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Application of molecular biological methods for studying probiotics and the gut flora

Published online by Cambridge University Press:  09 March 2007

A. L. McCartney*
Affiliation:
Food Microbial Sciences Unit, School of Food Biosciences, University of Reading, Whiteknights, Reading RG6 6AP, UK
*
*Corresponding author: Dr A. L. McCartney, fax +44 118 9357222, email a.l.mccartney@reading.ac.uk
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Abstract

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Increasingly, the microbiological scientific community is relying on molecular biology to define the complexity of the gut flora and to distinguish one organism from the next. This is particularly pertinent in the field of probiotics, and probiotic therapy, where identifying probiotics from the commensal flora is often warranted. Current techniques, including genetic fingerprinting, gene sequencing, oligonucleotide probes and specific primer selection, discriminate closely related bacteria with varying degrees of success. Additional molecular methods being employed to determine the constituents of complex microbiota in this area of research are community analysis, denaturing gradient gel electrophoresis (DGGE)/temperature gradient gel electrophoresis (TGGE), fluorescent in situ hybridisation (FISH) and probe grids. Certain approaches enable specific aetiological agents to be monitored, whereas others allow the effects of dietary intervention on bacterial populations to be studied. Other approaches demonstrate diversity, but may not always enable quantification of the population. At the heart of current molecular methods is sequence information gathered from culturable organisms. However, the diversity and novelty identified when applying these methods to the gut microflora demonstrates how little is known about this ecosystem. Of greater concern is the inherent bias associated with some molecular methods. As we understand more of the complexity and dynamics of this diverse microbiota we will be in a position to develop more robust molecular-based technologies to examine it. In addition to identification of the microbiota and discrimination of probiotic strains from commensal organisms, the future of molecular biology in the field of probiotics and the gut flora will, no doubt, stretch to investigations of functionality and activity of the microflora, and/or specific fractions. The quest will be to demonstrate the roles of probiotic strains in vivo and not simply their presence or absence.

