imkanı Koloni biası yoktur

Yuumlksek maliyet Homopolimerlerde

yuumlksek hata oranı Kısa sekans okuma Kapsamlı biyoinformatik

analize ihtiyaccedil duyar

Geri doumlnuumlşuumlmluuml terminatoumlr

dizileme

(HiSeq20002500 Illumina)

Etkili Okuma suumlrelerini iyileştiren

suumlreklilik Yuumlksek verimlilik Manuel iş azlığı

Uzun ccedilalışma suumlresi Kısa okuma uzunlukları Yuumlkseltme geliştirilme

Ligasyon

(5500xl SOLiD Life

Technologies)

Duumlşuumlk hata oranı Yuumlksek verimlilik

Ccedilok kısa uzunluklar Uzun ccedilalışma suumlresi

Gerccedilek zamanlı sekans (PacBio

RS Pacific Biosceince)

Oumlrnek hazırlaması kolay Reaktif maliyeti duumlşuumlk Ccedilok uzun okuma uzunluğu

Hata oranı yuumlksek Sistem maliyeti yuumlksek Sistem kurulumu zordur

Mikrobiyota Analiz Youmlntemleri

Bioresour Technol 2015 Sep192735-40 doi 101016jbiortech201505086 Epub 2015 Jun 9Use of PCR-DGGE based molecular methods to assessment of microbial diversity during anaerobic treatment of antibiotic combinationsAydin S1 Shahi A2 Ozbayram EG2 Ince B3 Ince O2Author information

AbstractAs it is currently often not know how anaerobic bioreactors eg for biogas production react if the substrate is contaminated by toxic compounds like antibiotics This study evaluated how anaerobic sequencing batch reactors were affected by amendments of different antibiotics and stepwise increasing concentrations The compositions of microbial community were determined in the seed sludge using 16S rRNA gene clone libraries and PCR-DGGE analyses were used for the detection of microbial community changes upon antibiotics additions According to PCR-DGGE results the syntrophic interaction of acetogens and methanogens is critical to the performance of the reactors Failure to maintain the stability of these microorganisms resulted in a decrease in the performance and stability of the anaerobic reactors Assessment of DGGE data is also useful for suggesting the potential to control ultimate microbial community structure especially derived from Gram-negative bacteria through bioaugmentation to successful for antibiotic biodegradationCopyright copy 2015 Elsevier Ltd All rights reserved

Mikrobiyota Analiz YoumlntemleriYontem Avantaj Dezavantaj

qPZR

Filogenetik tanımlama Kantitatif Hızlı Yuumlksek duyarlılık

PZR bias Bilinmeyen tuumlrlere

uygulanamaz Tek hedef

DGGETGGE

bull Hızlı bull Yarı kantitatif bull Oumlrneklerin ileri testler iccedilin

kullanımına olanak sağlar

Filogenetiktanımlama yapamaz

PZR bias

T-RFLP Hızlı Yarı kantitatif Maliyet etkin

Filogenetiktanımlama yapamaz

PZR bias

16S rRNA Filogenetik tanımlama Kantitatif

PZR bias Zahmetli Pahalı Klonlama bias

Mikrobiyota Analiz YoumlntemleriYontem (Uumlretici firma) Avantaj Dezavantaj

Pirodizileme

(454 GS FLX+ Roche)

Okuma suumlresi uzundur Yuumlksek verimlilik Duyarlı Aynı zamanda birden fazla

oumlrneği analiz etme imkanı Koloni biası yoktur

Yuumlksek maliyet Homopolimerlerde

yuumlksek hata oranı Kısa sekans okuma Kapsamlı biyoinformatik

analize ihtiyaccedil duyar

Geri doumlnuumlşuumlmluuml terminatoumlr

dizileme

(HiSeq20002500 Illumina)

Etkili Okuma suumlrelerini iyileştiren

suumlreklilik Yuumlksek verimlilik Manuel iş azlığı

Uzun ccedilalışma suumlresi Kısa okuma uzunlukları Yuumlkseltme geliştirilme

Ligasyon

(5500xl SOLiD Life

Technologies)

Duumlşuumlk hata oranı Yuumlksek verimlilik

Ccedilok kısa uzunluklar Uzun ccedilalışma suumlresi

Gerccedilek zamanlı sekans (PacBio

RS Pacific Biosceince)

Oumlrnek hazırlaması kolay Reaktif maliyeti duumlşuumlk Ccedilok uzun okuma uzunluğu

Hata oranı yuumlksek Sistem maliyeti yuumlksek Sistem kurulumu zordur

Mikrobiyota Analiz Youmlntemleri

Bioresour Technol 2015 Sep192735-40 doi 101016jbiortech201505086 Epub 2015 Jun 9Use of PCR-DGGE based molecular methods to assessment of microbial diversity during anaerobic treatment of antibiotic combinationsAydin S1 Shahi A2 Ozbayram EG2 Ince B3 Ince O2Author information

AbstractAs it is currently often not know how anaerobic bioreactors eg for biogas production react if the substrate is contaminated by toxic compounds like antibiotics This study evaluated how anaerobic sequencing batch reactors were affected by amendments of different antibiotics and stepwise increasing concentrations The compositions of microbial community were determined in the seed sludge using 16S rRNA gene clone libraries and PCR-DGGE analyses were used for the detection of microbial community changes upon antibiotics additions According to PCR-DGGE results the syntrophic interaction of acetogens and methanogens is critical to the performance of the reactors Failure to maintain the stability of these microorganisms resulted in a decrease in the performance and stability of the anaerobic reactors Assessment of DGGE data is also useful for suggesting the potential to control ultimate microbial community structure especially derived from Gram-negative bacteria through bioaugmentation to successful for antibiotic biodegradationCopyright copy 2015 Elsevier Ltd All rights reserved

Mikrobiyota Analiz YoumlntemleriYontem (Uumlretici firma) Avantaj Dezavantaj

Pirodizileme

(454 GS FLX+ Roche)

Okuma suumlresi uzundur Yuumlksek verimlilik Duyarlı Aynı zamanda birden fazla

oumlrneği analiz etme imkanı Koloni biası yoktur

Yuumlksek maliyet Homopolimerlerde

yuumlksek hata oranı Kısa sekans okuma Kapsamlı biyoinformatik

analize ihtiyaccedil duyar

Geri doumlnuumlşuumlmluuml terminatoumlr

dizileme

(HiSeq20002500 Illumina)

Etkili Okuma suumlrelerini iyileştiren

suumlreklilik Yuumlksek verimlilik Manuel iş azlığı

Uzun ccedilalışma suumlresi Kısa okuma uzunlukları Yuumlkseltme geliştirilme

Ligasyon

(5500xl SOLiD Life

Technologies)

Duumlşuumlk hata oranı Yuumlksek verimlilik

Ccedilok kısa uzunluklar Uzun ccedilalışma suumlresi

Gerccedilek zamanlı sekans (PacBio

RS Pacific Biosceince)

Oumlrnek hazırlaması kolay Reaktif maliyeti duumlşuumlk Ccedilok uzun okuma uzunluğu

Hata oranı yuumlksek Sistem maliyeti yuumlksek Sistem kurulumu zordur

Mikrobiyota Analiz Youmlntemleri

Bioresour Technol 2015 Sep192735-40 doi 101016jbiortech201505086 Epub 2015 Jun 9Use of PCR-DGGE based molecular methods to assessment of microbial diversity during anaerobic treatment of antibiotic combinationsAydin S1 Shahi A2 Ozbayram EG2 Ince B3 Ince O2Author information

AbstractAs it is currently often not know how anaerobic bioreactors eg for biogas production react if the substrate is contaminated by toxic compounds like antibiotics This study evaluated how anaerobic sequencing batch reactors were affected by amendments of different antibiotics and stepwise increasing concentrations The compositions of microbial community were determined in the seed sludge using 16S rRNA gene clone libraries and PCR-DGGE analyses were used for the detection of microbial community changes upon antibiotics additions According to PCR-DGGE results the syntrophic interaction of acetogens and methanogens is critical to the performance of the reactors Failure to maintain the stability of these microorganisms resulted in a decrease in the performance and stability of the anaerobic reactors Assessment of DGGE data is also useful for suggesting the potential to control ultimate microbial community structure especially derived from Gram-negative bacteria through bioaugmentation to successful for antibiotic biodegradationCopyright copy 2015 Elsevier Ltd All rights reserved

Mikrobiyota Analiz Youmlntemleri

Bioresour Technol 2015 Sep192735-40 doi 101016jbiortech201505086 Epub 2015 Jun 9Use of PCR-DGGE based molecular methods to assessment of microbial diversity during anaerobic treatment of antibiotic combinationsAydin S1 Shahi A2 Ozbayram EG2 Ince B3 Ince O2Author information

AbstractAs it is currently often not know how anaerobic bioreactors eg for biogas production react if the substrate is contaminated by toxic compounds like antibiotics This study evaluated how anaerobic sequencing batch reactors were affected by amendments of different antibiotics and stepwise increasing concentrations The compositions of microbial community were determined in the seed sludge using 16S rRNA gene clone libraries and PCR-DGGE analyses were used for the detection of microbial community changes upon antibiotics additions According to PCR-DGGE results the syntrophic interaction of acetogens and methanogens is critical to the performance of the reactors Failure to maintain the stability of these microorganisms resulted in a decrease in the performance and stability of the anaerobic reactors Assessment of DGGE data is also useful for suggesting the potential to control ultimate microbial community structure especially derived from Gram-negative bacteria through bioaugmentation to successful for antibiotic biodegradationCopyright copy 2015 Elsevier Ltd All rights reserved

Bioresour Technol 2015 Sep192735-40 doi 101016jbiortech201505086 Epub 2015 Jun 9Use of PCR-DGGE based molecular methods to assessment of microbial diversity during anaerobic treatment of antibiotic combinationsAydin S1 Shahi A2 Ozbayram EG2 Ince B3 Ince O2Author information

AbstractAs it is currently often not know how anaerobic bioreactors eg for biogas production react if the substrate is contaminated by toxic compounds like antibiotics This study evaluated how anaerobic sequencing batch reactors were affected by amendments of different antibiotics and stepwise increasing concentrations The compositions of microbial community were determined in the seed sludge using 16S rRNA gene clone libraries and PCR-DGGE analyses were used for the detection of microbial community changes upon antibiotics additions According to PCR-DGGE results the syntrophic interaction of acetogens and methanogens is critical to the performance of the reactors Failure to maintain the stability of these microorganisms resulted in a decrease in the performance and stability of the anaerobic reactors Assessment of DGGE data is also useful for suggesting the potential to control ultimate microbial community structure especially derived from Gram-negative bacteria through bioaugmentation to successful for antibiotic biodegradationCopyright copy 2015 Elsevier Ltd All rights reserved

Int J Food Microbiol 2012 Feb 15153(3)428-35 doi 101016jijfoodmicro201112008 Epub 2011 Dec 13

Combination of culture-dependent and culture-independent molecular methods for the determination of lactic microbiota in sucukKesmen Z1 Yetiman AE Gulluce A Kacmaz N Sagdic O Cetin B Adiguzel A Sahin F Yetim HAbstractIn this study the culture-dependent and culture-independent molecular methods were used for the identification of lactic acid bacteria (LAB) in sucuk a Turkish fermented dry sausage On the one hand the PCR-DGGE method targetting the V1 and V3 regions of 16S DNA was applied to DNA that was directly extracted from sucuk samples On the other hand rep-PCR fingerprinting was performed for the primary differentiation and grouping of the isolates and the results were confirmed by sequencing of the 16S rDNA and 16S-23S rDNA intergenic spacer region As a result of the PCR-DGGE analysis of all the samples total 8 different lactic acid bacteria were identified and Lactobacillus sakei Lactobacillus curvatus and Weissella viridescens were the dominant microbiota among these bacteria The culture-dependent approach indicated that the majority of the strains belonged to the Lactobacillus genera including Lb sakei Lactobacillus plantarum Lb curvatus Lactobacillus brevis Lactobacillus farciminisand Lactobacillus alimentarius However Leuconostoc and Weisella were also detected as minor genera Again Lactococcus piscium Weissella halotolerans Staphylococcus succinus and the comigratedStaphylococcus piscifermentansStaphylococcus condimentiStaphylococcus carnosus group were detected only with the culture-independent method while Lb plantarum Leuconostoc mesenteroidesand Leuconostoc citreum were identified only by using the culture-dependent method In the results it was concluded that the combination of culture-dependent and culture-independent methods was necessary for reliable and detailed investigation of LAB communities in fermented food products

Folia Microbiol (Praha) 2013 May58(3)201-10 doi 101007s12223-012-0199-1 Epub 2012 Oct 4

Culturable bacterial microbiota of Plagiodera versicolora (L) (Coleoptera Chrysomelidae) and virulence of the isolated strainsDemirci M1 Sevim E Demir İ Sevim AAuthor information

AbstractPlagiodera versicolora (Laicharting 1781) (Coleoptera Chrysomelidae) is an important forest pest which damages many trees such as willow poplar and hazelnut In order to find new microbes that can be utilized as a possible microbial control agent against this pest we investigated the culturablebacterial flora of it and tested the isolated bacteria against P versicolora larvae and adults We were able to isolate nine bacteria from larvae and adults The isolates were characterized using a combination of morphological biochemical and physiological methods Additionally we sequenced the partial sequence of the 16S rRNA gene to verify conventional identification results Based on characterization studies the isolates were identified as Staphylococcus sp Pv1 Rahnella sp Pv2 Rahnella sp Pv3 Rahnella sp Pv4 Rahnella sp Pv5 Pantoea agglomerans Pv6 Staphylococcus sp Pv7 Micrococcus luteus Pv8 and Rahnella sp Pv9 The highest insecticidal activity against larvae and adults was obtained from M luteus Pv8 with 50 and 40 mortalities within 10 days after treatment respectively Extracellular enzyme activity of the bacterial isolates such as amylase proteinase lipase cellulose and chitinase was also determined Consequently our results show that M luteus Pv8 might be a good candidate as a possible microbial control agent against P versicolora and were discussed with respect to biocontrol potential of the bacterial isolates

J Pediatr Gastroenterol Nutr 2013 Mar56(3)328-32 doi 101097MPG0b013e31827a964bBreast milk jaundice effect of bacteria present in breast milk and infant fecesTuzun F1 Kumral A Duman N Ozkan HAuthor information

AbstractOBJECTIVEBreast milk is an important source of bacteria in establishing the infantile intestinal microbiota that appear to influence the enterohepatic circulation of bilirubin The aim of the present study was to evaluate the effect of breast milks microbiological content on the development of breast milk jaundice (BMJ)METHODSA total number of 60 mother-infant pairs enrolled to the study Two groups were defined BMJ group (n=30) full-term otherwise healthy newborns who were considered BMJ control group (n=30) full-term healthy newborns without jaundice All newborns in the study were exclusively breast-fed The breast milk samples and the feces of infants were evaluated for content of selected bacterial populations (Bifidobacterium Lactobacillus Clostridium Staphylococcus and Streptococcus species) with real-time polymerase chain reactionRESULTSBifidobacterium bifidum content in the breast milk and B adolescentis B bifidum and B longum content in the fecal samples were higher in the control group than in the BMJ group The milk and fecal concentrations of B bifidum were significantly correlated The concentrations of breast milk B bifidum and fecal B bifidum B adolescentis and B longum were found to be negatively correlated with bilirubin levelsCONCLUSIONSOur results suggest that Bifidobacterium species in breast milk may protect against BMJ

Mikrobiyota ccedilalışmalarında molekuumlleryoumlntemler

1 PCR (qPCR ile 16S rRNA kantitasyonu)

2 DNA Parmak izi analizleri

1 Denaturating gradient jel elektroforezi

2 SSCP

3 T-RFLP

3 Gen kuumltuumlphanesi oluşturulması ve 16S rRNA sekans

1 Amplifikasyon uumlruumlnlerinin plazmid aracılığı iletransformasyonu dizi analizi

2 Yeni Nesil Dizileme

OumlZEL TEŞEKKUumlR

CEREN ERDOĞDU

SERAP SUumlZUumlK

Helicobacter pylori Tedavisinin Bağırsak Mikrobiyota Uumlzerine Etkisi

Serap Suzuk1 Meltem Yalınay2 Tarkan Karakan3

1 THSK Mikrobiyoloji referans Laboratuvarı DB2 GUumlTF Tıbbi Mikrobiyoloji Anabilim Dalı

3 GUumlTF Gastroenteroloji Bilim Dalı

3 KLİMUD Kongresi Antalya 19-22 Kasım 2015

Ccedilalışmanın Amacı

bull Helicobacter pylori (HP) tedavisi alan goumlnuumllluumllerde antibiyotik kullanımının bağırsak mikrobiyotası uumlzerindeki etkilerini qPZRyoumlntemi ile goumlstermek

Ccedilalışmanın Dizaynı

Etik Kurul Onayı Alınması

Goumlnuumllluumllerin Seccedililmesi

Antibiyotik kullanım oumlncesi gaita oumlrneğinin

alınması

10 guumlnluumlk PPI+TET+MET+BİZ

Tedavi bittikten 6 hafta sonra

Antibiyotik kullanım sonrası gaita oumlrneğinin

alınması

İki grup gaita oumlrneğinden qPZR ile seccedililen

bakterilerin kantitasyonunun

yapılması

İstatistiksel Analiz

Materyal Metod

bull Bu ccedilalışmada kullanılan standart suşlar

ndash Bifidobacterium breve ATCC 15700

ndash Bacteroides fragilis ATCC 25285

ndash Lactobacillus acidophilus ATCC 4356

ndash Akkermansia muciniphila ATCC BAA-835

ndash Faecalibacterium prausnitzii ATCC 27766

Materyal Metod

ATCC suşlarındankuumlltuumlr yapıldı

Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)

-20degC saklandı

Gram Boyama

A260dalga boyunda 3 okumaDNA kopya sayısı hesaplandı

Standart Eğriler oluşturuldu (110

1100 11000 110000 1100000 diluumlsyonlar )

Materyal Metod

39 Goumlnuumllluumlden Gaita Oumlrneği Alındı Tedaviden 6 Hafta Sonra

Goumlnuumllluumllerin 18rsquoinden Antibiyotik

Kullanım Sonrası Gaita Oumlrneği Alındı

200 mg gaita-80degC saklandı

200 mg gaita-80degC saklandı

Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)

-20degC saklandı

Ekstraksiyon(QIAmp DNA StoolMini Kit Qiagen Almanya)

-20degC saklandı

ordmC

72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96

Flu

ore

scence

7

6

5

4

3

2

1

ordmC

73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97

dF

dT

10

08

06

04

02

00

Bif

Threshold

Materyal Metod

bull Verilerin tuumlmuuml Microsoft Excele girilerek logbakteriyel miktarı hesaplandı

bull Veriler SPSS 220 paket programı ile analiz edildi

bull Antibiyotik kullanım oumlncesi ve antibiyotik kullanım sonrası karşılaştırmalar Mann-Whitney U Testi ile değerlendirildi

Bulgular

Bakteriler

Antibiyotik Kullanım

Oumlncesi OrtalamaplusmnSS

(log10g)

Antibiyotik Kullanım

Sonrası OrtalamaplusmnSS

(log10g)

p

Bifidobacterium spp 781plusmn112 611plusmn274 0000

B fragilis 824plusmn119 569plusmn289 0000

Lactobacillus spp 739plusmn061 632plusmn174 0000

A mucinophilia 808plusmn060 610plusmn086 0000

F prausnitzii 921plusmn209 632plusmn159 0000

SS Standart Sapma

Nonalkolik Karaciğer Yağlanması Olan Hastalar ve Sağlıklı Kontrollerde

Bağırsak Mikrobiyotasının MolekuumllerYoumlntemlerle Karşılaştırılması

Ceren ErdoğduDanışman ProfDr Meltem Yalınay

9 Eyluumll 2016Ankara

İntestinal mikrobiyota ve NAFLD ilişkisi

Lİ et al 2013 Journal of Parenteral and Enteral Nutrition

Hipotez

bull NAFLD ve kontrol mikrobiyotası arasındafarklılıklar bulunmaktadır

ndash Mikrobiyotadaki farklılıklar barsaktan dolaşımageccedilen endotoksin miktarını arttırabilir

ndash İnflamasyon artabilir

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

Metod

bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)

bull 38 sağlıklı kontrol

ge18 yaşında olan

artmış serum aminotransferaz seviyeleri goumlzlenen

alkol tuumlketimi lt20 grhafta olan

son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan

eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

Metod

bull US ve artmış karaciğer enzimleri

bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz

oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli

fibrozis olarak değerlendirilmiştir

bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)

Brunt EM 1999 Liver

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

qPCR analizleri iccedilin standartlarınhazırlanması

Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922

Kuumlltuumlr05 Mc Farland Suumlspansiyon

cfu sayım

Ekstraksiyon

Nanodrop Oumllccediluumlm

110 diluumlsyon

Standart eğri

0

5

10

15

20

25

30

35

1E+02 1E+04 1E+06 1E+08

Cro

ssin

g-p

oin

t (C

p)

valu

e

DNA copy number microL

A muciniphila

B fragilis group

Bifidobacterium spp

F prausnitzii

Enterobacteriaceae

Lactobacillus spp

Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml

Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml

Dışkı oumlrneklerinden qPCR analizleri

Dışkı oumlrnekleri

200 mg Ekstraksiyon Nanodrop Oumllccediluumlm

Roche LightCycler 20

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi

Hasta (n=49) Kontrol (n=35)

Her oumlrnekten 50 ngmicroL havuzlandı

LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR

Agaroz Jelde Goumlruumlntuumlleme

Purifikasyon

TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)

kullanarak amplifikasyon uumlruumlnlerinin ligasyonu

Ecoli JM 109 kullanarak transformasyon

Ampisilin iccedileren LB besiyerlerine kuumlltuumlr

Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi

HastaKontr

olHasta

Kontr

ol

Lac-F Lac-R Koloni PCR

Agaroz jelde goumlruumlntuumlleme

Purifikasyon

g-Bifid-F g-Bifid-R koloni PCR

Sekans PubMed Blast 97 seq similarity

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

İnflamasyon Belirteccedilleri

İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı

hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci

Yoneda et al 2007 J Gastroenterol

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması

bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

dış

kı

Kontrol

NAFLDp=0003

p=0001 plt0001

Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması

A m

ucinip

hilia

F pra

usnitz

ii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

dış

kı

F0-F1

F2

plt0001

Enterobacteriaceae ile BKİ arasındakipozitif korelasyon

20 30 400

5

10

15

BMI

En

tero

ba

cte

ria

ce

ae

lo

g1

0g

r d

ışk

ı

BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)

-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)

Kontr

ol

NAFLD

-05

00

05

10

15

20

mg

dL

Kontrol

NAFLD

hs-CRP

NAFLD ve kontrol grubu hs-CRP seviyeleri

-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)

Kontr

ol

NAFLD

-20

0

20

40

60E

Um

L

Endotoxin

NAFLD ve kontrol grubu serum endotoksin duumlzeyleri

NAFLD ve kontrol grubu sitokin duumlzeyleri

bull TNF-α ve IL-6 seviyeleri bakımından fark yok

bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Pat

ient

0

20

40

60

80

IL-6

pg

mL

Control

Patient

NAFLD ve kontrol grubundaLactobacillus tuumlrleri

L delbrueckii 9

Lruminis50

Lmucosae2

Lacidophilus4

Lgastricus2

Lsalivarius4

Uncultured Lactobacillus

spp18

L helveticus7

Lsakei4

Kontrol

L delbrueckii Lruminis

Lmucosae Lacidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L helveticus

Lsakei

Ldelbrueckii 3

Lruminis39

Lmucosae9

L acidophilus11

Lgastricus6

Lsalivarius3

Uncultured Lactobacillus

spp5

L Johsonii5

Lreuteri19

NAFLD

Ldelbrueckii Lruminis

Lmucosae L acidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L Johsonii

Lreuteri

NAFLD ve kontrol grubundaBifidobacterium tuumlrleri

B adolescentis55

Blongum15

Bbifidum6

Bkashiwanohense10

Bcatenulatum4

Bruminatium2

Uncultured Bifidobacterium

spp Clone8

Kontrol

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Bcatenulatum

Bruminatium

Uncultured Bifidobacterium spp Clone

B adolescentis42

Blongum36

Bbifidum2

Bkashiwanohense2

Binfantis8

Uncultured Bifidobacterium

spp clone10

NAFLD

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Binfantis

Uncultured Bifidobacterium spp clone

Sonuccedil

NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir

A muciniphila NAFLD grubunda daha duumlşuumlk orandadır

Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir

B fragilis kilo durumuna bağımlı bir değişken

Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir

Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir

F prausnitzii mikrobiyotanın dominant bir uumlyesi

Sonuccedil

bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı

bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı

bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi

Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25

CEREN ERDOĞDU

Meltem Yalınay

Tarkan Karakan

Martin J Blaser

Barış Otlu

Doruk Engin

Sinem Solmaz

Berrin Oumlztuumlrk

GUuml Endoskopi Uumlnitesi

GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)

