Identification of glucose-fermenting bacteria present in an in vitro model of the human intestine by RNA-stable isotope probing.
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-(13)C]-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 (13)C-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 (13)C-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.
Egert
Markus
M
Wageningen Centre for Food Sciences, Wageningen, The Netherlands.
de Graaf
Albert A
AA
Maathuis
Annet
A
de Waard
Pieter
P
Plugge
Caroline M
CM
Smidt
Hauke
H
Deutz
Nicolaas E P
NE
Dijkema
Cor
C
de Vos
Willem M
WM
Venema
Koen
K
D001421:Bacteria, Anaerobic / Q000145:classification / Q000235:genetics / Q000378:metabolism; D002247:Carbon Isotopes / Q000378:metabolism; D005285:Fermentation; D005947:Glucose / Q000378:metabolism*; D006094:Gram-Positive Bacteria / Q000145:classification* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007421:Intestine, Small / Q000382:microbiology*; D007553:Isotope Labeling / Q000379:methods; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008954:Models, Biological; D008969:Molecular Sequence Data; D010528:Peristalsis; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D017422:Sequence Analysis, DNA
D002247:Carbon Isotopes; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D005947:Glucose