Type
Research Article
Copyright
Copyright © The Nutrition Society 2002

References

Brigidi, P, Vitali, B, Swennen, E, Altomare, L, Rossi, M & Matteuzzi, D (2000) Specific detection of Bifidobacterium strains in a pharmaceutical probiotic product and in human faeces by PCR. Systematic and Applied Microbiology 23, 391399.CrossRefGoogle Scholar
Charteris, WP, Kelly, PM, Morelli, L & Collins, JK (1997) Review article: selective detection, enumeration and identification of potential probiotic Lactobacillus and Bifidobacterium species in mixed bacterial populations. International Journal of Food Microbiology 35, 127.CrossRefGoogle ScholarPubMed
Franks, AH, Harmsen, HJM, Raangs, GC, Jansen, GJ, Schut, F & Welling, GW (1998) Variations of bacterial populations in human faeces measured by fluorescent in situ hybridisation with group-specific 16S rRNA-targeted oligonucleotide probes. Applied and Environmental Microbiology 64, 33363345.CrossRefGoogle ScholarPubMed
Giraffa, G & Neviani, E (2000) Molecular identification and characterisation of food-associated lactobacilli. Italian Journal of Food Science 4, 403423.Google Scholar
Hozapfel, WH, Haberer, P, Geisen, R, Bjorkroth, J & Schillinger, U (2001) Taxonomy and important features of probiotic microorganisms in food and nutrition. American Journal of Clinical Nutrition 73(S), 365S373S.CrossRefGoogle Scholar
Jian, W, Zhu, L & Dong, X (2001) New approach to phylogenetic analysis of the genus Bifidobacterium based on partial HSP60 gene sequences. International Journal of Systematic and Evolutionary Microbiology 51, 16221638.CrossRefGoogle ScholarPubMed
Kimura, K, McCartney, AL, McConnell, MA & Tannock, GW (1997) Analysis of fecal populations of bifidobacteria and lactobacilli and investigation of the immunological responses of their human hosts to the predominant strains. Applied and Environmental Microbiology 63, 33943398.CrossRefGoogle Scholar
Klein, G, Pack, A, Bonaparte, C & Reuter, G (1998) Taxonomy and physiology of probiotic lactic acid bacteria. International Journal of Food Microbiology 41, 103125.CrossRefGoogle ScholarPubMed
Kullen, MJ, Brady, LJ & O'Sullivan, DJ (1997) Evaluation of using a short region of the recA gene for rapid and sensitive speciation of dominant bifidobacteria in the human large intestine. FEMS Microbiology Letters 154, 377383.CrossRefGoogle Scholar
Langendijk, PS, Schut, F, Jansen, GJ, Raangs, GC, Kamphuis, GR, Wilkinson, MHF & Welling, GW (1995) Quantitative fluorescence in situ hybridisation of Bifidobacterium spp with genus-specific 16S rRNA-targeted probes and its application in fecal samples. Applied and Environmental Microbiology 61, 30693075.CrossRefGoogle ScholarPubMed
Lebond-Bourget, N, Philippe, H, Mangin, I & Decaris, B (1996) 16S rRNA and 16S to 23S internal transcribed spacer sequence analyses reveal inter-and intraspecific Bifidobacterium phylogeny. International Journal of Systematic Bacteriology 46, 102111.CrossRefGoogle Scholar
Lucchinin, F, Kmet, V, Cesena, C, Coppi, L, Bottazzi, V & Morelli, L (1998) Specific detection of a probiotic Lactobacillus strain in faecal samples by using multiplex PCR. FEMS Microbiology Letters 158, 273278.CrossRefGoogle Scholar
Marsh, TL (1999) Terminal restriction fragment length polymorphism (T-RFLP): an emerging method for characterising diversity among homologous populations of amplification products. Current Opinions in Microbiology 2, 323324.CrossRefGoogle ScholarPubMed
Matsuki, T, Watanabe, K, Tanaka, R, Fukuda, M & Oyaizu, H (1999) Distribution of bifidobacterial species in human intestinal microflora examined with 16S rRNA-gene-targeted species-specific primers. Applied and Environmental Microbiology 65, 45064512.CrossRefGoogle ScholarPubMed
McCartney, AL, Wang, W & Tannock, GW (1996) Molecular analysis of the composition of the bifidobacterial and lactobacillus microflora of humans. Applied and Environmental Microbiology 62, 46084613.CrossRefGoogle ScholarPubMed
Muyzer, G (1999) DGGE/TGGE a method for identifying genes from natural ecosystems. Current Opinions in Microbiology 2, 317322.CrossRefGoogle ScholarPubMed
Muyzer, G, de Waal, EC & Uitterlinden, AG (1993) Profiling of complex microbial populations by DGGE analysis of PCR-amplified genes coding for 16S rRNA. Applied and Environmental Microbiology 59, 695700.CrossRefGoogle Scholar
O'Sullivan, DJ (1999) Methods for analysis of the intestinal microflora. In Probiotics. A Critical Review, pp. 2344 [Tannock, GW, editor]. Norfolk, UK: Horizon Scientific Press.Google Scholar