NYU Biyoistatistik Departmanı (Huilin Li)

Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)

Roche Diagnostics teknik ekibi

Teşekkuumlrler

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

OumlZEL TEŞEKKUumlR

CEREN ERDOĞDU

SERAP SUumlZUumlK

Helicobacter pylori Tedavisinin Bağırsak Mikrobiyota Uumlzerine Etkisi

Serap Suzuk1 Meltem Yalınay2 Tarkan Karakan3

1 THSK Mikrobiyoloji referans Laboratuvarı DB2 GUumlTF Tıbbi Mikrobiyoloji Anabilim Dalı

3 GUumlTF Gastroenteroloji Bilim Dalı

3 KLİMUD Kongresi Antalya 19-22 Kasım 2015

Ccedilalışmanın Amacı

bull Helicobacter pylori (HP) tedavisi alan goumlnuumllluumllerde antibiyotik kullanımının bağırsak mikrobiyotası uumlzerindeki etkilerini qPZRyoumlntemi ile goumlstermek

Ccedilalışmanın Dizaynı

Etik Kurul Onayı Alınması

Goumlnuumllluumllerin Seccedililmesi

Antibiyotik kullanım oumlncesi gaita oumlrneğinin

alınması

10 guumlnluumlk PPI+TET+MET+BİZ

Tedavi bittikten 6 hafta sonra

Antibiyotik kullanım sonrası gaita oumlrneğinin

alınması

İki grup gaita oumlrneğinden qPZR ile seccedililen

bakterilerin kantitasyonunun

yapılması

İstatistiksel Analiz

Materyal Metod

bull Bu ccedilalışmada kullanılan standart suşlar

ndash Bifidobacterium breve ATCC 15700

ndash Bacteroides fragilis ATCC 25285

ndash Lactobacillus acidophilus ATCC 4356

ndash Akkermansia muciniphila ATCC BAA-835

ndash Faecalibacterium prausnitzii ATCC 27766

Materyal Metod

ATCC suşlarındankuumlltuumlr yapıldı

Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)

-20degC saklandı

Gram Boyama

A260dalga boyunda 3 okumaDNA kopya sayısı hesaplandı

Standart Eğriler oluşturuldu (110

1100 11000 110000 1100000 diluumlsyonlar )

Materyal Metod

39 Goumlnuumllluumlden Gaita Oumlrneği Alındı Tedaviden 6 Hafta Sonra

Goumlnuumllluumllerin 18rsquoinden Antibiyotik

Kullanım Sonrası Gaita Oumlrneği Alındı

200 mg gaita-80degC saklandı

200 mg gaita-80degC saklandı

Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)

-20degC saklandı

Ekstraksiyon(QIAmp DNA StoolMini Kit Qiagen Almanya)

-20degC saklandı

ordmC

72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96

Flu

ore

scence

7

6

5

4

3

2

1

ordmC

73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97

dF

dT

10

08

06

04

02

00

Bif

Threshold

Materyal Metod

bull Verilerin tuumlmuuml Microsoft Excele girilerek logbakteriyel miktarı hesaplandı

bull Veriler SPSS 220 paket programı ile analiz edildi

bull Antibiyotik kullanım oumlncesi ve antibiyotik kullanım sonrası karşılaştırmalar Mann-Whitney U Testi ile değerlendirildi

Bulgular

Bakteriler

Antibiyotik Kullanım

Oumlncesi OrtalamaplusmnSS

(log10g)

Antibiyotik Kullanım

Sonrası OrtalamaplusmnSS

(log10g)

p

Bifidobacterium spp 781plusmn112 611plusmn274 0000

B fragilis 824plusmn119 569plusmn289 0000

Lactobacillus spp 739plusmn061 632plusmn174 0000

A mucinophilia 808plusmn060 610plusmn086 0000

F prausnitzii 921plusmn209 632plusmn159 0000

SS Standart Sapma

Nonalkolik Karaciğer Yağlanması Olan Hastalar ve Sağlıklı Kontrollerde

Bağırsak Mikrobiyotasının MolekuumllerYoumlntemlerle Karşılaştırılması

Ceren ErdoğduDanışman ProfDr Meltem Yalınay

9 Eyluumll 2016Ankara

İntestinal mikrobiyota ve NAFLD ilişkisi

Lİ et al 2013 Journal of Parenteral and Enteral Nutrition

Hipotez

bull NAFLD ve kontrol mikrobiyotası arasındafarklılıklar bulunmaktadır

ndash Mikrobiyotadaki farklılıklar barsaktan dolaşımageccedilen endotoksin miktarını arttırabilir

ndash İnflamasyon artabilir

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

Metod

bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)

bull 38 sağlıklı kontrol

ge18 yaşında olan

artmış serum aminotransferaz seviyeleri goumlzlenen

alkol tuumlketimi lt20 grhafta olan

son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan

eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

Metod

bull US ve artmış karaciğer enzimleri

bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz

oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli

fibrozis olarak değerlendirilmiştir

bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)

Brunt EM 1999 Liver

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

qPCR analizleri iccedilin standartlarınhazırlanması

Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922

Kuumlltuumlr05 Mc Farland Suumlspansiyon

cfu sayım

Ekstraksiyon

Nanodrop Oumllccediluumlm

110 diluumlsyon

Standart eğri

0

5

10

15

20

25

30

35

1E+02 1E+04 1E+06 1E+08

Cro

ssin

g-p

oin

t (C

p)

valu

e

DNA copy number microL

A muciniphila

B fragilis group

Bifidobacterium spp

F prausnitzii

Enterobacteriaceae

Lactobacillus spp

Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml

Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml

Dışkı oumlrneklerinden qPCR analizleri

Dışkı oumlrnekleri

200 mg Ekstraksiyon Nanodrop Oumllccediluumlm

Roche LightCycler 20

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi

Hasta (n=49) Kontrol (n=35)

Her oumlrnekten 50 ngmicroL havuzlandı

LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR

Agaroz Jelde Goumlruumlntuumlleme

Purifikasyon

TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)

kullanarak amplifikasyon uumlruumlnlerinin ligasyonu

Ecoli JM 109 kullanarak transformasyon

Ampisilin iccedileren LB besiyerlerine kuumlltuumlr

Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi

HastaKontr

olHasta

Kontr

ol

Lac-F Lac-R Koloni PCR

Agaroz jelde goumlruumlntuumlleme

Purifikasyon

g-Bifid-F g-Bifid-R koloni PCR

Sekans PubMed Blast 97 seq similarity

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

İnflamasyon Belirteccedilleri

İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı

hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci

Yoneda et al 2007 J Gastroenterol

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması

bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

dış

kı

Kontrol

NAFLDp=0003

p=0001 plt0001

Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması

A m

ucinip

hilia

F pra

usnitz

ii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

dış

kı

F0-F1

F2

plt0001

Enterobacteriaceae ile BKİ arasındakipozitif korelasyon

20 30 400

5

10

15

BMI

En

tero

ba

cte

ria

ce

ae

lo

g1

0g

r d

ışk

ı

BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)

-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)

Kontr

ol

NAFLD

-05

00

05

10

15

20

mg

dL

Kontrol

NAFLD

hs-CRP

NAFLD ve kontrol grubu hs-CRP seviyeleri

-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)

Kontr

ol

NAFLD

-20

0

20

40

60E

Um

L

Endotoxin

NAFLD ve kontrol grubu serum endotoksin duumlzeyleri

NAFLD ve kontrol grubu sitokin duumlzeyleri

bull TNF-α ve IL-6 seviyeleri bakımından fark yok

bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Pat

ient

0

20

40

60

80

IL-6

pg

mL

Control

Patient

NAFLD ve kontrol grubundaLactobacillus tuumlrleri

L delbrueckii 9

Lruminis50

Lmucosae2

Lacidophilus4

Lgastricus2

Lsalivarius4

Uncultured Lactobacillus

spp18

L helveticus7

Lsakei4

Kontrol

L delbrueckii Lruminis

Lmucosae Lacidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L helveticus

Lsakei

Ldelbrueckii 3

Lruminis39

Lmucosae9

L acidophilus11

Lgastricus6

Lsalivarius3

Uncultured Lactobacillus

spp5

L Johsonii5

Lreuteri19

NAFLD

Ldelbrueckii Lruminis

Lmucosae L acidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L Johsonii

Lreuteri

NAFLD ve kontrol grubundaBifidobacterium tuumlrleri

B adolescentis55

Blongum15

Bbifidum6

Bkashiwanohense10

Bcatenulatum4

Bruminatium2

Uncultured Bifidobacterium

spp Clone8

Kontrol

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Bcatenulatum

Bruminatium

Uncultured Bifidobacterium spp Clone

B adolescentis42

Blongum36

Bbifidum2

Bkashiwanohense2

Binfantis8

Uncultured Bifidobacterium

spp clone10

NAFLD

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Binfantis

Uncultured Bifidobacterium spp clone

Sonuccedil

NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir

A muciniphila NAFLD grubunda daha duumlşuumlk orandadır

Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir

B fragilis kilo durumuna bağımlı bir değişken

Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir

Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir

F prausnitzii mikrobiyotanın dominant bir uumlyesi

Sonuccedil

bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı

bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı

bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi

Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25

CEREN ERDOĞDU

Meltem Yalınay

Tarkan Karakan

Martin J Blaser

Barış Otlu

Doruk Engin

Sinem Solmaz

Berrin Oumlztuumlrk

GUuml Endoskopi Uumlnitesi

GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)

NYU Biyoistatistik Departmanı (Huilin Li)

Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)

Roche Diagnostics teknik ekibi

Teşekkuumlrler

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

Helicobacter pylori Tedavisinin Bağırsak Mikrobiyota Uumlzerine Etkisi

Serap Suzuk1 Meltem Yalınay2 Tarkan Karakan3

1 THSK Mikrobiyoloji referans Laboratuvarı DB2 GUumlTF Tıbbi Mikrobiyoloji Anabilim Dalı

3 GUumlTF Gastroenteroloji Bilim Dalı

3 KLİMUD Kongresi Antalya 19-22 Kasım 2015

Ccedilalışmanın Amacı

bull Helicobacter pylori (HP) tedavisi alan goumlnuumllluumllerde antibiyotik kullanımının bağırsak mikrobiyotası uumlzerindeki etkilerini qPZRyoumlntemi ile goumlstermek

Ccedilalışmanın Dizaynı

Etik Kurul Onayı Alınması

Goumlnuumllluumllerin Seccedililmesi

Antibiyotik kullanım oumlncesi gaita oumlrneğinin

alınması

10 guumlnluumlk PPI+TET+MET+BİZ

Tedavi bittikten 6 hafta sonra

Antibiyotik kullanım sonrası gaita oumlrneğinin

alınması

İki grup gaita oumlrneğinden qPZR ile seccedililen

bakterilerin kantitasyonunun

yapılması

İstatistiksel Analiz

Materyal Metod

bull Bu ccedilalışmada kullanılan standart suşlar

ndash Bifidobacterium breve ATCC 15700

ndash Bacteroides fragilis ATCC 25285

ndash Lactobacillus acidophilus ATCC 4356

ndash Akkermansia muciniphila ATCC BAA-835

ndash Faecalibacterium prausnitzii ATCC 27766

Materyal Metod

ATCC suşlarındankuumlltuumlr yapıldı

Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)

-20degC saklandı

Gram Boyama

A260dalga boyunda 3 okumaDNA kopya sayısı hesaplandı

Standart Eğriler oluşturuldu (110

1100 11000 110000 1100000 diluumlsyonlar )

Materyal Metod

39 Goumlnuumllluumlden Gaita Oumlrneği Alındı Tedaviden 6 Hafta Sonra

Goumlnuumllluumllerin 18rsquoinden Antibiyotik

Kullanım Sonrası Gaita Oumlrneği Alındı

200 mg gaita-80degC saklandı

200 mg gaita-80degC saklandı

Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)

-20degC saklandı

Ekstraksiyon(QIAmp DNA StoolMini Kit Qiagen Almanya)

-20degC saklandı

ordmC

72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96

Flu

ore

scence

7

6

5

4

3

2

1

ordmC

73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97

dF

dT

10

08

06

04

02

00

Bif

Threshold

Materyal Metod

bull Verilerin tuumlmuuml Microsoft Excele girilerek logbakteriyel miktarı hesaplandı

bull Veriler SPSS 220 paket programı ile analiz edildi

bull Antibiyotik kullanım oumlncesi ve antibiyotik kullanım sonrası karşılaştırmalar Mann-Whitney U Testi ile değerlendirildi

Bulgular

Bakteriler

Antibiyotik Kullanım

Oumlncesi OrtalamaplusmnSS

(log10g)

Antibiyotik Kullanım

Sonrası OrtalamaplusmnSS

(log10g)

p

Bifidobacterium spp 781plusmn112 611plusmn274 0000

B fragilis 824plusmn119 569plusmn289 0000

Lactobacillus spp 739plusmn061 632plusmn174 0000

A mucinophilia 808plusmn060 610plusmn086 0000

F prausnitzii 921plusmn209 632plusmn159 0000

SS Standart Sapma

Nonalkolik Karaciğer Yağlanması Olan Hastalar ve Sağlıklı Kontrollerde

Bağırsak Mikrobiyotasının MolekuumllerYoumlntemlerle Karşılaştırılması

Ceren ErdoğduDanışman ProfDr Meltem Yalınay

9 Eyluumll 2016Ankara

İntestinal mikrobiyota ve NAFLD ilişkisi

Lİ et al 2013 Journal of Parenteral and Enteral Nutrition

Hipotez

bull NAFLD ve kontrol mikrobiyotası arasındafarklılıklar bulunmaktadır

ndash Mikrobiyotadaki farklılıklar barsaktan dolaşımageccedilen endotoksin miktarını arttırabilir

ndash İnflamasyon artabilir

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

Metod

bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)

bull 38 sağlıklı kontrol

ge18 yaşında olan

artmış serum aminotransferaz seviyeleri goumlzlenen

alkol tuumlketimi lt20 grhafta olan

son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan

eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

Metod

bull US ve artmış karaciğer enzimleri

bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz

oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli

fibrozis olarak değerlendirilmiştir

bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)

Brunt EM 1999 Liver

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

qPCR analizleri iccedilin standartlarınhazırlanması

Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922

Kuumlltuumlr05 Mc Farland Suumlspansiyon

cfu sayım

Ekstraksiyon

Nanodrop Oumllccediluumlm

110 diluumlsyon

Standart eğri

0

5

10

15

20

25

30

35

1E+02 1E+04 1E+06 1E+08

Cro

ssin

g-p

oin

t (C

p)

valu

e

DNA copy number microL

A muciniphila

B fragilis group

Bifidobacterium spp

F prausnitzii

Enterobacteriaceae

Lactobacillus spp

Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml

Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml

Dışkı oumlrneklerinden qPCR analizleri

Dışkı oumlrnekleri

200 mg Ekstraksiyon Nanodrop Oumllccediluumlm

Roche LightCycler 20

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi

Hasta (n=49) Kontrol (n=35)

Her oumlrnekten 50 ngmicroL havuzlandı

LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR

Agaroz Jelde Goumlruumlntuumlleme

Purifikasyon

TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)

kullanarak amplifikasyon uumlruumlnlerinin ligasyonu

Ecoli JM 109 kullanarak transformasyon

Ampisilin iccedileren LB besiyerlerine kuumlltuumlr

Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi

HastaKontr

olHasta

Kontr

ol

Lac-F Lac-R Koloni PCR

Agaroz jelde goumlruumlntuumlleme

Purifikasyon

g-Bifid-F g-Bifid-R koloni PCR

Sekans PubMed Blast 97 seq similarity

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

İnflamasyon Belirteccedilleri

İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı

hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci

Yoneda et al 2007 J Gastroenterol

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması

bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

dış

kı

Kontrol

NAFLDp=0003

p=0001 plt0001

Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması

A m

ucinip

hilia

F pra

usnitz

ii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

dış

kı

F0-F1

F2

plt0001

Enterobacteriaceae ile BKİ arasındakipozitif korelasyon

20 30 400

5

10

15

BMI

En

tero

ba

cte

ria

ce

ae

lo

g1

0g

r d

ışk

ı

BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)

-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)

Kontr

ol

NAFLD

-05

00

05

10

15

20

mg

dL

Kontrol

NAFLD

hs-CRP

NAFLD ve kontrol grubu hs-CRP seviyeleri

-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)

Kontr

ol

NAFLD

-20

0

20

40

60E

Um

L

Endotoxin

NAFLD ve kontrol grubu serum endotoksin duumlzeyleri

NAFLD ve kontrol grubu sitokin duumlzeyleri

bull TNF-α ve IL-6 seviyeleri bakımından fark yok

bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Pat

ient

0

20

40

60

80

IL-6

pg

mL

Control

Patient

NAFLD ve kontrol grubundaLactobacillus tuumlrleri

L delbrueckii 9

Lruminis50

Lmucosae2

Lacidophilus4

Lgastricus2

Lsalivarius4

Uncultured Lactobacillus

spp18

L helveticus7

Lsakei4

Kontrol

L delbrueckii Lruminis

Lmucosae Lacidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L helveticus

Lsakei

Ldelbrueckii 3

Lruminis39

Lmucosae9

L acidophilus11

Lgastricus6

Lsalivarius3

Uncultured Lactobacillus

spp5

L Johsonii5

Lreuteri19

NAFLD

Ldelbrueckii Lruminis

Lmucosae L acidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L Johsonii

Lreuteri

NAFLD ve kontrol grubundaBifidobacterium tuumlrleri

B adolescentis55

Blongum15

Bbifidum6

Bkashiwanohense10

Bcatenulatum4

Bruminatium2

Uncultured Bifidobacterium

spp Clone8

Kontrol

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Bcatenulatum

Bruminatium

Uncultured Bifidobacterium spp Clone

B adolescentis42

Blongum36

Bbifidum2

Bkashiwanohense2

Binfantis8

Uncultured Bifidobacterium

spp clone10

NAFLD

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Binfantis

Uncultured Bifidobacterium spp clone

Sonuccedil

NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir

A muciniphila NAFLD grubunda daha duumlşuumlk orandadır

Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir

B fragilis kilo durumuna bağımlı bir değişken

Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir

Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir

F prausnitzii mikrobiyotanın dominant bir uumlyesi

Sonuccedil

bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı

bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı

bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi

Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25

CEREN ERDOĞDU

Meltem Yalınay

Tarkan Karakan

Martin J Blaser

Barış Otlu

Doruk Engin

Sinem Solmaz

Berrin Oumlztuumlrk

GUuml Endoskopi Uumlnitesi

GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)

NYU Biyoistatistik Departmanı (Huilin Li)

Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)

Roche Diagnostics teknik ekibi

Teşekkuumlrler

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

Ccedilalışmanın Amacı

bull Helicobacter pylori (HP) tedavisi alan goumlnuumllluumllerde antibiyotik kullanımının bağırsak mikrobiyotası uumlzerindeki etkilerini qPZRyoumlntemi ile goumlstermek

Ccedilalışmanın Dizaynı

Etik Kurul Onayı Alınması

Goumlnuumllluumllerin Seccedililmesi

Antibiyotik kullanım oumlncesi gaita oumlrneğinin

alınması

10 guumlnluumlk PPI+TET+MET+BİZ

Tedavi bittikten 6 hafta sonra

Antibiyotik kullanım sonrası gaita oumlrneğinin

alınması

İki grup gaita oumlrneğinden qPZR ile seccedililen

bakterilerin kantitasyonunun

yapılması

İstatistiksel Analiz

Materyal Metod

bull Bu ccedilalışmada kullanılan standart suşlar

ndash Bifidobacterium breve ATCC 15700

ndash Bacteroides fragilis ATCC 25285

ndash Lactobacillus acidophilus ATCC 4356

ndash Akkermansia muciniphila ATCC BAA-835

ndash Faecalibacterium prausnitzii ATCC 27766

Materyal Metod

ATCC suşlarındankuumlltuumlr yapıldı

Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)

-20degC saklandı

Gram Boyama

A260dalga boyunda 3 okumaDNA kopya sayısı hesaplandı

Standart Eğriler oluşturuldu (110

1100 11000 110000 1100000 diluumlsyonlar )

Materyal Metod

39 Goumlnuumllluumlden Gaita Oumlrneği Alındı Tedaviden 6 Hafta Sonra

Goumlnuumllluumllerin 18rsquoinden Antibiyotik

Kullanım Sonrası Gaita Oumlrneği Alındı

200 mg gaita-80degC saklandı

200 mg gaita-80degC saklandı

Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)

-20degC saklandı

Ekstraksiyon(QIAmp DNA StoolMini Kit Qiagen Almanya)

-20degC saklandı

ordmC

72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96

Flu

ore

scence

7

6

5

4

3

2

1

ordmC

73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97

dF

dT

10

08

06

04

02

00

Bif

Threshold

Materyal Metod

bull Verilerin tuumlmuuml Microsoft Excele girilerek logbakteriyel miktarı hesaplandı

bull Veriler SPSS 220 paket programı ile analiz edildi

bull Antibiyotik kullanım oumlncesi ve antibiyotik kullanım sonrası karşılaştırmalar Mann-Whitney U Testi ile değerlendirildi

Bulgular

Bakteriler

Antibiyotik Kullanım

Oumlncesi OrtalamaplusmnSS

(log10g)

Antibiyotik Kullanım

Sonrası OrtalamaplusmnSS

(log10g)

p

Bifidobacterium spp 781plusmn112 611plusmn274 0000

B fragilis 824plusmn119 569plusmn289 0000

Lactobacillus spp 739plusmn061 632plusmn174 0000

A mucinophilia 808plusmn060 610plusmn086 0000

F prausnitzii 921plusmn209 632plusmn159 0000

SS Standart Sapma

Nonalkolik Karaciğer Yağlanması Olan Hastalar ve Sağlıklı Kontrollerde

Bağırsak Mikrobiyotasının MolekuumllerYoumlntemlerle Karşılaştırılması

Ceren ErdoğduDanışman ProfDr Meltem Yalınay

9 Eyluumll 2016Ankara

İntestinal mikrobiyota ve NAFLD ilişkisi

Lİ et al 2013 Journal of Parenteral and Enteral Nutrition

Hipotez

bull NAFLD ve kontrol mikrobiyotası arasındafarklılıklar bulunmaktadır

ndash Mikrobiyotadaki farklılıklar barsaktan dolaşımageccedilen endotoksin miktarını arttırabilir

ndash İnflamasyon artabilir

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

Metod

bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)

bull 38 sağlıklı kontrol

ge18 yaşında olan

artmış serum aminotransferaz seviyeleri goumlzlenen

alkol tuumlketimi lt20 grhafta olan

son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan

eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

Metod

bull US ve artmış karaciğer enzimleri

bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz

oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli

fibrozis olarak değerlendirilmiştir

bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)

Brunt EM 1999 Liver

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

qPCR analizleri iccedilin standartlarınhazırlanması

Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922

Kuumlltuumlr05 Mc Farland Suumlspansiyon

cfu sayım

Ekstraksiyon

Nanodrop Oumllccediluumlm

110 diluumlsyon

Standart eğri

0

5

10

15

20

25

30

35

1E+02 1E+04 1E+06 1E+08

Cro

ssin

g-p

oin

t (C

p)

valu

e

DNA copy number microL

A muciniphila

B fragilis group

Bifidobacterium spp

F prausnitzii

Enterobacteriaceae

Lactobacillus spp

Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml

Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml

Dışkı oumlrneklerinden qPCR analizleri

Dışkı oumlrnekleri

200 mg Ekstraksiyon Nanodrop Oumllccediluumlm

Roche LightCycler 20

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi

Hasta (n=49) Kontrol (n=35)

