Identification of glucose-fermenting bacteria present in an in vitro model of the human intestine by RNA-stable isotope probing
article
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.
Topics
Biomedical ResearchCarbohydratesDigestionMetabolic profilingNMRStable isotope probingAcetic acidAlcoholBacterial RNAButyric acidCarbohydrateCarbon 13Formic acidGlucoseGlycerolLactic acidRNA 16SStable isotopeBacteriumCarbohydrateDigestionFermentationGlucoseInoculationMicrobial activityPhylogeneticsRNAStable isotopeBacterium identificationClostridium perfringensControlled studyDensity gradient centrifugationFermentationGastrointestinal tractIn vitro studyInoculationIntestineLactic acid bacteriumLactobacillus fermentumMicrofloraMolecular phylogenyMolecular probeNonhumanNuclear magnetic resonance spectroscopyNucleotide sequencePriority journalRNA fingerprintingRNA stabilitystrain differenceStreptococcus bovisBacteria, AnaerobicCarbon IsotopesFermentationGlucoseGram-Positive BacteriaHumansIntestine, SmallIsotope LabelingMagnetic Resonance SpectroscopyModels, BiologicalMolecular Sequence DataPeristalsisRNA, BacterialRNA, Ribosomal, 16SSequence Analysis, DNABacteria (microorganisms)Clostridium perfringensLactobacillus fermentumStreptococcus bovis
TNO Identifier
239922
ISSN
01686496
Source
FEMS Microbiology Ecology, 60(1), pp. 126-135.
Pages
126-135
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