16S rRNA-based stable isotope probing (SIP) and nuclear magnetic resonance (NMR) spectroscopy-based metabolic profiling were used to identify bacteria fermenting glucose under conditions simulating the human intestine. The TIM-2 in vitro model of the human intestine was inoculated with a GI tract microbiota resembling that of the small intestine, to which subsequently 4, 20 or 40 mM of [U-13C]-glucose were added. RNA was extracted from lumen samples after 0 (control), 1, 2 and 4 h and subjected to density-gradient ultracentrifugation. Phylogenetic analysis of unlabeled 16S rRNA revealed a microbial community dominated by lactic acid bacteria and Clostridium perfringens. Distinct 13C-incorporation into bacterial RNA was only observed for the 40-mM addition. 16S rRNA fingerprinting showed an activity drop of Lactobacillus fermentum after glucose addition, while Streptococcus bovis and C. perfringens were identified as the most active glucose-fermenters. Accordingly, NMR analysis identified lactate, acetate, butyrate and formate as the principal fermentation products, constituting up to 91% of the 13C-carbon balance. RNA-SIP combined with metabolic profiling allowed us to detect differential utilization of a general model carbohydrate, indicating that this approach holds great potential to identify bacteria involved in the fermentation of dietary relevant oligo- and polymeric carbohydrates in the human intestine.
- microbial community structure
- chain fatty-acids
- propionate oxidation
Egert, M. G. G., de Graaf, A. A., Maathuis, A., de Waard, P., Plugge, C. M., Smidt, H., Deutz, N. E. P., Dijkema, C., de Vos, W. M., & Venema, K. (2007). Identification of glucose-fermenting bacteria present in an in-vitro model of the human inetstine by RNA-stable isotope probing. FEMS microbiology ecology, 60(1), 126-135. https://doi.org/10.1111/j.1574-6941.2007.00281.x