Her oumlrnekten 50 ngmicroL havuzlandı

LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR

Agaroz Jelde Goumlruumlntuumlleme

Purifikasyon

TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)

kullanarak amplifikasyon uumlruumlnlerinin ligasyonu

Ecoli JM 109 kullanarak transformasyon

Ampisilin iccedileren LB besiyerlerine kuumlltuumlr

Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi

HastaKontr

olHasta

Kontr

ol

Lac-F Lac-R Koloni PCR

Agaroz jelde goumlruumlntuumlleme

Purifikasyon

g-Bifid-F g-Bifid-R koloni PCR

Sekans PubMed Blast 97 seq similarity

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

İnflamasyon Belirteccedilleri

İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı

hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci

Yoneda et al 2007 J Gastroenterol

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması

bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

dış

kı

Kontrol

NAFLDp=0003

p=0001 plt0001

Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması

A m

ucinip

hilia

F pra

usnitz

ii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

dış

kı

F0-F1

F2

plt0001

Enterobacteriaceae ile BKİ arasındakipozitif korelasyon

20 30 400

5

10

15

BMI

En

tero

ba

cte

ria

ce

ae

lo

g1

0g

r d

ışk

ı

BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)

-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)

Kontr

ol

NAFLD

-05

00

05

10

15

20

mg

dL

Kontrol

NAFLD

hs-CRP

NAFLD ve kontrol grubu hs-CRP seviyeleri

-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)

Kontr

ol

NAFLD

-20

0

20

40

60E

Um

L

Endotoxin

NAFLD ve kontrol grubu serum endotoksin duumlzeyleri

NAFLD ve kontrol grubu sitokin duumlzeyleri

bull TNF-α ve IL-6 seviyeleri bakımından fark yok

bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Pat

ient

0

20

40

60

80

IL-6

pg

mL

Control

Patient

NAFLD ve kontrol grubundaLactobacillus tuumlrleri

L delbrueckii 9

Lruminis50

Lmucosae2

Lacidophilus4

Lgastricus2

Lsalivarius4

Uncultured Lactobacillus

spp18

L helveticus7

Lsakei4

Kontrol

L delbrueckii Lruminis

Lmucosae Lacidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L helveticus

Lsakei

Ldelbrueckii 3

Lruminis39

Lmucosae9

L acidophilus11

Lgastricus6

Lsalivarius3

Uncultured Lactobacillus

spp5

L Johsonii5

Lreuteri19

NAFLD

Ldelbrueckii Lruminis

Lmucosae L acidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L Johsonii

Lreuteri

NAFLD ve kontrol grubundaBifidobacterium tuumlrleri

B adolescentis55

Blongum15

Bbifidum6

Bkashiwanohense10

Bcatenulatum4

Bruminatium2

Uncultured Bifidobacterium

spp Clone8

Kontrol

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Bcatenulatum

Bruminatium

Uncultured Bifidobacterium spp Clone

B adolescentis42

Blongum36

Bbifidum2

Bkashiwanohense2

Binfantis8

Uncultured Bifidobacterium

spp clone10

NAFLD

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Binfantis

Uncultured Bifidobacterium spp clone

Sonuccedil

NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir

A muciniphila NAFLD grubunda daha duumlşuumlk orandadır

Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir

B fragilis kilo durumuna bağımlı bir değişken

Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir

Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir

F prausnitzii mikrobiyotanın dominant bir uumlyesi

Sonuccedil

bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı

bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı

bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi

Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25

CEREN ERDOĞDU

Meltem Yalınay

Tarkan Karakan

Martin J Blaser

Barış Otlu

Doruk Engin

Sinem Solmaz

Berrin Oumlztuumlrk

GUuml Endoskopi Uumlnitesi

GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)

NYU Biyoistatistik Departmanı (Huilin Li)

Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)

Roche Diagnostics teknik ekibi

Teşekkuumlrler

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

Ccedilalışmanın Dizaynı

Etik Kurul Onayı Alınması

Goumlnuumllluumllerin Seccedililmesi

Antibiyotik kullanım oumlncesi gaita oumlrneğinin

alınması

10 guumlnluumlk PPI+TET+MET+BİZ

Tedavi bittikten 6 hafta sonra

Antibiyotik kullanım sonrası gaita oumlrneğinin

alınması

İki grup gaita oumlrneğinden qPZR ile seccedililen

bakterilerin kantitasyonunun

yapılması

İstatistiksel Analiz

Materyal Metod

bull Bu ccedilalışmada kullanılan standart suşlar

ndash Bifidobacterium breve ATCC 15700

ndash Bacteroides fragilis ATCC 25285

ndash Lactobacillus acidophilus ATCC 4356

ndash Akkermansia muciniphila ATCC BAA-835

ndash Faecalibacterium prausnitzii ATCC 27766

Materyal Metod

ATCC suşlarındankuumlltuumlr yapıldı

Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)

-20degC saklandı

Gram Boyama

A260dalga boyunda 3 okumaDNA kopya sayısı hesaplandı

Standart Eğriler oluşturuldu (110

1100 11000 110000 1100000 diluumlsyonlar )

Materyal Metod

39 Goumlnuumllluumlden Gaita Oumlrneği Alındı Tedaviden 6 Hafta Sonra

Goumlnuumllluumllerin 18rsquoinden Antibiyotik

Kullanım Sonrası Gaita Oumlrneği Alındı

200 mg gaita-80degC saklandı

200 mg gaita-80degC saklandı

Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)

-20degC saklandı

Ekstraksiyon(QIAmp DNA StoolMini Kit Qiagen Almanya)

-20degC saklandı

ordmC

72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96

Flu

ore

scence

7

6

5

4

3

2

1

ordmC

73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97

dF

dT

10

08

06

04

02

00

Bif

Threshold

Materyal Metod

bull Verilerin tuumlmuuml Microsoft Excele girilerek logbakteriyel miktarı hesaplandı

bull Veriler SPSS 220 paket programı ile analiz edildi

bull Antibiyotik kullanım oumlncesi ve antibiyotik kullanım sonrası karşılaştırmalar Mann-Whitney U Testi ile değerlendirildi

Bulgular

Bakteriler

Antibiyotik Kullanım

Oumlncesi OrtalamaplusmnSS

(log10g)

Antibiyotik Kullanım

Sonrası OrtalamaplusmnSS

(log10g)

p

Bifidobacterium spp 781plusmn112 611plusmn274 0000

B fragilis 824plusmn119 569plusmn289 0000

Lactobacillus spp 739plusmn061 632plusmn174 0000

A mucinophilia 808plusmn060 610plusmn086 0000

F prausnitzii 921plusmn209 632plusmn159 0000

SS Standart Sapma

Nonalkolik Karaciğer Yağlanması Olan Hastalar ve Sağlıklı Kontrollerde

Bağırsak Mikrobiyotasının MolekuumllerYoumlntemlerle Karşılaştırılması

Ceren ErdoğduDanışman ProfDr Meltem Yalınay

9 Eyluumll 2016Ankara

İntestinal mikrobiyota ve NAFLD ilişkisi

Lİ et al 2013 Journal of Parenteral and Enteral Nutrition

Hipotez

bull NAFLD ve kontrol mikrobiyotası arasındafarklılıklar bulunmaktadır

ndash Mikrobiyotadaki farklılıklar barsaktan dolaşımageccedilen endotoksin miktarını arttırabilir

ndash İnflamasyon artabilir

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

Metod

bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)

bull 38 sağlıklı kontrol

ge18 yaşında olan

artmış serum aminotransferaz seviyeleri goumlzlenen

alkol tuumlketimi lt20 grhafta olan

son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan

eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

Metod

bull US ve artmış karaciğer enzimleri

bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz

oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli

fibrozis olarak değerlendirilmiştir

bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)

Brunt EM 1999 Liver

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

qPCR analizleri iccedilin standartlarınhazırlanması

Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922

Kuumlltuumlr05 Mc Farland Suumlspansiyon

cfu sayım

Ekstraksiyon

Nanodrop Oumllccediluumlm

110 diluumlsyon

Standart eğri

0

5

10

15

20

25

30

35

1E+02 1E+04 1E+06 1E+08

Cro

ssin

g-p

oin

t (C

p)

valu

e

DNA copy number microL

A muciniphila

B fragilis group

Bifidobacterium spp

F prausnitzii

Enterobacteriaceae

Lactobacillus spp

Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml

Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml

Dışkı oumlrneklerinden qPCR analizleri

Dışkı oumlrnekleri

200 mg Ekstraksiyon Nanodrop Oumllccediluumlm

Roche LightCycler 20

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi

Hasta (n=49) Kontrol (n=35)

Her oumlrnekten 50 ngmicroL havuzlandı

LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR

Agaroz Jelde Goumlruumlntuumlleme

Purifikasyon

TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)

kullanarak amplifikasyon uumlruumlnlerinin ligasyonu

Ecoli JM 109 kullanarak transformasyon

Ampisilin iccedileren LB besiyerlerine kuumlltuumlr

Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi

HastaKontr

olHasta

Kontr

ol

Lac-F Lac-R Koloni PCR

Agaroz jelde goumlruumlntuumlleme

Purifikasyon

g-Bifid-F g-Bifid-R koloni PCR

Sekans PubMed Blast 97 seq similarity

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

İnflamasyon Belirteccedilleri

İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı

hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci

Yoneda et al 2007 J Gastroenterol

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması

bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

dış

kı

Kontrol

NAFLDp=0003

p=0001 plt0001

Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması

A m

ucinip

hilia

F pra

usnitz

ii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

dış

kı

F0-F1

F2

plt0001

Enterobacteriaceae ile BKİ arasındakipozitif korelasyon

20 30 400

5

10

15

BMI

En

tero

ba

cte

ria

ce

ae

lo

g1

0g

r d

ışk

ı

BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)

-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)

Kontr

ol

NAFLD

-05

00

05

10

15

20

mg

dL

Kontrol

NAFLD

hs-CRP

NAFLD ve kontrol grubu hs-CRP seviyeleri

-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)

Kontr

ol

NAFLD

-20

0

20

40

60E

Um

L

Endotoxin

NAFLD ve kontrol grubu serum endotoksin duumlzeyleri

NAFLD ve kontrol grubu sitokin duumlzeyleri

bull TNF-α ve IL-6 seviyeleri bakımından fark yok

bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Pat

ient

0

20

40

60

80

IL-6

pg

mL

Control

Patient

NAFLD ve kontrol grubundaLactobacillus tuumlrleri

L delbrueckii 9

Lruminis50

Lmucosae2

Lacidophilus4

Lgastricus2

Lsalivarius4

Uncultured Lactobacillus

spp18

L helveticus7

Lsakei4

Kontrol

L delbrueckii Lruminis

Lmucosae Lacidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L helveticus

Lsakei

Ldelbrueckii 3

Lruminis39

Lmucosae9

L acidophilus11

Lgastricus6

Lsalivarius3

Uncultured Lactobacillus

spp5

L Johsonii5

Lreuteri19

NAFLD

Ldelbrueckii Lruminis

Lmucosae L acidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L Johsonii

Lreuteri

NAFLD ve kontrol grubundaBifidobacterium tuumlrleri

B adolescentis55

Blongum15

Bbifidum6

Bkashiwanohense10

Bcatenulatum4

Bruminatium2

Uncultured Bifidobacterium

spp Clone8

Kontrol

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Bcatenulatum

Bruminatium

Uncultured Bifidobacterium spp Clone

B adolescentis42

Blongum36

Bbifidum2

Bkashiwanohense2

Binfantis8

Uncultured Bifidobacterium

spp clone10

NAFLD

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Binfantis

Uncultured Bifidobacterium spp clone

Sonuccedil

NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir

A muciniphila NAFLD grubunda daha duumlşuumlk orandadır

Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir

B fragilis kilo durumuna bağımlı bir değişken

Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir

Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir

F prausnitzii mikrobiyotanın dominant bir uumlyesi

Sonuccedil

bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı

bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı

bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi

Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25

CEREN ERDOĞDU

Meltem Yalınay

Tarkan Karakan

Martin J Blaser

Barış Otlu

Doruk Engin

Sinem Solmaz

Berrin Oumlztuumlrk

GUuml Endoskopi Uumlnitesi

GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)

NYU Biyoistatistik Departmanı (Huilin Li)

Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)

Roche Diagnostics teknik ekibi

Teşekkuumlrler

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

Materyal Metod

bull Bu ccedilalışmada kullanılan standart suşlar

ndash Bifidobacterium breve ATCC 15700

ndash Bacteroides fragilis ATCC 25285

ndash Lactobacillus acidophilus ATCC 4356

ndash Akkermansia muciniphila ATCC BAA-835

ndash Faecalibacterium prausnitzii ATCC 27766

Materyal Metod

ATCC suşlarındankuumlltuumlr yapıldı

Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)

-20degC saklandı

Gram Boyama

A260dalga boyunda 3 okumaDNA kopya sayısı hesaplandı

Standart Eğriler oluşturuldu (110

1100 11000 110000 1100000 diluumlsyonlar )

Materyal Metod

39 Goumlnuumllluumlden Gaita Oumlrneği Alındı Tedaviden 6 Hafta Sonra

Goumlnuumllluumllerin 18rsquoinden Antibiyotik

Kullanım Sonrası Gaita Oumlrneği Alındı

200 mg gaita-80degC saklandı

200 mg gaita-80degC saklandı

Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)

-20degC saklandı

Ekstraksiyon(QIAmp DNA StoolMini Kit Qiagen Almanya)

-20degC saklandı

ordmC

72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96

Flu

ore

scence

7

6

5

4

3

2

1

ordmC

73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97

dF

dT

10

08

06

04

02

00

Bif

Threshold

Materyal Metod

bull Verilerin tuumlmuuml Microsoft Excele girilerek logbakteriyel miktarı hesaplandı

bull Veriler SPSS 220 paket programı ile analiz edildi

bull Antibiyotik kullanım oumlncesi ve antibiyotik kullanım sonrası karşılaştırmalar Mann-Whitney U Testi ile değerlendirildi

Bulgular

Bakteriler

Antibiyotik Kullanım

Oumlncesi OrtalamaplusmnSS

(log10g)

Antibiyotik Kullanım

Sonrası OrtalamaplusmnSS

(log10g)

p

Bifidobacterium spp 781plusmn112 611plusmn274 0000

B fragilis 824plusmn119 569plusmn289 0000

Lactobacillus spp 739plusmn061 632plusmn174 0000

A mucinophilia 808plusmn060 610plusmn086 0000

F prausnitzii 921plusmn209 632plusmn159 0000

SS Standart Sapma

Nonalkolik Karaciğer Yağlanması Olan Hastalar ve Sağlıklı Kontrollerde

Bağırsak Mikrobiyotasının MolekuumllerYoumlntemlerle Karşılaştırılması

Ceren ErdoğduDanışman ProfDr Meltem Yalınay

9 Eyluumll 2016Ankara

İntestinal mikrobiyota ve NAFLD ilişkisi

Lİ et al 2013 Journal of Parenteral and Enteral Nutrition

Hipotez

bull NAFLD ve kontrol mikrobiyotası arasındafarklılıklar bulunmaktadır

ndash Mikrobiyotadaki farklılıklar barsaktan dolaşımageccedilen endotoksin miktarını arttırabilir

ndash İnflamasyon artabilir

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

Metod

bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)

bull 38 sağlıklı kontrol

ge18 yaşında olan

artmış serum aminotransferaz seviyeleri goumlzlenen

alkol tuumlketimi lt20 grhafta olan

son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan

eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

Metod

bull US ve artmış karaciğer enzimleri

bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz

oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli

fibrozis olarak değerlendirilmiştir

bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)

Brunt EM 1999 Liver

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

qPCR analizleri iccedilin standartlarınhazırlanması

Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922

Kuumlltuumlr05 Mc Farland Suumlspansiyon

cfu sayım

Ekstraksiyon

Nanodrop Oumllccediluumlm

110 diluumlsyon

Standart eğri

0

5

10

15

20

25

30

35

1E+02 1E+04 1E+06 1E+08

Cro

ssin

g-p

oin

t (C

p)

valu

e

DNA copy number microL

A muciniphila

B fragilis group

Bifidobacterium spp

F prausnitzii

Enterobacteriaceae

Lactobacillus spp

Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml

Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml

Dışkı oumlrneklerinden qPCR analizleri

Dışkı oumlrnekleri

200 mg Ekstraksiyon Nanodrop Oumllccediluumlm

Roche LightCycler 20

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi

Hasta (n=49) Kontrol (n=35)

Her oumlrnekten 50 ngmicroL havuzlandı

LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR

Agaroz Jelde Goumlruumlntuumlleme

Purifikasyon

TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)

kullanarak amplifikasyon uumlruumlnlerinin ligasyonu

Ecoli JM 109 kullanarak transformasyon

Ampisilin iccedileren LB besiyerlerine kuumlltuumlr

Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi

HastaKontr

olHasta

Kontr

ol

Lac-F Lac-R Koloni PCR

Agaroz jelde goumlruumlntuumlleme

Purifikasyon

g-Bifid-F g-Bifid-R koloni PCR

Sekans PubMed Blast 97 seq similarity

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

İnflamasyon Belirteccedilleri

İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı

hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci

Yoneda et al 2007 J Gastroenterol

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması

bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

dış

kı

Kontrol

NAFLDp=0003

p=0001 plt0001

Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması

A m

ucinip

hilia

F pra

usnitz

ii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

dış

kı

F0-F1

F2

plt0001

Enterobacteriaceae ile BKİ arasındakipozitif korelasyon

20 30 400

5

10

15

BMI

En

tero

ba

cte

ria

ce

ae

lo

g1

0g

r d

ışk

ı

BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)

-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)

Kontr

ol

NAFLD

-05

00

05

10

15

20

mg

dL

Kontrol

NAFLD

hs-CRP

NAFLD ve kontrol grubu hs-CRP seviyeleri

-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)

Kontr

ol

NAFLD

-20

0

20

40

60E

Um

L

Endotoxin

NAFLD ve kontrol grubu serum endotoksin duumlzeyleri

NAFLD ve kontrol grubu sitokin duumlzeyleri

bull TNF-α ve IL-6 seviyeleri bakımından fark yok

bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Pat

ient

0

20

40

60

80

IL-6

pg

mL

Control

Patient

NAFLD ve kontrol grubundaLactobacillus tuumlrleri

L delbrueckii 9

Lruminis50

Lmucosae2

Lacidophilus4

Lgastricus2

Lsalivarius4

Uncultured Lactobacillus

spp18

L helveticus7

Lsakei4

Kontrol

L delbrueckii Lruminis

Lmucosae Lacidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L helveticus

Lsakei

Ldelbrueckii 3

Lruminis39

Lmucosae9

L acidophilus11

Lgastricus6

Lsalivarius3

Uncultured Lactobacillus

spp5

L Johsonii5

Lreuteri19

NAFLD

Ldelbrueckii Lruminis

Lmucosae L acidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L Johsonii

Lreuteri

NAFLD ve kontrol grubundaBifidobacterium tuumlrleri

B adolescentis55

Blongum15

Bbifidum6

Bkashiwanohense10

Bcatenulatum4

Bruminatium2

Uncultured Bifidobacterium

spp Clone8

Kontrol

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Bcatenulatum

Bruminatium

Uncultured Bifidobacterium spp Clone

B adolescentis42

Blongum36

Bbifidum2

Bkashiwanohense2

Binfantis8

Uncultured Bifidobacterium

spp clone10

NAFLD

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Binfantis

Uncultured Bifidobacterium spp clone

Sonuccedil

NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir

A muciniphila NAFLD grubunda daha duumlşuumlk orandadır

Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir

B fragilis kilo durumuna bağımlı bir değişken

Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir

Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir

F prausnitzii mikrobiyotanın dominant bir uumlyesi

Sonuccedil

bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı

bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı

bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi

Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25

CEREN ERDOĞDU

Meltem Yalınay

Tarkan Karakan

Martin J Blaser

Barış Otlu

Doruk Engin

Sinem Solmaz

Berrin Oumlztuumlrk

GUuml Endoskopi Uumlnitesi

GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)

NYU Biyoistatistik Departmanı (Huilin Li)

Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)

Roche Diagnostics teknik ekibi

Teşekkuumlrler

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

Materyal Metod

ATCC suşlarındankuumlltuumlr yapıldı

Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)

-20degC saklandı

Gram Boyama

A260dalga boyunda 3 okumaDNA kopya sayısı hesaplandı

Standart Eğriler oluşturuldu (110

1100 11000 110000 1100000 diluumlsyonlar )

Materyal Metod

39 Goumlnuumllluumlden Gaita Oumlrneği Alındı Tedaviden 6 Hafta Sonra

Goumlnuumllluumllerin 18rsquoinden Antibiyotik

Kullanım Sonrası Gaita Oumlrneği Alındı

200 mg gaita-80degC saklandı

200 mg gaita-80degC saklandı

Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)

-20degC saklandı

Ekstraksiyon(QIAmp DNA StoolMini Kit Qiagen Almanya)

-20degC saklandı

ordmC

72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96

Flu

ore

scence

7

6

5

4

3

2

1

ordmC

73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97

dF

dT

10

08

06

04

02

00

Bif

Threshold

Materyal Metod

bull Verilerin tuumlmuuml Microsoft Excele girilerek logbakteriyel miktarı hesaplandı

bull Veriler SPSS 220 paket programı ile analiz edildi

bull Antibiyotik kullanım oumlncesi ve antibiyotik kullanım sonrası karşılaştırmalar Mann-Whitney U Testi ile değerlendirildi

Bulgular

Bakteriler

Antibiyotik Kullanım

Oumlncesi OrtalamaplusmnSS

(log10g)

Antibiyotik Kullanım

Sonrası OrtalamaplusmnSS

(log10g)

p

Bifidobacterium spp 781plusmn112 611plusmn274 0000

B fragilis 824plusmn119 569plusmn289 0000

Lactobacillus spp 739plusmn061 632plusmn174 0000

A mucinophilia 808plusmn060 610plusmn086 0000

F prausnitzii 921plusmn209 632plusmn159 0000

SS Standart Sapma

Nonalkolik Karaciğer Yağlanması Olan Hastalar ve Sağlıklı Kontrollerde

Bağırsak Mikrobiyotasının MolekuumllerYoumlntemlerle Karşılaştırılması

Ceren ErdoğduDanışman ProfDr Meltem Yalınay

9 Eyluumll 2016Ankara

İntestinal mikrobiyota ve NAFLD ilişkisi

Lİ et al 2013 Journal of Parenteral and Enteral Nutrition

Hipotez

bull NAFLD ve kontrol mikrobiyotası arasındafarklılıklar bulunmaktadır

ndash Mikrobiyotadaki farklılıklar barsaktan dolaşımageccedilen endotoksin miktarını arttırabilir

ndash İnflamasyon artabilir

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

Metod

bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)

bull 38 sağlıklı kontrol

ge18 yaşında olan

artmış serum aminotransferaz seviyeleri goumlzlenen

alkol tuumlketimi lt20 grhafta olan

son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan

eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

Metod

bull US ve artmış karaciğer enzimleri

bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz

oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli

fibrozis olarak değerlendirilmiştir

bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)

Brunt EM 1999 Liver

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

qPCR analizleri iccedilin standartlarınhazırlanması

Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922

Kuumlltuumlr05 Mc Farland Suumlspansiyon

cfu sayım

Ekstraksiyon

Nanodrop Oumllccediluumlm

110 diluumlsyon

Standart eğri

0

5

10

15

20

25

30

35

1E+02 1E+04 1E+06 1E+08

Cro

ssin

g-p

oin

t (C

p)

valu

e

DNA copy number microL

A muciniphila

B fragilis group

Bifidobacterium spp

F prausnitzii

Enterobacteriaceae

Lactobacillus spp

Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml

Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml

Dışkı oumlrneklerinden qPCR analizleri

Dışkı oumlrnekleri

200 mg Ekstraksiyon Nanodrop Oumllccediluumlm

Roche LightCycler 20

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi

Hasta (n=49) Kontrol (n=35)

Her oumlrnekten 50 ngmicroL havuzlandı

LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR

Agaroz Jelde Goumlruumlntuumlleme

Purifikasyon

TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)

kullanarak amplifikasyon uumlruumlnlerinin ligasyonu

Ecoli JM 109 kullanarak transformasyon

Ampisilin iccedileren LB besiyerlerine kuumlltuumlr

Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi

HastaKontr

olHasta

Kontr

ol

Lac-F Lac-R Koloni PCR

Agaroz jelde goumlruumlntuumlleme

Purifikasyon

g-Bifid-F g-Bifid-R koloni PCR

Sekans PubMed Blast 97 seq similarity

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

İnflamasyon Belirteccedilleri

İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı

hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci

Yoneda et al 2007 J Gastroenterol

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması

bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

dış

kı

Kontrol

NAFLDp=0003

p=0001 plt0001

Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması

A m

ucinip

hilia

F pra

usnitz

ii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

dış

kı

F0-F1

F2

plt0001

Enterobacteriaceae ile BKİ arasındakipozitif korelasyon

20 30 400

5

10

15

BMI

En

tero

ba

cte

ria

ce

ae

lo

g1

0g

r d

ışk

ı

BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)

