Tolerance to Oxygen and Asaccharolytic
Metabolism Distinguish the Human MucosallyAssociated Gut Microbiota from Stool
Lindsey Albenberg, DO
The Children’s Hospital of Philadelphia
The University of Pennsylvania
DISCLOSURE
• Nothing to disclose
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
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
Enrichment of “Aerotolerant” Catalase Positive
Bacteria on the Rectal Mucosa
p=0.002
p=0.004
Phosphorescence Quenching: A form of Biological Oximetry
Phosphorescent Nanoprobe Oxyphor G4
Me
O
O
O
O
O
Me
O
O
O
O
O
O
O
O
O
Me
O
O
O
O
HN
Me
O
O
O
O
O
Me
O
O
O
O O O
O
Me
O
O
Me
CO
O
O
O
O
CO
O
O
O
H
N
O
O CO
O
O
O
O
O
O
O O
O
O
Me
O
CO
O
O C
O
O
O O
O
Me O
O
O
O
O
H
N
O
H
N
HN
NH
O
O O O
O O O
O
O
O
O
O
O
Me
Me
OC
O
O
O
O
O
O
O O O
Me
O
C
O
H
N
O
C
O
O
O O
O
O
O
O O
O
Me
O
Me
Me
O
O
O
O
O
Me
O
O O
Me
O
O
O
Me
Pd
O
PEG
Poly(arylglycine)
• Non-toxic
• Does not interact with the
environment
• Does not cross biologic
membranes
• Unlimited water solubility
O
O
O O
O
Me
O
O
O
O
O
O O
O
O
O
O
O
O
O
O
CO
O
O
O
O
Me
O
O
OC
O
CO
O
HN
CO
O
HN
HN
O
O
OC
O
O
O
O
Me
O
O
OC O
NH O
O
NH
OC
O
O
O
O
O
O
O O
OC
H
N
N
H
O
NH
O
N
H
O
CO
O
OC
O
H
N
O
CO
HN
NH
HN
O
O
O
OC O
O
Me
O
O
HN
NH
O
O
C
O
NH O
N
H
HN
CO
N
Pd
N
OC
O
OC
O
O
NH
O
Porphyrin
N
O
O
Me O
N
OC
H HN
N
O
O
O
O
O
N
H
N
H HN
OC
O
O
C
O
O
CO
OC
O O
O
O
O
O
O
O
O
HN
NH
HN
O
O
O
O
O
O HN O
O
C
O
O
O
N
H
Me
O
O
O
HN
O
H
N
N
H
O
O Me
O
O
OC
HN
NH
O
O
O
O
NH
O
O
NH
NH
CO
O
O
O
O
O HN O
O
O
O
C
O
Me
O
O
O
CO
O
O
O
O
O
N
H
C
O
Me
O O
HN
NH
O
OC
O
O O
O
OC
O
O
O
O
O
OC
O
CO
O
CO
O
O O
O
O
O
O
O
O
Me
O
O
O
O
O
O
O O
O
O
O O
Me
Me
O
O
O
O O
O
Me
Me
O
Methods: Phosphorescence Quenching
a
c
detector (APD)
I(t)
emission
excitation
LED
excitation pulse
phosphorescence
decay
0
200
400
time (ms)
d
, M-1cm-1
optical
fiber
b
sampled
volume
428
448
2x10
5
1x10
5
637
530
excit
phos
Injected G4
Oxyphor
R4
0
G4
excitation:
400
500
600
700
eλmax = 635nm, nm
G4 detection:
λmax = 810 nm
intestinal wall
fecal material
Emission intensity, AU
813
G4
in drinking
Oxyphor
G4
water
698
600
800
, nm
1000
Vinogradov, SA et al. Rev Sci Instrum, 2001. 72(8): 3396-3406.
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
time (s)
800
1000
1200
0
500
1000
1500
time (s)
2000
2500
3000
Bacterial Taxonomy in Human Stool is Different
from Either Rectal Biopsies or Swabs
The Mucosally-Associated Microbiota in Humans
Mucosally
Associated
Consortium
Stool
Biopsy
Swab
The Assacharolytic and Oxygen Tolerance Bacterial Signature
at the Mucosal Interface
Ulger-Toprak et al. Int. J. System and Evol. Micro. 2010;60:1013
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)
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.
Thank You
Dr. Gary D. Wu
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
Bob Baldassano
Transition Award in IBD Research
Jim Lewis
Honghze Li
Jun Chen