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

• Published in: FEMS microbiology ecology Vol. 60; no. 1; pp. 126 - 135
• Main Authors: Egert, Markus, de Graaf, Albert A, Maathuis, Annet, de Waard, Pieter, Plugge, Caroline M, Smidt, Hauke, Deutz, Nicolaas E.P, Dijkema, Cor, de Vos, Willem M, Venema, Koen
• Format: Journal Article
• Language: English
• Published: Oxford, UK Oxford, UK : Blackwell Publishing Ltd 01.04.2007; Blackwell Publishing Ltd; Blackwell; Oxford University Press
• Subjects: Acetic acid; Bacteria; Bacteria, Anaerobic - classification; Bacteria, Anaerobic - genetics; Bacteria, Anaerobic - metabolism; Bacteriology; Biological and medical sciences; Carbohydrates; Carbon 13; Carbon Isotopes - metabolism; Clostridium perfringens; Computer simulation; digestion; Ecology; Fermentation; Fermenters; Fingerprinting; Fundamental and applied biological sciences. Psychology; Gastrointestinal tract; Glucose; Glucose - metabolism; Gram-Positive Bacteria - classification; Gram-Positive Bacteria - genetics; Gram-Positive Bacteria - metabolism; Humans; Intestine; Intestine, Small - microbiology; Isotope Labeling - methods; Lactic acid; Lactic acid bacteria; Magnetic Resonance Spectroscopy - methods; metabolic profiling; Metabolism; metabolomics; Microbiology; Microbiota; Microorganisms; Miscellaneous; Models, Biological; Molecular Sequence Data; NMR; Nuclear magnetic resonance; Peristalsis; Phylogeny; RNA, Bacterial - genetics; RNA, Bacterial - metabolism; RNA, Ribosomal, 16S - genetics; rRNA 16S; Sequence Analysis, DNA; Small intestine; Spectroscopy; stable isotope probing; Stable isotopes; Ultracentrifugation; carbohydrates; digestion; stable isotope probing; NMR; metabolic profiling; Lactic acid bacteria; Clostridiales; Microflora; Identification; NMR spectrometry; Glucose; Phylogeny; Clostridium perfringens; Streptococcus C; Ribosomal RNA; Streptococcal infection; 16S-RNA; Bacteria; Lactobacillaceae; Micrococcales; Microbial community; Human; Lactobacillus fermentum; Digestive system; Gut; Fermenter; Fermentation; Streptococcus bovis; Infection; Streptococcaceae; Fingerprint method; Clostridiaceae; Bacteriosis; Models
• ISSN: 0168-6496; 1574-6941
• DOI: 10.1111/j.1574-6941.2007.00281.x

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.