-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)

Kontr

ol

NAFLD

-05

00

05

10

15

20

mg

dL

Kontrol

NAFLD

hs-CRP

NAFLD ve kontrol grubu hs-CRP seviyeleri

-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)

Kontr

ol

NAFLD

-20

0

20

40

60E

Um

L

Endotoxin

NAFLD ve kontrol grubu serum endotoksin duumlzeyleri

NAFLD ve kontrol grubu sitokin duumlzeyleri

bull TNF-α ve IL-6 seviyeleri bakımından fark yok

bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Pat

ient

0

20

40

60

80

IL-6

pg

mL

Control

Patient

NAFLD ve kontrol grubundaLactobacillus tuumlrleri

L delbrueckii 9

Lruminis50

Lmucosae2

Lacidophilus4

Lgastricus2

Lsalivarius4

Uncultured Lactobacillus

spp18

L helveticus7

Lsakei4

Kontrol

L delbrueckii Lruminis

Lmucosae Lacidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L helveticus

Lsakei

Ldelbrueckii 3

Lruminis39

Lmucosae9

L acidophilus11

Lgastricus6

Lsalivarius3

Uncultured Lactobacillus

spp5

L Johsonii5

Lreuteri19

NAFLD

Ldelbrueckii Lruminis

Lmucosae L acidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L Johsonii

Lreuteri

NAFLD ve kontrol grubundaBifidobacterium tuumlrleri

B adolescentis55

Blongum15

Bbifidum6

Bkashiwanohense10

Bcatenulatum4

Bruminatium2

Uncultured Bifidobacterium

spp Clone8

Kontrol

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Bcatenulatum

Bruminatium

Uncultured Bifidobacterium spp Clone

B adolescentis42

Blongum36

Bbifidum2

Bkashiwanohense2

Binfantis8

Uncultured Bifidobacterium

spp clone10

NAFLD

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Binfantis

Uncultured Bifidobacterium spp clone

Sonuccedil

NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir

A muciniphila NAFLD grubunda daha duumlşuumlk orandadır

Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir

B fragilis kilo durumuna bağımlı bir değişken

Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir

Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir

F prausnitzii mikrobiyotanın dominant bir uumlyesi

Sonuccedil

bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı

bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı

bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi

Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25

CEREN ERDOĞDU

Meltem Yalınay

Tarkan Karakan

Martin J Blaser

Barış Otlu

Doruk Engin

Sinem Solmaz

Berrin Oumlztuumlrk

GUuml Endoskopi Uumlnitesi

GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)

NYU Biyoistatistik Departmanı (Huilin Li)

Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)

Roche Diagnostics teknik ekibi

Teşekkuumlrler

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

Materyal Metod

39 Goumlnuumllluumlden Gaita Oumlrneği Alındı Tedaviden 6 Hafta Sonra

Goumlnuumllluumllerin 18rsquoinden Antibiyotik

Kullanım Sonrası Gaita Oumlrneği Alındı

200 mg gaita-80degC saklandı

200 mg gaita-80degC saklandı

Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)

-20degC saklandı

Ekstraksiyon(QIAmp DNA StoolMini Kit Qiagen Almanya)

-20degC saklandı

ordmC

72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96

Flu

ore

scence

7

6

5

4

3

2

1

ordmC

73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97

dF

dT

10

08

06

04

02

00

Bif

Threshold

Materyal Metod

bull Verilerin tuumlmuuml Microsoft Excele girilerek logbakteriyel miktarı hesaplandı

bull Veriler SPSS 220 paket programı ile analiz edildi

bull Antibiyotik kullanım oumlncesi ve antibiyotik kullanım sonrası karşılaştırmalar Mann-Whitney U Testi ile değerlendirildi

Bulgular

Bakteriler

Antibiyotik Kullanım

Oumlncesi OrtalamaplusmnSS

(log10g)

Antibiyotik Kullanım

Sonrası OrtalamaplusmnSS

(log10g)

p

Bifidobacterium spp 781plusmn112 611plusmn274 0000

B fragilis 824plusmn119 569plusmn289 0000

Lactobacillus spp 739plusmn061 632plusmn174 0000

A mucinophilia 808plusmn060 610plusmn086 0000

F prausnitzii 921plusmn209 632plusmn159 0000

SS Standart Sapma

Nonalkolik Karaciğer Yağlanması Olan Hastalar ve Sağlıklı Kontrollerde

Bağırsak Mikrobiyotasının MolekuumllerYoumlntemlerle Karşılaştırılması

Ceren ErdoğduDanışman ProfDr Meltem Yalınay

9 Eyluumll 2016Ankara

İntestinal mikrobiyota ve NAFLD ilişkisi

Lİ et al 2013 Journal of Parenteral and Enteral Nutrition

Hipotez

bull NAFLD ve kontrol mikrobiyotası arasındafarklılıklar bulunmaktadır

ndash Mikrobiyotadaki farklılıklar barsaktan dolaşımageccedilen endotoksin miktarını arttırabilir

ndash İnflamasyon artabilir

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

Metod

bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)

bull 38 sağlıklı kontrol

ge18 yaşında olan

artmış serum aminotransferaz seviyeleri goumlzlenen

alkol tuumlketimi lt20 grhafta olan

son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan

eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

Metod

bull US ve artmış karaciğer enzimleri

bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz

oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli

fibrozis olarak değerlendirilmiştir

bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)

Brunt EM 1999 Liver

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

qPCR analizleri iccedilin standartlarınhazırlanması

Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922

Kuumlltuumlr05 Mc Farland Suumlspansiyon

cfu sayım

Ekstraksiyon

Nanodrop Oumllccediluumlm

110 diluumlsyon

Standart eğri

0

5

10

15

20

25

30

35

1E+02 1E+04 1E+06 1E+08

Cro

ssin

g-p

oin

t (C

p)

valu

e

DNA copy number microL

A muciniphila

B fragilis group

Bifidobacterium spp

F prausnitzii

Enterobacteriaceae

Lactobacillus spp

Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml

Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml

Dışkı oumlrneklerinden qPCR analizleri

Dışkı oumlrnekleri

200 mg Ekstraksiyon Nanodrop Oumllccediluumlm

Roche LightCycler 20

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi

Hasta (n=49) Kontrol (n=35)

Her oumlrnekten 50 ngmicroL havuzlandı

LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR

Agaroz Jelde Goumlruumlntuumlleme

Purifikasyon

TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)

kullanarak amplifikasyon uumlruumlnlerinin ligasyonu

Ecoli JM 109 kullanarak transformasyon

Ampisilin iccedileren LB besiyerlerine kuumlltuumlr

Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi

HastaKontr

olHasta

Kontr

ol

Lac-F Lac-R Koloni PCR

Agaroz jelde goumlruumlntuumlleme

Purifikasyon

g-Bifid-F g-Bifid-R koloni PCR

Sekans PubMed Blast 97 seq similarity

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

İnflamasyon Belirteccedilleri

İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı

hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci

Yoneda et al 2007 J Gastroenterol

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması

bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

dış

kı

Kontrol

NAFLDp=0003

p=0001 plt0001

Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması

A m

ucinip

hilia

F pra

usnitz

ii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

dış

kı

F0-F1

F2

plt0001

Enterobacteriaceae ile BKİ arasındakipozitif korelasyon

20 30 400

5

10

15

BMI

En

tero

ba

cte

ria

ce

ae

lo

g1

0g

r d

ışk

ı

BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)

-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)

Kontr

ol

NAFLD

-05

00

05

10

15

20

mg

dL

Kontrol

NAFLD

hs-CRP

NAFLD ve kontrol grubu hs-CRP seviyeleri

-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)

Kontr

ol

NAFLD

-20

0

20

40

60E

Um

L

Endotoxin

NAFLD ve kontrol grubu serum endotoksin duumlzeyleri

NAFLD ve kontrol grubu sitokin duumlzeyleri

bull TNF-α ve IL-6 seviyeleri bakımından fark yok

bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Pat

ient

0

20

40

60

80

IL-6

pg

mL

Control

Patient

NAFLD ve kontrol grubundaLactobacillus tuumlrleri

L delbrueckii 9

Lruminis50

Lmucosae2

Lacidophilus4

Lgastricus2

Lsalivarius4

Uncultured Lactobacillus

spp18

L helveticus7

Lsakei4

Kontrol

L delbrueckii Lruminis

Lmucosae Lacidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L helveticus

Lsakei

Ldelbrueckii 3

Lruminis39

Lmucosae9

L acidophilus11

Lgastricus6

Lsalivarius3

Uncultured Lactobacillus

spp5

L Johsonii5

Lreuteri19

NAFLD

Ldelbrueckii Lruminis

Lmucosae L acidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L Johsonii

Lreuteri

NAFLD ve kontrol grubundaBifidobacterium tuumlrleri

B adolescentis55

Blongum15

Bbifidum6

Bkashiwanohense10

Bcatenulatum4

Bruminatium2

Uncultured Bifidobacterium

spp Clone8

Kontrol

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Bcatenulatum

Bruminatium

Uncultured Bifidobacterium spp Clone

B adolescentis42

Blongum36

Bbifidum2

Bkashiwanohense2

Binfantis8

Uncultured Bifidobacterium

spp clone10

NAFLD

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Binfantis

Uncultured Bifidobacterium spp clone

Sonuccedil

NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir

A muciniphila NAFLD grubunda daha duumlşuumlk orandadır

Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir

B fragilis kilo durumuna bağımlı bir değişken

Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir

Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir

F prausnitzii mikrobiyotanın dominant bir uumlyesi

Sonuccedil

bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı

bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı

bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi

Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25

CEREN ERDOĞDU

Meltem Yalınay

Tarkan Karakan

Martin J Blaser

Barış Otlu

Doruk Engin

Sinem Solmaz

Berrin Oumlztuumlrk

GUuml Endoskopi Uumlnitesi

GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)

NYU Biyoistatistik Departmanı (Huilin Li)

Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)

Roche Diagnostics teknik ekibi

Teşekkuumlrler

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

Materyal Metod

bull Verilerin tuumlmuuml Microsoft Excele girilerek logbakteriyel miktarı hesaplandı

bull Veriler SPSS 220 paket programı ile analiz edildi

bull Antibiyotik kullanım oumlncesi ve antibiyotik kullanım sonrası karşılaştırmalar Mann-Whitney U Testi ile değerlendirildi

Bulgular

Bakteriler

Antibiyotik Kullanım

Oumlncesi OrtalamaplusmnSS

(log10g)

Antibiyotik Kullanım

Sonrası OrtalamaplusmnSS

(log10g)

p

Bifidobacterium spp 781plusmn112 611plusmn274 0000

B fragilis 824plusmn119 569plusmn289 0000

Lactobacillus spp 739plusmn061 632plusmn174 0000

A mucinophilia 808plusmn060 610plusmn086 0000

F prausnitzii 921plusmn209 632plusmn159 0000

SS Standart Sapma

Nonalkolik Karaciğer Yağlanması Olan Hastalar ve Sağlıklı Kontrollerde

Bağırsak Mikrobiyotasının MolekuumllerYoumlntemlerle Karşılaştırılması

Ceren ErdoğduDanışman ProfDr Meltem Yalınay

9 Eyluumll 2016Ankara

İntestinal mikrobiyota ve NAFLD ilişkisi

Lİ et al 2013 Journal of Parenteral and Enteral Nutrition

Hipotez

bull NAFLD ve kontrol mikrobiyotası arasındafarklılıklar bulunmaktadır

ndash Mikrobiyotadaki farklılıklar barsaktan dolaşımageccedilen endotoksin miktarını arttırabilir

ndash İnflamasyon artabilir

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

Metod

bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)

bull 38 sağlıklı kontrol

ge18 yaşında olan

artmış serum aminotransferaz seviyeleri goumlzlenen

alkol tuumlketimi lt20 grhafta olan

son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan

eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

Metod

bull US ve artmış karaciğer enzimleri

bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz

oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli

fibrozis olarak değerlendirilmiştir

bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)

Brunt EM 1999 Liver

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

qPCR analizleri iccedilin standartlarınhazırlanması

Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922

Kuumlltuumlr05 Mc Farland Suumlspansiyon

cfu sayım

Ekstraksiyon

Nanodrop Oumllccediluumlm

110 diluumlsyon

Standart eğri

0

5

10

15

20

25

30

35

1E+02 1E+04 1E+06 1E+08

Cro

ssin

g-p

oin

t (C

p)

valu

e

DNA copy number microL

A muciniphila

B fragilis group

Bifidobacterium spp

F prausnitzii

Enterobacteriaceae

Lactobacillus spp

Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml

Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml

Dışkı oumlrneklerinden qPCR analizleri

Dışkı oumlrnekleri

200 mg Ekstraksiyon Nanodrop Oumllccediluumlm

Roche LightCycler 20

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi

Hasta (n=49) Kontrol (n=35)

Her oumlrnekten 50 ngmicroL havuzlandı

LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR

Agaroz Jelde Goumlruumlntuumlleme

Purifikasyon

TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)

kullanarak amplifikasyon uumlruumlnlerinin ligasyonu

Ecoli JM 109 kullanarak transformasyon

Ampisilin iccedileren LB besiyerlerine kuumlltuumlr

Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi

HastaKontr

olHasta

Kontr

ol

Lac-F Lac-R Koloni PCR

Agaroz jelde goumlruumlntuumlleme

Purifikasyon

g-Bifid-F g-Bifid-R koloni PCR

Sekans PubMed Blast 97 seq similarity

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

İnflamasyon Belirteccedilleri

İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı

hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci

Yoneda et al 2007 J Gastroenterol

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması

bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

dış

kı

Kontrol

NAFLDp=0003

p=0001 plt0001

Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması

A m

ucinip

hilia

F pra

usnitz

ii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

dış

kı

F0-F1

F2

plt0001

Enterobacteriaceae ile BKİ arasındakipozitif korelasyon

20 30 400

5

10

15

BMI

En

tero

ba

cte

ria

ce

ae

lo

g1

0g

r d

ışk

ı

BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)

-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)

Kontr

ol

NAFLD

-05

00

05

10

15

20

mg

dL

Kontrol

NAFLD

hs-CRP

NAFLD ve kontrol grubu hs-CRP seviyeleri

-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)

Kontr

ol

NAFLD

-20

0

20

40

60E

Um

L

Endotoxin

NAFLD ve kontrol grubu serum endotoksin duumlzeyleri

NAFLD ve kontrol grubu sitokin duumlzeyleri

bull TNF-α ve IL-6 seviyeleri bakımından fark yok

bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Pat

ient

0

20

40

60

80

IL-6

pg

mL

Control

Patient

NAFLD ve kontrol grubundaLactobacillus tuumlrleri

L delbrueckii 9

Lruminis50

Lmucosae2

Lacidophilus4

Lgastricus2

Lsalivarius4

Uncultured Lactobacillus

spp18

L helveticus7

Lsakei4

Kontrol

L delbrueckii Lruminis

Lmucosae Lacidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L helveticus

Lsakei

Ldelbrueckii 3

Lruminis39

Lmucosae9

L acidophilus11

Lgastricus6

Lsalivarius3

Uncultured Lactobacillus

spp5

L Johsonii5

Lreuteri19

NAFLD

Ldelbrueckii Lruminis

Lmucosae L acidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L Johsonii

Lreuteri

NAFLD ve kontrol grubundaBifidobacterium tuumlrleri

B adolescentis55

Blongum15

Bbifidum6

Bkashiwanohense10

Bcatenulatum4

Bruminatium2

Uncultured Bifidobacterium

spp Clone8

Kontrol

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Bcatenulatum

Bruminatium

Uncultured Bifidobacterium spp Clone

B adolescentis42

Blongum36

Bbifidum2

Bkashiwanohense2

Binfantis8

Uncultured Bifidobacterium

spp clone10

NAFLD

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Binfantis

Uncultured Bifidobacterium spp clone

Sonuccedil

NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir

A muciniphila NAFLD grubunda daha duumlşuumlk orandadır

Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir

B fragilis kilo durumuna bağımlı bir değişken

Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir

Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir

F prausnitzii mikrobiyotanın dominant bir uumlyesi

Sonuccedil

bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı

bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı

bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi

Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25

CEREN ERDOĞDU

Meltem Yalınay

Tarkan Karakan

Martin J Blaser

Barış Otlu

Doruk Engin

Sinem Solmaz

Berrin Oumlztuumlrk

GUuml Endoskopi Uumlnitesi

GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)

NYU Biyoistatistik Departmanı (Huilin Li)

Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)

Roche Diagnostics teknik ekibi

Teşekkuumlrler

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

Bulgular

Bakteriler

Antibiyotik Kullanım

Oumlncesi OrtalamaplusmnSS

(log10g)

Antibiyotik Kullanım

Sonrası OrtalamaplusmnSS

(log10g)

p

Bifidobacterium spp 781plusmn112 611plusmn274 0000

B fragilis 824plusmn119 569plusmn289 0000

Lactobacillus spp 739plusmn061 632plusmn174 0000

A mucinophilia 808plusmn060 610plusmn086 0000

F prausnitzii 921plusmn209 632plusmn159 0000

SS Standart Sapma

Nonalkolik Karaciğer Yağlanması Olan Hastalar ve Sağlıklı Kontrollerde

Bağırsak Mikrobiyotasının MolekuumllerYoumlntemlerle Karşılaştırılması

Ceren ErdoğduDanışman ProfDr Meltem Yalınay

9 Eyluumll 2016Ankara

İntestinal mikrobiyota ve NAFLD ilişkisi

Lİ et al 2013 Journal of Parenteral and Enteral Nutrition

Hipotez

bull NAFLD ve kontrol mikrobiyotası arasındafarklılıklar bulunmaktadır

ndash Mikrobiyotadaki farklılıklar barsaktan dolaşımageccedilen endotoksin miktarını arttırabilir

ndash İnflamasyon artabilir

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

Metod

bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)

bull 38 sağlıklı kontrol

ge18 yaşında olan

artmış serum aminotransferaz seviyeleri goumlzlenen

alkol tuumlketimi lt20 grhafta olan

son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan

eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

Metod

bull US ve artmış karaciğer enzimleri

bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz

oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli

fibrozis olarak değerlendirilmiştir

bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)

Brunt EM 1999 Liver

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

qPCR analizleri iccedilin standartlarınhazırlanması

Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922

Kuumlltuumlr05 Mc Farland Suumlspansiyon

cfu sayım

Ekstraksiyon

Nanodrop Oumllccediluumlm

110 diluumlsyon

Standart eğri

0

5

10

15

20

25

30

35

1E+02 1E+04 1E+06 1E+08

Cro

ssin

g-p

oin

t (C

p)

valu

e

DNA copy number microL

A muciniphila

B fragilis group

Bifidobacterium spp

F prausnitzii

Enterobacteriaceae

Lactobacillus spp

Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml

Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml

Dışkı oumlrneklerinden qPCR analizleri

Dışkı oumlrnekleri

200 mg Ekstraksiyon Nanodrop Oumllccediluumlm

Roche LightCycler 20

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi

Hasta (n=49) Kontrol (n=35)

Her oumlrnekten 50 ngmicroL havuzlandı

LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR

Agaroz Jelde Goumlruumlntuumlleme

Purifikasyon

TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)

kullanarak amplifikasyon uumlruumlnlerinin ligasyonu

Ecoli JM 109 kullanarak transformasyon

Ampisilin iccedileren LB besiyerlerine kuumlltuumlr

Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi

HastaKontr

olHasta

Kontr

ol

Lac-F Lac-R Koloni PCR

Agaroz jelde goumlruumlntuumlleme

Purifikasyon

g-Bifid-F g-Bifid-R koloni PCR

Sekans PubMed Blast 97 seq similarity

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

İnflamasyon Belirteccedilleri

İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı

hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci

Yoneda et al 2007 J Gastroenterol

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması

bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

dış

kı

Kontrol

NAFLDp=0003

p=0001 plt0001

Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması

A m

ucinip

hilia

F pra

usnitz

ii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

dış

kı

F0-F1

F2

plt0001

Enterobacteriaceae ile BKİ arasındakipozitif korelasyon

20 30 400

5

10

15

BMI

En

tero

ba

cte

ria

ce

ae

lo

g1

0g

r d

ışk

ı

BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)

-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)

Kontr

ol

NAFLD

-05

00

05

10

15

20

mg

dL

Kontrol

NAFLD

hs-CRP

NAFLD ve kontrol grubu hs-CRP seviyeleri

-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)

Kontr

ol

NAFLD

-20

0

20

40

60E

Um

L

Endotoxin

NAFLD ve kontrol grubu serum endotoksin duumlzeyleri

NAFLD ve kontrol grubu sitokin duumlzeyleri

bull TNF-α ve IL-6 seviyeleri bakımından fark yok

bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Pat

ient

0

20

40

60

80

IL-6

pg

mL

Control

Patient

NAFLD ve kontrol grubundaLactobacillus tuumlrleri

L delbrueckii 9

Lruminis50

Lmucosae2

Lacidophilus4

Lgastricus2

Lsalivarius4

Uncultured Lactobacillus

spp18

L helveticus7

Lsakei4

Kontrol

L delbrueckii Lruminis

Lmucosae Lacidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L helveticus

Lsakei

Ldelbrueckii 3

Lruminis39

Lmucosae9

L acidophilus11

Lgastricus6

Lsalivarius3

Uncultured Lactobacillus

spp5

L Johsonii5

Lreuteri19

NAFLD

Ldelbrueckii Lruminis

Lmucosae L acidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L Johsonii

Lreuteri

NAFLD ve kontrol grubundaBifidobacterium tuumlrleri

B adolescentis55

Blongum15

Bbifidum6

Bkashiwanohense10

Bcatenulatum4

Bruminatium2

Uncultured Bifidobacterium

spp Clone8

Kontrol

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Bcatenulatum

Bruminatium

Uncultured Bifidobacterium spp Clone

B adolescentis42

Blongum36

Bbifidum2

Bkashiwanohense2

Binfantis8

Uncultured Bifidobacterium

spp clone10

NAFLD

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Binfantis

Uncultured Bifidobacterium spp clone

Sonuccedil

NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir

A muciniphila NAFLD grubunda daha duumlşuumlk orandadır

Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir

B fragilis kilo durumuna bağımlı bir değişken

Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir

Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir

F prausnitzii mikrobiyotanın dominant bir uumlyesi

Sonuccedil

bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı

bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı

bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi

Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25

CEREN ERDOĞDU

Meltem Yalınay

Tarkan Karakan

Martin J Blaser

Barış Otlu

Doruk Engin

Sinem Solmaz

Berrin Oumlztuumlrk

GUuml Endoskopi Uumlnitesi

GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)

NYU Biyoistatistik Departmanı (Huilin Li)

Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)

Roche Diagnostics teknik ekibi

Teşekkuumlrler

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

Nonalkolik Karaciğer Yağlanması Olan Hastalar ve Sağlıklı Kontrollerde

Bağırsak Mikrobiyotasının MolekuumllerYoumlntemlerle Karşılaştırılması

Ceren ErdoğduDanışman ProfDr Meltem Yalınay

9 Eyluumll 2016Ankara

İntestinal mikrobiyota ve NAFLD ilişkisi

Lİ et al 2013 Journal of Parenteral and Enteral Nutrition

Hipotez

bull NAFLD ve kontrol mikrobiyotası arasındafarklılıklar bulunmaktadır

ndash Mikrobiyotadaki farklılıklar barsaktan dolaşımageccedilen endotoksin miktarını arttırabilir

ndash İnflamasyon artabilir

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

Metod

bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)

bull 38 sağlıklı kontrol

ge18 yaşında olan

artmış serum aminotransferaz seviyeleri goumlzlenen

alkol tuumlketimi lt20 grhafta olan

son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan

eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

Metod

bull US ve artmış karaciğer enzimleri

bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz

oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli

fibrozis olarak değerlendirilmiştir

bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)

Brunt EM 1999 Liver

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

qPCR analizleri iccedilin standartlarınhazırlanması

Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922

Kuumlltuumlr05 Mc Farland Suumlspansiyon

cfu sayım

Ekstraksiyon

Nanodrop Oumllccediluumlm

110 diluumlsyon

Standart eğri

0

5

10

15

20

25

30

35

1E+02 1E+04 1E+06 1E+08

Cro

ssin

g-p

oin

t (C

p)

valu

e

DNA copy number microL

A muciniphila

B fragilis group

Bifidobacterium spp

F prausnitzii

Enterobacteriaceae

Lactobacillus spp

Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml

Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml

Dışkı oumlrneklerinden qPCR analizleri

Dışkı oumlrnekleri

200 mg Ekstraksiyon Nanodrop Oumllccediluumlm

Roche LightCycler 20

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi

Hasta (n=49) Kontrol (n=35)

