Microbes vs. chemistry in the origin of the anaerobic gut lumen

Abstract

The succession from aerobic and facultative anaerobic bacteria to obligate anaerobes in the infant gut along with the differences between the compositions of the mucosally adherent vs. luminal microbiota suggests that the gut microbes consume oxygen, which diffuses into the lumen from the intestinal tissue, maintaining the lumen in a deeply anaerobic state. Remarkably, measurements of luminal oxygen levels show nearly identical pO₂ (partial pressure of oxygen) profiles in conventional and germ-free mice, pointing to the existence of oxygen consumption mechanisms other than microbial respiration. In vitro experiments confirmed that the luminal contents of germ-free mice are able to chemically consume oxygen (e.g., via lipid oxidation reactions), although at rates significantly lower than those observed in the case of conventionally housed mice. For conventional mice, we also show that the taxonomic composition of the gut microbiota adherent to the gut mucosa and in the lumen throughout the length of the gut correlates with oxygen levels. At the same time, an increase in the biomass of the gut microbiota provides an explanation for the reduction of luminal oxygen in the distal vs. proximal gut. These results demonstrate how oxygen from the mammalian host is used by the gut microbiota, while both the microbes and the oxidative chemical reactions regulate luminal oxygen levels, shaping the composition of the microbial community throughout different regions of the gut.

Keywords: gut microbiota; luminal oxygen; microbial ecology; oxygen probes; phosphorescence quenching.

Fig. 1.

Microsphere-based phosphorescent probe Oxyphor MicroS for quantification of oxygen levels in the gut lumen. (A) Schematic illustration of the process of fabrication of Oxyphor MicroS using a microfluidic device. Wide-field images of the microspheres (B) in transmitted light and (C) phosphorescence in time-gated mode after excitation by a 20 μs-long light pulse (λ_ex = 630 nm; for imaging setup, see ref. 12). (D) Oxygen calibration plots of Oxyphor MicroS in substances of varying viscosities. Images of samples of murine cecal material after feeding Oxyphor MicroS: (E) transmitted light and (F) integrated luminescence intensity under continuous wave illumination (including back-scattered light and autofluorescence). (G) Phosphorescence in time-gated mode, showing signals only from the microspheres. Arrows indicate the location of Oxyphor MicroS probes (E and F).

Fig. 2.

Levels of oxygen in intestinal tissue vs. the lumen throughout the length of the intestinal tract. (A) Oxygen levels in conventionally housed mice. (B) Comparison of luminal oxygen levels throughout the length of the intestinal tract in conventionally housed vs. germ-free mice. For conventionally housed mice (intravascular): n = 10 for stomach, n = 10 for duodenum, n = 6 for ileum, and n = 6 for cecum. For conventionally housed mice (luminal): n = 24 for stomach, n = 20 for duodenum, n = 10 for ileum, and n = 13 for cecum. For germ-free mice: n = 3 for stomach and duodenum, n = 9 for ileum, and n = 8 for cecum. Mean ± SEM. *P < 0.05; ***P < 0.001 (two-tailed unpaired Student’s t test).

Fig. 3.

Dynamics of oxygen consumption by cecal contents in vitro and the effects of oxygen-involving reactions on the protein and lipid compositions. (A) Oxygen consumption by cecal contents of conventionally housed and germ-free mice. Cecal contents from conventionally housed mice consumed oxygen within 0.3–1 h, while cecal contents from germ-free mice consumed oxygen within 2.5–18 h, depending on the mass of the cecal contents used in these in vitro experiments. The x axis (time) is adjusted to account for the mass of the cecal contents: time (h) × mass of cecal contents (mg) to facilitate comparison. (B) Results of the lipidomic analysis of germ-free cecal material before and after oxygen consumption as well as the identification of lipid oxidation products (13-HODE) using deuterium-labeled LA (Inset). Mean ± SEM. *P < 0.01; **P < 0.01; ***P < 0.001. Two-tailed Student’s t test (unpaired, main figure; paired, Inset).

Fig. 4.

Oxygen consumption by bacteria along the intestinal tract. (A) Proportion of the four major bacterial phyla comprising the gut microbiota in luminal and mucosal samples throughout the length of the intestinal tract, as determined by 16S rRNA gene tag sequencing. (B) Total bacterial abundance in luminal and mucosal samples, as quantified by real-time qPCR of 16S rRNA gene copies. (C) Estimated oxygen consumption rate by luminal and mucosally adherent bacteria along the intestinal tract. Cec, cecum; Fec, feces; LI.D, large intestine–distal; LI.M, large Intestine–mid; LI.P, large intestine–proximal; SI.D, small intestine–distal; SI.M, small intestine–mid; SI.P, small intestine–proximal.