tolerance to oxygen and asaccharolytic metabolism distinguish the human mucosally- associated gut...
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Tolerance to Oxygen and Asaccharolytic Metabolism Distinguish the Human Mucosally-
Associated Gut Microbiota from Stool
Lindsey Albenberg, DOThe Children’s Hospital of Philadelphia
The University of Pennsylvania
DISCLOSURE
• Nothing to disclose
Dysbiosis in Inflammatory Bowel Disease (IBD)
Peterson et al. Cell Host & Microbe. 2008.
• Increases in Proteobacteria and Actinobacteria– Generally aerotolerant– Better able to manage
oxidative stress in the setting of inflammation?
• Host inflammatory response leads to production of oxidation products which serve as electron acceptors supporting anaerobic respiration by facultative anaerobes (Winter et al. EMBO Reports. 2013.)
The Anaerobic Intestinal Lumen
• The intestinal lumen in humans is thought to be strictly anaerobic, but the reason for this largely unknown
• Current technology is unable to dynamically quantify oxygen in the intestinal tract, so the mechanisms that maintain this anaerobic environment remain unclear
Oxygen Gradient
O2
Biopsy
Stool
Unweighted Unifrac
Biopsy and Stool Communities Cluster Separately Independent of Individual, Diet, and Time
CAFÉ Study Days 1 and 10 (Stool and Biopsy Samples)
Wu et al. Science 2011;334:105-8
• 10 healthy volunteers• Randomized to high
fat vs. low fat diet• 10 day inpatient stay • Daily stool sample
collection• Biopsy specimens
obtained on day 1 and day 10 (un-prepped flexible sigmoidoscopy)
• Focus on 73 bacterial genera with maximum proportion > 0.002• Classify each genus as either “facultative anaerobe or aerotolerant”,
“aerobe or microaerophile” or “obligate anaerobe”• Two groups
– Obligate anaerobe– All others• Classify the genera into Stool or Biopsy-dominant
CAFE biopsy vs. stool analysis
Classify the genera based on oxygen preference
Bacteria adherent to the rectal mucosa is enriched in aerotolerant bacteria relative to the feces where most organisms are obligate anaerobes.
Hypothesis:
p=0.002 p=0.004
Enrichment of “Aerotolerant” Catalase Positive Bacteria on the Rectal Mucosa
N N
N N
Pd
OC CO
OC
OC
OC CO
CO
CO
HNO
HN
NHO
ONH
ONH
HNO
OCCO
OC
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NHO
NH
HNO
OHN
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• Non-toxic• Does not interact with the
environment • Does not cross biologic
membranes• Unlimited water solubility
Phosphorescence Quenching: A form of Biological Oximetry
Phosphorescent Nanoprobe Oxyphor G4
Pd
Poly(arylglycine)PEG
Porphyrin
400 500 600 700
0
1x105
2x105
530
428
637
448
,
M-1cm
-1
, nm
600 800 1000
698
813
Em
issi
on
inte
nsi
ty,
AU
, nm
LED
detector (APD)
optical fiber
0 200 400
emissionexcitation
I(t)
time (ms)
excitation pulse
phosphorescencedecay
a
b
c
d
eintestinal wall
fecal material
sampled volume
excit phos
Oxyphor R4
Oxyphor G4
Injected G4
G4 in drinking water
G4 excitation: λmax = 635nm
G4 detection: λmax = 810 nm
Vinogradov, SA et al. Rev Sci Instrum, 2001. 72(8): 3396-3406.
Methods: Phosphorescence Quenching
Oxygenation of the Host and in the Gut Lumen
Probe Delivered IV Probe Delivered Orally
Oxygen on Oxygen off
Oxygen on
Oxygen off
Bacterial Taxonomy in Human Stool is Different from Either Rectal Biopsies or Swabs
Stool Biopsy Swab
Mu
cosally
Asso
ciated
C
on
sortiu
mThe Mucosally-Associated Microbiota in Humans
Murdochiella (Firmicute)Finegoldia (Firmicute)Anaerococcus (Firmicute)Peptoniphilus (Firmicute)Porphyromonas (Bacteroidetes) Campylobacter (Proteobacteria)Propionibacterium (Actinobacteria)Corynebacterium (Actinobacteria)Enterobacteriaceae (Proteobacteria)
Rectal Biopsy/Swab Specific Taxa
Assacharolytic A
erobic and F
acultative A
naerobic
Ulger-Toprak et al. Int. J. System and Evol. Micro. 2010;60:1013
The Assacharolytic and Oxygen Tolerance Bacterial Signature at the Mucosal Interface
Spatial Segregation of the Intestinal Microbiota at the Mucosal Interface
•Using phosphorescence quenching, we confirm the oxygen-poor environment of the gut lumen
•Composition of the gut microbiota is spatially-segregated along the radial axis of the gut
•The intestinal mucosal surface is enriched for oxygen tolerant organisms that may serve as “founding” communities for the development of the dysbiotic microbiota associated with intestinal inflammation
•By comparing the microbiota in rectal biopsies and swabs to the feces, we also show that a consortium of asaccharolytic bacteria that primarily metabolize amino acids are associated with mucus.
Dr. Gary D. Wu
The Wu LabLillian ChauChristel ChehoudYing-Yu ChenColleen JudgeSue KeilbaughJudith KelsenAndrew LinHelen Pauly-HubbardDavid ShenMike ShengSarah Smith
NASPGHAN Fellow to Faculty Transition Award in IBD Research
Support:
Thank You
Rick Bushman and the Bushman lab:- Stephanie Grunberg-Kyle Bittinger-Rohini Sinha
Sergei VinogradovTatiana EsipovaBob BaldassanoJim LewisHonghze LiJun Chen