Her oumlrnekten 50 ngmicroL havuzlandı

LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR

Agaroz Jelde Goumlruumlntuumlleme

Purifikasyon

TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)

kullanarak amplifikasyon uumlruumlnlerinin ligasyonu

Ecoli JM 109 kullanarak transformasyon

Ampisilin iccedileren LB besiyerlerine kuumlltuumlr

Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi

HastaKontr

olHasta

Kontr

ol

Lac-F Lac-R Koloni PCR

Agaroz jelde goumlruumlntuumlleme

Purifikasyon

g-Bifid-F g-Bifid-R koloni PCR

Sekans PubMed Blast 97 seq similarity

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

İnflamasyon Belirteccedilleri

İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı

hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci

Yoneda et al 2007 J Gastroenterol

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması

bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

dış

kı

Kontrol

NAFLDp=0003

p=0001 plt0001

Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması

A m

ucinip

hilia

F pra

usnitz

ii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

dış

kı

F0-F1

F2

plt0001

Enterobacteriaceae ile BKİ arasındakipozitif korelasyon

20 30 400

5

10

15

BMI

En

tero

ba

cte

ria

ce

ae

lo

g1

0g

r d

ışk

ı

BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)

-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)

Kontr

ol

NAFLD

-05

00

05

10

15

20

mg

dL

Kontrol

NAFLD

hs-CRP

NAFLD ve kontrol grubu hs-CRP seviyeleri

-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)

Kontr

ol

NAFLD

-20

0

20

40

60E

Um

L

Endotoxin

NAFLD ve kontrol grubu serum endotoksin duumlzeyleri

NAFLD ve kontrol grubu sitokin duumlzeyleri

bull TNF-α ve IL-6 seviyeleri bakımından fark yok

bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Pat

ient

0

20

40

60

80

IL-6

pg

mL

Control

Patient

NAFLD ve kontrol grubundaLactobacillus tuumlrleri

L delbrueckii 9

Lruminis50

Lmucosae2

Lacidophilus4

Lgastricus2

Lsalivarius4

Uncultured Lactobacillus

spp18

L helveticus7

Lsakei4

Kontrol

L delbrueckii Lruminis

Lmucosae Lacidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L helveticus

Lsakei

Ldelbrueckii 3

Lruminis39

Lmucosae9

L acidophilus11

Lgastricus6

Lsalivarius3

Uncultured Lactobacillus

spp5

L Johsonii5

Lreuteri19

NAFLD

Ldelbrueckii Lruminis

Lmucosae L acidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L Johsonii

Lreuteri

NAFLD ve kontrol grubundaBifidobacterium tuumlrleri

B adolescentis55

Blongum15

Bbifidum6

Bkashiwanohense10

Bcatenulatum4

Bruminatium2

Uncultured Bifidobacterium

spp Clone8

Kontrol

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Bcatenulatum

Bruminatium

Uncultured Bifidobacterium spp Clone

B adolescentis42

Blongum36

Bbifidum2

Bkashiwanohense2

Binfantis8

Uncultured Bifidobacterium

spp clone10

NAFLD

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Binfantis

Uncultured Bifidobacterium spp clone

Sonuccedil

NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir

A muciniphila NAFLD grubunda daha duumlşuumlk orandadır

Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir

B fragilis kilo durumuna bağımlı bir değişken

Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir

Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir

F prausnitzii mikrobiyotanın dominant bir uumlyesi

Sonuccedil

bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı

bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı

bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi

Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25

CEREN ERDOĞDU

Meltem Yalınay

Tarkan Karakan

Martin J Blaser

Barış Otlu

Doruk Engin

Sinem Solmaz

Berrin Oumlztuumlrk

GUuml Endoskopi Uumlnitesi

GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)

NYU Biyoistatistik Departmanı (Huilin Li)

Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)

Roche Diagnostics teknik ekibi

Teşekkuumlrler

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

İntestinal mikrobiyota ve NAFLD ilişkisi

Lİ et al 2013 Journal of Parenteral and Enteral Nutrition

Hipotez

bull NAFLD ve kontrol mikrobiyotası arasındafarklılıklar bulunmaktadır

ndash Mikrobiyotadaki farklılıklar barsaktan dolaşımageccedilen endotoksin miktarını arttırabilir

ndash İnflamasyon artabilir

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

Metod

bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)

bull 38 sağlıklı kontrol

ge18 yaşında olan

artmış serum aminotransferaz seviyeleri goumlzlenen

alkol tuumlketimi lt20 grhafta olan

son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan

eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

Metod

bull US ve artmış karaciğer enzimleri

bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz

oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli

fibrozis olarak değerlendirilmiştir

bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)

Brunt EM 1999 Liver

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

qPCR analizleri iccedilin standartlarınhazırlanması

Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922

Kuumlltuumlr05 Mc Farland Suumlspansiyon

cfu sayım

Ekstraksiyon

Nanodrop Oumllccediluumlm

110 diluumlsyon

Standart eğri

0

5

10

15

20

25

30

35

1E+02 1E+04 1E+06 1E+08

Cro

ssin

g-p

oin

t (C

p)

valu

e

DNA copy number microL

A muciniphila

B fragilis group

Bifidobacterium spp

F prausnitzii

Enterobacteriaceae

Lactobacillus spp

Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml

Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml

Dışkı oumlrneklerinden qPCR analizleri

Dışkı oumlrnekleri

200 mg Ekstraksiyon Nanodrop Oumllccediluumlm

Roche LightCycler 20

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi

Hasta (n=49) Kontrol (n=35)

Her oumlrnekten 50 ngmicroL havuzlandı

LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR

Agaroz Jelde Goumlruumlntuumlleme

Purifikasyon

TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)

kullanarak amplifikasyon uumlruumlnlerinin ligasyonu

Ecoli JM 109 kullanarak transformasyon

Ampisilin iccedileren LB besiyerlerine kuumlltuumlr

Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi

HastaKontr

olHasta

Kontr

ol

Lac-F Lac-R Koloni PCR

Agaroz jelde goumlruumlntuumlleme

Purifikasyon

g-Bifid-F g-Bifid-R koloni PCR

Sekans PubMed Blast 97 seq similarity

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

İnflamasyon Belirteccedilleri

İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı

hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci

Yoneda et al 2007 J Gastroenterol

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması

bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

dış

kı

Kontrol

NAFLDp=0003

p=0001 plt0001

Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması

A m

ucinip

hilia

F pra

usnitz

ii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

dış

kı

F0-F1

F2

plt0001

Enterobacteriaceae ile BKİ arasındakipozitif korelasyon

20 30 400

5

10

15

BMI

En

tero

ba

cte

ria

ce

ae

lo

g1

0g

r d

ışk

ı

BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)

-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)

Kontr

ol

NAFLD

-05

00

05

10

15

20

mg

dL

Kontrol

NAFLD

hs-CRP

NAFLD ve kontrol grubu hs-CRP seviyeleri

-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)

Kontr

ol

NAFLD

-20

0

20

40

60E

Um

L

Endotoxin

NAFLD ve kontrol grubu serum endotoksin duumlzeyleri

NAFLD ve kontrol grubu sitokin duumlzeyleri

bull TNF-α ve IL-6 seviyeleri bakımından fark yok

bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Pat

ient

0

20

40

60

80

IL-6

pg

mL

Control

Patient

NAFLD ve kontrol grubundaLactobacillus tuumlrleri

L delbrueckii 9

Lruminis50

Lmucosae2

Lacidophilus4

Lgastricus2

Lsalivarius4

Uncultured Lactobacillus

spp18

L helveticus7

Lsakei4

Kontrol

L delbrueckii Lruminis

Lmucosae Lacidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L helveticus

Lsakei

Ldelbrueckii 3

Lruminis39

Lmucosae9

L acidophilus11

Lgastricus6

Lsalivarius3

Uncultured Lactobacillus

spp5

L Johsonii5

Lreuteri19

NAFLD

Ldelbrueckii Lruminis

Lmucosae L acidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L Johsonii

Lreuteri

NAFLD ve kontrol grubundaBifidobacterium tuumlrleri

B adolescentis55

Blongum15

Bbifidum6

Bkashiwanohense10

Bcatenulatum4

Bruminatium2

Uncultured Bifidobacterium

spp Clone8

Kontrol

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Bcatenulatum

Bruminatium

Uncultured Bifidobacterium spp Clone

B adolescentis42

Blongum36

Bbifidum2

Bkashiwanohense2

Binfantis8

Uncultured Bifidobacterium

spp clone10

NAFLD

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Binfantis

Uncultured Bifidobacterium spp clone

Sonuccedil

NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir

A muciniphila NAFLD grubunda daha duumlşuumlk orandadır

Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir

B fragilis kilo durumuna bağımlı bir değişken

Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir

Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir

F prausnitzii mikrobiyotanın dominant bir uumlyesi

Sonuccedil

bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı

bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı

bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi

Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25

CEREN ERDOĞDU

Meltem Yalınay

Tarkan Karakan

Martin J Blaser

Barış Otlu

Doruk Engin

Sinem Solmaz

Berrin Oumlztuumlrk

GUuml Endoskopi Uumlnitesi

GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)

NYU Biyoistatistik Departmanı (Huilin Li)

Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)

Roche Diagnostics teknik ekibi

Teşekkuumlrler

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

Hipotez

bull NAFLD ve kontrol mikrobiyotası arasındafarklılıklar bulunmaktadır

ndash Mikrobiyotadaki farklılıklar barsaktan dolaşımageccedilen endotoksin miktarını arttırabilir

ndash İnflamasyon artabilir

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

Metod

bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)

bull 38 sağlıklı kontrol

ge18 yaşında olan

artmış serum aminotransferaz seviyeleri goumlzlenen

alkol tuumlketimi lt20 grhafta olan

son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan

eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

Metod

bull US ve artmış karaciğer enzimleri

bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz

oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli

fibrozis olarak değerlendirilmiştir

bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)

Brunt EM 1999 Liver

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

qPCR analizleri iccedilin standartlarınhazırlanması

Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922

Kuumlltuumlr05 Mc Farland Suumlspansiyon

cfu sayım

Ekstraksiyon

Nanodrop Oumllccediluumlm

110 diluumlsyon

Standart eğri

0

5

10

15

20

25

30

35

1E+02 1E+04 1E+06 1E+08

Cro

ssin

g-p

oin

t (C

p)

valu

e

DNA copy number microL

A muciniphila

B fragilis group

Bifidobacterium spp

F prausnitzii

Enterobacteriaceae

Lactobacillus spp

Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml

Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml

Dışkı oumlrneklerinden qPCR analizleri

Dışkı oumlrnekleri

200 mg Ekstraksiyon Nanodrop Oumllccediluumlm

Roche LightCycler 20

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi

Hasta (n=49) Kontrol (n=35)

Her oumlrnekten 50 ngmicroL havuzlandı

LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR

Agaroz Jelde Goumlruumlntuumlleme

Purifikasyon

TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)

kullanarak amplifikasyon uumlruumlnlerinin ligasyonu

Ecoli JM 109 kullanarak transformasyon

Ampisilin iccedileren LB besiyerlerine kuumlltuumlr

Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi

HastaKontr

olHasta

Kontr

ol

Lac-F Lac-R Koloni PCR

Agaroz jelde goumlruumlntuumlleme

Purifikasyon

g-Bifid-F g-Bifid-R koloni PCR

Sekans PubMed Blast 97 seq similarity

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

İnflamasyon Belirteccedilleri

İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı

hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci

Yoneda et al 2007 J Gastroenterol

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması

bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

dış

kı

Kontrol

NAFLDp=0003

p=0001 plt0001

Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması

A m

ucinip

hilia

F pra

usnitz

ii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

dış

kı

F0-F1

F2

plt0001

Enterobacteriaceae ile BKİ arasındakipozitif korelasyon

20 30 400

5

10

15

BMI

En

tero

ba

cte

ria

ce

ae

lo

g1

0g

r d

ışk

ı

BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)

-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)

Kontr

ol

NAFLD

-05

00

05

10

15

20

mg

dL

Kontrol

NAFLD

hs-CRP

NAFLD ve kontrol grubu hs-CRP seviyeleri

-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)

Kontr

ol

NAFLD

-20

0

20

40

60E

Um

L

Endotoxin

NAFLD ve kontrol grubu serum endotoksin duumlzeyleri

NAFLD ve kontrol grubu sitokin duumlzeyleri

bull TNF-α ve IL-6 seviyeleri bakımından fark yok

bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Pat

ient

0

20

40

60

80

IL-6

pg

mL

Control

Patient

NAFLD ve kontrol grubundaLactobacillus tuumlrleri

L delbrueckii 9

Lruminis50

Lmucosae2

Lacidophilus4

Lgastricus2

Lsalivarius4

Uncultured Lactobacillus

spp18

L helveticus7

Lsakei4

Kontrol

L delbrueckii Lruminis

Lmucosae Lacidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L helveticus

Lsakei

Ldelbrueckii 3

Lruminis39

Lmucosae9

L acidophilus11

Lgastricus6

Lsalivarius3

Uncultured Lactobacillus

spp5

L Johsonii5

Lreuteri19

NAFLD

Ldelbrueckii Lruminis

Lmucosae L acidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L Johsonii

Lreuteri

NAFLD ve kontrol grubundaBifidobacterium tuumlrleri

B adolescentis55

Blongum15

Bbifidum6

Bkashiwanohense10

Bcatenulatum4

Bruminatium2

Uncultured Bifidobacterium

spp Clone8

Kontrol

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Bcatenulatum

Bruminatium

Uncultured Bifidobacterium spp Clone

B adolescentis42

Blongum36

Bbifidum2

Bkashiwanohense2

Binfantis8

Uncultured Bifidobacterium

spp clone10

NAFLD

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Binfantis

Uncultured Bifidobacterium spp clone

Sonuccedil

NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir

A muciniphila NAFLD grubunda daha duumlşuumlk orandadır

Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir

B fragilis kilo durumuna bağımlı bir değişken

Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir

Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir

F prausnitzii mikrobiyotanın dominant bir uumlyesi

Sonuccedil

bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı

bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı

bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi

Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25

CEREN ERDOĞDU

Meltem Yalınay

Tarkan Karakan

Martin J Blaser

Barış Otlu

Doruk Engin

Sinem Solmaz

Berrin Oumlztuumlrk

GUuml Endoskopi Uumlnitesi

GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)

NYU Biyoistatistik Departmanı (Huilin Li)

Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)

Roche Diagnostics teknik ekibi

Teşekkuumlrler

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

Metod

bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)

bull 38 sağlıklı kontrol

ge18 yaşında olan

artmış serum aminotransferaz seviyeleri goumlzlenen

alkol tuumlketimi lt20 grhafta olan

son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan

eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

Metod

bull US ve artmış karaciğer enzimleri

bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz

oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli

fibrozis olarak değerlendirilmiştir

bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)

Brunt EM 1999 Liver

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

qPCR analizleri iccedilin standartlarınhazırlanması

Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922

Kuumlltuumlr05 Mc Farland Suumlspansiyon

cfu sayım

Ekstraksiyon

Nanodrop Oumllccediluumlm

110 diluumlsyon

Standart eğri

0

5

10

15

20

25

30

35

1E+02 1E+04 1E+06 1E+08

Cro

ssin

g-p

oin

t (C

p)

valu

e

DNA copy number microL

A muciniphila

B fragilis group

Bifidobacterium spp

F prausnitzii

Enterobacteriaceae

Lactobacillus spp

Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml

Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml

Dışkı oumlrneklerinden qPCR analizleri

Dışkı oumlrnekleri

200 mg Ekstraksiyon Nanodrop Oumllccediluumlm

Roche LightCycler 20

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi

Hasta (n=49) Kontrol (n=35)

Her oumlrnekten 50 ngmicroL havuzlandı

LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR

Agaroz Jelde Goumlruumlntuumlleme

Purifikasyon

TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)

kullanarak amplifikasyon uumlruumlnlerinin ligasyonu

Ecoli JM 109 kullanarak transformasyon

Ampisilin iccedileren LB besiyerlerine kuumlltuumlr

Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi

HastaKontr

olHasta

Kontr

ol

Lac-F Lac-R Koloni PCR

Agaroz jelde goumlruumlntuumlleme

Purifikasyon

g-Bifid-F g-Bifid-R koloni PCR

Sekans PubMed Blast 97 seq similarity

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

İnflamasyon Belirteccedilleri

İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı

hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci

Yoneda et al 2007 J Gastroenterol

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması

bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

dış

kı

Kontrol

NAFLDp=0003

p=0001 plt0001

Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması

A m

ucinip

hilia

F pra

usnitz

ii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

dış

kı

F0-F1

F2

plt0001

Enterobacteriaceae ile BKİ arasındakipozitif korelasyon

20 30 400

5

10

15

BMI

En

tero

ba

cte

ria

ce

ae

lo

g1

0g

r d

ışk

ı

BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)

-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)

Kontr

ol

NAFLD

-05

00

05

10

15

20

mg

dL

Kontrol

NAFLD

hs-CRP

NAFLD ve kontrol grubu hs-CRP seviyeleri

-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)

Kontr

ol

NAFLD

-20

0

20

40

60E

Um

L

Endotoxin

NAFLD ve kontrol grubu serum endotoksin duumlzeyleri

NAFLD ve kontrol grubu sitokin duumlzeyleri

bull TNF-α ve IL-6 seviyeleri bakımından fark yok

bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Pat

ient

0

20

40

60

80

IL-6

pg

mL

Control

Patient

NAFLD ve kontrol grubundaLactobacillus tuumlrleri

L delbrueckii 9

Lruminis50

Lmucosae2

Lacidophilus4

Lgastricus2

Lsalivarius4

Uncultured Lactobacillus

spp18

L helveticus7

Lsakei4

Kontrol

L delbrueckii Lruminis

Lmucosae Lacidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L helveticus

Lsakei

Ldelbrueckii 3

Lruminis39

Lmucosae9

L acidophilus11

Lgastricus6

Lsalivarius3

Uncultured Lactobacillus

spp5

L Johsonii5

Lreuteri19

NAFLD

Ldelbrueckii Lruminis

Lmucosae L acidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L Johsonii

Lreuteri

NAFLD ve kontrol grubundaBifidobacterium tuumlrleri

B adolescentis55

Blongum15

Bbifidum6

Bkashiwanohense10

Bcatenulatum4

Bruminatium2

Uncultured Bifidobacterium

spp Clone8

Kontrol

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Bcatenulatum

Bruminatium

Uncultured Bifidobacterium spp Clone

B adolescentis42

Blongum36

Bbifidum2

Bkashiwanohense2

Binfantis8

Uncultured Bifidobacterium

spp clone10

NAFLD

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Binfantis

Uncultured Bifidobacterium spp clone

Sonuccedil

NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir

A muciniphila NAFLD grubunda daha duumlşuumlk orandadır

Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir

B fragilis kilo durumuna bağımlı bir değişken

Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir

Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir

F prausnitzii mikrobiyotanın dominant bir uumlyesi

Sonuccedil

bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı

bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı

bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi

Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25

CEREN ERDOĞDU

Meltem Yalınay

Tarkan Karakan

Martin J Blaser

Barış Otlu

Doruk Engin

Sinem Solmaz

Berrin Oumlztuumlrk

GUuml Endoskopi Uumlnitesi

GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)

NYU Biyoistatistik Departmanı (Huilin Li)

Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)

Roche Diagnostics teknik ekibi

Teşekkuumlrler

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

Metod

bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)

bull 38 sağlıklı kontrol

ge18 yaşında olan

artmış serum aminotransferaz seviyeleri goumlzlenen

alkol tuumlketimi lt20 grhafta olan

son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan

eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

Metod

bull US ve artmış karaciğer enzimleri

bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz

oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli

fibrozis olarak değerlendirilmiştir

bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)

Brunt EM 1999 Liver

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

qPCR analizleri iccedilin standartlarınhazırlanması

Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922

Kuumlltuumlr05 Mc Farland Suumlspansiyon

cfu sayım

Ekstraksiyon

Nanodrop Oumllccediluumlm

110 diluumlsyon

Standart eğri

0

5

10

15

20

25

30

35

1E+02 1E+04 1E+06 1E+08

Cro

ssin

g-p

oin

t (C

p)

valu

e

DNA copy number microL

A muciniphila

B fragilis group

Bifidobacterium spp

F prausnitzii

Enterobacteriaceae

Lactobacillus spp

Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml

Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml

Dışkı oumlrneklerinden qPCR analizleri

Dışkı oumlrnekleri

200 mg Ekstraksiyon Nanodrop Oumllccediluumlm

Roche LightCycler 20

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi

Hasta (n=49) Kontrol (n=35)

Her oumlrnekten 50 ngmicroL havuzlandı

LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR

Agaroz Jelde Goumlruumlntuumlleme

Purifikasyon

TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)

kullanarak amplifikasyon uumlruumlnlerinin ligasyonu

Ecoli JM 109 kullanarak transformasyon

Ampisilin iccedileren LB besiyerlerine kuumlltuumlr

Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi

HastaKontr

olHasta

Kontr

ol

Lac-F Lac-R Koloni PCR

Agaroz jelde goumlruumlntuumlleme

Purifikasyon

g-Bifid-F g-Bifid-R koloni PCR

Sekans PubMed Blast 97 seq similarity

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

İnflamasyon Belirteccedilleri

İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı

hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci

Yoneda et al 2007 J Gastroenterol

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması

bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

dış

kı

Kontrol

NAFLDp=0003

p=0001 plt0001

Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması

A m

ucinip

hilia

F pra

usnitz

ii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

dış

kı

F0-F1

F2

plt0001

Enterobacteriaceae ile BKİ arasındakipozitif korelasyon

20 30 400

5

10

15

BMI

En

tero

ba

cte

ria

ce

ae

lo

g1

0g

r d

ışk

ı

BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)

-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)

Kontr

ol

NAFLD

-05

00

05

10

15

20

mg

dL

Kontrol

NAFLD

hs-CRP

NAFLD ve kontrol grubu hs-CRP seviyeleri

-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)

Kontr

ol

NAFLD

-20

0

20

40

60E

Um

L

Endotoxin

NAFLD ve kontrol grubu serum endotoksin duumlzeyleri

NAFLD ve kontrol grubu sitokin duumlzeyleri

bull TNF-α ve IL-6 seviyeleri bakımından fark yok

bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Pat

ient

0

20

40

60

80

IL-6

pg

mL

Control

Patient

NAFLD ve kontrol grubundaLactobacillus tuumlrleri

L delbrueckii 9

Lruminis50

Lmucosae2

Lacidophilus4

Lgastricus2

Lsalivarius4

Uncultured Lactobacillus

spp18

L helveticus7

Lsakei4

Kontrol

L delbrueckii Lruminis

Lmucosae Lacidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L helveticus

Lsakei

Ldelbrueckii 3

Lruminis39

Lmucosae9

L acidophilus11

Lgastricus6

Lsalivarius3

Uncultured Lactobacillus

spp5

L Johsonii5

Lreuteri19

NAFLD

Ldelbrueckii Lruminis

Lmucosae L acidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L Johsonii

Lreuteri

NAFLD ve kontrol grubundaBifidobacterium tuumlrleri

B adolescentis55

Blongum15

Bbifidum6

Bkashiwanohense10

Bcatenulatum4

Bruminatium2

Uncultured Bifidobacterium

spp Clone8

Kontrol

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Bcatenulatum

Bruminatium

Uncultured Bifidobacterium spp Clone

B adolescentis42

Blongum36

Bbifidum2

Bkashiwanohense2

Binfantis8

Uncultured Bifidobacterium

spp clone10

NAFLD

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Binfantis

Uncultured Bifidobacterium spp clone

Sonuccedil

NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir

A muciniphila NAFLD grubunda daha duumlşuumlk orandadır

Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir

B fragilis kilo durumuna bağımlı bir değişken

Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir

Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir

F prausnitzii mikrobiyotanın dominant bir uumlyesi

Sonuccedil

bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı

bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı

bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi

Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25

CEREN ERDOĞDU

Meltem Yalınay

Tarkan Karakan

Martin J Blaser

Barış Otlu

Doruk Engin

Sinem Solmaz

Berrin Oumlztuumlrk

GUuml Endoskopi Uumlnitesi

GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)

NYU Biyoistatistik Departmanı (Huilin Li)

Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)

