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) • 10 healthy volunteers • Randomized to high fat vs. low fat diet • 10 day inpatient stay • Daily stool sample collection Biopsy • 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 Bacteria adherent to the rectal mucosa is enriched in Hypothesis: 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=0.002 p=0.004 Phosphorescence Quenching: A form of Biological Oximetry Phosphorescent Nanoprobe Oxyphor G4 Me Me Me O O O O Me O O • Non-toxic O O O O O O Me O O O O O O O O Me O O O O O O O O O O O O O O O O O O O O O Me • Does not interact with the O O O Me O O CO OC O O O OC CO O Me O O O O O Me O O Me NH HN O O O CO OC O O environment O O O O O O O O O O O O O O O O O HN NH O O O O C N O O N C O O O O H NH HN H O O O O OC OC O O O O Me • Does not cross biologic O NH HN O O C NH HN C O O O O O O O Me O HN NH O O O O O HN NH O O CO O O O CO O OC O OC O O O O O O Me membranes O H N N H Me N N O N H HN HN H N O O H O O H O O CO OC CO OC O O N N O O O O O Pd Me O O N N O • Unlimited water solubility Me OC CO CO OC O O O O O O H O O H O N H HN HN H N O N N O O O N N H O O H O O Me O CO O O OC O OC CO O O O O O Me O HN O NH O O O HN NH O O O O O O O C NH HN C Me O O O O NH HN O O O O O O O Me CO OC O O NH HN O Me O O H H O O O O C N O O N C O O HN NH O O O O O O O O O O O O O O O O O O OC CO O O O O NH HN O O O O O O O Me Me CO O O OC O O O O O O Me O CO OC O O O O O O O O O O O Me O O O Me O O O O O O O O O O Pd O O O O O O O O O O O O O O O O O Me O Me O O Me O Me Me PEG Porphyrin Poly(arylglycine) Methods: Phosphorescence Quenching G4 excitation: Injected G4 λmax = 635nm G4 in drinking water 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 Probe Delivered Orally 2013-01-09 Oxygen off Oxygen on mouth cecum 90 liver Probe: PdTBP/PMMA (calibrated in stool) ctrl 3 8 80 70 6 Oxygen off 60 (mmHg) 2 4 (mm Hg) (mm 2 pO pO 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 Consortium Associated Associated Mucosally 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 Rectal Biopsy/Swab Specific Taxa Murdochiella (Firmicute) Assacharolytic Finegoldia (Firmicute) Anaerococcus (Firmicute) Peptoniphilus (Firmicute) Porphyromonas (Bacteroidetes) Aerobic and Facultative Campylobacter (Proteobacteria) Anaerobic 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. Thank You Dr. Gary D. Wu Bob Baldassano 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 Sergei Vinogradov Support: Rick Bushman and the Tatiana Esipova NASPGHAN Fellow to Faculty Bushman lab: Jim Lewis Transition Award in IBD Research - Stephanie Grunberg Honghze Li - Kyle Bittinger Jun Chen - Rohini Sinha 5.