genes encoding toxin of clostridium difficile in children ... et al. 2014.pdf · genes encoding...

Research ArticleGenes Encoding Toxin of Clostridium difficile in Children withand without Diarrhea

Victor R C Merino1 Viviane Nakano1 Sydney M Finegold234 and Mario J Avila-Campos1

1 Anaerobe Laboratory Department of Microbiology Institute of Biomedical Science University of Sao Paulo Sao Paulo SP Brazil2 Veterans Affairs West Los Angeles Medical Center Los Angeles CA USA3Department of Medicine School of Medicine University of California Los Angeles CA USA4Department of Microbiology Immunology and Molecular Genetics School of Medicine University of CaliforniaLos Angeles CA USA

Correspondence should be addressed to Mario J Avila-Campos mariojacuspbr

Received 4 February 2014 Accepted 17 April 2014 Published 29 April 2014

Academic Editor Katsumi Doi

Copyright copy 2014 Victor R C Merino et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The presence of gene 16S rRNA and genes encoding toxin A (tcdA) toxin B (tcdB) and binary toxin (cdtAcdtB) of Clostridiumdifficile in stool samples from children with (110) and without (150) diarrhea was determined by using a TaqMan system Fifty-seven(219) out of 260 stool samples harbored the 16S rRNA gene The genetic profile of tcdA+tcdBminus and cdtA+cdtB+ was verifiedin one C difficile-positive diarrhea sample and of tcdA+tcdB+ in three C difficile-positive nondiarrhea samples The presence oftcdA+tcdB+ in stools obtained from children without diarrhea suggests that they were asymptomatic carriers of toxigenic strains

1 Introduction

Clostridium difficile is the primary etiological agent ofantibiotic-associated diarrhea and pseudomembranous col-itis and is recognized as a cause of nosocomial diarrheaworldwide [1] The pathogenic effects of C difficile aremucosal damage to the colon that is caused by the productionof toxin A (308 kDa) andor toxin B (270 kDa) [2]

Some C difficile strains also produce a binary toxinencoded by cdtA and cdtB genes and both genes have beenobserved in strains producing C difficile-associated diarrhea(CDAD) [3] In Brazil few studies have shown the presenceof toxigenicC difficile harboring tcdA andor tcdB but not thebinary toxin genes in stool samples from children with acutediarrhea [4 5]

The cytotoxic assays performed from stool samples orisolated strains for detecting toxigenic C difficile are consid-ered the ldquogold standardrdquo [6] Other methods such as cultureand enzyme immunoassay (EIA) are not specific for theidentification of these toxigenic strains but EIA method isused in most laboratories worldwide [7]

In this study a quantitative detection of genes encodingtoxins A and B and binary toxin directly from C difficile-positive stool samples of children with and without acutediarrhea was determined

2 Methods

21 Sample Collection and DNA Extraction Two hundredsixty stool samples were obtained from 110 children withdiarrhea and from 150 children without diarrhea aged from1 month to 8 years old Stool samples were collected fromMarch 2008 through November 2010 Children did not dis-play other illnesses or comorbidities than diarrhea and theywere not under antibiotic treatment for at least three monthsprior to the sample collection Diarrhea was defined as threeor more unformed stools in the 24 h prior to enrollmentThe Ethics Committee of the Biomedical Science Institute atthe University of Sao Paulo (number 743CEP) approved thisstudy Total bacterial genomic DNAwas obtained from stoolsby using a QIAamp DNA stool minikit (Qiagen Germany)according to the manufacturerrsquos instructions

Hindawi Publishing CorporationScientificaVolume 2014 Article ID 594014 4 pageshttpdxdoiorg1011552014594014

2 Scientifica

Table 1 Genes and primerprobe sets used to detect C difficile and genes encoding toxins

Genes Oligonucleotides51015840 rarr 31015840 Melting temperature (∘C) References

16S rRNAlowastF-TTG AGC GAT TTA CTT CGG TAA AGAR-TGT ACT GGC TCA CCT TTG ATA TTC AP-CCA CGC GTT ACT CAC CCG TCC G

60 [16]

tcdAlowastlowastF-CAG GGC TAA TAG TTT GTT TAC AGA ACAR-CAA CAT CTA AAT ATA CTC CGC CAA AAP-TTA TAG TCA GCA GCT AGG ATT TCC ACG ATT TAA CAA CTC C

