Abstract
We isolated a total of 49 strains of lactic acid bacteria from the faeces of healthy donors. The species in that group were determined as L. plantarum (11 strains), L. casei (11 strains), L. rhamnosus (seven strains), L. fermentum (seven strains), L. gasseri (six strains), L. delbrueckii ssp. lactis (four strains), L. salivarius (two strains), and L. acidophilus (one strain). Genotyping at strain level was performed using random amplification of polymorphic DNA (RAPD), pulsed field gel electrophoresis (PFGE) with endonucleases ApaI and XhoI and amplified fragment length polymorphism (AFLP) with enzymes XhoI and TaqI. The main objective was the comparison of three molecular typing techniques: AFLP, PFGE and RAPD in their applicability to determine the genetic diversity among the isolates. RAPD was the easiest, comparatively rapid and fairly strain discriminative tool. PFGE was the most laborious method but producing the most stable profiles with satisfactory discriminatory power. AFLP proved to be the most discriminative approach for typing of the strains. AFLP could differentiate strains with the same PFGE profiles. Therefore, AFLP successfully could replace the labor consuming PFGE. The specially developed AFLP and PFGE proved very high potential to evaluate the strain diversity of Lactobacillus spp. with human origin.
Similar content being viewed by others
References
Dimitrov Z, Michaylova M, Minkova S (2005) Characterization of Lactobacillus helveticus strains isolated from Bulgarian yoghurt, cheese, plants and human faecal samples by sodium dodecilsulfate polyacrylamide gel electrophoresis of cell-wall proteins, ribotyping and pulsed field gel fingerprinting. Int Dairy J 15:998–1005
Dykes GA, von Holy A (1994) Strain typing in the genus Lactobacillus. Lett Appl Microbiol 19:63–66
Gatti M, Fornasari E, Neviani E (1997) Cell-wall protein profiles of dairy thermophilic. Lett Appl Microbiol 25:345–348
Goldin BR, Gorbach SL (1992) Probiotics for humans. In: Fuller R (ed) Probiotics. The scientific basis. Chapman and Hall, London, pp 355–376
Janssen P, Coopman R, Huys G, Swings J, Bleeker M, de Vos P, Zabeau M, Kersters K (1996) Evaluation of the DNA fingerprinting method AFLP as a new tool in bacterial taxonomy. Microbiology 142:1881–1893
Marteau P, Rambaud JC (1993) Potential of using lactic acid bacteria for therapy and immunomodulation in man. FEMS Microbiol Rev 12:207–220
Miteva V, Boudakov I, Ivanova-Stoyancheva G, Marinova B, Mitev V, Mengaud J (2001) Differentiation of Lactobacillus delbrueckii subspecies by ribotyping and amplified ribosomal DNA restriction analysis (ARDRA). J Appl Microbiol 90:909–918
Pineiro M, Stanton C (2007) Probiotic bacteria: legislative framework—requirements to evidence basis. J Nutr 137:850–853
Pot B, Hertel C, Ludwig W, Descheemaeker P, Kersters K, Schleifer KH (1993) Identification and classification of Lb. acidophilus, Lb. gasseri and Lb. johnsonii strains by SDS-PAGE and rRNA-targeted oligonucleotide probe hybridization. J Gen Microbiol 139:513–517
Rodtong S, Tannock GW (1993) Differentiation of Lactobacillus strains by ribotyping. Appl Environ Microbiol 59:3480–3484
Roussel Y, Colmin C, Simonet JM, Decaris B (1993) Strain characterization, genome size and plasmid content in the Lactobacillus acidophilus group (Hansen and Mocquot). J Appl Bacteriol 74:549–556
Salminen S, Isolauri E, Salminen E (1996) Clinical uses of probiotics for stabilizing the gut mucosal barrier: successful strains and future challenges. Antonie Leeuwenhoek 70:347–358
Schleifer KH (1990) DNA probes in food microbiology. Food Biotechnol 4:585–598
Schleifer KH, Ehrmann M, Beimfohr C, Brockmann E, Ludwig W, Amann R (1995) Application of molecular methods for the classification and identification of lactic acid bacteria. Int Dairy J 5:1081–1094
Tynkkynen S, Satokari R, Saarela M, Mattila-Sandholm T, Saxelin M (1999) Comparison of ribotyping, randomly amplified polymorphic DNA analysis, and pulsed-field gel electrophoresis in typing of L. rhamnosus and L. casei strains. Appl Environ Microbiol 65:3908–3914
Vandamme P, Pot B, Gillis M, de Vos P, Kersters K, Swings J (1996) Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiol Rev 60:407–438
Vauterin L, Vauterin P (1992) Computer-aided objective comparison of electrophoresis patterns for grouping and identification of microorganisms. Eur Microbiol 1:37–41
Vos P, Hogers R, Bleeker M, Reijans M, van de Lee T, Horners M, Frijters A, Pot J, Peleman J, Muiper M, Zabeau M (1995) AFLP: a new concept for DNA fingerprinting. Nucleic Acids Res 21:4407–4414
Walter J Tannock GW, Tilsala-Timisjarvi A, Rodtong S, Loach DM, Munro K, Alatossava T (2000) Detection and identification of gastrointestinal Lactobacillus species by using denaturing gradient gel electrophoresis and species-specific PCR primers. Appl Environ Microbiol 66:297–303
Xanthopoulos V, Ztaliou I, Gaier W, Tzanetakis N, Litopoulou-Tzanetaki E (1999) Differentiation of Lactobacillus isolates from infant faeces by SDS-PAGE and rRNA-targeted oligonucleotide probes. J Appl Micrbiol 87:743–749
Zhong W, Millsap K, Bialkowska-Hobrzanska H, Reid G (1998) Differentiation of Lactobacillus species by molecular typing. Appl Environ Microbiol 64:2418–2423
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Dimitrov, Z.P., Minkova, S. & Michaylova, M. Comparative evaluation of three molecular typing methods in their applicability to differentiate Lactobacillus strains with human origin. World J Microbiol Biotechnol 24, 1305–1312 (2008). https://doi.org/10.1007/s11274-007-9603-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11274-007-9603-4