Self-reinoculation with fecal flora changes microbiota density and composition leading to an altered bile-acid profile in the mouse small intestine

Abstract

Background: The upper gastrointestinal tract plays a prominent role in human physiology as the primary site for enzymatic digestion and nutrient absorption, immune sampling, and drug uptake. Alterations to the small intestine microbiome have been implicated in various human diseases, such as non-alcoholic steatohepatitis and inflammatory bowel conditions. Yet, the physiological and functional roles of the small intestine microbiota in humans remain poorly characterized because of the complexities associated with its sampling. Rodent models are used extensively in microbiome research and enable the spatial, temporal, compositional, and functional interrogation of the gastrointestinal microbiota and its effects on the host physiology and disease phenotype. Classical, culture-based studies have documented that fecal microbial self-reinoculation (via coprophagy) affects the composition and abundance of microbes in the murine proximal gastrointestinal tract. This pervasive selfreinoculation behavior could be a particularly relevant study factor when investigating small intestine microbiota. Modern microbiome studies either do not take self-reinoculation into account, or assume that approaches such as single housing mice or housing on wire mesh floors eliminate it. These assumptions have not been rigorously tested with modern tools. Here, we used quantitative 16S rRNA gene amplicon sequencing, quantitative microbial functional gene content inference, and metabolomic analyses of bile acids to evaluate the effects of selfreinoculation on microbial loads, composition, and function in the murine upper gastrointestinal tract.Results: In coprophagic mice, continuous self-exposure to the fecal flora had substantial quantitative and qualitative effects on the upper gastrointestinal microbiome. These differences in microbial abundance and community composition were associated with an altered profile of the small intestine bile acid pool, and, importantly, could not be inferred from analyzing large intestine or stool samples. Overall, the patterns observed in the small intestine of non-coprophagic mice (reduced total microbial load, low abundance of anaerobic microbiota, and bile acids predominantly in the conjugated form) resemble those typically seen in the human small intestine.Conclusions: Future studies need to take self-reinoculation into account when using mouse models to evaluate gastrointestinal microbial colonization and function in relation to xenobiotic transformation and pharmacokinetics or in the context of physiological states and diseases linked to small intestine microbiome and to small intestine dysbiosis.

Quantitative microbiome profiling in lumenal and tissue samples with broad coverage and dynamic range via a single-step 16S rRNA gene DNA copy quantification and amplicon barcoding

“…5). These results are in agreement with data previously obtained using a two-step approach (utilizing absolute 16S rRNA gene copy quantification with a dedicated qPCR assay performed separately from the barcoding PCR reaction) described in [6,7].…”

“…The Cq values obtained based on the real-time fluorescence measurements during the BC-qPCR reaction were in a good agreement with the absolute 16S rRNA gene DNA copy values ( Fig. 4C) estimated in the same samples (samples and data were from [6,7]) using the previously optimized qPCR assay (Fig. 3C).…”

“…2C) for a number of taxa ( Fig. 5) among samples from four experimental groups of mice described in [6,7] using the NGS data from [6,7] and the absolute fold difference data for the total 16S rRNA gene abundance in the corresponding samples from BC-qPCR assay (this study).…”

“…NGS data obtained from [6,7] were used to calculate the fold difference values among samples using the single-step fold-difference approach (this study) for each individual taxon (order level). Multiple comparisons between the four experimental groups of mice were performed for each taxon using the Kruskal-Wallis test.…”

“…Fecal samples were collected from SPF C57BL6/J mice of 2-12 months of age originally purchased from Jackson Laboratory (Sacramento, CA, USA) and housed in the Caltech animal facility for up to 10 months. Germ free mouse intestinal mucosal samples from germ free C57BL6/J mice of 2-5 months of age obtained from the germfree mouse colony maintained in the Caltech animal facility were collected and processed as in [6,7].…”