Roche Diagnostics teknik ekibi

Teşekkuumlrler

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

Metod

bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)

bull 38 sağlıklı kontrol

ge18 yaşında olan

artmış serum aminotransferaz seviyeleri goumlzlenen

alkol tuumlketimi lt20 grhafta olan

son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan

eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

Metod

bull US ve artmış karaciğer enzimleri

bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz

oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli

fibrozis olarak değerlendirilmiştir

bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)

Brunt EM 1999 Liver

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

qPCR analizleri iccedilin standartlarınhazırlanması

Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922

Kuumlltuumlr05 Mc Farland Suumlspansiyon

cfu sayım

Ekstraksiyon

Nanodrop Oumllccediluumlm

110 diluumlsyon

Standart eğri

0

5

10

15

20

25

30

35

1E+02 1E+04 1E+06 1E+08

Cro

ssin

g-p

oin

t (C

p)

valu

e

DNA copy number microL

A muciniphila

B fragilis group

Bifidobacterium spp

F prausnitzii

Enterobacteriaceae

Lactobacillus spp

Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml

Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml

Dışkı oumlrneklerinden qPCR analizleri

Dışkı oumlrnekleri

200 mg Ekstraksiyon Nanodrop Oumllccediluumlm

Roche LightCycler 20

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi

Hasta (n=49) Kontrol (n=35)

Her oumlrnekten 50 ngmicroL havuzlandı

LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR

Agaroz Jelde Goumlruumlntuumlleme

Purifikasyon

TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)

kullanarak amplifikasyon uumlruumlnlerinin ligasyonu

Ecoli JM 109 kullanarak transformasyon

Ampisilin iccedileren LB besiyerlerine kuumlltuumlr

Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi

HastaKontr

olHasta

Kontr

ol

Lac-F Lac-R Koloni PCR

Agaroz jelde goumlruumlntuumlleme

Purifikasyon

g-Bifid-F g-Bifid-R koloni PCR

Sekans PubMed Blast 97 seq similarity

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

İnflamasyon Belirteccedilleri

İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı

hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci

Yoneda et al 2007 J Gastroenterol

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması

bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

dış

kı

Kontrol

NAFLDp=0003

p=0001 plt0001

Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması

A m

ucinip

hilia

F pra

usnitz

ii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

dış

kı

F0-F1

F2

plt0001

Enterobacteriaceae ile BKİ arasındakipozitif korelasyon

20 30 400

5

10

15

BMI

En

tero

ba

cte

ria

ce

ae

lo

g1

0g

r d

ışk

ı

BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)

-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)

Kontr

ol

NAFLD

-05

00

05

10

15

20

mg

dL

Kontrol

NAFLD

hs-CRP

NAFLD ve kontrol grubu hs-CRP seviyeleri

-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)

Kontr

ol

NAFLD

-20

0

20

40

60E

Um

L

Endotoxin

NAFLD ve kontrol grubu serum endotoksin duumlzeyleri

NAFLD ve kontrol grubu sitokin duumlzeyleri

bull TNF-α ve IL-6 seviyeleri bakımından fark yok

bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Pat

ient

0

20

40

60

80

IL-6

pg

mL

Control

Patient

NAFLD ve kontrol grubundaLactobacillus tuumlrleri

L delbrueckii 9

Lruminis50

Lmucosae2

Lacidophilus4

Lgastricus2

Lsalivarius4

Uncultured Lactobacillus

spp18

L helveticus7

Lsakei4

Kontrol

L delbrueckii Lruminis

Lmucosae Lacidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L helveticus

Lsakei

Ldelbrueckii 3

Lruminis39

Lmucosae9

L acidophilus11

Lgastricus6

Lsalivarius3

Uncultured Lactobacillus

spp5

L Johsonii5

Lreuteri19

NAFLD

Ldelbrueckii Lruminis

Lmucosae L acidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L Johsonii

Lreuteri

NAFLD ve kontrol grubundaBifidobacterium tuumlrleri

B adolescentis55

Blongum15

Bbifidum6

Bkashiwanohense10

Bcatenulatum4

Bruminatium2

Uncultured Bifidobacterium

spp Clone8

Kontrol

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Bcatenulatum

Bruminatium

Uncultured Bifidobacterium spp Clone

B adolescentis42

Blongum36

Bbifidum2

Bkashiwanohense2

Binfantis8

Uncultured Bifidobacterium

spp clone10

NAFLD

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Binfantis

Uncultured Bifidobacterium spp clone

Sonuccedil

NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir

A muciniphila NAFLD grubunda daha duumlşuumlk orandadır

Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir

B fragilis kilo durumuna bağımlı bir değişken

Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir

Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir

F prausnitzii mikrobiyotanın dominant bir uumlyesi

Sonuccedil

bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı

bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı

bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi

Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25

CEREN ERDOĞDU

Meltem Yalınay

Tarkan Karakan

Martin J Blaser

Barış Otlu

Doruk Engin

Sinem Solmaz

Berrin Oumlztuumlrk

GUuml Endoskopi Uumlnitesi

GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)

NYU Biyoistatistik Departmanı (Huilin Li)

Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)

Roche Diagnostics teknik ekibi

Teşekkuumlrler

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

Metod

bull US ve artmış karaciğer enzimleri

bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz

oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli

fibrozis olarak değerlendirilmiştir

bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)

Brunt EM 1999 Liver

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

qPCR analizleri iccedilin standartlarınhazırlanması

Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922

Kuumlltuumlr05 Mc Farland Suumlspansiyon

cfu sayım

Ekstraksiyon

Nanodrop Oumllccediluumlm

110 diluumlsyon

Standart eğri

0

5

10

15

20

25

30

35

1E+02 1E+04 1E+06 1E+08

Cro

ssin

g-p

oin

t (C

p)

valu

e

DNA copy number microL

A muciniphila

B fragilis group

Bifidobacterium spp

F prausnitzii

Enterobacteriaceae

Lactobacillus spp

Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml

Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml

Dışkı oumlrneklerinden qPCR analizleri

Dışkı oumlrnekleri

200 mg Ekstraksiyon Nanodrop Oumllccediluumlm

Roche LightCycler 20

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi

Hasta (n=49) Kontrol (n=35)

Her oumlrnekten 50 ngmicroL havuzlandı

LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR

Agaroz Jelde Goumlruumlntuumlleme

Purifikasyon

TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)

kullanarak amplifikasyon uumlruumlnlerinin ligasyonu

Ecoli JM 109 kullanarak transformasyon

Ampisilin iccedileren LB besiyerlerine kuumlltuumlr

Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi

HastaKontr

olHasta

Kontr

ol

Lac-F Lac-R Koloni PCR

Agaroz jelde goumlruumlntuumlleme

Purifikasyon

g-Bifid-F g-Bifid-R koloni PCR

Sekans PubMed Blast 97 seq similarity

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

İnflamasyon Belirteccedilleri

İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı

hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci

Yoneda et al 2007 J Gastroenterol

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması

bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

dış

kı

Kontrol

NAFLDp=0003

p=0001 plt0001

Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması

A m

ucinip

hilia

F pra

usnitz

ii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

dış

kı

F0-F1

F2

plt0001

Enterobacteriaceae ile BKİ arasındakipozitif korelasyon

20 30 400

5

10

15

BMI

En

tero

ba

cte

ria

ce

ae

lo

g1

0g

r d

ışk

ı

BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)

-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)

Kontr

ol

NAFLD

-05

00

05

10

15

20

mg

dL

Kontrol

NAFLD

hs-CRP

NAFLD ve kontrol grubu hs-CRP seviyeleri

-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)

Kontr

ol

NAFLD

-20

0

20

40

60E

Um

L

Endotoxin

NAFLD ve kontrol grubu serum endotoksin duumlzeyleri

NAFLD ve kontrol grubu sitokin duumlzeyleri

bull TNF-α ve IL-6 seviyeleri bakımından fark yok

bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Pat

ient

0

20

40

60

80

IL-6

pg

mL

Control

Patient

NAFLD ve kontrol grubundaLactobacillus tuumlrleri

L delbrueckii 9

Lruminis50

Lmucosae2

Lacidophilus4

Lgastricus2

Lsalivarius4

Uncultured Lactobacillus

spp18

L helveticus7

Lsakei4

Kontrol

L delbrueckii Lruminis

Lmucosae Lacidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L helveticus

Lsakei

Ldelbrueckii 3

Lruminis39

Lmucosae9

L acidophilus11

Lgastricus6

Lsalivarius3

Uncultured Lactobacillus

spp5

L Johsonii5

Lreuteri19

NAFLD

Ldelbrueckii Lruminis

Lmucosae L acidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L Johsonii

Lreuteri

NAFLD ve kontrol grubundaBifidobacterium tuumlrleri

B adolescentis55

Blongum15

Bbifidum6

Bkashiwanohense10

Bcatenulatum4

Bruminatium2

Uncultured Bifidobacterium

spp Clone8

Kontrol

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Bcatenulatum

Bruminatium

Uncultured Bifidobacterium spp Clone

B adolescentis42

Blongum36

Bbifidum2

Bkashiwanohense2

Binfantis8

Uncultured Bifidobacterium

spp clone10

NAFLD

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Binfantis

Uncultured Bifidobacterium spp clone

Sonuccedil

NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir

A muciniphila NAFLD grubunda daha duumlşuumlk orandadır

Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir

B fragilis kilo durumuna bağımlı bir değişken

Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir

Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir

F prausnitzii mikrobiyotanın dominant bir uumlyesi

Sonuccedil

bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı

bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı

bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi

Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25

CEREN ERDOĞDU

Meltem Yalınay

Tarkan Karakan

Martin J Blaser

Barış Otlu

Doruk Engin

Sinem Solmaz

Berrin Oumlztuumlrk

GUuml Endoskopi Uumlnitesi

GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)

NYU Biyoistatistik Departmanı (Huilin Li)

Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)

Roche Diagnostics teknik ekibi

Teşekkuumlrler

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

qPCR analizleri iccedilin standartlarınhazırlanması

Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922

Kuumlltuumlr05 Mc Farland Suumlspansiyon

cfu sayım

Ekstraksiyon

Nanodrop Oumllccediluumlm

110 diluumlsyon

Standart eğri

0

5

10

15

20

25

30

35

1E+02 1E+04 1E+06 1E+08

Cro

ssin

g-p

oin

t (C

p)

valu

e

DNA copy number microL

A muciniphila

B fragilis group

Bifidobacterium spp

F prausnitzii

Enterobacteriaceae

Lactobacillus spp

Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml

Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml

Dışkı oumlrneklerinden qPCR analizleri

Dışkı oumlrnekleri

200 mg Ekstraksiyon Nanodrop Oumllccediluumlm

Roche LightCycler 20

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi

Hasta (n=49) Kontrol (n=35)

Her oumlrnekten 50 ngmicroL havuzlandı

LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR

Agaroz Jelde Goumlruumlntuumlleme

Purifikasyon

TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)

kullanarak amplifikasyon uumlruumlnlerinin ligasyonu

Ecoli JM 109 kullanarak transformasyon

Ampisilin iccedileren LB besiyerlerine kuumlltuumlr

Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi

HastaKontr

olHasta

Kontr

ol

Lac-F Lac-R Koloni PCR

Agaroz jelde goumlruumlntuumlleme

Purifikasyon

g-Bifid-F g-Bifid-R koloni PCR

Sekans PubMed Blast 97 seq similarity

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

İnflamasyon Belirteccedilleri

İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı

hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci

Yoneda et al 2007 J Gastroenterol

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması

bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

dış

kı

Kontrol

NAFLDp=0003

p=0001 plt0001

Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması

A m

ucinip

hilia

F pra

usnitz

ii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

dış

kı

F0-F1

F2

plt0001

Enterobacteriaceae ile BKİ arasındakipozitif korelasyon

20 30 400

5

10

15

BMI

En

tero

ba

cte

ria

ce

ae

lo

g1

0g

r d

ışk

ı

BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)

-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)

Kontr

ol

NAFLD

-05

00

05

10

15

20

mg

dL

Kontrol

NAFLD

hs-CRP

NAFLD ve kontrol grubu hs-CRP seviyeleri

-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)

Kontr

ol

NAFLD

-20

0

20

40

60E

Um

L

Endotoxin

NAFLD ve kontrol grubu serum endotoksin duumlzeyleri

NAFLD ve kontrol grubu sitokin duumlzeyleri

bull TNF-α ve IL-6 seviyeleri bakımından fark yok

bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Pat

ient

0

20

40

60

80

IL-6

pg

mL

Control

Patient

NAFLD ve kontrol grubundaLactobacillus tuumlrleri

L delbrueckii 9

Lruminis50

Lmucosae2

Lacidophilus4

Lgastricus2

Lsalivarius4

Uncultured Lactobacillus

spp18

L helveticus7

Lsakei4

Kontrol

L delbrueckii Lruminis

Lmucosae Lacidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L helveticus

Lsakei

Ldelbrueckii 3

Lruminis39

Lmucosae9

L acidophilus11

Lgastricus6

Lsalivarius3

Uncultured Lactobacillus

spp5

L Johsonii5

Lreuteri19

NAFLD

Ldelbrueckii Lruminis

Lmucosae L acidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L Johsonii

Lreuteri

NAFLD ve kontrol grubundaBifidobacterium tuumlrleri

B adolescentis55

Blongum15

Bbifidum6

Bkashiwanohense10

Bcatenulatum4

Bruminatium2

Uncultured Bifidobacterium

spp Clone8

Kontrol

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Bcatenulatum

Bruminatium

Uncultured Bifidobacterium spp Clone

B adolescentis42

Blongum36

Bbifidum2

Bkashiwanohense2

Binfantis8

Uncultured Bifidobacterium

spp clone10

NAFLD

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Binfantis

Uncultured Bifidobacterium spp clone

Sonuccedil

NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir

A muciniphila NAFLD grubunda daha duumlşuumlk orandadır

Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir

B fragilis kilo durumuna bağımlı bir değişken

Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir

Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir

F prausnitzii mikrobiyotanın dominant bir uumlyesi

Sonuccedil

bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı

bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı

bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi

Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25

CEREN ERDOĞDU

Meltem Yalınay

Tarkan Karakan

Martin J Blaser

Barış Otlu

Doruk Engin

Sinem Solmaz

Berrin Oumlztuumlrk

GUuml Endoskopi Uumlnitesi

GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)

NYU Biyoistatistik Departmanı (Huilin Li)

Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)

Roche Diagnostics teknik ekibi

Teşekkuumlrler

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

qPCR analizleri iccedilin standartlarınhazırlanması

Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922

Kuumlltuumlr05 Mc Farland Suumlspansiyon

cfu sayım

Ekstraksiyon

Nanodrop Oumllccediluumlm

110 diluumlsyon

Standart eğri

0

5

10

15

20

25

30

35

1E+02 1E+04 1E+06 1E+08

Cro

ssin

g-p

oin

t (C

p)

valu

e

DNA copy number microL

A muciniphila

B fragilis group

Bifidobacterium spp

F prausnitzii

Enterobacteriaceae

Lactobacillus spp

Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml

Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml

Dışkı oumlrneklerinden qPCR analizleri

Dışkı oumlrnekleri

200 mg Ekstraksiyon Nanodrop Oumllccediluumlm

Roche LightCycler 20

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi

Hasta (n=49) Kontrol (n=35)

Her oumlrnekten 50 ngmicroL havuzlandı

LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR

Agaroz Jelde Goumlruumlntuumlleme

Purifikasyon

TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)

kullanarak amplifikasyon uumlruumlnlerinin ligasyonu

Ecoli JM 109 kullanarak transformasyon

Ampisilin iccedileren LB besiyerlerine kuumlltuumlr

Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi

HastaKontr

olHasta

Kontr

ol

Lac-F Lac-R Koloni PCR

Agaroz jelde goumlruumlntuumlleme

Purifikasyon

g-Bifid-F g-Bifid-R koloni PCR

Sekans PubMed Blast 97 seq similarity

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

İnflamasyon Belirteccedilleri

İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı

hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci

Yoneda et al 2007 J Gastroenterol

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması

bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

dış

kı

Kontrol

NAFLDp=0003

p=0001 plt0001

Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması

A m

ucinip

hilia

F pra

usnitz

ii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

dış

kı

F0-F1

F2

plt0001

Enterobacteriaceae ile BKİ arasındakipozitif korelasyon

20 30 400

5

10

15

BMI

En

tero

ba

cte

ria

ce

ae

lo

g1

0g

r d

ışk

ı

BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)

-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)

Kontr

ol

NAFLD

-05

00

05

10

15

20

mg

dL

Kontrol

NAFLD

hs-CRP

NAFLD ve kontrol grubu hs-CRP seviyeleri

-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)

Kontr

ol

NAFLD

-20

0

20

40

60E

Um

L

Endotoxin

NAFLD ve kontrol grubu serum endotoksin duumlzeyleri

NAFLD ve kontrol grubu sitokin duumlzeyleri

bull TNF-α ve IL-6 seviyeleri bakımından fark yok

bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Pat

ient

0

20

40

60

80

IL-6

pg

mL

Control

Patient

NAFLD ve kontrol grubundaLactobacillus tuumlrleri

L delbrueckii 9

Lruminis50

Lmucosae2

Lacidophilus4

Lgastricus2

Lsalivarius4

Uncultured Lactobacillus

spp18

L helveticus7

Lsakei4

Kontrol

L delbrueckii Lruminis

Lmucosae Lacidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L helveticus

Lsakei

Ldelbrueckii 3

Lruminis39

Lmucosae9

L acidophilus11

Lgastricus6

Lsalivarius3

Uncultured Lactobacillus

spp5

L Johsonii5

Lreuteri19

NAFLD

Ldelbrueckii Lruminis

Lmucosae L acidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L Johsonii

Lreuteri

NAFLD ve kontrol grubundaBifidobacterium tuumlrleri

B adolescentis55

Blongum15

Bbifidum6

Bkashiwanohense10

Bcatenulatum4

Bruminatium2

Uncultured Bifidobacterium

spp Clone8

Kontrol

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Bcatenulatum

Bruminatium

Uncultured Bifidobacterium spp Clone

B adolescentis42

Blongum36

Bbifidum2

Bkashiwanohense2

Binfantis8

Uncultured Bifidobacterium

spp clone10

NAFLD

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Binfantis

Uncultured Bifidobacterium spp clone

Sonuccedil

NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir

A muciniphila NAFLD grubunda daha duumlşuumlk orandadır

Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir

B fragilis kilo durumuna bağımlı bir değişken

Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir

Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir

F prausnitzii mikrobiyotanın dominant bir uumlyesi

Sonuccedil

bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı

bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı

bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi

Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25

CEREN ERDOĞDU

Meltem Yalınay

Tarkan Karakan

Martin J Blaser

Barış Otlu

Doruk Engin

Sinem Solmaz

Berrin Oumlztuumlrk

GUuml Endoskopi Uumlnitesi

GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)

NYU Biyoistatistik Departmanı (Huilin Li)

Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)

Roche Diagnostics teknik ekibi

Teşekkuumlrler

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml

Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml

Dışkı oumlrneklerinden qPCR analizleri

Dışkı oumlrnekleri

200 mg Ekstraksiyon Nanodrop Oumllccediluumlm

Roche LightCycler 20

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi

Hasta (n=49) Kontrol (n=35)

Her oumlrnekten 50 ngmicroL havuzlandı

LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR

Agaroz Jelde Goumlruumlntuumlleme

Purifikasyon

TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)

kullanarak amplifikasyon uumlruumlnlerinin ligasyonu

Ecoli JM 109 kullanarak transformasyon

Ampisilin iccedileren LB besiyerlerine kuumlltuumlr

Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi

HastaKontr

olHasta

Kontr

ol

Lac-F Lac-R Koloni PCR

Agaroz jelde goumlruumlntuumlleme

Purifikasyon

g-Bifid-F g-Bifid-R koloni PCR

Sekans PubMed Blast 97 seq similarity

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

İnflamasyon Belirteccedilleri

İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı

hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci

Yoneda et al 2007 J Gastroenterol

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması

bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

dış

kı

Kontrol

NAFLDp=0003

p=0001 plt0001

Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması

A m

ucinip

hilia

F pra

usnitz

ii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

dış

kı

F0-F1

F2

plt0001

Enterobacteriaceae ile BKİ arasındakipozitif korelasyon

20 30 400

5

10

15

BMI

En

tero

ba

cte

ria

ce

ae

lo

g1

0g

r d

ışk

ı

BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)

-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)

Kontr

ol

NAFLD

-05

00

05

10

15

20

mg

dL

Kontrol

NAFLD

hs-CRP

NAFLD ve kontrol grubu hs-CRP seviyeleri

-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)

Kontr

ol

NAFLD

-20

0

20

40

60E

Um

L

Endotoxin

NAFLD ve kontrol grubu serum endotoksin duumlzeyleri

NAFLD ve kontrol grubu sitokin duumlzeyleri

bull TNF-α ve IL-6 seviyeleri bakımından fark yok

bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Pat

ient

0

20

40

60

80

IL-6

pg

mL

Control

Patient

NAFLD ve kontrol grubundaLactobacillus tuumlrleri

L delbrueckii 9

Lruminis50

Lmucosae2

Lacidophilus4

Lgastricus2

Lsalivarius4

Uncultured Lactobacillus

spp18

L helveticus7

Lsakei4

Kontrol

L delbrueckii Lruminis

Lmucosae Lacidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L helveticus

Lsakei

Ldelbrueckii 3

Lruminis39

Lmucosae9

L acidophilus11

Lgastricus6

Lsalivarius3

Uncultured Lactobacillus

spp5

L Johsonii5

Lreuteri19

NAFLD

Ldelbrueckii Lruminis

Lmucosae L acidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L Johsonii

Lreuteri

NAFLD ve kontrol grubundaBifidobacterium tuumlrleri

B adolescentis55

Blongum15

Bbifidum6

Bkashiwanohense10

Bcatenulatum4

Bruminatium2

Uncultured Bifidobacterium

spp Clone8

Kontrol

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Bcatenulatum

Bruminatium

Uncultured Bifidobacterium spp Clone

B adolescentis42

Blongum36

Bbifidum2

Bkashiwanohense2

Binfantis8

Uncultured Bifidobacterium

spp clone10

NAFLD

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Binfantis

Uncultured Bifidobacterium spp clone

Sonuccedil

NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir

A muciniphila NAFLD grubunda daha duumlşuumlk orandadır

Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir

B fragilis kilo durumuna bağımlı bir değişken

Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir

Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir

F prausnitzii mikrobiyotanın dominant bir uumlyesi

Sonuccedil

bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı

bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı

bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi

Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25

CEREN ERDOĞDU

Meltem Yalınay

Tarkan Karakan

Martin J Blaser

Barış Otlu

Doruk Engin

Sinem Solmaz

Berrin Oumlztuumlrk

GUuml Endoskopi Uumlnitesi

GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)

NYU Biyoistatistik Departmanı (Huilin Li)

Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)

Roche Diagnostics teknik ekibi

Teşekkuumlrler

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml

Dışkı oumlrneklerinden qPCR analizleri

Dışkı oumlrnekleri

200 mg Ekstraksiyon Nanodrop Oumllccediluumlm

Roche LightCycler 20

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi

Hasta (n=49) Kontrol (n=35)

Her oumlrnekten 50 ngmicroL havuzlandı

LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR

Agaroz Jelde Goumlruumlntuumlleme

Purifikasyon

TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)

kullanarak amplifikasyon uumlruumlnlerinin ligasyonu

Ecoli JM 109 kullanarak transformasyon

Ampisilin iccedileren LB besiyerlerine kuumlltuumlr

Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi

HastaKontr

olHasta

Kontr

ol

Lac-F Lac-R Koloni PCR

Agaroz jelde goumlruumlntuumlleme

Purifikasyon

g-Bifid-F g-Bifid-R koloni PCR

Sekans PubMed Blast 97 seq similarity

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

İnflamasyon Belirteccedilleri

İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı

hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci

Yoneda et al 2007 J Gastroenterol

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması

bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

dış

kı

Kontrol

NAFLDp=0003

p=0001 plt0001

Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması

A m

ucinip

hilia

F pra

usnitz

ii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

dış

kı

F0-F1

F2

plt0001

Enterobacteriaceae ile BKİ arasındakipozitif korelasyon

20 30 400

5

10

15

BMI

En

tero

ba

cte

ria

ce

ae

lo

g1

0g

r d

ışk

ı

BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)

-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)

Kontr

ol

NAFLD

-05

00

05

10

15

20

mg

dL

Kontrol

NAFLD

hs-CRP

NAFLD ve kontrol grubu hs-CRP seviyeleri

-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)