51 [17]

tcdBlowastlowastF-AGC AGT TGA ATA TAG TGG TTT AGT TAG AGT TGR-CAT GCT TTT TTA GTT TCT GGA TTG AAP-CAT CCA GTC TCA ATT GTA TAT GTT TCT CCA

58 [17]

cdtAlowastlowastF-GAT CTG GTC CTC AAG AAT TTG GTTR-GCT TGT CCT TCC CAT TTT CGA TTP-AAC TCT TAC TTC CCC TGA AT

51 [17]

cdtBlowastlowastF-AAA AGCT TCA GGT TCT TTT GAC AAGR-TGA TCA GTA GAG GCA TGT TCA TTT GP-CAA GAG ATC CGT TAG TTG CAG CAT ATC CAA TTG T

58 [17]

lowastUsed to detect C difficilelowastlowastUsed to detect toxigenic C difficile

Table 2 Quantitative detection of Clostridium difficile and target genes in diarrhea and nondiarrhea samples

Target gene Diarrhea (119899 = 110) Nondiarrhea (119899 = 150)119875

Number (range of log10) Mean plusmn SD Number (range of log

10) Mean plusmn SD

16S rRNAlowast 20 (12ndash69) 40 plusmn 17 37 (13ndash80) 48 plusmn 15 0063tcdA 1 (21) ND 3 (13ndash76) 29 plusmn 13 NDtcdB 0 (0) ND 3 (32ndash53) 46 plusmn 12 NDcdtA 1 (27) ND 0 (0) ND NDcdtB 1 (37) ND 0 (0) ND NDlowastUsed to detect C difficileND not determined

All toxigenic C difficile-positive stool samples were eval-uated for the presence of the toxins AB with an enzymeimmunoassay (EIA) by using a commercial kit Xpect Cdifficile Toxin AB Test (Remel USA)

22 PCR Amplification Real-time PCR assays were car-ried out in duplicate and performed in a total volume of25 120583L containing 2X TaqManUniversalMasterMix (AppliedBiosystems USA) 100 120583Mof each primer 100 120583Mof TaqManprobe and 2 ng of DNA Amplifications were performed in athermal cycler programmed as follows denaturation at 95∘Cfor 10min followed by 45 cycles of two steps denaturationat 95∘C for 15 s and an annealing temperature at 60∘C for1min for detecting C difficile 16S rRNA gene and annealingtemperatures for different toxin-encoding genes were asfollows at 51∘C for detecting tcdA and cdtA genes and at 58∘Cfor detecting tcdB and cdtB genesThe primerprobe sets usedare shown in Table 1

Bacterial DNA amplifications were adjusted using 1198772values gt 0900 A sample was considered positive for atarget gene when the detected fluorescence generates a curveabove the background fluorescence which was established byRotor Gene 6000 analytical software (Corbett Life ScienceAustralia)

23 Statistical Analysis Mean valuesplusmn SDwere calculated foreach toxin gene using a Studentrsquos 119905-test A difference of 119875 lt005was considered statistically significant (GraphPad PrismSoftware Inc)

3 Results

By using a commercial kit Xpect no toxin-positive stoolsample was observed Fifty-seven (219) out of 260 stoolsamples analyzed harbored C difficile (20 from children withdiarrhea and 37 from children without diarrhea) TwentyC difficile-positive diarrhea samples were obtained fromchildren between six months and 7 years old and amongthem a 1-year-old child harbored the tcdA gene and another1-year-old child (tcdAminustcdBminus) harbored both binary toxingenes (cdtA and cdtB) Also the 37 (246) C difficile-positive nondiarrhea samples were obtained from 1-to-5-yearold children and of them three children (a 1 year old and 2years old) harbored both genes tcdA and tcdB (Table 2) Noneof the C difficile-positive nondiarrhea samples harbored thebinary toxin genes

The presence of the target genes and their respective copynumbers are shown in Table 2 The tcdA was observed indiarrhea sample with a value of log

