Probiotics and Antimicrobial Proteins

, Volume 11, Issue 1, pp 325–331 | Cite as

Screening of the Enterocin-Encoding Genes and Their Genetic Determinism in the Bacteriocinogenic Enterococcus faecium GHB21

  • Mohamed MerzougEmail author
  • Khédidja Mosbahi
  • Daniel Walker
  • Nour-Eddine Karam


Enterococci are well-known for their ability to produce a variety of antimicrobial peptides called enterocins. Most of these enterocins withstand extreme conditions and are very effective against a broad spectrum of undesirable bacteria including some Gram-negative bacteria. The same enterococci strain can produce multiple enterocins simultaneously. The genetic determinants of these bacteriocins can either be located on plasmids or on bacterial chromosome. Digestion of Enterococcus faecium GHB21 plasmids with various restriction endonucleases suggests the presence of two plasmids named pGHB-21.1 and pGHB-21.2 whose respective sizes are ~ 10.0 kb and ~ 3.3 kb. The screening of enterocin-encoding genes among E. faecium GHB21 genome by PCR followed by amplicon sequencing indicated the presence of three different enterocin structural genes similar to entA, entB, and entP genes previously detected in other E. faecium strains. These enterocin genes were, subsequently, localized on the bacterial chromosome based on PCR-targeted screening using total DNA and plasmids of E. faecium GHB21 as separate templates.


E. faecium GHB21 Plasmids PCR Enterocin genes Genetic determinism 



This work was funded by the Algerian Ministry of Higher Education and Scientific Research (MESRS), General Directorate for Scientific Research and Technological Development (DGRSDT), and the University of Glasgow.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Ethical Approval

This article does not contain any studies with human participants or animals performed by any of the authors.

For this type of study, formal consent is not required.

This manuscript has not been published and is not under consideration for publication elsewhere.


