Identification of glucose-fermenting bacteria present in an in vitro model of the human intestine by RNA-stable isotope probing
de Graaf, A.A.
de Waard, P.
de Vos, W.M.
TNO Kwaliteit van Leven
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. © 2007 Federation of European Microbiological Societies.
To reference this document use:
Stable isotope probing
density gradient centrifugation
in vitro study
lactic acid bacterium
nuclear magnetic resonance spectroscopy
Magnetic Resonance Spectroscopy
Molecular Sequence Data
RNA, Ribosomal, 16S
Sequence Analysis, DNA
FEMS Microbiology Ecology, 60 (1), 126-135