Abstract
The present study focused on identification and genotypic characterization of Lactic acid bacteria (LAB) in the intestine of freshwater fish. 76 strains of LAB were isolated and identified by 16S rRNA gene sequences and hsp60 gene sequences as different strains of Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus fermentum, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus brevis, Lactobacillus reuteri, Lactobacillus salivarius, Pediococcus pentosaceus, Pediococcus acidilactici, Weissella paramesenteroides, Weissella cibaria, Enterococcus faecium, and Enterococcus durans. The hsp60 gene showed a higher level of sequence variation among the isolates examined, with lower interspecies sequence similarity providing more resolutions at the species level than the 16S rRNA gene. Phylogenetic tree derived from hsp60 gene sequences with higher bootstrap values at the nodal branches was more consistent as compared to phylogenetic tree constructed from 16S rRNA gene sequences. Closely related species L. plantarum and L. pentosus as well as species L. delbrueckii subsp. bulgaricus and L. fermentum were segregated in different cluster in hsp60 phylogenetic tree whereas such a distribution was not apparent in 16S rRNA phylogenetic tree. In silico restriction analysis revealed a high level of polymorphism within hsp60 gene sequences. Restriction pattern with enzymes AgsI and MseI in hsp60 gene sequences allowed differentiation of all the species including closely related species L. plantarum and L. pentosus, E. faecium and E. durans. In general, hsp60 gene with higher evolutionary divergence proved to be a better phylogenetic marker for the group LAB.
Similar content being viewed by others
References
Abed, T.A., Evaluation of methods for the extraction and purification of DNA of cultured Lactobacillus colony isolated from dairy products, Int. J. Appl. Microbiol. Biotechnol. Res., 2013, vol. 1, pp. 20–25.
Baffoni, L., Stenico, V., Strahsburger, E., Gaggia, F., Di Gioia, D., Modesto, M., Mattarelli, P., and Biavati, B., Identification of species belonging to the Bifidobacterium genus by PCR-RFLP analysis of a hsp60 gene fragment. BMC Microbiol., 2013, vol. 13, p. 149.
Balcazar, J.L., Vendrell, D., de Bias, I., Ruiz-Zarzuela, I., Muzquiz, J.L., and Girones, O., Characterization of probiotic properties of lactic acid bacteria isolated from intestinal microbiota of fish, Aquaculture, 2008, vol. 278, pp. 188–191.
Blaiotta, G., Fusco, V., Ercolini, D., Aponte, M., Pepe, O., and Villani, F., Lactobacillus strain diversity based on partial hsp60 gene sequences and design of PCR-restriction fragment length polymorphism assays for species identification and differentiation, Appl. Environ. Microbiol., 2008, vol. 74, no. 1, pp. 208–215.
Chang, Y.H., Shangkuan, Y.H., Lin, H.C., and Liu, H.W., PCR assay of the groEL gene for detection and differentiation of Bacillus cereus group cells, Appl. Environ. Microbiol., 2003, vol. 69, no. 8, pp. 4502–4510.
Denev, S., Staykov, Y., Moutafchieva, R., and Beev, G., Microbial ecology of the gastrointestinal tract of fish and the potential application of probiotics and prebiotics in finfish aquaculture, Int. Aquat. Res., 2003, vol. 1, pp. 1–29.
Felis, G.E., Vancanneyt, M., Snauwaert, C., Swings, J., Torriani, S., Castioni, A., and Dellaglio, F., Reclassification of Lactobacillus thermotolerans Niamsup et al. 2003 as a later synonym of Lactobacillus ingluviei Baele et al. 2003, Int. J. Syst. Evol. Microbiol., 2006, vol. 56, pp. 793–795.
Goh, S.H., Facklam, R.R., Chang, M., Hill, J.E., Tyrrell, G.J., Burns, E.C., Chan, D., He, C., Rahim, T., Shaw, C., and Hemmingsen, S.M., Identification of Enterococcus species and phenotypically similar Lactococcus and Vagococcus species by reverse checkerboard hybridization to chaperonin 60 gene sequences, J. Clin. Microbiol., 2000, vol. 38, no. 11, pp. 3953–3959.