  1. 1.
    Jack RW, Tagg JR, Ray B (1995) Bacteriocins of Gram positive bacteria. Microbiol Rev 59:171–200Google Scholar
  2. 2.
    Klaenhammer TR (1988) Bacteriocins of lactic acid bacteria. Biochimie 70:337–349CrossRefGoogle Scholar
  3. 3.
    Yang SC, Lin CH, Sung CT, Fang JY (2014) Antibacterial activities of bacteriocins: application in foods and pharmaceuticals. Front Microbiol 5:241Google Scholar
  4. 4.
    Pieniz S, Andreazza R, Anghinoni T, Camargo F, Brandelli A (2014) Probiotic potential, antimicrobial and antioxidant activities of Enterococcus durans strain LAB18s. Food Control 37:251–256CrossRefGoogle Scholar
  5. 5.
    Svetoch E, Eruslanov B, Perelygin V, Mitsevich E, Mitsevich I, Borzenkov V, Levchuk V, Svetoch O, Kovalev Y, Stepanshin Y, Siragusa G, Seal B, Stern N (2008) Diverse antimicrobial killing by Enterococcus faecium E 50-52 bacteriocin. J Agric Food Chem 56:1942–1948CrossRefGoogle Scholar
  6. 6.
    Belkum MJ, Hayema BJ, Geis A, Kok J, Venema G (1989) Cloning of two bacteriocin genes from a lactococcal bacteriocin plasmid. Appl Environ Microbiol 55:1187–1191Google Scholar
  7. 7.
    Cleveland J, Montville TJ, Nes IF, Chikindas ML (2001) Bacteriocins: safe, natural antimicrobials for food preservation. Int J Food Microbiol 71:1–20CrossRefGoogle Scholar
  8. 8.
    Luquet FM, Corrieu G (2005) Bactéries lactiques et probiotiques. Tec & Doc Lavoisier, ParisGoogle Scholar
  9. 9.
    Bonacina JS, Saavedra LH (2015) Genome mining and transcriptional analysis of bacteriocin genes in Enterococcus faecium CRL1879. J Data Mining Genomics Proteomics 6:1–8CrossRefGoogle Scholar
  10. 10.
    García de Fernando G (2011) Enterococcus in milk and dairy products. In: Fuquay JW, Fox PF, McSweeney PLH (eds) Encyclopedia of dairy sciences, 2nd edn. Elsevier, Boston, pp 153–159CrossRefGoogle Scholar
  11. 11.
    Hassan M, Brede D, Diep D, Nes I, Lotfipour F, Hojabri Z (2015) Efficient inactivation of multi-antibiotics resistant nosocomial enterococci by purified hiracin bacteriocin. Adv Pharm Bull 5:393–401CrossRefGoogle Scholar
  12. 12.
    Mojsova S, Krstevski K, Dzadzovski I, Popova Z, Sekulovski P (2015) Phenotypic and genotypic characteristics of enterocin producing Enterococci against pathogenic bacteria. Mac Vet Rev 38:209–216CrossRefGoogle Scholar
  13. 13.
    Yildirim M, Sahingil D, Tokatli K, Isleroglu H, Bilgin H, Yildirim Z (2014) Enterocin HZ produced by a wild Enterococcus faecium strain isolated from a traditional, starter-free pickled cheese. J Dairy Res 81:164–172CrossRefGoogle Scholar
  14. 14.
    Franz CMAP, Van Belkum M, Holzapfel W, Abriouel H, Gálvez A (2007) Diversity of enterococcal bacteriocins and their grouping in a new classification scheme. FEMS Microbiol Rev 31:293–310CrossRefGoogle Scholar
  15. 15.
    Nes IF, Diep DB, Holo H (2007) Bacteriocin diversity in Streptococcus and Enterococcus. J Bacteriol 189:1189–1198CrossRefGoogle Scholar
  16. 16.
    Nes I, Diep D, Ike Y (2014) Enterococcal bacteriocins and antimicrobial proteins that contribute to niche control. In: Gilmore MS, Clewell DB, Ike Y, Shankar N (eds) Enterococci: from commensals to leading causes of drug resistant infection, 1st edn. Massachusetts Eye and Ear Infirmary, Boston, pp 637–668Google Scholar
  17. 17.
    Franz CMAP, Schillinger U, Holzapfel WH (1996) Production and characterization of enterocin 900, a bacteriocin produced by Enterococcus faecium BFE 900 from black olives. Int J Food Microbiol 29:255–270CrossRefGoogle Scholar
  18. 18.
    Casaus P, Nilsen T, Cintas L, Nes I, Hernandez P, Holo H (1997) Enterocin B, a new bacteriocin from Enterococcus faecium T136 which can act synergistically with enterocin A. Microbiology 143:2287–2294CrossRefGoogle Scholar
  19. 19.
