Analyzing the functionality of the human intestinal microbiota by stable isotope probing.

The human gastro-intestinal (GI) tract comprises a series of complex and dynamic organs ranging from the stomach to the distal colon, which harbor immense microbial assemblages, with considerable diversity and significant metabolic activity that are vital for human health. For understanding the functionality of microbial communities, it is necessary to elucidate the role of individual species within a community. The aim of the current study was to explore the functionality of the human GI tract microbiota, particularly in the conversions of dietary carbohydrates, in the human large intestine using in vitro as well as in vivo experiments. The functional capacity of the human intestinal microbiota in the fermentation of relevant [13-C]-labeled dietary carbohydrates was analyzed using 16S ribosomal RNA (rRNA)-based Stable Isotope Probing (SIP). Integrated application of RNA-based SIP with high-throughput diagnostic microarray-based phylogenetic profiling and metabolic flux analysis resulted in identification of the primary degraders of potato starch, inulin and lactose under human colon-like conditions. Furthermore, metabolic cross-feeding networks were proposed, involving secondary fermentation processes. Ruminococcus bromii was identified as the key player in the degradation of potato starch based on molecular analysis of samples taken during the fermentation under human colon like conditions, but also through fermentation studies in mono-culture or co-culture with Eubacterium rectale. The latter experiments revealed a crossfeeding relationship between R. bromii and E. rectale that involved the H2 produced by R. bromii from the starch degradation to increase production of propionate and acetate by E. rectale. Furthermore, species related to Dorea longicatena and Bifidobacterium adolescentis were identified as key members in inulin degradation, and Actinobacteria, particularly Bifidobacterium spp. and Collinsella spp. , were found to be the main taxa involved in lactose fermentation under human colon like conditions. Moving beyond in vitro studies we explored the role of the gut microbiota in lactose fermentation with a pioneering study, where [U-13C]-lactose was delivered directly to the terminal ileum of healthy human volunteers using a multi-lumen catheter that was also used for sampling of luminal content, which was further examined by RNA-SIP and phylogenetic microarray analysis. Individual patterns of the microbiota involved in lactose fermentation were observed, showing that in both volunteers different microbial populations were involved, which could be attributed to differences in the sampling location. A population related to Lactobacillus plantarum was found to be most actively involved in fermentation in one of the volunteers during the trial, while in the other volunteer most probably members of the genera Bifidobacterium and Colinsella were the primary lactose degraders, in agreement with the in vitro model study. Further analysis on the metabolites that accumulated during the lactose degradation will help to reconstruct the metabolic pathways involved in intestinal metabolism. Based on our exploratory research using RNA-based stable isotope probing together with high throughput phylogenetic analysis and metabolite profiling we could expand our knowledge on key microbial functions related to a healthy gut status.