1021 Similarly binary

Scientifica 3

toxin genes were observed in log10

27 (cdtA) and log10

37(cdtB) Moreover in nondiarrhea samples tcdA was detectedfrom log

1013 to log

1036 and tcdB from log

1032 to log

1053

The specific primerprobe sets here used showed an accuratestandard curve over a 5-log-unit linear range that permittedthe copy number determination from standard amplificationsand detection to a limit of the 100 copies of the target DNAin unknown samples In this study C difficilewas detected indiarrhea (182) and nondiarrhea (246) stool samples

4 Discussion

In Brazil there are few epidemiological studies involvingthis pathogen as a cause of diarrhea in children [4 8]Since a stool sample harbored the profile of toxigenicitytcdA+tcdBminus and another cdtA+cdtB+ it suggested a lowcirculation of toxigenic C difficile in Brazilian children andthe acute diarrhea in children could have been caused byother enteropathogens than C difficile like typical viral orbacterial pathogens such asNorovirusRotavirus Salmonellaor Campylobacter and this needs to be investigated

The presence of toxins AB and binary toxin-producingC difficile in diarrhea outbreak has been associated to severeCDAD processes [9] On the other hand it is possible thatstrains can harbor toxins AB andor binary toxin geneswithout any expression and it could explain the failure todetect the presence of toxins when the EIA was used

The quantitative PCR by using the TaqMan systemprovided a direct detection of tcdAtcdB and cdtAcdtBfrom diarrhea and nondiarrhea stool samples and it coulddetermine a rapid bacterial screening in patients with acutediarrhea and due to the high sensitivity to detecting Cdifficile strains this assay has been useful in surveillance ofnosocomial diarrhea when required [10 11]

The presence of toxin genes (tcdA+tcdB+) in stool sam-ples obtained from three children without diarrhea suggeststhat they were asymptomatic carriers of toxigenic strainsIn addition an asymptomatic colonization by toxigenic Cdifficile was recently described in neonates suggesting thathealthy children display some tolerance to toxigenic strainsand it could play a role in the development of the intestinalimmune system [12ndash15]

Our results suggest that more studies are necessary todetermine the real role of toxigenic C difficile strains andtheir asymptomatic colonization in the human intestinaltract mainly in children

Conflict of Interests

All authors declare that they have no financialcommercialconflict of interests regarding the publication of this paper

Acknowledgments

The authors thank Marcia Harumi Fukugaiti for her tech-nical support and Dr Jon E Rosenblatt (Mayo ClinicUSA) and Lynne Sloan that kindly donated C diffi-cile CDC (tcdA+tcdB+ and cdtA+cdtB+) strain Viviane

Nakanowas supported by FAPESP (Grants 200857330-4 and200903792-0) This study was supported by Fundacao deAmparo a Pesquisa do Estado de Sao Paulo (FAPESP Grantnos 0703655-7 and CAPES 247209)

References

[1] J G Bartlett ldquoAntibiotic-associated diarrheardquoClinical InfectiousDiseases vol 15 no 4 pp 573ndash581 1992

[2] D E Voth and J D Ballard ldquoClostridium difficile toxinsmechanism of action and role in diseaserdquo Clinical MicrobiologyReviews vol 18 no 2 pp 247ndash263 2005

[3] H Pituch M Rupnik P Obuch-Woszczatynski A GrubesicF Meisel-Mikołajczyk and M Łuczak ldquoDetection of binary-toxin genes (cdtA and cdtB) among Clostridium difficile strainsisolated from patients with C difficile-associated diarrhoea(CDAD) in Polandrdquo Journal of Medical Microbiology vol 54no 2 pp 143ndash147 2005

[4] C E Ferreira V Nakano E L Durigon and M J Avila-Campos ldquoPrevalence of Clostridium spp and Clostridium dif-ficile in Children with Acute Diarrhea in Sao Paulo City BrazilrdquoMemorias do Instituto Oswaldo Cruz vol 98 no 4 pp 451ndash4542003

[5] A P Alcides J S Brazier L J F Pinto et al ldquoNew PCRribotypes of Clostridium difficile detected in children in BrazilrdquoAntonie van Leeuwenhoek International Journal of General andMolecular Microbiology vol 92 no 1 pp 53ndash59 2007

