Optimization of PCR-RFLP Method to Confirm the Identification of Enterococcus faecalis and Enterococcus Faecium Isolated from Food Samples
Keywords:
PCR-RFLP, 16S rRNA, multiplex PCR, Enterococcus faecalis, Enterococcus faeciumAbstract
Commercial kits for species identification of Enterococcus faecalis and Enterococcus faecium are in some cases (environmental isolates) incorrect to distinguish this species of enterococci. Also the positive results of multiplex PCR are subjected by amount and purity of genomic DNA. This study aimed to optimize the identification of Enterococcus faecalis and Enterococcus faecium isolated from food samples and from the culture collection by
PCR-RFLP. The strains identified by conventional biochemical EN-COCCUS test (reactions to arginine, sorbose, arabinose, manitol, sorbitol, melibiose, raffinose, melezitose) were submitted to multiplex PCR. The PCR amplification of 16S rRNA with universal primers generated a 433 bp product. Restriction fragment length polymorphism (RFLP) was analysed by restriction endonuclease HinfI. The sizes of restriction fragments for Enterococcus faecalis were 285bp, 85bp and 63 bp and for Enterococcus faecium were 370 bp and 63bp.
References
Wiggins, B. A., Discriminant analysis of antibiotic resistance patterns in fecal streptococci, a method to differentiate human and animal sources of fecal pollution in natural waters, Applied and Environmental Microbiology, 1996, 62, 3997-4002
Harwood, V. J., Wiggins, B., Hagedorn, C., Ellender, R. D., Gooch, J., Kern, J., Samadpour, M., Chapman, A. C. H., Robinson, B. J., Thompson, B. C., Phenotypic library-based microbial source tracking methods: efficacy in the California collaborative study, Journal of Water and Health, 2003, 1, 153-166
Facklam, R. R., Elliott, J. A., Identification, classification, and clinical relevance of catalase-negative, gram-positive cocci, excluding the streptococci and enterococci, Clinical Microbiology Review, 1995, 8, 479-495
Moore, D. F., Zhowandai, M. H., Ferguson, D. M., McGee, C., Mott, J. B., Stewart, J. C., Comparison of 16S rRNA sequencing with conventional and commercial phenotypic techniques for identification of enterococci from the marine environment, Journal of Applied Microbiology, 2006, 100, 1272-1281
Eigner, U., Fahr, A., Weizenegger, M., Witte, W., Evaluation of a new molecular system for simultaneous identification of four Enterococcus species and their glycopeptide resistance genotypes, Journal of Clinical Microbiology, 2005, 43, 2920-2922
Carvalho, M. S., Teixeira, L. M., Facklam, R. R., Use of tests for acidification of methyl-a-D-glucopyranoside and susceptibility to efrotomycin for differentiation of strains of Enterococcus and some related genera, Journal of Clinical Microbiology, 1998, 36, 1584-1587
Willey, B. M., Jones, R. N., McGeer, A., Witte, W., French, G., Roberts, R. B., Jenkins, S. G., Nadler, H., Low, D. E. Practical approach to the identification of clinically relevant Enterococcus species, Diagnostic Microbiology and Infectious Disease, 1999, 34, 165-171
Menichetti, F., Current and emerging serious Gram-positive infections, In Clinical Microbiological Infection, 2005, 11, 22-28
Murray, B. E. 1990. The life and times of the Enterococcus, Clinical Microbiology Review, 3, 46-65
Wellinghausen, N., Bartel, M., Essig, A., Poppert, S., Rapid identification of clinically relevant Enterococcus species by fluorescence in situ hybridization, Journal of Clinical Microbiology, 2007, 45, 3424-3426
Fracalanzza, S. A. P., Scheidegger, E. M. D., Santos, P.F., Cardarelli-Leite, P., Teixeira, L. M., Antimicrobial resistance profiles of entero¬cocci isolated from poultry meat and pasteurized milk in Rio de Janeiro, Brazil, Memórias do Instituto Oswaldo Cruz, 2007, 102, 853-859
