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Development of a Rapid Identification Method for the Differentiation of Enterococcus Species Using a Species-Specific Multiplex PCR Based on Comparative Genomics

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Abstract

Enterococci are lactic acid bacteria that are commonly found in food and in animal gut. Since 16 S ribosomal RNA (rRNA) sequences, genetic markers for bacterial identification, are similar among several Enterococcus species, it is very difficult to determine the correct species based on only 16 S rRNA sequences. Therefore, we developed a rapid method for the identification of different Enterococcus species using comparative genomics. We compared 38 genomes of 13 Enterococcus species retrieved from the National Center of Biotechnology Information database and identified 25,623 orthologs. Among the orthologs, four genes were specific to four Enterococcus species (Enterococcus faecalis, Enterococcus faecium, Enterococcus hirae, and Enterococcus durans). We designed species-specific primer sets targeting the genes and developed a multiplex PCR using primer sets that could distinguish the four Enterococcus species among the nine strains of Enterococcus species that were available locally. The multiplex PCR method also distinguished the four species isolated from various environments, such as feces of chicken and cow, meat of chicken, cow, and pigs, and fermented soybeans (Cheonggukjang and Doenjang). These results indicated that our novel multiplex PCR using species-specific primers could identify the four Enterococcus species in a rapid and easy way. This method will be useful to distinguish Enterococcus species in food, feed, and clinical settings.

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Acknowledgements

This study was supported by a grant from the Strategic Initiative for Microbiomes in Agriculture and Food, Ministry of Agriculture, Food and Rural Affairs, Republic of Korea as part of the multi-ministerial Genome Technology to Business Translation Program (Grant ID: 914005–04) and the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2016R1C1B2016246). We appreciate KACC (Korean Agricultural Culture Collection) for donation of E. durans, E. hirae, E. mundtii, E. avium, E. gallinarum, E. silesiacus, E. termitis strains for this study. Jongbin Park was supported by the CK-II Program from NRF (CALSIS, Center for Aniaml Life Senary Industry Specialization).

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Authors and Affiliations

  1. Department of Animal Life System, College of Animal Life Sciences, Kangwon National University, Chuncheon, Kangwon-do, Republic of Korea

    Jongbin Park

  2. Department of Animal Life Science, College of Animal Life Sciences, Kangwon National University, Chuncheon, Kangwon-do, Republic of Korea

    Gwi-Deuk Jin, Jae In Pak, Jihyun Won & Eun Bae Kim

  3. Division of Applied Animal Science, College of Animal Life Sciences, Kangwon National University, Chuncheon, Kangwon-do, Republic of Korea

    Jae In Pak & Eun Bae Kim

  4. Institute of Animal Resources, College of Animal Life Sciences, Kangwon National University, Chuncheon, Kangwon-do, Republic of Korea

    Jihyun Won

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  1. Jongbin Park
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  2. Gwi-Deuk Jin
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Correspondence to Eun Bae Kim.

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Park, J., Jin, GD., Pak, J.I. et al. Development of a Rapid Identification Method for the Differentiation of Enterococcus Species Using a Species-Specific Multiplex PCR Based on Comparative Genomics. Curr Microbiol 74, 476–483 (2017). https://doi.org/10.1007/s00284-017-1210-5

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  • DOI: https://doi.org/10.1007/s00284-017-1210-5

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