O-016 Tolerance to Oxygen and Asaccharolytic Metabolism Distinguishes the Human Mucosally-associated Gut Microbiota from Stool (2013)

The gut microbiota exists within a dynamic environment determined by host physiology. Examples include the effects of diet on bile acid secretion, the production of mucus by the intestinal epithelium, and the delivery of urea to the colon. The response of the gut microbiota to these factors helps to shape its structure and function, which may show spatial segregation. Here, we characterize factors that distinguish the fecal from the mucosally-adherent microbiota. Additionally, we provide support for the importance of an oxygen gradient in the gut lumen in the development of the dysbiosis seen in inflammatory bowel disease (IBD). Partial pressure of oxygen (pO2) was measured using phosphorescence quenching. Excitation of a phosphorescent probe by light produces phosphorescence decay, whose rate is proportional to the concentration of oxygen in the environment. A water-soluble probe, Oxyphor G4, was administered IV for tissue pO2 measurements. To quantify luminal pO2 we used Oxyphor Micro, a water-insoluble probe. To characterize the microbial oxygen signature in human samples, we examined the oxygen preference of bacterial taxa found in biopsy specimens versus stool samples from a study previously published by our group1. We also examined the bacterial taxa found in paired stool and rectal swab samples from 7 pediatric subjects. Microbiota composition was determined by 454 pyrosequencing of 16S rRNA genes analyzed by UniFrac. IV injection of Oxyphor G4 led to its distribution throughout the vasculature. By directing excitation at the intestinal wall, pO2 in intestinal tissue was measured. In contrast, ingested Oxyphor Micro remained in the feces, providing selective measurement of luminal pO2. The pO2 was much lower in the feces than in the tissue. By characterizing the oxygen preference of the bacterial genera in the paired stool and biopsy samples in our previously published dataset1, we found that aerotolerant organisms were greatly enriched in biopsy samples relative to stool (P < 5.4e–05). An analysis, which included the results of the rectal swab study, revealed that the microbiota obtained by swab and biopsy, were more similar to each other than to stool (Fig. 1). Additionally, the mucosally-adherent microbiota could be distinguished from that in stool by the presence of aerotolerant taxa belonging to the Proteobacteria and Actinobacteria phyla as well as assacharolytic taxa from the Firmicutes and Bacteroidetes phyla. Using phosphorescence quenching, we confirm the oxygen-poor environment of the gut lumen. Using 16S rRNA gene sequencing, we reveal an oxygen gradient by demonstrating enrichment of aerotolerant organisms associated with the mucosa relative to the feces. Since the observed phyla contain taxa that are consistently found in the dysbiotic signature associated with IBD, this supports the hypothesis that dysbiosis is the result of the oxidative nature of the inflammatory response. By comparing rectal biopsies and swabs to feces, we also show that a consortium of asaccharolytic bacteria that primarily metabolize amino acids are mucosally-adherent, a possible consequence of the protein content of mucus and the shedding of the intestinal epithelium. These findings reveal a previously unappreciated complexity of the gut microbiota. 1. Wu GD, et al. Science 2011;334:105–8.