    Ennahar S, Asou Y, Zendo T, Sonomoto K, Ishizaki A (2001) Biochemical and genetic evidence for production of enterocins A and B by Enterococcus faecium WHE 81. Int J Food Microbiol 70:291–301CrossRefGoogle Scholar
  20. 20.
    Foulquié Moreno MR, Callewaert R, Devreese B, Van Beeumen J, De Vuyst L (2003) Isolation and biochemical characterisation of enterocins produced by Enterococci from different sources. J Appl Microbiol 94:214–229CrossRefGoogle Scholar
  21. 21.
    Sonsa-Ard N, Rodtong S, Chikindas ML, Yongsawatdigul J (2015) Characterization of bacteriocin produced by Enterococcus faecium CN-25 isolated from traditionally Thai fermented fish roe. Food Control 54:308–316CrossRefGoogle Scholar
  22. 22.
    Van den Berghe E, De Winter T, De Vuyst L (2006) Enterocin A production by Enterococcus faecium FAIR-E 406 is characterised by a temperature- and pH-dependent switch-off mechanism when growth is limited due to nutrient depletion. Int J Food Microbiol 107:159–170CrossRefGoogle Scholar
  23. 23.
    Huang Y, Ye K, Yu K, Wang K, Zhou G (2016) The potential influence of two Enterococcus faecium on the growth of Listeria monocytogenes. Food Control 67:18–24CrossRefGoogle Scholar
  24. 24.
    Park SH, Itoh K, Fujisawa T (2003) Characteristics and identification of enterocins produced by Enterococcus faecium JCM 5804T. J Appl Microbiol 95:294–300CrossRefGoogle Scholar
  25. 25.
    Merzoug M, Dalache F, Zadi Karam H, Karam NE (2016) Isolation and preliminary characterisation of bacteriocin produced by Enterococcus faecium GHB21 isolated from Algerian paste of dates “ghars”. Ann Microbiol 66:795–805CrossRefGoogle Scholar
  26. 26.
    De Man JC, Rogosa M, Sharpe ME (1960) A medium for the cultivation of Lactobacilli. J Appl Bacteriol 23:130–135CrossRefGoogle Scholar
  27. 27.
    De Vuyst L, Foulquié Moreno MR, Revets H (2003) Screening for enterocins and detection of hemolysin and vancomycin resistance in enterococci of different origins. Int J Food Microbiol 84:299–318CrossRefGoogle Scholar
  28. 28.
    Gutiérrez J, Criado R, Citti R, Martín M, Herranz C, Nes I, Cintas L, Hernández P (2005) Cloning, production and functional expression of enterocin P, a sec-dependent bacteriocin produced by Enterococcus faecium P13, in Escherichia coli. Int J Food Microbiol 103:239–250CrossRefGoogle Scholar
  29. 29.
    Cintas L, Casaus P, Herranz C, Havarstein L, Holo H, Hernandez P, Nes I (2000) Biochemical and genetic evidence that Enterococcus faecium L50 produces enterocins L50A and L50B, the sec-dependent enterocin P, and a novel bacteriocin secreted without an N-terminal extension termed enterocin Q. J Bacteriol 182:6806–6814CrossRefGoogle Scholar
  30. 30.
    Saavedra L, Minahk C, de Ruiz Holgado A, Sesma F (2004) Enhancement of the enterocin CRL35 activity by a synthetic peptide derived from the NH2-terminal sequence. Antimicrob Agents Chemother 48:2778–2781CrossRefGoogle Scholar
  31. 31.
    Zendo T, Eungruttanagorn N, Fujioka S, Tashiro Y, Nomura K, Sera Y, Kobayashi G, Nakayama J, Ishizaki A, Sonomoto K (2005) Identification and production of a bacteriocin from Enterococcus mundtii QU 2 isolated from soybean. J Appl Microbiol 99:1181–1190CrossRefGoogle Scholar
  32. 32.
    Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, New YorkGoogle Scholar
  33. 33.
    Garcia-Migura L, Hasman H, Jensen LB (2009) Presence of pRI1: a small cryptic mobilizable plasmid isolated from Enterococcus faecium of human and animal origin. Curr Microbiol 58:95–100CrossRefGoogle Scholar
  34. 34.
    Kumar N, Ponnaluri C, Putarjunan A, Ranganathan S, Roy U, Das A (2012) Characterization of temperature inducible promoters from a novel rolling circle replicating plasmid of Enterococcus faecium DJ1. Plasmid 67:211–226CrossRefGoogle Scholar
  35. 35.