Goh, S.H., Potter, S., Wood, J.O., Hemmingsen, S.M., Reynolds, R.P., and Chow, A.W., HSP60 gene sequences as universal targets for microbial species identification: studies with coagulase-negative staphylococci, J. Clin. Microbiol., 1996, vol. 34, no. 4, pp. 818–823.
Huang, C.H. and Lee, F.L., The dnaK gene as a molecular marker for the classification and discrimination of the Lactobacillus casei group, Antonie Van Leeuwenhoek, 2011, vol. 99, no. 2, pp. 319–327.
Kimura, M., The Neutral Theory of Molecular Evolution, Cambridge: Cambridge Univ. Press, 1983, pp. 28–31.
Kwok, A.Y. and Chow, A.W., Phylogenetic study of Staphylococcus and Macrococcus species based on partial hsp60 gene sequences, Int. J. Syst. Evol. Microbiol., 2003, vol. 53, pp. 87–92.
Mikkonen, T.P., Karenlampi, R.I., and Hanninen, M.L., Phylogenetic analysis of gastric and enterohepatic Helicobacter species based on partial HSP60 gene sequences, Int. J. Syst. Evol. Microbiol., 2004, vol. 54, pp. 753–758.
Muto, A. and Osawa, S., The guanine and cytosine content of genomic DNA and bacterial evolution, Proc. Natl. Acad. Sci. U. S. A., 1987, vol. 84, no. 1, pp. 166–169.
Park, S.-H., Jung, J.-H., Seo, D.-H., Lee, H.-L., Kim, G.-W., Park, S.-Y., Shin, W.-C., Hong, S., and Park, C.-S., Differentiation of lactic acid bacteria based on RFLP analysis of the tuf gene, Food. Sci. Biotechnol., 2012, vol. 21, no. 3, pp. 911–915.
Sen, R. and Maiti, N.K., Genomic and functional diversity of bacteria isolated from hot springs in Odisha, India, Geomicrobiol. J., 2014, vol. 31, no. 7, pp. 541–550.
Soto, L.P., Frizzo, L.S., Bertozzi, E., Avataneo, E., Sequeira, G.J., and Rosmini, M.R., Molecular microbial analysis of Lactobacillus strains isolated from the gut of calves for potential probiotic use, Vet. Med. Intern., 2010. doi: doi 10.4061/2010/274987
Stanley, J., Baquar, N., and Burnens, A., Molecular subtyping scheme for Salmonella panama, J. Clin. Microbiol., 1995, vol. 33, no. 5, pp. 1206–1211.
Stepkowski, T., Czaplinska, M., Miedzinska, K., and Moulin, L., The variable part of the dnaK gene as an alternative marker for phylogenetic studies of rhizobia and related alpha Proteobacteria, Syst. Appl. Microbiol., 2003, vol. 26, no. 4, pp. 483–494.
Yang, Z. and Yoder, A.D., Estimation of the transition/transversion rate bias and species sampling, J. Mol. Evol., 1999, vol. 48, no. 3, pp. 274–283.
Yu, J., Sun, Z., Liu, W., Bao, Q., Zhang, J., and Zhang, H., Phylogenetic study of Lactobacillus acidophilus group, L. casei group and L. plantarum group based on partial hsp60, pheS and tuf gene sequences. Eur. Food. Res. Technol., 2012, vol. 234, no. 6, pp. 927–934.
Author information
Authors and Affiliations
Corresponding author
Additional information
The article is published in the original.
Rights and permissions
About this article
Cite this article
Maji, U.J., Mohanty, S. Genotypic characterization of Lactic acid bacteria in gut microbiome of freshwater fish. Microbiology 86, 276–285 (2017). https://doi.org/10.1134/S0026261717020138
Received:
Published:
Issue Date:
DOI: https://doi.org/10.1134/S0026261717020138