Hostname: page-component-8448b6f56d-wq2xx Total loading time: 0 Render date: 2024-04-17T23:37:17.169Z Has data issue: false hasContentIssue false

Molecular biological methods for studying the gut microbiota: the EU human gut flora project

Published online by Cambridge University Press:  09 March 2007

M. Blaut*
Affiliation:
German Institute of Human Nutrition, Potsdam-Rehbruecke, Department of Gastrointestinal Microbiology, Arthur-Scheunert-Allee 114–116, 14558 Bergholz-Rehbruecke, Germany
M.D. Collins
Affiliation:
Food Microbial Sciences Unit, School of Food Biosciences, The University of Reading, PO Box 226, Whiteknights, Reading RG6 6AP, UK
G. W. Welling
Affiliation:
Rijksuniversiteit Groningen, Laboratorium voor Medische Microbiologie, Hanzeplein 1, 9713 GZ Groningen, The Netherlands
J. Doré
Affiliation:
Institut National de la Recherche Agronomique, Laboratoire d'Ecologie et de Physiologie du Système Digestif, Domaine de Vilvert, 78352 Jouy-en-Josas Cedex, France
J. van Loo
Affiliation:
ORAFTI, Aandorenstraat 1, 3300 Tienen, Belgium
W. de Vos
Affiliation:
Wageningen University, Laboratory of Microbiology, Hesselink van Suchtelenweg 4, Wageningen 6703 CT, The Netherlands
*
*Corresponding author: Professor M. Blaut, tel +49 33 20 08 84 70, fax +49 33 20 08 84 07, email Blaut@www.dife.de
Rights & Permissions [Opens in a new window]

Abstract

Core share and HTML view are not available for this content. However, as you have access to this content, a full PDF is available via the ‘Save PDF’ action button.

Seven European laboratories co-operated in a joint project (FAIR CT97-3035) to develop, refine and apply molecular methods towards facilitating elucidation of the complex composition of the human intestinal microflora and to devise robust methodologies for monitoring the gut flora in response to diet. An extensive database of 16S rRNA sequences for tracking intestinal bacteria was generated by sequencing the 16S rRNA genes of new faecal isolates and of clones obtained by amplification with polymerase chain reaction (PCR) on faecal DNA from subjects belonging to different age groups. The analyses indicated that the number of different species (diversity) present in the human gut increased with age. The sequence information generated, provided the basis for design of 16S rRNA-directed oligonucleotide probes to specifically detect bacteria at various levels of phylogenetic hierarchy. The probes were tested for their specificity and used in whole-cell and dot-blot hybridisations. The applicability of the developed methods was demonstrated in several studies and the major outcomes are described.

