Volume 147, Issue 5, Pages 1055-1063.e8 (November 2014) Correlation Between Intraluminal Oxygen Gradient and Radial Partitioning of Intestinal Microbiota  Lindsey Albenberg, Tatiana V. Esipova, Colleen P. Judge, Kyle Bittinger, Jun Chen, Alice Laughlin, Stephanie Grunberg, Robert N. Baldassano, James D. Lewis, Hongzhe Li, Stephen R. Thom, Frederic D. Bushman, Sergei A. Vinogradov, Gary D. Wu  Gastroenterology  Volume 147, Issue 5, Pages 1055-1063.e8 (November 2014) DOI: 10.1053/j.gastro.2014.07.020 Copyright © 2014 AGA Institute Terms and Conditions

Figure 1 Application of phosphorescence oximetry to intestinal oxygen measurements. (A) Intestinal oxygen measurements by phosphorescence quenching (the scheme does not represent correct proportions of physical dimensions). (B) Phosphorescence decays typically observed in tissue (Oxyphor G4) and intraluminal (OxyphorMicro) oxygen measurements. (C) Structure, optical absorption, and phosphorescence spectra of palladium (II) tetrabenzoporphyrin used as an oxygen-sensing element in both Oxyphor G4 and OxyphorMicro. (D) Calibration curves for Oxyphor G4 in physiological saline and OxyphorMicro directly in mouse fecal material at 36.5°C. Data for OxyphorMicro are a superposition of several animal samples. Gastroenterology 2014 147, 1055-1063.e8DOI: (10.1053/j.gastro.2014.07.020) Copyright © 2014 AGA Institute Terms and Conditions

Figure 2 Effect of altering the oxygen concentration in the inhaled gas mixture on host tissue and gut luminal oxygen levels. Arrows indicate switching pure O2 on (↓) and returning to ambient air (↑). (A) Changes in intestinal tissue Po2 in the cecum. Data for the liver tissue are shown for comparison. Changes in intestinal luminal Po2 using (B) trans-abdominal measurements and (C) in the cecum after laparotomy. (D) Relative time-dependent changes in tissue and luminal (cecum) Po2s: Δ Po2 = Po2 (t) - Po2 (0), where Po2 (t) and Po2 (0) are the Po2 values measured at time t and in the beginning of the experiment (time zero), respectively. Gastroenterology 2014 147, 1055-1063.e8DOI: (10.1053/j.gastro.2014.07.020) Copyright © 2014 AGA Institute Terms and Conditions

Figure 3 Differences in oxygen tolerance and community membership between the mucosally associated and fecal microbiota. Bacterial communities were profiled using 16S rRNA gene sequencing. (A) Separation of murine bacterial communities, analyzed using 16S rRNA gene sequencing of V1V2 tags, after day 6 of HBOT. An unweighted UniFrac plot is shown (P = .008; permanova). (B) Number of samples with operational taxonomic units annotating as Anaerostipes. The difference achieves P < 10e-10 (logistic mixed-effects model). (C) Number of bacterial genera classified as obligate anaerobes vs more oxygen-tolerant “all others” in paired human rectal biopsy and stool samples. (D) Number of bacterial genera classified as catalase positive vs catalase negative in paired human rectal biopsy and stool samples. (E) Relationship between mucosally and stool-associated microbiota using an unweighted UniFrac PCoA ordination (ellipses represent 95% confidence intervals for a multivariate normal distribution). (F) Pairwise distance in microbiota composition between stool, rectal biopsy, and rectal swab samples. Gastroenterology 2014 147, 1055-1063.e8DOI: (10.1053/j.gastro.2014.07.020) Copyright © 2014 AGA Institute Terms and Conditions

Figure 4 Heat map of the microbiota in stool, biopsy, and rectal swab samples from 16S rRNA gene sequencing. In the Mucosally Associated Consortium, 1 = asaccharolytic bacteria and 2 = aerobic or facultative anaerobic bacteria. Gastroenterology 2014 147, 1055-1063.e8DOI: (10.1053/j.gastro.2014.07.020) Copyright © 2014 AGA Institute Terms and Conditions

Figure 5 Gene content in gut bacteria inferred from 16S rRNA gene data. Gene content was inferred by using 16S rRNA gene tag data to access whole-genome sequences of bacterial relatives, and this information was used to infer potential gene content in the bacterial communities studied. Gastroenterology 2014 147, 1055-1063.e8DOI: (10.1053/j.gastro.2014.07.020) Copyright © 2014 AGA Institute Terms and Conditions

Supplementary Figure 1 Set-up used for calibration of the phosphorescent probe OxyphorMicro in semisolid samples. Gastroenterology 2014 147, 1055-1063.e8DOI: (10.1053/j.gastro.2014.07.020) Copyright © 2014 AGA Institute Terms and Conditions

Supplementary Figure 2 Typical calibration plot obtained using the set-up shown in Supplementary Figure 1. Blue arrows indicate time points when readings of phosphorescence lifetimes of OxyphorMicro were taken, and red arrows show the matching lifetimes for the solution of Oxyphor G4, from which Po2 values were calculated. Gastroenterology 2014 147, 1055-1063.e8DOI: (10.1053/j.gastro.2014.07.020) Copyright © 2014 AGA Institute Terms and Conditions

Supplementary Figure 3 Bacterial lineages showing significant changes over time after HBOT vs controls (n = 5 in each group). Longitudinal behavior was analyzed using a generalized linear mixed-effects model, allowing a random intercept for each mouse, and tested for an interaction between treatment and time point. Shown are lineages achieving P < .05 after correction for multiple comparisons. Gastroenterology 2014 147, 1055-1063.e8DOI: (10.1053/j.gastro.2014.07.020) Copyright © 2014 AGA Institute Terms and Conditions

Supplementary Figure 4 Unassigned reads at each rank in taxonomy based on sample type. An analysis using a Kruskal–Wallis test of ranked proportions was performed to determine if there was a difference in unassigned reads between sample types. There was no statistically significant difference (P = .4848). Gastroenterology 2014 147, 1055-1063.e8DOI: (10.1053/j.gastro.2014.07.020) Copyright © 2014 AGA Institute Terms and Conditions

Supplementary Figure 5 Heat map of gene abundance assigned to major metabolic pathways inferred from 16S rRNA gene sequence information in stool, biopsy, and swab samples using the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States. Gastroenterology 2014 147, 1055-1063.e8DOI: (10.1053/j.gastro.2014.07.020) Copyright © 2014 AGA Institute Terms and Conditions