[6] P D Stamper W Babiker R Alcabasa et al ldquoEvaluation of anew commercial TaqMan PCR assay for direct detection of theClostridium difficile toxin B gene in clinical stool specimensrdquoJournal of Clinical Microbiology vol 47 no 12 pp 3846ndash38502009

[7] K C Carroll and M Loeffelholz ldquoConventional versus molec-ular methods for the detection of Clostridium difficilerdquo Journalof Clinical Microbiology vol 49 pp S49ndashS52 2011

[8] L B Garcia and M de Uzeda ldquoOccurrence of Clostridiumdifficile in the feces of children of Rio de Janeiro RJ BrazilrdquoRevista do Instituto de Medicina Tropical de Sao Paulo vol 30no 6 pp 419ndash423 1988

[9] F Barbut D Decre V Lalande et al ldquoClinical features ofClostridium difficile-associated diarrhoea due to binary toxin(actin-specific ADP-ribosyltransferase)-producing strainsrdquoJournal of Medical Microbiology vol 54 no 2 pp 181ndash185 2005

[10] S D Belanger M Boissinot N Clairoux F J Picard andM GBergeron ldquoRapid detection of Clostridium difficile in feces byreal-time PCRrdquo Journal of Clinical Microbiology vol 41 no 2pp 730ndash734 2003

[11] R J van den Berg N Vaessen H P Endtz T Schulin ER Van Der Vorm and E J Kuijper ldquoEvaluation of real-timePCR and conventional diagnostic methods for the detectionof Clostridium difficile-associated diarrhoea in a prospectivemulticentre studyrdquo Journal of Medical Microbiology vol 56 no1 pp 36ndash42 2007

[12] R P Bolton S K Tait P R F Dear and M S LosowskyldquoAsymptomatic neonatal colonisation by Clostridium difficilerdquoArchives of Disease in Childhood vol 59 no 5 pp 466ndash4721984

[13] S Matsuki E Ozaki M Shozu et al ldquoColonization by Clostrid-ium difficile of neonates in a hospital and infants and childrenin three day-care facilities of Kanazawa Japanrdquo InternationalMicrobiology vol 8 no 1 pp 43ndash48 2005

4 Scientifica

[14] S Jangi and J T Lamont ldquoAsymptomatic colonization byClostridium difficile in infants Implications for disease in laterliferdquo Journal of Pediatric Gastroenterology and Nutrition vol 51no 1 pp 2ndash7 2010

[15] C Rousseau I Poilane L de Pontual A C Maherault Ale Monnier and A Collignon ldquoClostridium difficile carriagein healthy infants in the community a potential reservoir forpathogenic strainsrdquo Clinical Infectious Diseases vol 55 pp1209ndash1215 2012

[16] J Penders C Vink C Driessen N London C Thijs andE E Stobberingh ldquoQuantification of Bifidobacterium sppEscherichia coli and Clostridium difficile in faecal samples ofbreast-fed and formula-fed infants by real-time PCRrdquo FEMSMicrobiology Letters vol 243 no 1 pp 141ndash147 2005

[17] D Wroblewski G E Hannett D J Bopp et al ldquoRapid molec-ular characterization of Clostridium difficile and assessment ofpopulations ofC difficile in stool specimensrdquo Journal of ClinicalMicrobiology vol 47 no 7 pp 2142ndash2148 2009

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Advances in

Virolog y


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Stem CellsInternational



Enzyme Research



Microbiology

2 Scientifica

Table 1 Genes and primerprobe sets used to detect C difficile and genes encoding toxins

Genes Oligonucleotides51015840 rarr 31015840 Melting temperature (∘C) References

16S rRNAlowastF-TTG AGC GAT TTA CTT CGG TAA AGAR-TGT ACT GGC TCA CCT TTG ATA TTC AP-CCA CGC GTT ACT CAC CCG TCC G

60 [16]

tcdAlowastlowastF-CAG GGC TAA TAG TTT GTT TAC AGA ACAR-CAA CAT CTA AAT ATA CTC CGC CAA AAP-TTA TAG TCA GCA GCT AGG ATT TCC ACG ATT TAA CAA CTC C

51 [17]

tcdBlowastlowastF-AGC AGT TGA ATA TAG TGG TTT AGT TAG AGT TGR-CAT GCT TTT TTA GTT TCT GGA TTG AAP-CAT CCA GTC TCA ATT GTA TAT GTT TCT CCA