O'Sullivan, DJ & Kullen, MJ (1998) Tracking of probiotic bifidobacteria in the intestine. International Dairy Journal 8, 513525.CrossRefGoogle Scholar
Prassad, J, Gill, H, Smart, J & Gopal, PK (1998) Selection and characterisation of Lactobacillus and Bifidobacterium strains for use as probiotics. International Dairy Journal 8, 9931002.CrossRefGoogle Scholar
Qiu, X, Wu, L, Huang, H, McDonel, PE, Palumbo, AV, Tiedje, JM & Zhou, J (2001) Evaluation of PCR-generated chimeras, mutations, and heteroduplexes with 16S rRNA gene-based cloning. Applied and Environmental Microbiology 67, 880887.CrossRefGoogle ScholarPubMed
Roy, D & Sirois, S (2000) Molecular differentiation of Bifidobacterium species with amplified ribosomal DNA restriction analysis and alignment of short regions of the ldh gene. FEMS Microbiology Letters 191, 1724.CrossRefGoogle ScholarPubMed
Roy, D, Sirois, S & Vincent, D (2001) Molecular discrimination of lactobacilli used as starter and probiotic cultures by amplified ribosomal DNA restriction analysis. Current Microbiology 42, 282289.CrossRefGoogle ScholarPubMed
Satokari, RM, Vaughan, EE, Akkermans, ADL, Saarela, M & de Vos, WM (2001) Bifidobacterial diversity in human faeces detected by genus-specific PCR and denaturing gradient gel electrophoresis. Applied and Environmental Microbiology 67, 504513.CrossRefGoogle ScholarPubMed
Spratt, BG (1999) Multilocus sequence typing: molecular typing of bacterial pathogens in an era of rapid DNA sequencing and the Internet. Current Opinions in Microbiology 2, 312316.CrossRefGoogle Scholar
Suau, A, Bonnet, R, Sutren, M, Godon, J-J, Gibson, GR, Collins, MD & Dore, 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
Tannock, GW, Tilsala-Timisjarvi, A, Rodtong, S, Ng, J, Munro, K & Alatossav, T (1999) Identification of Lactobacillus isolates from the gastrointestinal tract, silage, and yoghurt by 16S–23S rRNA gene intergenic spacer region sequence comparisons. Applied and Environmental Microbiology 65, 42644267.CrossRefGoogle ScholarPubMed
Tilsala-Timisjarvi, A & Alatossava, T (1997) Development of oligonucleotide primers from the 16S–23S rRNA intergenic sequences for identifying different dairy and probiotic lactic acid bacteria by PCR. International Journal of Food Microbiology 35, 4956.CrossRefGoogle ScholarPubMed
Tilsala-Timisjarvi, A & Alatossava, T (1998) Strain-specific identification of probiotic Lactobacillus rhamnosus with randomly amplified polymorphic DNA-derived PCR primers. Applied and Environmental Microbiology 64, 48164819.CrossRefGoogle ScholarPubMed
Torriani, S, Zapparoli, G & Dellaglio, F (1999) Use of PCR-based methods for rapid differentiation of L delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. Applied and Environmental Microbiology 65, 43514356.CrossRefGoogle ScholarPubMed
Tynkkynen, S, Satokari, R, Saarela, M, Mattila-Sandholm, M & Saxelin, M (1999) Comparison of ribotyping, randomly amplified polymorphic DNA analysis, and pulsed-field gel electrophoresis in typing of Lactobacillus rhamnosus and L casei strains. Applied and Environmental Microbiology 65, 39083914.CrossRefGoogle ScholarPubMed
Vos, P, Hogers, R, Bleeker, M, Reijans, M, van de Lee, T, Hornes, M, Frijters, A, Pot, J, Peleman, J, Kuiper, M & Zabeau, M (1995) AFLP: a new technique for DNA fingerprinting. Nucleic Acids Research 23, 44074414.CrossRefGoogle ScholarPubMed
Walter, J, Tannock, GW, Tilsala-Timisjarvi, A, Rodtong, S, Loach, DM, Munro, K & Alatossava, T (2000) Detection and identification of gastrointestinal Lactobacillus species by DGGE and species-specific PCR primers. Applied and Environmental Microbiology 66, 297303.CrossRefGoogle ScholarPubMed
Wang, R-F, Cao, W-W & Cerniglia, CE (1996) PCR detection and quantification of predominant anaerobic bacteria in human and animal faecal samples. Applied and Environmental Microbiology 62, 12421247.CrossRefGoogle Scholar
Wayne, LG, Brenner, DJ, Colwell, RR, Grimont, PAD, Kandler, O, Krichevsky, MI, Moore, LH, Moore, WEC, Murray, RGE, Stackebrandt, E, Starr, MP & Truper, HG (1987) Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. International Journal of Systematic Bacteriology 37, 463464.Google Scholar
Wintzingerode, FV, Gobel, UB & Stackebrandt, E (1997) Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. FEMS Microbiology Reviews 21, 213229.CrossRefGoogle Scholar
Zoetendal, EG, Akkermans, ADL & de Vos, WM (1998) Temperature gradient gel electrophoresis analysis of 16S rRNA from human fecal samples reveals stable and host-specific communities of active bacteria. Applied and Environmental Microbiology 64, 38543859.CrossRefGoogle ScholarPubMed