Application of Repetitive Extragenic Palindromic Elements Based on PCR in Detection of Genetic Relationship of Lactic Acid Bacteria Species Isolated from Traditional Fermented Food Products

Authors
F. Tafvizi 1
M. Tajabadi Ebrahimi 2
1 Department of Biology, Islamic Azad University, Parand Branch, Tehran, Islamic Republic of Iran.
2 Department of Biology, Faculty of Science, Islamic Azad University, Central Tehran Branch, Tehran, Islamic Republic of Iran.
Abstract
Tarkhineh is one of the traditional foods in Iran and a rich source of probiotic bacteria. The objective of this study was to identify the probiotic bacteria isolated from Tarkhineh using 16S rRNA gene sequencing and molecular typing with repetitive extragenic palindromic–PCR (REP-PCR). In total, 20 different bacteria were isolated from traditional dairy products and Tarkhineh. Molecular identification of the isolates was carried out by 16S rRNA gene sequencing, and DNA sequences of isolates deposited in GenBank. The REP-PCR reaction by REP1R-I, REP2-I and REP1R-I+REP2-I markers was performed for fingerprinting and characterization of the isolates. Un-weighted pair group method with arithmetic mean (UPGMA) clustering methods were performed based on Dice similarity. The REP1R-I primer grouped isolates into three, and REP2-I and REP1R-I+REP2-I grouped all isolates into four main clusters in dendrograms. In all analyses, isolates of Lactobacillus casei, Lactobacillus brevis, Lactobacillus plantarum, and Entrococcus facium formed separate clusters. The results of sequencing corresponded to clustering in the dendrogram. According to the results, REP-PCR is an accurate technique for determining the genetic diversity of lactic acid bacteria species.

Keywords

1. Ben Amor, K., Vaughan, E. E. and De Vos, W. M. 2007. Advanced Molecular Tools for the Identification of Lactic Acid Bacteria. J. Nutr., 137: 741–747.
2. Busse, H.J., Denner, E.B.M. and Lubitz, W. 1996. Classification and Identification of Bacteria-Current Approaches to an Old Problem: Overview of Methods Used in Bacterial Systematics. J. Biotechnol., 47: 3-38.
3. Callon, C., Millet, L. and Montel, M. C. 2004. Diversity of Lactic Acid Bacteria Isolated from AOC Salers Cheese. J. Dairy Res., 71: 231–244.
4. Collins, M. D., Rodrigues, U., Ash, C., Aguirre, M., Farrow, J. A. E., Martinez-Murcia, A., Phillips, B. A., Williams, A. M., Wallbanks, S. 1991. Phylogenetic Analysis of the Genus Lactobacillus and Related Lactic Acid Bacteria as Determined by Reverse Transcriptase Sequencing of 16S rRNA. FEMS Microbiol. Lett., 77: 5-12.
5. De Urraza, P. J., Gómez-Zavaglia, A., Lozano, M. E., Romanowski, V. and De Antoni, G. L. 2000. DNA Fingerprinting of Thermophilic Lactic Acid Bacteria Using Repetitive Sequence Based Polymerase Chain Reaction. J. Dairy. Res., 67: 381–392.
6. Du Plessis, E. M. and Dicks, L. M. T. 1995. Evaluation of Random Amplified Polymorphic DNA (RAPD)-PCR as a Method to Differentiate Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus gallinarum, Lactobacillus gasseri and Lactobacillus johnsonii. Curr. Microbiol., 31: 114-118.
7. Ehrmann, M. A., Mu¨ller, M. R. A. and Vogel, R. F. 2003. Molecular Analysis of Sourdough Reveals Lactobacillus mindensis sp. nov. Int. J. Syst. Evol. Microbiol., 53: 7–13.
8. El Soda, M. 1999. The Magic World of Lactic Acid Bacteria. Proceedings of the Symposium on Starter Cultures and Their Use in Dairy Industry. RoMEO, Alexandria, PP. 1–12.
