In Vivo Assessment of Resistant Starch Degradation by the Caecal Microbiota of Mice Using RNA-Based Stable Isotope Probing-A Proof-of-Principle Study.

Resistant starch (RS) is the digestion resistant fraction of complex polysaccharide starch. By reaching the large bowel, RS can function as a prebiotic carbohydrate, i.e., it can shape the structure and activity of bowel bacterial communities towards a profile that confers health benefits. However, knowledge about the fate of RS in complex intestinal communities and the microbial members involved in its degradation is limited. In this study, 16S ribosomal RNA (rRNA)-based stable isotope probing (RNA-SIP) was used to identify mouse bowel bacteria involved in the assimilation of RS or its derivatives directly in their natural gut habitat. Stable-isotope [U13C]-labeled native potato starch was administrated to mice, and caecal contents were collected before 0 h and 2 h and 4 h after administration. 'Heavy', isotope-labeled [13C]RNA species, presumably derived from bacteria that have metabolized the labeled starch, were separated from 'light', unlabeled [12C]RNA species by fractionation of isolated total RNA in isopycnic-density gradients. Inspection of different density gradients showed a continuous increase in 'heavy' 16S rRNA in caecal samples over the course of the experiment. Sequencing analyses of unlabeled and labeled 16S amplicons particularly suggested a group of unclassified Clostridiales, Dorea, and a few other taxa (Bacteroides, Turicibacter) to be most actively involved in starch assimilation in vivo. In addition, metabolic product analyses revealed that the predominant 13C-labeled short chain fatty acid (SCFA) in caecal contents produced from the [U13C] starch was butyrate. For the first time, this study provides insights into the metabolic transformation of RS by intestinal bacterial communities directly within a gut ecosystem, which will finally help to better understand its prebiotic potential and possible applications in human health.

RNA-Based Stable Isotope Probing Suggests Allobaculum spp. as Particularly Active Glucose Assimilators in a Complex Murine Microbiota Cultured In Vitro.

RNA-based stable isotope probing (RNA-SIP) and metabolic profiling were used to detect actively glucose-consuming bacteria in a complex microbial community obtained from a murine model system. A faeces-derived microbiota was incubated under anaerobic conditions for 0, 2, and 4 h with 40 mM [U13C]glucose. Isopycnic density gradient ultracentrifugation and fractionation of isolated RNA into labeled and unlabeled fractions followed by 16S rRNA sequencing showed a quick adaptation of the bacterial community in response to the added sugar, which was dominated by unclassified Lachnospiraceae species. Inspection of distinct fractions of isotope-labeled RNA revealed Allobaculum spp. as particularly active glucose utilizers in the system, as the corresponding RNA showed significantly higher proportions among the labeled RNA. With time, the labeled sugar was used by a wider spectrum of faecal bacteria. Metabolic profiling indicated rapid fermentation of [U13C]glucose, with lactate, acetate, and propionate being the principal 13C-labeled fermentation products, and suggested that "cross-feeding" occurred in the system. RNA-SIP combined with metabolic profiling of 13C-labeled products allowed insights into the microbial assimilation of a general model substrate, demonstrating the appropriateness of this technology to study assimilation processes of nutritionally more relevant substrates, for example, prebiotic carbohydrates, in the gut microbiota of mice as a model system.