Species identification of enterococci by biochemical test and molecular-genetic methods

  • Monika Lavová Slovak University of Agriculture, Faculty of Biotechnology and Food Sciences, Department of evaluation and processing of animal products, Tr. A. Hlinku 2, 949 76 Nitra
  • Jana Bezeková Slovak University of Agriculture, Faculty of Biotechnology and Food Sciences, Department of evaluation and processing of animal products, Tr. A. Hlinku 2, 949 76 Nitra
  • Margita Čanigová Slovak University of Agriculture, Faculty of Biotechnology and Food Sciences, Department of evaluation and processing of animal products, Tr. A. Hlinku 2, 949 76 Nitra
  • Miroslav Kročko Slovak University of Agriculture, Faculty of Biotechnology and Food Sciences, Department of evaluation and processing of animal products, Tr. A. Hlinku 2, 949 76 Nitra
  • Konrad Domig University of Natural Resources and Applied Life Sciences Vienna, Institute of Food Science, Food Microbiology and Hygiene Laboratory, Muthgasse 18, A-1190 Vienna, Austria
DOI: https://doi.org/10.5219/364
Keywords: enterococci, identification, EN-COCCUS test, PCR method

Abstract

The aim of this study was comparison different methods of species identification of enterococci. One hundred and fifty three suspected colonies were isolated from milk and dairy products (cheeses from cow´s, ewe´s and goat´s milk). On the bases of their growth on BEA agar, microscopic characteristic, results of Gram staining, catalase test and PYRAtest was thirty four isolates assigned to the genus Enterococcus. These isolates were identified by commercial biochemical test EN-COCCUS. 52.9% of them were included in species E. faecalis, 29.4% in E. faecium, 14.7% in E. durans and 2.9% in E. group III. This group includes 3 species: E. durans, E. hirae, E. faecalis asaccharolytic var. Then 16S rRNA sequencing nucleotide of all isolates was realized. Results of sequencing were compared with NCBI database. Only 14.7% of isolates were in 100% accordance. One from them was species E. durans and others were designated as E. faecium. For 20.6% of detected isolates was in accordance with more reference strains. Other isolates were identical with reference strain on 99%. For verification of all results species-specific PCR was used and 52.9% isolates were identified as species E. faecalis, 32.4% as E. faecium and 14.7% as E. durans. Strains belonging to the species E. faecalis were identified the most reliable by all used methods.

References

Arias, A. C., Robredo, B., Singh, K. V., Torres, K., Panesso, D., Murray, B. E. 2006. Rapid identification of E. hirae and E. durans by PCR and detection of a homologue of the E. hirae mur-2 gene in E. durans. Journal of Clinical Microbiology, vol. 44, no. 4, p. 1567-1570. http://dx.doi.org/10.1128/JCM.44.4.1567-1570.2006  PMid: 16597896


Aymerich, T., Martin, B., Garriga, M., Hugas, M. 2003. Microbial quality and direct PCR identification of lactic acid bacteria and non-pathogenic staphylococci from artisanal low-acid sausages. Applied and Environmental Microbiology, vol. 69, no. 8, p. 4583-4594. http://dx.doi.org/10.1128/AEM.69.8.4583-4594.2003  PMid: 12902246


Brtkova, A., Filipova, M., Drahovska, H., Bujdakova, H. 2010. Characterization of enterococci of animal and environmental origin using phenotypic methods and comparison with PCR based methods. Veterinarni Medicina, vol. 55, no. 3, p. 97-105. [cit. 2014-02-16]. Retrieved from the web: http://www.agriculturejournals.cz/publicFiles/18656.pdf


Citak, S., Gundogan, N., Mend, A. 2006. Occurence, isolation and antibiotic resistance of Enterococcus species isolated from raw milk samples in Turkey. Milchwissenschaft, vol. 61, no. 2, p. 150-152.


Čanigová, M., Ducková, V., Kročko, M., Račková, J., Bezeková, J. 2012. Enterococci and their ability to form a biofilm. Potravinarstvo, vol. 6, no. 2, p. 15-18. http://dx.doi.org/10.5219/200


di Cello, F., Bevivino, A., Chiarini, L., Fani, R., Pafferri, D., Tabacchioni, S., Dalmastri, C. 1997. Biodiversity of Burkholderia cepacia population isolated from the maize rhizosphere at different plant growth stages. Applied and Environmental Microbiology, vol. 63, no. 11, p. 4485-4493. PMid: 9361434


Domig, K. J., Mayer, H. K., Kneifel, W. 2003. Methods used for the isolation, enumeration, characterisation and identification of Enterococcus spp.: 1. Media for isolation and enumeration. International Journal of Food Microbiology, vol. 88, no. 2-3, p. 147-164. http://dx.doi.org/10.1016/S0168-1605(03)00177-6  PMid: 14596987


Ducková, V., Čanigová, M., Kročko, M., Bezeková, J. 2012. Antimicrobial activity of thyme and rosemary essential oil. Potravinarstvo, vol. 6, no. 1, p. 10-13. http://dx.doi.org/10.5219/178