    Clewell DB, Weaver KE, Dunny GM, Coque TM, Francia MV, Hayes F (2014) Extrachromosomal and mobile elements in enterococci: transmission, maintenance, and epidemiology. In: Gilmore MS, Clewell DB, Ike Y, Shankar N (eds) Enterococci: from commensals to leading causes of drug resistant infection, 1st edn. Massachusetts Eye and Ear Infirmary, Boston, pp 309–420Google Scholar
  36. 36.
    Francia MV, Clewell DB (2002) Amplification of the tetracycline resistance determinant of pAMα1 in Enterococcus faecalis requires a site-specific recombination event involving relaxase. J Bacteriol 184:5187–5193CrossRefGoogle Scholar
  37. 37.
    Nilsen T, Nes IF, Holo H (1998) An exported inducer peptide regulates bacteriocin production in Enterococcus faecium CTC492. J Bacteriol 180:1848–1854Google Scholar
  38. 38.
    Aymerich T, Holo H, Havarstein LS, Hugas M, Garriga M, Nes I (1996) Biochemical and genetic characterization of enterocin A from Enterococcus faecium, a new antilisterial bacteriocin in the pediocin family of bacteriocins. Appl Environ Microbiol 62:1676–1682Google Scholar
  39. 39.
    Cintas L, Casaus P, Håvarstein L, Hernández P, Nes I (1997) Biochemical and genetic characterization of enterocin P, a novel sec-dependent bacteriocin from Enterococcus faecium P13 with a broad antimicrobial spectrum. Appl Environ Microbiol 63:4321–4330Google Scholar
  40. 40.
    Brandão A, Almeida T, Muñoz-Atienza E, Torres C, Igrejas G, Hernández P, Cintas L, Poeta P, Herranz C (2010) Antimicrobial activity and occurrence of bacteriocin structural genes in Enterococcus spp. of human and animal origin isolated in Portugal. Arch Microbiol 192:927–936CrossRefGoogle Scholar
  41. 41.
    Javaherzadeh V, Jamshidian M, Zahraei M, Youseftabar A, Milani M, Hassan M, Lotfipour F (2015) Evaluation of bacteriocin activities among enterococcal poultry isolates from east Azarbaijan Iran. Pharm Sci 21:72–76CrossRefGoogle Scholar
  42. 42.
    Izquierdo E, Wagner C, Marchioni E, Aoude-Werner D, Ennahar S (2009) Enterocin 96, a novel class II bacteriocin produced by Enterococcus faecalis WHE 96, isolated from Munster cheese. Appl Environ Microbiol 75:4273–4276CrossRefGoogle Scholar
  43. 43.
    Ogaki MB, Rocha KR, Terra MR, Furlaneto MC, Maia L (2016) Screening of the enterocin-encoding genes and antimicrobial activity in Enterococcus species. J Microbiol Biotechnol 26:1026–1034CrossRefGoogle Scholar
  44. 44.
    Özdemir GB, Oryaşın E, Bıyık HH, Özteber M, Bozdoğan B (2011) Phenotypic and genotypic characterization of bacteriocins in enterococcal isolates of different sources. Indian J Microbiol 51:182–187CrossRefGoogle Scholar
  45. 45.
    Poeta P, Costa D, Rojo-Bezares B, Zarazaga M, Klibi N, Rodrigues J, Torres C (2007) Detection of antimicrobial activities and bacteriocin structural genes in faecal Enterococci of wild animals. Microbiol Res 162:257–263CrossRefGoogle Scholar
  46. 46.
    Hu C, Malaphan W, Zendo T, Nakayama J, Sonomoto K (2010) Enterocin X, a novel two-peptide bacteriocin from Enterococcus faecium KU-B5, has an antibacterial spectrum entirely different from those of its component peptides. Appl Environ Microbiol 76:4542–4545CrossRefGoogle Scholar
  47. 47.
    Achemchem F, Cebrián R, Abrini J, Martínez-Bueno M, Valdivia E, Maqueda M (2012) Antimicrobial characterization and safety aspects of the bacteriocinogenic Enterococcus hirae F420 isolated from Moroccan raw goat milk. Can J Microbiol 58:596–604CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Laboratoire de Biologie des Microorganismes et Biotechnologie, BP1524, Oran El MnaouerUniversité d’Oran 1 Ahmed Ben BellaOranAlgeria
  2. 2.Département des Sciences Agronomiques et BiotechnologieUniversité de ChlefChlefAlgeria
  3. 3.Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life SciencesfUniversity of GlasgowGlasgowUK

Personalised recommendations