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Correlation Between Intraluminal Oxygen
Gradient and Radial Partitioning of Intestinal
Microbiota
Lindsey Albenberg, DO
The Children’s Hospital of Philadelphia
The University of Pennsylvania
Dysbiosis in Inflammatory Bowel Disease (IBD)
• 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.)
Peterson et al. Cell Host
& Microbe. 2008.
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
1
Oxygen Gradient
O2
Biopsy and Stool Communities Cluster Separately
Independent of Individual, Diet, and Time
CAFÉ Study Days 1 and 10
(Stool and Biopsy Samples)
•
•
•
•
Biopsy
•
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)
Stool
Unweighted Unifrac
Wu et al. Science 2011;334:105-8
CAFE biopsy vs. stool analysis
Hypothesis:
Bacteria adherent to the rectal mucosa is enriched in
aerotolerant bacteria relative to the feces where most
organisms are obligate anaerobes.
Classify the genera based on oxygen preference
• 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
2
Enrichment of “Aerotolerant” Catalase Positive
Bacteria on the Rectal Mucosa
p=0.004
p=0.002
Phosphorescence Quenching: A form of Biological Oximetry
Phosphorescent Nanoprobe Oxyphor G4
Me
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H
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NH O
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Me
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NH O
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Porphyrin
OC
HN
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N
Pd
N N
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NH
H
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Me
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O Me
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NH
• Non-toxic
• Does not interact with the
environment
• Does not cross biologic
membranes
• Unlimited water solubility
O
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OC
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CO
CO
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PEG
Poly(arylglycine)
Methods: Phosphorescence Quenching
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.
3
Oxygenation of the Host and in the Gut Lumen
Probe Delivered IV
2013-01-09
Oxygen on
Probe Delivered Orally
Oxygen off
mouth
cecum
liver
90
Probe: PdTBP/PMMA
(calibrated in stool)
ctrl 3
8
6
70
pO2 (mm Hg)
pO2 (mmHg)
80
60
Oxygen off
4
Oxygen on
50
2
40
0
30
0
200
400
600
800
1000
1200
0
500
1000
1500
2000
2500
3000
time (s)
time (s)
Albenberg, L et al. Gastroenterology, 2014. 147(5): 1055-63.
Bacterial Taxonomy in Human Stool is Different
from Either Rectal Biopsies or Swabs
Albenberg, L et al. Gastroenterology, 2014. 147(5): 1055-63.
The Mucosally-Associated Microbiota in Humans
Mucosally
Associated
Consortium
Stool
Biopsy
Swab
Albenberg, L et al. Gastroenterology, 2014. 147(5): 1055-63.
4
The Assacharolytic and Oxygen Tolerance Bacterial Signature
at the Mucosal Interface
Aerobic and
Facultative
Anaerobic
Assacharolytic
Rectal Biopsy/Swab Specific
Taxa
Murdochiella (Firmicute)
Finegoldia (Firmicute)
Anaerococcus (Firmicute)
Peptoniphilus (Firmicute)
Porphyromonas (Bacteroidetes)
Campylobacter (Proteobacteria)
Propionibacterium (Actinobacteria)
Corynebacterium (Actinobacteria)
Enterobacteriaceae (Proteobacteria)
Ulger-Toprak et al. Int. J. System and Evol. Micro. 2010;60:1013
Albenberg, L et al. Gastroenterology, 2014. 147(5): 1055-63.
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
Bob Baldassano
Thank You
The Wu Lab
Lillian Chau
Christel Chehoud
Ying-Yu Chen
Colleen Judge
Sue Keilbaugh
Judith Kelsen
Andrew Lin
Helen Pauly-Hubbard
David Shen
Mike Sheng
Sarah Smith
Rick Bushman and the
Bushman lab:
- Stephanie Grunberg
- Kyle Bittinger
- Rohini Sinha
Sergei Vinogradov
Support:
Tatiana Esipova
NASPGHAN Fellow to Faculty
Jim Lewis
Transition Award in IBD Research
Honghze Li
Jun Chen
5
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