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BACKGROUND Intestinal bacterial communities are not homogenous throughout the gastrointestinal tract. Human research on the gut microbiome often neglects intra-intestinal variability by relying on a single measurement from stool samples. One source of complexity is the adherence to undigested, residual fiber. Currently, no procedure exists to extract RNA from distinct bacterial subpopulations in stool samples. MATERIAL AND METHODS A serial centrifugation procedure was developed in which bacterial RNA could be extracted from distinct stool-fractions - fiber-adherent and non-fiber-adherent bacteria. To test whether the separation procedure yielded distinct bacterial subpopulations, a set of RT-qPCR assays were developed for a fiber-adherent bacterial species, Bifidobacterium adolescentis, then a within-subject repeated-measures study was conducted with 3 human subjects undergoing 4 dietary regimens. At each timepoint, between-fraction differences in gene expression were evaluated. RESULTS The RNA isolation procedure was able to isolate intact RNA in 20 of 24 samples in the fiber-adherent fraction. PurB and sdh were identified as suitable reference genes for B. adolescentis RT-qPCR assays. When subjects were provided a high resistant starch diet, bacterial fractions exhibited different expression of the trp operon (p=0.031). CONCLUSIONS Our study provides human gut microbiome researchers a novel tool for evaluating functional characteristics of bacterial subpopulations in human stool. Moreover, these experiments provide modest support for the existence of a functionally unique fiber-adherent subpopulation of B. adolescentis. Until a more thorough evaluation of the adherent and non-adherent fraction can be performed, researchers should be cautious when generalizing functional data derived solely from unfractionated stool samples.

Development and Preliminary Validation of a Feasible Procedure for Isolating RNA from Fiber-Adherent Bacteria in Human Stool