Kontr

ol

NAFLD

-20

0

20

40

60E

Um

L

Endotoxin

NAFLD ve kontrol grubu serum endotoksin duumlzeyleri

NAFLD ve kontrol grubu sitokin duumlzeyleri

bull TNF-α ve IL-6 seviyeleri bakımından fark yok

bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Pat

ient

0

20

40

60

80

IL-6

pg

mL

Control

Patient

NAFLD ve kontrol grubundaLactobacillus tuumlrleri

L delbrueckii 9

Lruminis50

Lmucosae2

Lacidophilus4

Lgastricus2

Lsalivarius4

Uncultured Lactobacillus

spp18

L helveticus7

Lsakei4

Kontrol

L delbrueckii Lruminis

Lmucosae Lacidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L helveticus

Lsakei

Ldelbrueckii 3

Lruminis39

Lmucosae9

L acidophilus11

Lgastricus6

Lsalivarius3

Uncultured Lactobacillus

spp5

L Johsonii5

Lreuteri19

NAFLD

Ldelbrueckii Lruminis

Lmucosae L acidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L Johsonii

Lreuteri

NAFLD ve kontrol grubundaBifidobacterium tuumlrleri

B adolescentis55

Blongum15

Bbifidum6

Bkashiwanohense10

Bcatenulatum4

Bruminatium2

Uncultured Bifidobacterium

spp Clone8

Kontrol

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Bcatenulatum

Bruminatium

Uncultured Bifidobacterium spp Clone

B adolescentis42

Blongum36

Bbifidum2

Bkashiwanohense2

Binfantis8

Uncultured Bifidobacterium

spp clone10

NAFLD

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Binfantis

Uncultured Bifidobacterium spp clone

Sonuccedil

NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir

A muciniphila NAFLD grubunda daha duumlşuumlk orandadır

Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir

B fragilis kilo durumuna bağımlı bir değişken

Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir

Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir

F prausnitzii mikrobiyotanın dominant bir uumlyesi

Sonuccedil

bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı

bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı

bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi

Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25

CEREN ERDOĞDU

Meltem Yalınay

Tarkan Karakan

Martin J Blaser

Barış Otlu

Doruk Engin

Sinem Solmaz

Berrin Oumlztuumlrk

GUuml Endoskopi Uumlnitesi

GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)

NYU Biyoistatistik Departmanı (Huilin Li)

Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)

Roche Diagnostics teknik ekibi

Teşekkuumlrler

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

Dışkı oumlrneklerinden qPCR analizleri

Dışkı oumlrnekleri

200 mg Ekstraksiyon Nanodrop Oumllccediluumlm

Roche LightCycler 20

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi

Hasta (n=49) Kontrol (n=35)

Her oumlrnekten 50 ngmicroL havuzlandı

LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR

Agaroz Jelde Goumlruumlntuumlleme

Purifikasyon

TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)

kullanarak amplifikasyon uumlruumlnlerinin ligasyonu

Ecoli JM 109 kullanarak transformasyon

Ampisilin iccedileren LB besiyerlerine kuumlltuumlr

Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi

HastaKontr

olHasta

Kontr

ol

Lac-F Lac-R Koloni PCR

Agaroz jelde goumlruumlntuumlleme

Purifikasyon

g-Bifid-F g-Bifid-R koloni PCR

Sekans PubMed Blast 97 seq similarity

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

İnflamasyon Belirteccedilleri

İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı

hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci

Yoneda et al 2007 J Gastroenterol

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması

bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

dış

kı

Kontrol

NAFLDp=0003

p=0001 plt0001

Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması

A m

ucinip

hilia

F pra

usnitz

ii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

dış

kı

F0-F1

F2

plt0001

Enterobacteriaceae ile BKİ arasındakipozitif korelasyon

20 30 400

5

10

15

BMI

En

tero

ba

cte

ria

ce

ae

lo

g1

0g

r d

ışk

ı

BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)

-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)

Kontr

ol

NAFLD

-05

00

05

10

15

20

mg

dL

Kontrol

NAFLD

hs-CRP

NAFLD ve kontrol grubu hs-CRP seviyeleri

-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)

Kontr

ol

NAFLD

-20

0

20

40

60E

Um

L

Endotoxin

NAFLD ve kontrol grubu serum endotoksin duumlzeyleri

NAFLD ve kontrol grubu sitokin duumlzeyleri

bull TNF-α ve IL-6 seviyeleri bakımından fark yok

bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Pat

ient

0

20

40

60

80

IL-6

pg

mL

Control

Patient

NAFLD ve kontrol grubundaLactobacillus tuumlrleri

L delbrueckii 9

Lruminis50

Lmucosae2

Lacidophilus4

Lgastricus2

Lsalivarius4

Uncultured Lactobacillus

spp18

L helveticus7

Lsakei4

Kontrol

L delbrueckii Lruminis

Lmucosae Lacidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L helveticus

Lsakei

Ldelbrueckii 3

Lruminis39

Lmucosae9

L acidophilus11

Lgastricus6

Lsalivarius3

Uncultured Lactobacillus

spp5

L Johsonii5

Lreuteri19

NAFLD

Ldelbrueckii Lruminis

Lmucosae L acidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L Johsonii

Lreuteri

NAFLD ve kontrol grubundaBifidobacterium tuumlrleri

B adolescentis55

Blongum15

Bbifidum6

Bkashiwanohense10

Bcatenulatum4

Bruminatium2

Uncultured Bifidobacterium

spp Clone8

Kontrol

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Bcatenulatum

Bruminatium

Uncultured Bifidobacterium spp Clone

B adolescentis42

Blongum36

Bbifidum2

Bkashiwanohense2

Binfantis8

Uncultured Bifidobacterium

spp clone10

NAFLD

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Binfantis

Uncultured Bifidobacterium spp clone

Sonuccedil

NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir

A muciniphila NAFLD grubunda daha duumlşuumlk orandadır

Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir

B fragilis kilo durumuna bağımlı bir değişken

Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir

Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir

F prausnitzii mikrobiyotanın dominant bir uumlyesi

Sonuccedil

bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı

bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı

bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi

Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25

CEREN ERDOĞDU

Meltem Yalınay

Tarkan Karakan

Martin J Blaser

Barış Otlu

Doruk Engin

Sinem Solmaz

Berrin Oumlztuumlrk

GUuml Endoskopi Uumlnitesi

GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)

NYU Biyoistatistik Departmanı (Huilin Li)

Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)

Roche Diagnostics teknik ekibi

Teşekkuumlrler

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi

Hasta (n=49) Kontrol (n=35)

Her oumlrnekten 50 ngmicroL havuzlandı

LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR

Agaroz Jelde Goumlruumlntuumlleme

Purifikasyon

TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)

kullanarak amplifikasyon uumlruumlnlerinin ligasyonu

Ecoli JM 109 kullanarak transformasyon

Ampisilin iccedileren LB besiyerlerine kuumlltuumlr

Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi

HastaKontr

olHasta

Kontr

ol

Lac-F Lac-R Koloni PCR

Agaroz jelde goumlruumlntuumlleme

Purifikasyon

g-Bifid-F g-Bifid-R koloni PCR

Sekans PubMed Blast 97 seq similarity

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

İnflamasyon Belirteccedilleri

İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı

hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci

Yoneda et al 2007 J Gastroenterol

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması

bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

dış

kı

Kontrol

NAFLDp=0003

p=0001 plt0001

Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması

A m

ucinip

hilia

F pra

usnitz

ii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

dış

kı

F0-F1

F2

plt0001

Enterobacteriaceae ile BKİ arasındakipozitif korelasyon

20 30 400

5

10

15

BMI

En

tero

ba

cte

ria

ce

ae

lo

g1

0g

r d

ışk

ı

BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)

-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)

Kontr

ol

NAFLD

-05

00

05

10

15

20

mg

dL

Kontrol

NAFLD

hs-CRP

NAFLD ve kontrol grubu hs-CRP seviyeleri

-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)

Kontr

ol

NAFLD

-20

0

20

40

60E

Um

L

Endotoxin

NAFLD ve kontrol grubu serum endotoksin duumlzeyleri

NAFLD ve kontrol grubu sitokin duumlzeyleri

bull TNF-α ve IL-6 seviyeleri bakımından fark yok

bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Pat

ient

0

20

40

60

80

IL-6

pg

mL

Control

Patient

NAFLD ve kontrol grubundaLactobacillus tuumlrleri

L delbrueckii 9

Lruminis50

Lmucosae2

Lacidophilus4

Lgastricus2

Lsalivarius4

Uncultured Lactobacillus

spp18

L helveticus7

Lsakei4

Kontrol

L delbrueckii Lruminis

Lmucosae Lacidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L helveticus

Lsakei

Ldelbrueckii 3

Lruminis39

Lmucosae9

L acidophilus11

Lgastricus6

Lsalivarius3

Uncultured Lactobacillus

spp5

L Johsonii5

Lreuteri19

NAFLD

Ldelbrueckii Lruminis

Lmucosae L acidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L Johsonii

Lreuteri

NAFLD ve kontrol grubundaBifidobacterium tuumlrleri

B adolescentis55

Blongum15

Bbifidum6

Bkashiwanohense10

Bcatenulatum4

Bruminatium2

Uncultured Bifidobacterium

spp Clone8

Kontrol

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Bcatenulatum

Bruminatium

Uncultured Bifidobacterium spp Clone

B adolescentis42

Blongum36

Bbifidum2

Bkashiwanohense2

Binfantis8

Uncultured Bifidobacterium

spp clone10

NAFLD

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Binfantis

Uncultured Bifidobacterium spp clone

Sonuccedil

NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir

A muciniphila NAFLD grubunda daha duumlşuumlk orandadır

Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir

B fragilis kilo durumuna bağımlı bir değişken

Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir

Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir

F prausnitzii mikrobiyotanın dominant bir uumlyesi

Sonuccedil

bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı

bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı

bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi

Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25

CEREN ERDOĞDU

Meltem Yalınay

Tarkan Karakan

Martin J Blaser

Barış Otlu

Doruk Engin

Sinem Solmaz

Berrin Oumlztuumlrk

GUuml Endoskopi Uumlnitesi

GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)

NYU Biyoistatistik Departmanı (Huilin Li)

Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)

Roche Diagnostics teknik ekibi

Teşekkuumlrler

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi

Hasta (n=49) Kontrol (n=35)

Her oumlrnekten 50 ngmicroL havuzlandı

LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR

Agaroz Jelde Goumlruumlntuumlleme

Purifikasyon

TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)

kullanarak amplifikasyon uumlruumlnlerinin ligasyonu

Ecoli JM 109 kullanarak transformasyon

Ampisilin iccedileren LB besiyerlerine kuumlltuumlr

Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi

HastaKontr

olHasta

Kontr

ol

Lac-F Lac-R Koloni PCR

Agaroz jelde goumlruumlntuumlleme

Purifikasyon

g-Bifid-F g-Bifid-R koloni PCR

Sekans PubMed Blast 97 seq similarity

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

İnflamasyon Belirteccedilleri

İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı

hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci

Yoneda et al 2007 J Gastroenterol

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması

bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

dış

kı

Kontrol

NAFLDp=0003

p=0001 plt0001

Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması

A m

ucinip

hilia

F pra

usnitz

ii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

dış

kı

F0-F1

F2

plt0001

Enterobacteriaceae ile BKİ arasındakipozitif korelasyon

20 30 400

5

10

15

BMI

En

tero

ba

cte

ria

ce

ae

lo

g1

0g

r d

ışk

ı

BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)

-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)

Kontr

ol

NAFLD

-05

00

05

10

15

20

mg

dL

Kontrol

NAFLD

hs-CRP

NAFLD ve kontrol grubu hs-CRP seviyeleri

-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)

Kontr

ol

NAFLD

-20

0

20

40

60E

Um

L

Endotoxin

NAFLD ve kontrol grubu serum endotoksin duumlzeyleri

NAFLD ve kontrol grubu sitokin duumlzeyleri

bull TNF-α ve IL-6 seviyeleri bakımından fark yok

bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Pat

ient

0

20

40

60

80

IL-6

pg

mL

Control

Patient

NAFLD ve kontrol grubundaLactobacillus tuumlrleri

L delbrueckii 9

Lruminis50

Lmucosae2

Lacidophilus4

Lgastricus2

Lsalivarius4

Uncultured Lactobacillus

spp18

L helveticus7

Lsakei4

Kontrol

L delbrueckii Lruminis

Lmucosae Lacidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L helveticus

Lsakei

Ldelbrueckii 3

Lruminis39

Lmucosae9

L acidophilus11

Lgastricus6

Lsalivarius3

Uncultured Lactobacillus

spp5

L Johsonii5

Lreuteri19

NAFLD

Ldelbrueckii Lruminis

Lmucosae L acidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L Johsonii

Lreuteri

NAFLD ve kontrol grubundaBifidobacterium tuumlrleri

B adolescentis55

Blongum15

Bbifidum6

Bkashiwanohense10

Bcatenulatum4

Bruminatium2

Uncultured Bifidobacterium

spp Clone8

Kontrol

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Bcatenulatum

Bruminatium

Uncultured Bifidobacterium spp Clone

B adolescentis42

Blongum36

Bbifidum2

Bkashiwanohense2

Binfantis8

Uncultured Bifidobacterium

spp clone10

NAFLD

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Binfantis

Uncultured Bifidobacterium spp clone

Sonuccedil

NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir

A muciniphila NAFLD grubunda daha duumlşuumlk orandadır

Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir

B fragilis kilo durumuna bağımlı bir değişken

Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir

Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir

F prausnitzii mikrobiyotanın dominant bir uumlyesi

Sonuccedil

bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı

bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı

bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi

Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25

CEREN ERDOĞDU

Meltem Yalınay

Tarkan Karakan

Martin J Blaser

Barış Otlu

Doruk Engin

Sinem Solmaz

Berrin Oumlztuumlrk

GUuml Endoskopi Uumlnitesi

GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)

NYU Biyoistatistik Departmanı (Huilin Li)

Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)

Roche Diagnostics teknik ekibi

Teşekkuumlrler

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

İnflamasyon Belirteccedilleri

İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı

hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci

Yoneda et al 2007 J Gastroenterol

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması

bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

dış

kı

Kontrol

NAFLDp=0003

p=0001 plt0001

Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması

A m

ucinip

hilia

F pra

usnitz

ii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

dış

kı

F0-F1

F2

plt0001

Enterobacteriaceae ile BKİ arasındakipozitif korelasyon

20 30 400

5

10

15

BMI

En

tero

ba

cte

ria

ce

ae

lo

g1

0g

r d

ışk

ı

BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)

-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)

Kontr

ol

NAFLD

-05

00

05

10

15

20

mg

dL

Kontrol

NAFLD

hs-CRP

NAFLD ve kontrol grubu hs-CRP seviyeleri

-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)

Kontr

ol

NAFLD

-20

0

20

40

60E

Um

L

Endotoxin

NAFLD ve kontrol grubu serum endotoksin duumlzeyleri

NAFLD ve kontrol grubu sitokin duumlzeyleri

bull TNF-α ve IL-6 seviyeleri bakımından fark yok

bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Pat

ient

0

20

40

60

80

IL-6

pg

mL

Control

Patient

NAFLD ve kontrol grubundaLactobacillus tuumlrleri

L delbrueckii 9

Lruminis50

Lmucosae2

Lacidophilus4

Lgastricus2

Lsalivarius4

Uncultured Lactobacillus

spp18

L helveticus7

Lsakei4

Kontrol

L delbrueckii Lruminis

Lmucosae Lacidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L helveticus

Lsakei

Ldelbrueckii 3

Lruminis39

Lmucosae9

L acidophilus11

Lgastricus6

Lsalivarius3

Uncultured Lactobacillus

spp5

L Johsonii5

Lreuteri19

NAFLD

Ldelbrueckii Lruminis

Lmucosae L acidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L Johsonii

Lreuteri

NAFLD ve kontrol grubundaBifidobacterium tuumlrleri

B adolescentis55

Blongum15

Bbifidum6

Bkashiwanohense10

Bcatenulatum4

Bruminatium2

Uncultured Bifidobacterium

spp Clone8

Kontrol

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Bcatenulatum

Bruminatium

Uncultured Bifidobacterium spp Clone

B adolescentis42

Blongum36

Bbifidum2

Bkashiwanohense2

Binfantis8

Uncultured Bifidobacterium

spp clone10

NAFLD

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Binfantis

Uncultured Bifidobacterium spp clone

Sonuccedil

NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir

A muciniphila NAFLD grubunda daha duumlşuumlk orandadır

Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir

B fragilis kilo durumuna bağımlı bir değişken

Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir

Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir

F prausnitzii mikrobiyotanın dominant bir uumlyesi

Sonuccedil

bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı

bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı

bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi

Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25

CEREN ERDOĞDU

Meltem Yalınay

Tarkan Karakan

Martin J Blaser

Barış Otlu

Doruk Engin

Sinem Solmaz

Berrin Oumlztuumlrk

GUuml Endoskopi Uumlnitesi

GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)

NYU Biyoistatistik Departmanı (Huilin Li)

Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)

Roche Diagnostics teknik ekibi

Teşekkuumlrler

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

İnflamasyon Belirteccedilleri

İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı

hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci

Yoneda et al 2007 J Gastroenterol

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması

bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

dış

kı

Kontrol

NAFLDp=0003

p=0001 plt0001

Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması

A m

ucinip

hilia

F pra

usnitz

ii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

dış

kı

F0-F1

F2

plt0001

Enterobacteriaceae ile BKİ arasındakipozitif korelasyon

20 30 400

5

10

15

BMI

En

tero

ba

cte

ria

ce

ae

lo

g1

0g

r d

ışk

ı

BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)

-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)

Kontr

ol

NAFLD

-05

00

05

10

15

20

mg

dL

Kontrol

NAFLD

hs-CRP

NAFLD ve kontrol grubu hs-CRP seviyeleri

-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)

Kontr

ol

NAFLD

-20

0

20

40

60E

Um

L

Endotoxin

NAFLD ve kontrol grubu serum endotoksin duumlzeyleri

NAFLD ve kontrol grubu sitokin duumlzeyleri

bull TNF-α ve IL-6 seviyeleri bakımından fark yok

bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Pat

ient

0

20

40

60

80

IL-6

pg

mL

Control

Patient

NAFLD ve kontrol grubundaLactobacillus tuumlrleri

L delbrueckii 9

Lruminis50

Lmucosae2

Lacidophilus4

Lgastricus2

Lsalivarius4

Uncultured Lactobacillus

spp18

L helveticus7

Lsakei4

Kontrol

L delbrueckii Lruminis

Lmucosae Lacidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L helveticus

Lsakei

Ldelbrueckii 3

Lruminis39

Lmucosae9

L acidophilus11

Lgastricus6

Lsalivarius3

Uncultured Lactobacillus

spp5

L Johsonii5

Lreuteri19

NAFLD

Ldelbrueckii Lruminis

Lmucosae L acidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L Johsonii

Lreuteri

NAFLD ve kontrol grubundaBifidobacterium tuumlrleri

B adolescentis55

Blongum15

Bbifidum6

Bkashiwanohense10

Bcatenulatum4

Bruminatium2

Uncultured Bifidobacterium

spp Clone8

Kontrol

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Bcatenulatum

Bruminatium

Uncultured Bifidobacterium spp Clone

B adolescentis42

Blongum36

Bbifidum2

Bkashiwanohense2

Binfantis8

Uncultured Bifidobacterium

spp clone10

NAFLD

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Binfantis

Uncultured Bifidobacterium spp clone

Sonuccedil

NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir

A muciniphila NAFLD grubunda daha duumlşuumlk orandadır

Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir

B fragilis kilo durumuna bağımlı bir değişken

Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir

Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir

F prausnitzii mikrobiyotanın dominant bir uumlyesi

Sonuccedil

bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı

bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı

bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi

Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25

CEREN ERDOĞDU

Meltem Yalınay

Tarkan Karakan

Martin J Blaser

Barış Otlu

Doruk Engin

Sinem Solmaz

Berrin Oumlztuumlrk

GUuml Endoskopi Uumlnitesi

GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)

NYU Biyoistatistik Departmanı (Huilin Li)

Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)

Roche Diagnostics teknik ekibi

Teşekkuumlrler

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

NAFLD (n=52) KONTROL (n=38)

Dışkı

ELISA (TNF-α IL-6)Hs-CRP

DNA

qPCR

Kromojenik LAL Testi(Endotoksin)

A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae

Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi

Kan

Serum

Metod

Dizi analizi

Rutin Biyokimyasaltetkikler

NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması

bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

dış

kı

Kontrol

NAFLDp=0003

p=0001 plt0001

Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması

A m

ucinip

hilia

F pra

usnitz

ii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

dış

kı

F0-F1

F2

plt0001

Enterobacteriaceae ile BKİ arasındakipozitif korelasyon

20 30 400

5

10

15

BMI

En

tero

ba

cte

ria

ce

ae

lo

g1

0g

r d

ışk

ı

BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)

-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)

Kontr

ol

NAFLD

-05

00

05

10

15

20

mg

dL

Kontrol

NAFLD

hs-CRP

NAFLD ve kontrol grubu hs-CRP seviyeleri

-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)

Kontr

ol

NAFLD

-20

0

20

40

60E

Um

L

Endotoxin

NAFLD ve kontrol grubu serum endotoksin duumlzeyleri

NAFLD ve kontrol grubu sitokin duumlzeyleri

bull TNF-α ve IL-6 seviyeleri bakımından fark yok

bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Pat

ient

0

20

40

60

80

IL-6

pg

mL

Control

Patient

NAFLD ve kontrol grubundaLactobacillus tuumlrleri

L delbrueckii 9

Lruminis50

Lmucosae2

Lacidophilus4

Lgastricus2

Lsalivarius4

Uncultured Lactobacillus

spp18

L helveticus7

Lsakei4

Kontrol

L delbrueckii Lruminis

Lmucosae Lacidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L helveticus

Lsakei

Ldelbrueckii 3

Lruminis39

Lmucosae9

L acidophilus11

Lgastricus6

Lsalivarius3

Uncultured Lactobacillus

spp5

L Johsonii5

Lreuteri19

NAFLD

Ldelbrueckii Lruminis

Lmucosae L acidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L Johsonii

Lreuteri

NAFLD ve kontrol grubundaBifidobacterium tuumlrleri

B adolescentis55

Blongum15

Bbifidum6

Bkashiwanohense10

Bcatenulatum4

Bruminatium2

Uncultured Bifidobacterium

spp Clone8

Kontrol

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Bcatenulatum

Bruminatium

Uncultured Bifidobacterium spp Clone

B adolescentis42

Blongum36

Bbifidum2

Bkashiwanohense2

Binfantis8

Uncultured Bifidobacterium

spp clone10

NAFLD

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Binfantis

Uncultured Bifidobacterium spp clone

Sonuccedil

NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir

A muciniphila NAFLD grubunda daha duumlşuumlk orandadır

Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir

B fragilis kilo durumuna bağımlı bir değişken

Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir

Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir

F prausnitzii mikrobiyotanın dominant bir uumlyesi

Sonuccedil

bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı

bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı

bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi

Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25

CEREN ERDOĞDU

Meltem Yalınay

Tarkan Karakan

Martin J Blaser

Barış Otlu

Doruk Engin

Sinem Solmaz

Berrin Oumlztuumlrk

GUuml Endoskopi Uumlnitesi

GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)

NYU Biyoistatistik Departmanı (Huilin Li)

Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)

Roche Diagnostics teknik ekibi

Teşekkuumlrler

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması

bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

dış

kı

Kontrol

NAFLDp=0003

p=0001 plt0001

Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması

A m

ucinip

hilia

F pra

usnitz

ii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

dış

kı

F0-F1

F2

plt0001

Enterobacteriaceae ile BKİ arasındakipozitif korelasyon

20 30 400

5

10

15

BMI

En

tero

ba

cte

ria

ce

ae

lo

g1

0g

r d

ışk

ı

BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)

-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)

Kontr

ol

NAFLD

-05

00

05

10

15

20

mg

dL

Kontrol

NAFLD

hs-CRP

NAFLD ve kontrol grubu hs-CRP seviyeleri

-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)