9. Gancheva, A., Pot, B., Vanhonacker, K., Hoste, B. and Kersters, K. 1999. A Polyphasic Approach towards the Identification of Strains Belonging to Lactobacillus acidophilus and Related Species. Syst. Appl. Microbiol., 22: 573-585.
10. Gevers, D., Huys, G. and Swings, H. 2001. Applicability of Rep-PCR Fingerprinting for Identification of Lactobacillus Species. FEMS Microbiol. Lett., 205: 31–36.
11. Giorgio, B. C., De Dea Lindner, J., Lazzi, C., Gatti, M. and Nevian, E. 2011. Evaluation of Genetic Polymorphism among Lactobacillus rhamnosus Non-starter Parmigiano Reggiano Cheese Strains. Int. J. Food. Microbiol., 144: 569–572.
12. Hurtado, A., Reguant, C., Bordons, A. and Rozès, N. 2010. Evaluation of a Single and Combined Inoculation of a Lactobacillus pentosus Starter for Processing cv. Arbequina Natural Green Olives. Food Microbiol., 27: 731-740.
13. Kesmen, Z., Yetiman, A. E., Gulluce, A., Kacmaz, N., Sagdic, O., Cetin, B., Adiguzel, A., Sahin, F., Yetim, H. 2012. Combination of Culture-dependent and Culture-independent Molecular Methods for the Determination of Lactic Microbiota in Sucuk. Int. J. Food Microbiol., 153: 428–435.
14. Krizova, J., Spanova, A. and Rittich, B. 2008. RAPD and Rep PCR Fingerprinting for Characterization of Bifidobacterium Species. Folia. Microbiol., 53(2): 99-104.
15. Kullen, M. J., Sanozky-Dawes, R. B., Crowell, D.C., Klaenhammer, T. R. 2000. Use of the DNA Sequence of Variable Regions of the 16S rRNA for Rapid and Accurate Identification of Bacteria in the Lactobacillus acidophilus Complex. J. Appl. Microbiol., 89: 511-516.
16. Lick, S. 2003. Typing systems for Lactobacilli: Review. Milchwissenschaft, 58: 256–260.
17. Lo´pez, A. and Alippi A. M. 2009. Diversity of Bacillus megaterium Isolates Cultured from Honeys. LWT-Food Sci. Technol., 42: 212–219.
18. Marco, V. and Ralf, Z. 2002. Specific Identification and Molecular Typing Analysis of Lactobacillus johnsonii by using PCR-based Methods and Pulsed-fied Gel Electrophoresis. FEMS Microbiol. Lett., 217: 141-154.
19. Manzano, M., Giusto, C., Iacumin, L., Cantoni, C. and Comi, G. 2009. Molecular Methods to Evaluate Biodiversity in Bacillus cereus and Bacillus thuringiensis strains from Different Origins. Food Microbiol., 26: 259–264.
20. Meintanis, C., Chalkou, K. I., Kormas, K. A., Lymperopoulou, D. S., Katsifas, E. A., Hatzinikolaou, D. G., Karagouni, A. D. 2008. Application of rpoB Sequence Similarity Snalysis, REP-PCR and BOX-PCR for the Differentiation of Species within the Genus Geobacillus. Lett. Appl. Microbiol., 46: 395–401.
21. Mohammed, M., Abd El-Aziz, H., Omran, N., Anwar, S. H., Awad, S. and El-Soda, M. 2009. Rep-PCR Characterization and Biochemical Selection of Lactic Acid Bacteria Isolated from the Delta Area of Egypt. Int. J. Food Microbiol., 128: 417–423.
22. Olive, D. M. and Bean, P. 1999. Principles and Applications of Methods for DNA-based Typing of Microbial Organisms. J. Clin. Microbiol., 37: 1661–1669.
23. Pot, B., Hertel, C., Ludwig, W., Descheemaeker, P., Kersters, K. and Schleifer, K. H. 1993. Identification and Classification of Lactobacillus acidophilus, L. gasseri and L. johnsonii strains by SDS-PAGE and rRNA Target Oligonucleotide Probe Hybridisation. J. Gen. Microbiol., 139: 513-517.