Type
Research Article
Copyright
Copyright © The Nutrition Society 2002

References

Amann, RI, Binder, BJ, Olson, RJ, Chisholm, SW, Devereux, R & Stahl, DA (1990 a) Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Applied and Environmental Microbiology 56, 19191925.CrossRefGoogle ScholarPubMed
Amann, RI, Krumholz, L & Stahl, DA (1990 b) Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. Journal of Bacteriology 172, 762770.CrossRefGoogle ScholarPubMed
Amann, RI, Ludwig, W & Schleifer, K-H (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiological Reviews 59, 143169.CrossRefGoogle ScholarPubMed
Berg, RD (1996) The indigenous gastrointestinal microflora. Trends in Microbiology 4, 430435.CrossRefGoogle ScholarPubMed
Bouhnik, Y, Flourie, B, Andrieux, C, Bisetti, N, Briet, F & Rambaud, JC (1996) Effects of Bifidobacterium sp fermented milk ingested with or without inulin on colonic bifidobacteria and enzymatic activities in healthy humans. European Journal of Clinical Nutrition 50, 269273.Google ScholarPubMed
Cummings, JH & Macfarlane, GT (1997) Colonic microflora: nutrition and health. Nutrition 13, 476478.CrossRefGoogle ScholarPubMed
Doré, J, Sghir, A, Hannequart-Gramet, G, Corthier, G & Pochart, P (1998) Design and evaluation of a 16S rRNA-targeted oligonucleotide probe for specific detection and quantitation of human faecal Bacteroides populations. Systematics and Applied Microbiology 21, 6571.CrossRefGoogle ScholarPubMed
Finegold, SM, Sutter, VL & Mathisen, GE (1983) Normal indigenous intestinal flora. In Human intestinal microflora in health and disease, pp. 331 [Hentges, DJ, editor]. New York: Academic Press.CrossRefGoogle Scholar
Franks, AH, Harmsen, HJM, Raangs, GC, Jansen, GJ, Schut, F & Welling, GW (1998) Variations of bacterial populations in human feces measured by fluorescent in situ hybridization with group-specific 16S rRNA-targeted oligonucleotide probes. Applied and Environmental Microbiology 64, 33363345.CrossRefGoogle ScholarPubMed
Gibson, GR & Roberfroid, MB (1995) Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. Journal of Nutrition 125, 14011412.CrossRefGoogle ScholarPubMed
Harmsen, HJ, Wildeboer-Veloo, AC, Grijpstra, J, Knol, J, Degener, JE & Welling, GW (2000) Development of 16S rRNA-based probes for the Coriobacterium group and the Atopobium cluster and their application for enumeration of Coriobacteriaceae in human feces from volunteers of different age groups. Applied and Environmental Microbiology 66, 45234527.CrossRefGoogle ScholarPubMed
Holdeman, LV, Good, IJ & Moore, WEC (1976) Human fecal flora: variation in bacterial composition within individuals and a possible effect of emotional stress. Applied and Environmental Microbiology 31, 359375.CrossRefGoogle Scholar
Kruse, H-P, Kleessen, B & Blaut, M (1999) Effects of inulin on faecal bifidobacteria in human subjects. British Journal of Nutrition 82, 375382.CrossRefGoogle ScholarPubMed
Langendijk, PS, Schut, F, Jansen, GJ, Raangs, GC, Kamphuis, GR, Wilkinson, MH & Welling, GW (1995) Quantitative fluorescence in situ hybridization of Bifidobacterium spp with genus-specific 16S rRNA-targeted probes and its application in fecal samples. Applied and Environmental Microbiology 61, 30693075.CrossRefGoogle ScholarPubMed
Ludwig, W, Strunk, O, Klugbauer, S, Klugbauer, N, Weizenegger, M, Neumaier, J, Bachleitner, M & Schleifer, KH (1998) Bacterial phylogeny based on comparative sequence analysis. Electrophoresis 19, 554568.CrossRefGoogle ScholarPubMed
Maidak, BL, Olsen, GJ, Larsen, N, Overbeek, R, McCaughey, MJ & Woese, CR (1997) The RDP (Ribosomal Database Project). Nucleic Acids Research 25, 109110.CrossRefGoogle ScholarPubMed
Manz, W, Amann, R, Ludwig, W, Vancanneyt, M & Schleifer, KH (1996) Application of a suite of 16S rRNA-specific oligonucleotide probes designed to investigate bacteria of the phylum Cytophaga-Flavobacter-Bacteroides in the natural environment. Microbiology 142, 10971106.CrossRefGoogle ScholarPubMed
Moore, WE & Holdeman, LV (1974) Human fecal flora: the normal flora of 20 Japanese-Hawaiians. Applied Microbiology 27, 961979.CrossRefGoogle ScholarPubMed
Olsen, GJ, Lane, DJ, Giovannoni, SJ, Pace, NR & Stahl, DA (1986) Microbial ecology and evolution: a ribosomal RNA approach. Annual Reviews of Microbiology 40, 337365.CrossRefGoogle ScholarPubMed
Roberfroid, MB (1998) Prebiotics and synbiotics: concepts and nutritional properties. British Journal of Nutrition 80, S197S202.CrossRefGoogle ScholarPubMed
Rowland, IR (1988) Interactions of the gut microflora and the host in toxicology. Toxicology and Pathology 16, 147153.CrossRefGoogle ScholarPubMed
Schneider, H, Schwiertz, A, Collins, MD & Blaut, M (1999) Anaerobic transformation of quercetin-3-glucoside by bacteria from the human intestinal tract. Archives of Microbiology 171, 8191.CrossRefGoogle ScholarPubMed
Schneider, H, Simmering, R, Hartmann, L, Pforte, H & Blaut, M (2000) Degradation of quercetin-3-glucoside in gnotobiotic rats associated with human intestinal bacteria. Journal of Applied Microbiology 89, 10271037.CrossRefGoogle ScholarPubMed
Schwiertz, A, Le Blay, G & Blaut, M (2000) Quantification of different Eubacterium spp in human fecal samples with species-specific 16S rRNA-targeted oligonucleotide probes. Applied and Environmental Microbiology 66, 375382.CrossRefGoogle ScholarPubMed
Sghir, A, Gramet, G, Suau, A, Rochet, V, Pochart, P & Doré, J (2000) Quantification of bacterial groups within human fecal flora by oligonucleotide probe hybridization. Applied and Environmental Microbiology 66, 22632266.CrossRefGoogle ScholarPubMed
Simmering, R, Kleessen, B & Blaut, M (1999) Quantification of the flavonoid-degrading bacterium Eubacterium ramulus in human fecal samples with a species-specific oligonucleotide hybridization probe. Applied and Environmental Microbiology 65, 37053709.CrossRefGoogle ScholarPubMed
Stahl, DA, Flesher, B, Mansfield, HR & Montgomery, L (1988) Use of phylogenetically based hybridization probes for studies of ruminal microbial ecology. Applied and Environmental Microbiology 54, 10791084.CrossRefGoogle ScholarPubMed
Suau, A, Bonnet, R, Sutren, M, Godon, JJ, Gibson, GR, Collins, MD & Doré, J (1999) Direct analysis of genes encoding 16S rRNA from complex communities reveals many novel molecular species within the human gut. Applied and Environmental Microbiology 65, 47994807.CrossRefGoogle ScholarPubMed
Suolinna, EM, Buchsbaum, RN & Racker, E (1975) The effect of flavonoids on aerobic glycolysis and growth of tumor cells. Cancer Research 35, 18651872.Google ScholarPubMed
Wilson, KH & Blitchington, RB (1996) Human colonic biota studied by ribosomal DNA sequence analysis. Applied and Environmental Microbiology 62, 22732278.CrossRefGoogle ScholarPubMed
Woese, CR (1987) Bacterial evolution. Microbiological Reviews 51, 221271.CrossRefGoogle ScholarPubMed
Woese, CR, Kandler, O & Wheelis, ML (1990) Towards a natural system of organisms: proposal for the domains Archaea, Bacteria and Eucarya. Proeedings of the National Academy of Sciences USA 87, 45764579.CrossRefGoogle ScholarPubMed
Zheng, D, Alm, EW, Stahl, DA & Raskin, L (1996) Characterization of universal small-subunit rRNA hybridization probes for quantitative molecular microbial ecology studies. Applied and Environmental Microbiology 62, 45044513.CrossRefGoogle ScholarPubMed