Culture independent analysis of the human gut microbiota and its activities

The human gut is colonized by a diverse collection of microorganisms which make up our intestinal microbiota. This predominantly anaerobic and largely uncultured microbiota is intimately involved in diverse host physiological processes and constitutes an important confounding factor when studying dietary interactions with host health and disease. Moreover, the gut microbiota effectively extends the metabolic potential of the human genome and contributes significantly to inter-individual metabolic variation. However, the fact that between 70 and 80% of the gut micro biota is recalcitrant to growth in pure culture under laboratory conditions has traditionally proven a considerable hurdle to studying these important host-microbiota-diet interactions. In this chapter, we will present current and commonly used molecular microbiology techniques for measuring both relative abundance of different intestinal bacteria in a culture independent manner, and more novel approaches to assess species richness within this complex microbial consortia. Culture independent molecular techniques commonly used to accurately enumerate bacteria within the gut microbiota in a culture independent manner include fluorescent in situ hybridization (FISH) and quantitative PCR (qPCR) with 16S rRNA targeted oligonucleotide probes and primers respectively. Species richness within the gut microbiota is commonly monitored using denaturing gradient gel electrophoresis or similar techniques and more recently, high through-put 16S rRNA community sequencing. The advantages and limitations of these different techniques as they relate to studying the microbial ecology of the gut microbiota will be addressed. We will also discuss the application of high throughput DNA sequencing to explore the metabolic potential encoded by the gut microbial metagenome. The complementary approach of metabonomics, which allows profiling metabolite fluxes present in whole human biofluids will also be discussed as a means of following the metabolic kinetic of human-microbiota-diet interactions at the systems level. Finally, we will present a detailed protocol for coanalysis of culture independent microbiological data with data derived from UPCL-MS and 1H-NMR based metabolite profiling. This approach has the potential to link bacterial populations in faecal or intestinal samples with specific metabolites in human biofluids, potentially elucidating novel interactions between human metabolism and the complex collection of bacteria in our gut.