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

  • M. Blaut (a1), M.D. Collins (a2), G. W. Welling (a3), J. Doré (a4), J. van Loo (a5) and W. de Vos (a6)...


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.


Corresponding author

*Corresponding author: Professor M. Blaut, tel +49 33 20 08 84 70, fax +49 33 20 08 84 07, email


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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.
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.
Amann, RI, Ludwig, W & Schleifer, K-H (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiological Reviews 59, 143169.
Berg, RD (1996) The indigenous gastrointestinal microflora. Trends in Microbiology 4, 430435.
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.
Cummings, JH & Macfarlane, GT (1997) Colonic microflora: nutrition and health. Nutrition 13, 476478.
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.
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.
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.
Gibson, GR & Roberfroid, MB (1995) Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. Journal of Nutrition 125, 14011412.
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.
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.
Kruse, H-P, Kleessen, B & Blaut, M (1999) Effects of inulin on faecal bifidobacteria in human subjects. British Journal of Nutrition 82, 375382.
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.
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.
Maidak, BL, Olsen, GJ, Larsen, N, Overbeek, R, McCaughey, MJ & Woese, CR (1997) The RDP (Ribosomal Database Project). Nucleic Acids Research 25, 109110.
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.
Moore, WE & Holdeman, LV (1974) Human fecal flora: the normal flora of 20 Japanese-Hawaiians. Applied Microbiology 27, 961979.
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.
Roberfroid, MB (1998) Prebiotics and synbiotics: concepts and nutritional properties. British Journal of Nutrition 80, S197S202.
Rowland, IR (1988) Interactions of the gut microflora and the host in toxicology. Toxicology and Pathology 16, 147153.
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.
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.
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.
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.
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.
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.
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.
Suolinna, EM, Buchsbaum, RN & Racker, E (1975) The effect of flavonoids on aerobic glycolysis and growth of tumor cells. Cancer Research 35, 18651872.
Wilson, KH & Blitchington, RB (1996) Human colonic biota studied by ribosomal DNA sequence analysis. Applied and Environmental Microbiology 62, 22732278.
Woese, CR (1987) Bacterial evolution. Microbiological Reviews 51, 221271.
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.
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.



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