24. Rodtong, S. and Tannock, G. W. 1993. Differentiation of Lactobacillus Strains by Ribotyping. Appl. Environ. Microbiol., 59: 3480-3484.
25. Roy, D., Ward, P., Vincent, D. and Mondou, F. 2000. Molecular Identification of Potentially Probiotic Lactobacilli. Curr. Microbiol., 40: 40-46.
26. Singh, S., Goswami, P., Singh, R. and Heller, K. J. 2009. Application of Molecular Identification Tools for Lactobacillus, with a Focus on Discrimination between Closely Related Species: A Review. LWT-Food. Sci. Technol., 42: 448–457.
27. Stackebrandt, E. and Rainey, F. A. 1995. Partial and Complete 16S rDNA Sequences: Their Use in Generation of 16S rDNA Phylogenetic Trees and Their Implications in Molecular Ecological Studies. In: “Molecular Microbial Ecology Manual”, (Eds.): Akkeramns, A. D. L. van Elsas, J. D. and de Bruijn, F. J.. Kluwer Academic Publishers, Dordrecht, PP. 1-17.
28. ˇSvec, P., Vancanneyt, M., Seman, M., Snauwaert, C., Lefebvre, K., Sedlaˇcek, I., Swings, J. 2005. Evaluation of (GTG)5-PCR for Identification of Enterococcus spp. FEMS Microbiol. Lett., 247: 59–63.
29. Tabatabaei Yazdi, F., Alizadeh Behbahani, B., Mohebbi, M., Mortazavi, S. A., Ghaitaranpour, A. 2012. Identification of Lactic Acid Bacteria Isolated from Tarkhineh, a Traditional Iranian Fermented Food. Sci. J. Microbiol., 1(7): 152-159.
30. Tafvizi, F. and Tajabadi Ebrahimi, M. 2012. Detection of Genetic Diversity and Classification of Lactobacillus Species Isolated from Iranian Traditional Dairy Products by RAPD Fingerprinting and POPGENE Analysis. Ann. Biol. Res., 3(10): 4904-4911.
31. Tajabadi Ebrahimi, M., Bahrami, H. and Ziary, Z. 2011 a, Tarkhineh Source of Probiotic Lactic Acid Bacteria. The Quarterly Journal of Biological Sci., 4(12): 1-9. [in Persian]
32. Tajabadi Ebrahimi, M., Ouwehand, A. C., Hejazi, M. A. and Jafari, P. 2011b. Traditional Iranian Dairy Products: A Source of Potential Probiotic Lactobacilli. Afr. J. Microbiol. Res., 5(1): 20-27.
33. Tajabadi Ebrahimi, M., Ouwehand, A. C., Jafari, P., Bahrami, H. and Heidary Nasrabadi, M. 2011. Evaluation Probiotic Effects of Lactic Acid Bacteria Isolated from Tarkhineh, International Scientific Conference on Probiotic and Prebiotic; June 14-16, Slovakia, PP???
34. Temmerman, R., Scheirlinck, I., Huys, G. and Swings, J. 2003. Culture-independent Analysis of Probiotic Products by Denaturing Gradient Gel Electrophoresis. Appl. Environ. Microbiol., 69: 220–226.
35. Vandamme, P., Pot, B., Gillis, V., de Vos, P., Kersters, K. and Swings, J. 1996. Polyphasic Taxonomy, a Consensus Approach to Bacterial Systematics. Microb. Rev., 60: 407-438.
36. Versalovic, J., Koeuth, T. and Lupski, J. R. 1991. Distribution of Repetitive DNA sequences in Eubacteria and Application to Fingerprinting of Bacterial Genomes. Nucleic. Acids. Res., 19(24): 6823 -6831.
37. Woese, C. R. 1987. Bacterial Evolution. Microbial. Rev., 51: 221-71.
Journal of Agricultural Science and Technology
Volume 17, Issue 1
January and February 2015
Pages 87-98