Dutka-Malen, S., Evers, S., Courvalin, P. 1995. Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant Enterococci by PCR. Journal of Clinical Microbiology, vol. 33, no. 1, p. 24-27. PMid: 7699051


Fabianová, J., Ducková, V., Čanigová, M., Kročko, M. 2010. Presence of enterococci in cow milk and their antibiotic resistance. Potravinarstvo, vol. 4, no. 2, p. 17-21. http://dx.doi.org/10.5219/45


Fei, C., Lu, X., Qian, Y., Zhang, H., Mahmood, Q. 2006. Identification of Enterococcus sp. from midgut of silkworm based on biochemical and 16S rDNA sequencing analysis. Annals of Microbiology, vol. 56, no. 3, p. 201-205. http://dx.doi.org/10.1007/BF03175006


Jamaly, N., Benjouad, A., Comunian, R., Daga, E., Bouksaim, M. 2010.  Characterization of Enterococci isolated from Moroccan dairy products. African Journal of Microbiology Research, vol. 4, no. 16, p. 1768-1774. [cit. 2011-02-17]. Retrieved from the web: http://www.researchgate.net/publication/228627157_Characterization_of_Enterococci_isolated_from_Moroccan_dairy_products 

 

Jurkovič, D., Križková, L., Dušinský, R., Belicová, A., Sojka, M., Krajčovič, J., Ebringer, L. 2006. Identification and characterization of enterococci from bryndza cheese. Letters in Applied Microbiology, vol. 42, p. 553–559, ISSN 0266-8254.

 

Krebs-Artimová, A., Ducková, V., Kročko, M. 2013. Occurrence of antibiotic resistance enterococci on skin of teats and teat cups of milking machine. Potravinarstvo, vol. 7, no. 1, p. 181-185. http://dx.doi.org/10.5219/310

 

Kročko, M., Čanigová, M., Ducková, V., Artimová, A., Bezeková, J., Poston, P. 2011. Antibiotic resistance of Enterococcus Species Isolated from Raw Foods of Animal Origin in South West Part of Slovakia. Czech J. Food Sci., vol. 29, no. 6, p. 654-659. [cit. 2011-02-16]. Retrieved from the web: http://www.agriculturejournals.cz/publicFiles/51707.pdf

 

Nikolic, M., Terzic-Vidojevic, A., Jovcic, B., Begovic, J., Golic, N., Topisirovic, L. 2008. Characterization of lactic acid bacteria isolated from Bukuljac, a homemade goat's milk cheese. International Journal of Food Microbiology, vol. 122, no. 1-2, p. 162-170. http://dx.doi.org/10.1016/j.ijfoodmicro.2007.11.075  PMid: 18177967

 

Nieto-Arribas, P., Seseña, S., Poveda, J. M., Chicón, R., Cabezas, L., Palop, L. 2011. Enterococcus populations in artisanal Manchego cheese: Biodiversity, technological and safety aspects. Food Microbiology, vol. 28, no. 5, p. 891-899. http://dx.doi.org/10.1016/j.fm.2010.12.005  PMid: 21569931

 

Pesavento, G., Calonico, C., Ducci, B., Magnanini, A., Lo Nostro, A. 2014. Prevalence and antibiotic resistance of Enterococcus spp. isolated from retail cheese, ready-to-eat salads, ham, and raw meat. Food Microbiology, vol. 41, p. 1-7. http://dx.doi.org/10.1016/j.fm.2014.01.008

 

Račková, J. 2012. Rezistenica enterokokov izolovaných z mlieka voči antibiotikám a sanitačným prostriedkom: Disertation work. Slovak University of Agriculture in Nitra, 2012, 118 p.

 

Serio, A., Chaves-López, C., Paparella, A., Suzzi, G. 2010. Evaluation of metabolic activities of enterococci isolated from Pecorino Abruzzese cheese. International Dairy Journal, vol. 20, no. 7, p. 459-464. http://dx.doi.org/10.1016/j.idairyj.2010.02.005

 

STN 56 0100: 1970. Mikrobiologické skúšanie požívatín, predmetov bežnej spotreby a prostredia potravinárskych prevádzok. Bratislava : ÚNMS, 239 s. 

 

URL 1:  http://www.ncbi.nlm.nih.gov/

 

Valenzuela, A. S., Benomar, N., Abriouel, H., López, R. L., Veljovic, K., Cañamero, M. M., Topisirovic,  M. K. L., Gálvez, A. 2009. Virulence factors, antibiotic resistance, and bacteriocins in enterococci from artisan foods of animal origin. Food Control, vol. 20, no. 4, p. 381-385. http://dx.doi.org/10.1016/j.foodcont.2008.06.004

Published
2014-05-02
How to Cite
Lavová, M., Bezeková, J., Čanigová, M., Kročko, M., & Domig, K. (2014). Species identification of enterococci by biochemical test and molecular-genetic methods. Potravinarstvo Slovak Journal of Food Sciences, 8(1), 124-129. https://doi.org/10.5219/364
Issue
Vol. 8 No. 1 (2014): Potravinarstvo
Section
Articles
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