58 [17]

cdtAlowastlowastF-GAT CTG GTC CTC AAG AAT TTG GTTR-GCT TGT CCT TCC CAT TTT CGA TTP-AAC TCT TAC TTC CCC TGA AT

51 [17]

cdtBlowastlowastF-AAA AGCT TCA GGT TCT TTT GAC AAGR-TGA TCA GTA GAG GCA TGT TCA TTT GP-CAA GAG ATC CGT TAG TTG CAG CAT ATC CAA TTG T

58 [17]

lowastUsed to detect C difficilelowastlowastUsed to detect toxigenic C difficile

Table 2 Quantitative detection of Clostridium difficile and target genes in diarrhea and nondiarrhea samples

Target gene Diarrhea (119899 = 110) Nondiarrhea (119899 = 150)119875

Number (range of log10) Mean plusmn SD Number (range of log

10) Mean plusmn SD

16S rRNAlowast 20 (12ndash69) 40 plusmn 17 37 (13ndash80) 48 plusmn 15 0063tcdA 1 (21) ND 3 (13ndash76) 29 plusmn 13 NDtcdB 0 (0) ND 3 (32ndash53) 46 plusmn 12 NDcdtA 1 (27) ND 0 (0) ND NDcdtB 1 (37) ND 0 (0) ND NDlowastUsed to detect C difficileND not determined

All toxigenic C difficile-positive stool samples were eval-uated for the presence of the toxins AB with an enzymeimmunoassay (EIA) by using a commercial kit Xpect Cdifficile Toxin AB Test (Remel USA)

22 PCR Amplification Real-time PCR assays were car-ried out in duplicate and performed in a total volume of25 120583L containing 2X TaqManUniversalMasterMix (AppliedBiosystems USA) 100 120583Mof each primer 100 120583Mof TaqManprobe and 2 ng of DNA Amplifications were performed in athermal cycler programmed as follows denaturation at 95∘Cfor 10min followed by 45 cycles of two steps denaturationat 95∘C for 15 s and an annealing temperature at 60∘C for1min for detecting C difficile 16S rRNA gene and annealingtemperatures for different toxin-encoding genes were asfollows at 51∘C for detecting tcdA and cdtA genes and at 58∘Cfor detecting tcdB and cdtB genesThe primerprobe sets usedare shown in Table 1

Bacterial DNA amplifications were adjusted using 1198772values gt 0900 A sample was considered positive for atarget gene when the detected fluorescence generates a curveabove the background fluorescence which was established byRotor Gene 6000 analytical software (Corbett Life ScienceAustralia)

23 Statistical Analysis Mean valuesplusmn SDwere calculated foreach toxin gene using a Studentrsquos 119905-test A difference of 119875 lt005was considered statistically significant (GraphPad PrismSoftware Inc)

3 Results

By using a commercial kit Xpect no toxin-positive stoolsample was observed Fifty-seven (219) out of 260 stoolsamples analyzed harbored C difficile (20 from children withdiarrhea and 37 from children without diarrhea) TwentyC difficile-positive diarrhea samples were obtained fromchildren between six months and 7 years old and amongthem a 1-year-old child harbored the tcdA gene and another1-year-old child (tcdAminustcdBminus) harbored both binary toxingenes (cdtA and cdtB) Also the 37 (246) C difficile-positive nondiarrhea samples were obtained from 1-to-5-yearold children and of them three children (a 1 year old and 2years old) harbored both genes tcdA and tcdB (Table 2) Noneof the C difficile-positive nondiarrhea samples harbored thebinary toxin genes

The presence of the target genes and their respective copynumbers are shown in Table 2 The tcdA was observed indiarrhea sample with a value of log

1021 Similarly binary

Scientifica 3


27 (cdtA) and log10


1013 to log


1032 to log

1053


4 Discussion








Acknowledgments



References














4 Scientifica












Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Scientifica 3


27 (cdtA) and log10


1013 to log


1032 to log

1053


4 Discussion








Acknowledgments



References














4 Scientifica












Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

4 Scientifica












Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

genes encoding toxin of clostridium difficile in children ... et al. 2014.pdf · genes encoding...

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