Tsai, C. C., Liu, T. H., Chen, M. H., Tsai, C. C., Tsen, H. Y., Toxicity evaluation for an Enterococcus faecium strain TM39 in vitro and in vivo, Food and Chemical Toxicology, 2004, 42, 1601-1609
Gomes, B. C., Esteves, C. T., Palazzo, I. C. V., Darini, A. L. C., Felis, G. E., Sechi, L. A., Franco, B. D. G. M., De Martinis, E. C. P., Prevalence and characterization of Enterococcus spp. isolated from Brazilian foods, Food Microbiology, 2008, 25, 668-675
Nubel, U., Engelen, B., Felske, A., Snaidr, J., Wieshuber, A., Amann, R. I., Ludwig, W., Backhaus, H., Sequence heterogeneities of genes encoding 16s rRNAs in Paenibacillus polymyxa detected by temperature gradient gel electrophoresis, Journal of Bacteriology, 1996, 178, 5636-5643
Vincze, T., Posfai, J., Roberts, R. J., NEBcutter: a program to cleave DNA with restriction enzymes, Nucleic Acids Research, 2003, 31, 3688-3691
Fortina, M. G., Ricci, G., Borgo, F., Manachini, P. L., Rapid identification of Enterococcus italicus by PCR with primers targeted to 16S rRNA gene, Letters in Applied Microbiology, 2007, 44, 443-446
Harwood, V. J., Delahoya, N. C., Ulrich, R. M., Kramer, M. F., Whitlock, J. E., Garey, J. R., Lim, D. V., Molecular confirmation of Enterococcus faecalis and E. faecium from clinical, faecal and environmental sources, Letters in Applied Microbiology, 2004, 38, 476-482
Jurkovič, D., Križková, L., Dušinský, R., Belicová, A., Sojka, M., Krajčovič, J., Ebringer, L., Identification and characterization of enterococci from bryndza cheese, Letters in Applied Microbiology, 2006, 42, 553-559
Angeletti, S., Lorino, G., Gherardi, G., Battistoni, F., De Cesaris, M., Dicuonzo, G., Routine molecular identification of enterococci by gene-specific PCR and 16S ribosomal DNA sequencing, Journal of clinical microbiology, 2001, 39, 794-797
Monstein, H. J., Johansson, Y., Jonasson, J., Detection of vancomycin resistance genes combined with typing of Enterococci by means of multiplex PCR amplification and multiple primer DNA sequencing, Acta Pathologica et Microbiologica Scandinavica, 2000, 108, 67-73
Kariyama, R., Mitsuhata, R., Chow, J. W., Clewell, D. B., Kumon, H., Simple a reliable multiplex PCR assay for surveillance isolates of vancomycin-resistant enterococci, Journal of Clinical Microbiology, 2000, 38, 3092-3095
Patel, R., Piper, K. E., Rouse, M. S., Steckelberg, J. M., Uhl, J. R., Kohner, P., Hopkins, M. K., Cockerill III, F. R., Kline, B. C., Determination of 16S rRNA sequences of enterococci and application to species identification of nonmotile Enterococcus gallinarum isolates, Journal of Clinical Microbiology, 1998, 36, 3399-3407
Dutka-Malen, S., Evers, S., Courvalin, P., Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR, Journal of Clinical Microbiology, 1995, 33, 1434
Gurtler, V., Stanisich, V. A. New approaches to typing and identi¬fication of bacteria using the 16S-23S rDNA spacer region, Mi¬crobiology, 1996, 142, 3-16
Williams, A. M., Rodrigues, U. M., Collins, M. D. Intrageneric relationships of Enterococci as determined by reverse transcriptase sequencing of small-subunit rRNA, Research in Microbiology, 1991, 142, 67-74
Leclerc, H., Devriese, L. A., Mossel, D.A., Taxonomical changes in intestinal (faecal) enterococci and streptococci: consequences on their use as indicators of faecal contamination in drinking water, Journal of Applied Bacteriology, 1996, 81, 459-466
Scheidegger, E. M. D., Fracalanzza, S. A. P., Teixeira, L. M., Cardarelli-Leite, P., RFLP analysis of a PCR-amplified fragment of the 16S rRNA gene as a tool to identify Enterococcus strains, Memórias do Instituto Oswaldo Cruz, 2009, 104, 1003-1008