Kontr

ol

NAFLD

-20

0

20

40

60E

Um

L

Endotoxin

NAFLD ve kontrol grubu serum endotoksin duumlzeyleri

NAFLD ve kontrol grubu sitokin duumlzeyleri

bull TNF-α ve IL-6 seviyeleri bakımından fark yok

bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Pat

ient

0

20

40

60

80

IL-6

pg

mL

Control

Patient

NAFLD ve kontrol grubundaLactobacillus tuumlrleri

L delbrueckii 9

Lruminis50

Lmucosae2

Lacidophilus4

Lgastricus2

Lsalivarius4

Uncultured Lactobacillus

spp18

L helveticus7

Lsakei4

Kontrol

L delbrueckii Lruminis

Lmucosae Lacidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L helveticus

Lsakei

Ldelbrueckii 3

Lruminis39

Lmucosae9

L acidophilus11

Lgastricus6

Lsalivarius3

Uncultured Lactobacillus

spp5

L Johsonii5

Lreuteri19

NAFLD

Ldelbrueckii Lruminis

Lmucosae L acidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L Johsonii

Lreuteri

NAFLD ve kontrol grubundaBifidobacterium tuumlrleri

B adolescentis55

Blongum15

Bbifidum6

Bkashiwanohense10

Bcatenulatum4

Bruminatium2

Uncultured Bifidobacterium

spp Clone8

Kontrol

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Bcatenulatum

Bruminatium

Uncultured Bifidobacterium spp Clone

B adolescentis42

Blongum36

Bbifidum2

Bkashiwanohense2

Binfantis8

Uncultured Bifidobacterium

spp clone10

NAFLD

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Binfantis

Uncultured Bifidobacterium spp clone

Sonuccedil

NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir

A muciniphila NAFLD grubunda daha duumlşuumlk orandadır

Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir

B fragilis kilo durumuna bağımlı bir değişken

Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir

Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir

F prausnitzii mikrobiyotanın dominant bir uumlyesi

Sonuccedil

bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı

bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı

bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi

Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25

CEREN ERDOĞDU

Meltem Yalınay

Tarkan Karakan

Martin J Blaser

Barış Otlu

Doruk Engin

Sinem Solmaz

Berrin Oumlztuumlrk

GUuml Endoskopi Uumlnitesi

GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)

NYU Biyoistatistik Departmanı (Huilin Li)

Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)

Roche Diagnostics teknik ekibi

Teşekkuumlrler

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması

A m

ucinip

hilia

F pra

usnitz

ii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

dış

kı

F0-F1

F2

plt0001

Enterobacteriaceae ile BKİ arasındakipozitif korelasyon

20 30 400

5

10

15

BMI

En

tero

ba

cte

ria

ce

ae

lo

g1

0g

r d

ışk

ı

BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)

-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)

Kontr

ol

NAFLD

-05

00

05

10

15

20

mg

dL

Kontrol

NAFLD

hs-CRP

NAFLD ve kontrol grubu hs-CRP seviyeleri

-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)

Kontr

ol

NAFLD

-20

0

20

40

60E

Um

L

Endotoxin

NAFLD ve kontrol grubu serum endotoksin duumlzeyleri

NAFLD ve kontrol grubu sitokin duumlzeyleri

bull TNF-α ve IL-6 seviyeleri bakımından fark yok

bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Pat

ient

0

20

40

60

80

IL-6

pg

mL

Control

Patient

NAFLD ve kontrol grubundaLactobacillus tuumlrleri

L delbrueckii 9

Lruminis50

Lmucosae2

Lacidophilus4

Lgastricus2

Lsalivarius4

Uncultured Lactobacillus

spp18

L helveticus7

Lsakei4

Kontrol

L delbrueckii Lruminis

Lmucosae Lacidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L helveticus

Lsakei

Ldelbrueckii 3

Lruminis39

Lmucosae9

L acidophilus11

Lgastricus6

Lsalivarius3

Uncultured Lactobacillus

spp5

L Johsonii5

Lreuteri19

NAFLD

Ldelbrueckii Lruminis

Lmucosae L acidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L Johsonii

Lreuteri

NAFLD ve kontrol grubundaBifidobacterium tuumlrleri

B adolescentis55

Blongum15

Bbifidum6

Bkashiwanohense10

Bcatenulatum4

Bruminatium2

Uncultured Bifidobacterium

spp Clone8

Kontrol

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Bcatenulatum

Bruminatium

Uncultured Bifidobacterium spp Clone

B adolescentis42

Blongum36

Bbifidum2

Bkashiwanohense2

Binfantis8

Uncultured Bifidobacterium

spp clone10

NAFLD

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Binfantis

Uncultured Bifidobacterium spp clone

Sonuccedil

NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir

A muciniphila NAFLD grubunda daha duumlşuumlk orandadır

Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir

B fragilis kilo durumuna bağımlı bir değişken

Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir

Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir

F prausnitzii mikrobiyotanın dominant bir uumlyesi

Sonuccedil

bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı

bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı

bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi

Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25

CEREN ERDOĞDU

Meltem Yalınay

Tarkan Karakan

Martin J Blaser

Barış Otlu

Doruk Engin

Sinem Solmaz

Berrin Oumlztuumlrk

GUuml Endoskopi Uumlnitesi

GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)

NYU Biyoistatistik Departmanı (Huilin Li)

Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)

Roche Diagnostics teknik ekibi

Teşekkuumlrler

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

Enterobacteriaceae ile BKİ arasındakipozitif korelasyon

20 30 400

5

10

15

BMI

En

tero

ba

cte

ria

ce

ae

lo

g1

0g

r d

ışk

ı

BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)

-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)

Kontr

ol

NAFLD

-05

00

05

10

15

20

mg

dL

Kontrol

NAFLD

hs-CRP

NAFLD ve kontrol grubu hs-CRP seviyeleri

-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)

Kontr

ol

NAFLD

-20

0

20

40

60E

Um

L

Endotoxin

NAFLD ve kontrol grubu serum endotoksin duumlzeyleri

NAFLD ve kontrol grubu sitokin duumlzeyleri

bull TNF-α ve IL-6 seviyeleri bakımından fark yok

bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Pat

ient

0

20

40

60

80

IL-6

pg

mL

Control

Patient

NAFLD ve kontrol grubundaLactobacillus tuumlrleri

L delbrueckii 9

Lruminis50

Lmucosae2

Lacidophilus4

Lgastricus2

Lsalivarius4

Uncultured Lactobacillus

spp18

L helveticus7

Lsakei4

Kontrol

L delbrueckii Lruminis

Lmucosae Lacidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L helveticus

Lsakei

Ldelbrueckii 3

Lruminis39

Lmucosae9

L acidophilus11

Lgastricus6

Lsalivarius3

Uncultured Lactobacillus

spp5

L Johsonii5

Lreuteri19

NAFLD

Ldelbrueckii Lruminis

Lmucosae L acidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L Johsonii

Lreuteri

NAFLD ve kontrol grubundaBifidobacterium tuumlrleri

B adolescentis55

Blongum15

Bbifidum6

Bkashiwanohense10

Bcatenulatum4

Bruminatium2

Uncultured Bifidobacterium

spp Clone8

Kontrol

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Bcatenulatum

Bruminatium

Uncultured Bifidobacterium spp Clone

B adolescentis42

Blongum36

Bbifidum2

Bkashiwanohense2

Binfantis8

Uncultured Bifidobacterium

spp clone10

NAFLD

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Binfantis

Uncultured Bifidobacterium spp clone

Sonuccedil

NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir

A muciniphila NAFLD grubunda daha duumlşuumlk orandadır

Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir

B fragilis kilo durumuna bağımlı bir değişken

Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir

Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir

F prausnitzii mikrobiyotanın dominant bir uumlyesi

Sonuccedil

bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı

bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı

bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi

Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25

CEREN ERDOĞDU

Meltem Yalınay

Tarkan Karakan

Martin J Blaser

Barış Otlu

Doruk Engin

Sinem Solmaz

Berrin Oumlztuumlrk

GUuml Endoskopi Uumlnitesi

GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)

NYU Biyoistatistik Departmanı (Huilin Li)

Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)

Roche Diagnostics teknik ekibi

Teşekkuumlrler

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)

Kontr

ol

NAFLD

-05

00

05

10

15

20

mg

dL

Kontrol

NAFLD

hs-CRP

NAFLD ve kontrol grubu hs-CRP seviyeleri

-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)

Kontr

ol

NAFLD

-20

0

20

40

60E

Um

L

Endotoxin

NAFLD ve kontrol grubu serum endotoksin duumlzeyleri

NAFLD ve kontrol grubu sitokin duumlzeyleri

bull TNF-α ve IL-6 seviyeleri bakımından fark yok

bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Pat

ient

0

20

40

60

80

IL-6

pg

mL

Control

Patient

NAFLD ve kontrol grubundaLactobacillus tuumlrleri

L delbrueckii 9

Lruminis50

Lmucosae2

Lacidophilus4

Lgastricus2

Lsalivarius4

Uncultured Lactobacillus

spp18

L helveticus7

Lsakei4

Kontrol

L delbrueckii Lruminis

Lmucosae Lacidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L helveticus

Lsakei

Ldelbrueckii 3

Lruminis39

Lmucosae9

L acidophilus11

Lgastricus6

Lsalivarius3

Uncultured Lactobacillus

spp5

L Johsonii5

Lreuteri19

NAFLD

Ldelbrueckii Lruminis

Lmucosae L acidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L Johsonii

Lreuteri

NAFLD ve kontrol grubundaBifidobacterium tuumlrleri

B adolescentis55

Blongum15

Bbifidum6

Bkashiwanohense10

Bcatenulatum4

Bruminatium2

Uncultured Bifidobacterium

spp Clone8

Kontrol

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Bcatenulatum

Bruminatium

Uncultured Bifidobacterium spp Clone

B adolescentis42

Blongum36

Bbifidum2

Bkashiwanohense2

Binfantis8

Uncultured Bifidobacterium

spp clone10

NAFLD

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Binfantis

Uncultured Bifidobacterium spp clone

Sonuccedil

NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir

A muciniphila NAFLD grubunda daha duumlşuumlk orandadır

Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir

B fragilis kilo durumuna bağımlı bir değişken

Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir

Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir

F prausnitzii mikrobiyotanın dominant bir uumlyesi

Sonuccedil

bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı

bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı

bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi

Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25

CEREN ERDOĞDU

Meltem Yalınay

Tarkan Karakan

Martin J Blaser

Barış Otlu

Doruk Engin

Sinem Solmaz

Berrin Oumlztuumlrk

GUuml Endoskopi Uumlnitesi

GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)

NYU Biyoistatistik Departmanı (Huilin Li)

Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)

Roche Diagnostics teknik ekibi

Teşekkuumlrler

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)

Kontr

ol

NAFLD

-20

0

20

40

60E

Um

L

Endotoxin

NAFLD ve kontrol grubu serum endotoksin duumlzeyleri

NAFLD ve kontrol grubu sitokin duumlzeyleri

bull TNF-α ve IL-6 seviyeleri bakımından fark yok

bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Pat

ient

0

20

40

60

80

IL-6

pg

mL

Control

Patient

NAFLD ve kontrol grubundaLactobacillus tuumlrleri

L delbrueckii 9

Lruminis50

Lmucosae2

Lacidophilus4

Lgastricus2

Lsalivarius4

Uncultured Lactobacillus

spp18

L helveticus7

Lsakei4

Kontrol

L delbrueckii Lruminis

Lmucosae Lacidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L helveticus

Lsakei

Ldelbrueckii 3

Lruminis39

Lmucosae9

L acidophilus11

Lgastricus6

Lsalivarius3

Uncultured Lactobacillus

spp5

L Johsonii5

Lreuteri19

NAFLD

Ldelbrueckii Lruminis

Lmucosae L acidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L Johsonii

Lreuteri

NAFLD ve kontrol grubundaBifidobacterium tuumlrleri

B adolescentis55

Blongum15

Bbifidum6

Bkashiwanohense10

Bcatenulatum4

Bruminatium2

Uncultured Bifidobacterium

spp Clone8

Kontrol

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Bcatenulatum

Bruminatium

Uncultured Bifidobacterium spp Clone

B adolescentis42

Blongum36

Bbifidum2

Bkashiwanohense2

Binfantis8

Uncultured Bifidobacterium

spp clone10

NAFLD

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Binfantis

Uncultured Bifidobacterium spp clone

Sonuccedil

NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir

A muciniphila NAFLD grubunda daha duumlşuumlk orandadır

Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir

B fragilis kilo durumuna bağımlı bir değişken

Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir

Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir

F prausnitzii mikrobiyotanın dominant bir uumlyesi

Sonuccedil

bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı

bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı

bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi

Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25

CEREN ERDOĞDU

Meltem Yalınay

Tarkan Karakan

Martin J Blaser

Barış Otlu

Doruk Engin

Sinem Solmaz

Berrin Oumlztuumlrk

GUuml Endoskopi Uumlnitesi

GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)

NYU Biyoistatistik Departmanı (Huilin Li)

Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)

Roche Diagnostics teknik ekibi

Teşekkuumlrler

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

NAFLD ve kontrol grubu sitokin duumlzeyleri

bull TNF-α ve IL-6 seviyeleri bakımından fark yok

bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Pat

ient

0

20

40

60

80

IL-6

pg

mL

Control

Patient

NAFLD ve kontrol grubundaLactobacillus tuumlrleri

L delbrueckii 9

Lruminis50

Lmucosae2

Lacidophilus4

Lgastricus2

Lsalivarius4

Uncultured Lactobacillus

spp18

L helveticus7

Lsakei4

Kontrol

L delbrueckii Lruminis

Lmucosae Lacidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L helveticus

Lsakei

Ldelbrueckii 3

Lruminis39

Lmucosae9

L acidophilus11

Lgastricus6

Lsalivarius3

Uncultured Lactobacillus

spp5

L Johsonii5

Lreuteri19

NAFLD

Ldelbrueckii Lruminis

Lmucosae L acidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L Johsonii

Lreuteri

NAFLD ve kontrol grubundaBifidobacterium tuumlrleri

B adolescentis55

Blongum15

Bbifidum6

Bkashiwanohense10

Bcatenulatum4

Bruminatium2

Uncultured Bifidobacterium

spp Clone8

Kontrol

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Bcatenulatum

Bruminatium

Uncultured Bifidobacterium spp Clone

B adolescentis42

Blongum36

Bbifidum2

Bkashiwanohense2

Binfantis8

Uncultured Bifidobacterium

spp clone10

NAFLD

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Binfantis

Uncultured Bifidobacterium spp clone

Sonuccedil

NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir

A muciniphila NAFLD grubunda daha duumlşuumlk orandadır

Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir

B fragilis kilo durumuna bağımlı bir değişken

Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir

Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir

F prausnitzii mikrobiyotanın dominant bir uumlyesi

Sonuccedil

bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı

bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı

bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi

Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25

CEREN ERDOĞDU

Meltem Yalınay

Tarkan Karakan

Martin J Blaser

Barış Otlu

Doruk Engin

Sinem Solmaz

Berrin Oumlztuumlrk

GUuml Endoskopi Uumlnitesi

GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)

NYU Biyoistatistik Departmanı (Huilin Li)

Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)

Roche Diagnostics teknik ekibi

Teşekkuumlrler

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

NAFLD ve kontrol grubundaLactobacillus tuumlrleri

L delbrueckii 9

Lruminis50

Lmucosae2

Lacidophilus4

Lgastricus2

Lsalivarius4

Uncultured Lactobacillus

spp18

L helveticus7

Lsakei4

Kontrol

L delbrueckii Lruminis

Lmucosae Lacidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L helveticus

Lsakei

Ldelbrueckii 3

Lruminis39

Lmucosae9

L acidophilus11

Lgastricus6

Lsalivarius3

Uncultured Lactobacillus

spp5

L Johsonii5

Lreuteri19

NAFLD

Ldelbrueckii Lruminis

Lmucosae L acidophilus

Lgastricus Lsalivarius

Uncultured Lactobacillus spp L Johsonii

Lreuteri

NAFLD ve kontrol grubundaBifidobacterium tuumlrleri

B adolescentis55

Blongum15

Bbifidum6

Bkashiwanohense10

Bcatenulatum4

Bruminatium2

Uncultured Bifidobacterium

spp Clone8

Kontrol

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Bcatenulatum

Bruminatium

Uncultured Bifidobacterium spp Clone

B adolescentis42

Blongum36

Bbifidum2

Bkashiwanohense2

Binfantis8

Uncultured Bifidobacterium

spp clone10

NAFLD

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Binfantis

Uncultured Bifidobacterium spp clone

Sonuccedil

NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir

A muciniphila NAFLD grubunda daha duumlşuumlk orandadır

Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir

B fragilis kilo durumuna bağımlı bir değişken

Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir

Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir

F prausnitzii mikrobiyotanın dominant bir uumlyesi

Sonuccedil

bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı

bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı

bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi

Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25

CEREN ERDOĞDU

Meltem Yalınay

Tarkan Karakan

Martin J Blaser

Barış Otlu

Doruk Engin

Sinem Solmaz

Berrin Oumlztuumlrk

GUuml Endoskopi Uumlnitesi

GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)

NYU Biyoistatistik Departmanı (Huilin Li)

Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)

Roche Diagnostics teknik ekibi

Teşekkuumlrler

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

NAFLD ve kontrol grubundaBifidobacterium tuumlrleri

B adolescentis55

Blongum15

Bbifidum6

Bkashiwanohense10

Bcatenulatum4

Bruminatium2

Uncultured Bifidobacterium

spp Clone8

Kontrol

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Bcatenulatum

Bruminatium

Uncultured Bifidobacterium spp Clone

B adolescentis42

Blongum36

Bbifidum2

Bkashiwanohense2

Binfantis8

Uncultured Bifidobacterium

spp clone10

NAFLD

B adolescentis

Blongum

Bbifidum

Bkashiwanohense

Binfantis

Uncultured Bifidobacterium spp clone

Sonuccedil

NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir

A muciniphila NAFLD grubunda daha duumlşuumlk orandadır

Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir

B fragilis kilo durumuna bağımlı bir değişken

Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir

Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir

F prausnitzii mikrobiyotanın dominant bir uumlyesi

Sonuccedil

bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı

bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı

bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi

Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25

CEREN ERDOĞDU

Meltem Yalınay

Tarkan Karakan

Martin J Blaser

Barış Otlu

Doruk Engin

Sinem Solmaz

Berrin Oumlztuumlrk

GUuml Endoskopi Uumlnitesi

GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)

NYU Biyoistatistik Departmanı (Huilin Li)

Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)

Roche Diagnostics teknik ekibi

Teşekkuumlrler

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

Sonuccedil

NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir

A muciniphila NAFLD grubunda daha duumlşuumlk orandadır

Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir

B fragilis kilo durumuna bağımlı bir değişken

Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir

Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir

F prausnitzii mikrobiyotanın dominant bir uumlyesi

Sonuccedil

bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı

bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı

bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi

Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25

CEREN ERDOĞDU

Meltem Yalınay

Tarkan Karakan

Martin J Blaser

Barış Otlu

Doruk Engin

Sinem Solmaz

Berrin Oumlztuumlrk

GUuml Endoskopi Uumlnitesi

GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)

NYU Biyoistatistik Departmanı (Huilin Li)

Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)

Roche Diagnostics teknik ekibi

Teşekkuumlrler

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

Sonuccedil

bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı

bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı

bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi

Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25

CEREN ERDOĞDU

Meltem Yalınay

Tarkan Karakan

Martin J Blaser

Barış Otlu

Doruk Engin

Sinem Solmaz

Berrin Oumlztuumlrk

GUuml Endoskopi Uumlnitesi

GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)

NYU Biyoistatistik Departmanı (Huilin Li)

Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)

Roche Diagnostics teknik ekibi

Teşekkuumlrler

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

CEREN ERDOĞDU

Meltem Yalınay

Tarkan Karakan

Martin J Blaser

Barış Otlu

Doruk Engin

Sinem Solmaz

Berrin Oumlztuumlrk

GUuml Endoskopi Uumlnitesi

GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)

NYU Biyoistatistik Departmanı (Huilin Li)

Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)

Roche Diagnostics teknik ekibi

Teşekkuumlrler

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

Ceren Erdogdu17112016

Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak

Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması

Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak

mikrobiyotası iccedilerik ve yapı bakımından farklıdır

ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı

ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu

ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

Metod

bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik

tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir

bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis

bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans

NAFLD (n=43) KONTROL (n=23)

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri

Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin

kullanılan metriklerdir

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)

F0-F1

F2

Kontrol

Hasta

Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

NAFLD ve kontrol grubunda beta ccedileşitlilik

Kontrol (n=23)

NAFLD (n=43)

P=0004 method ANOSIM

Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

Fibrozis derecelerine goumlre beta ccedileşitlilik

F0-F1 (mild fibrosis) n=23

F2 (severe fibrosis) n=20

P=0005 method ANOSIM

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

NAFLD ve kontrol grubunda relatif dağılımlar

Kontrol NAFLD

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

Sonuccedil

bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır

bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

Blaser Lab

NYU Langone Biyoistatistik Boumluumlmuuml

NYU Langone Genom Merkezi

Teşekkuumlrler

Funding

T-RO1DK090989NIH

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance

p values were determined by t-test except male which was determined by chi square test

Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls

FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr

Characteristic

Patients

(n=52)

MeanplusmnSD

Control

(n=38)

MeanplusmnSD

p

Male () 478 324 0100

Age (years) 48 plusmn 12 36 plusmn 10 lt0001

BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001

Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001

ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001

AST (UL) 36 plusmn 18 21 plusmn 4 lt0001

ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001

Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613

HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001

LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065

Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001

Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001

Table 1 Demographic and Laboratory Results of the Participants

AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us sp

p

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae

0

5

10

15

log

10

gr

fec

es

Control

Patient

A m

ucinip

hilia

F pra

usnitzii

Lacto

bacill

us spp

Bifi

dobacte

rium

spp

B f

ragili

s gro

up

Entero

bacte

riace

ae0

5

10

15

log

10

gr

fec

es

F0-F1

F2

Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test

Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH

Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls

Contr

ol

Patie

nt-20

0

20

40

60

EU

mL

Control

Patient

Endotoxin

Contr

ol

Patie

nt0

20

40

60

80

TNF-alpha

pg

mL

Control

Patient

Contr

ol

Patie

nt-05

00

05

10

15

20

mg

dL

hs-CRP

Contr

ol

Patie

nt0

20

40

60

80

IL-6

pg

mL

Control

Patient

BA

C D

bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels

bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies

bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels

bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha

Results

Methods

Conclusions

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

Increased Akkermansia muciniphila and Bacteroides fragilis group

Abundance After Islamic Fasting

Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3

Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey

Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey

FRIDAY Hacettepe Universitycerenozkulhacettepeedutr

Table 2 Laboratory Data of the participants measured before andafter Islamic fasting

Abstract

Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members

BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test

Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively

bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition

bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values

bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan

bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition

bull

A mucin

iphila

F pra

usnitzii

Lactobacill

us spp

Bifidobacte

rium

spp

B fra

gilis g

roup

Entero

bacteria

ceae0

5

10

15

log1

0gr

fec

es

BF

AF

Characteristic

Before Fasting

(n=9)

MeanplusmnSD

After Fasting

(n=9)

MeanplusmnSD

p

BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810

Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821

ALT (UL) 246 plusmn 124 256 plusmn 112 0868

AST (UL) 228 plusmn 48 233 plusmn 48 0837

ALP (UL) 793 plusmn 228 779 plusmn 226 0904

Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009

HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876

LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914

Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879

Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006

Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing

(oC) Tm

Bifidobacterium

spp

B breve ATCC

15700

CTCCTGGAAACGGGTGG 550

g-Bifid-F 56 90

GGTGTTCTTCCCGATATCTACA g-Bifid-R

B fragilis group B fragilis ATCC

25285

ATAGCCTTTCGAAAGRAAGAT

495

g-Bfra-F

50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R

Lactobacillus spp L acidophilus

ATCC 4356

AGCAGTAGGGAATCTTCCA 341

Lact-F 55 86

CACCGCTACACATGGAG Lact-R

Amuciniphila Amuciniphila

ATCC BAA-835

CAGCACGTGAAGGTGGGGAC

327

AM-1

60 90 CCTTGCGGTTGGCTTCAGAT AM-2

F prausnitzii F prausnitzii

ATCC 27766

GATGGCCTCGCGTCCGATTAG 199

Fprau223F 60 88

CCGAAGACCTTCTTCCTCC Fprau420R

Enterobacteriaceae E coli ATCC

25922

CATTGACGTTACCCGCAGAAGAAGC

195

Eco1457F

63 87 CTCTACGAGACTCAAGCTTGC Eco1652R

Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards

Before IF After IF0

5

10

15

A muciniphila

log

10

gr

fec

es

Before IF After IF0

2

4

6

8

10

B fragilis

log

10

gr

fec

es

Results

Methods Conclusions

Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması

Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser

Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi

Analizi İle Karşılaştırılması