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Identification of glucose-fermenting bacteria present in an in vitro model of the human intestine by RNA-stable isotope probing

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Author: Egert, M. · Graaf, A.A. de · Maathuis, A. · Waard, P. de · Plugge, C.M. · Smidt, H. · Deutz, N.E.P. · Dijkema, C. · Vos, W.M. de · Venema, K.
Institution: TNO Kwaliteit van Leven
Source:FEMS Microbiology Ecology, 1, 60, 126-135
Identifier: 239922
doi: doi:10.1111/j.1574-6941.2007.00281.x
Keywords: Biology · Biomedical Research · Carbohydrates · Digestion · Metabolic profiling · NMR · Stable isotope probing · acetic acid · alcohol · bacterial RNA · butyric acid · carbohydrate · carbon 13 · formic acid · glucose · glycerol · lactic acid · RNA 16S · stable isotope · bacterium · carbohydrate · digestion · fermentation · glucose · inoculation · microbial activity · phylogenetics · RNA · stable isotope · article · bacterium identification · Clostridium perfringens · controlled study · density gradient centrifugation · fermentation · gastrointestinal tract · in vitro study · inoculation · intestine · lactic acid bacterium · Lactobacillus fermentum · microflora · molecular phylogeny · molecular probe · nonhuman · nuclear magnetic resonance spectroscopy · nucleotide sequence · priority journal · RNA fingerprinting · RNA stability · strain difference · Streptococcus bovis · Bacteria, Anaerobic · Carbon Isotopes · Fermentation · Glucose · Gram-Positive Bacteria · Humans · Intestine, Small · Isotope Labeling · Magnetic Resonance Spectroscopy · Models, Biological · Molecular Sequence Data · Peristalsis · RNA, Bacterial · RNA, Ribosomal, 16S · Sequence Analysis, DNA · Bacteria (microorganisms) · Clostridium perfringens · Lactobacillus fermentum · Streptococcus bovis


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.