Folia Microbiologica

, Volume 64, Issue 4, pp 535–545 | Cite as

Characterization of the bacteriocin produced by Enterococcus italicus ONU547 isolated from Thai fermented cabbage

  • Andrii MerlichEmail author
  • Mykola Galkin
  • Yvan Choiset
  • Nataliia Limanska
  • Nataliia Vasylieva
  • Volodymyr Ivanytsia
  • Thomas Haertlé
Original Article


Seventy-eight isolates of lactic acid bacteria from Ukraine and Thailand were screened for bacteriocinogenic activity against indicator strain Lactobacillus sakei subsp. sakei JCM 1157. One isolate showed an antagonistic activity of cell-free supernatant eliminated after the treatment with Proteinase K. Based on 16S rRNA gene sequence, this isolate was identified as Enterococcus italicus. Bacteriocin produced by this strain showed antimicrobial activity against L. sakei subsp. sakei JCM 1157, Brochothrix thermosphacta DSMZ 20171, and Listeria ivanovii subsp. ivanovii DSMZ 20750 in agar well diffusion assay. This bacteriocin was cationic and hydrophobic. The partially purified bacteriocin was thermostable, while heating of cell-free supernatant increased its activity more than twofold. Molecular mass of the partially purified bacteriocin as determined by SDS-PAGE differed from enterocin A and B previously known for E. italicus. Concentrated bacteriocin decreased the level of biofilm formation in L. sakei subsp. sakei JCM 1157 and Pseudomonas aeruginosa PAO1 in 52.5 and 48.0%, respectively (p < 0.05). We suggest that the studied bacteriocin could be a perspective antibiofilm agent in food conservation and medicine.



Andrii Merlich was supported by PhD scholarship from Campus France. We are grateful to Dr. Noraphat Hwanhlem for his kind advice during the work. We thank Hanitra Rabesona for her kind assistance.

Funding information

The work was supported by Bilateral French-Ukrainian project «Dnipro» granted by the Ministry of Foreign and European Affairs of France and the Ministry of Education and Science of Ukraine.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Aguilar C, Klotz B (2010) Effect of the temperature on the antagonistic activity of lactic acid bacteria against Escherichia coli and Listeria monocytogenes. J Food Saf 30:996–1015. CrossRefGoogle Scholar
  2. Al-Mathkhury HJ, Ali AS, Ghafil JA (2011) Antagonistic effect of bacteriocin against urinary catheter associated Pseudomonas aeruginosa biofilm. N Am J Med Sci 3:367–370. CrossRefPubMedPubMedCentralGoogle Scholar
  3. Aymerich T, Holo H, Håvarstein LS, Hugas M, Garriga M, Nes IF (1996) Biochemical and genetic characterization of enterocin A from Enterococcus faecium, a new antilisterial bacteriocin in the pediocin family of bacteriocins. Appl Environ Microbiol 62:1676–1682PubMedPubMedCentralGoogle Scholar
  4. Bajpai VK, Han J-H, Rather IA, Park C, Lim J, Paek WK, Lu JS, Yoon J-I, Park Y-H (2016) Characterization and antibacterial potential of lactic acid bacterium Pediococcus pentosaceous 4I1 isolated from freshwater fish Zacco koreanus. Front Microbiol 7:1–15. CrossRefGoogle Scholar
  5. Belgacem ZB, Rehaiem A, Bernárdez PF, Manai M, Castro PL (2012) Interactive effects of pH and temperature on the bacteriocin stability by response surface analysis. Microbiologiia 81:195–200. CrossRefGoogle Scholar
  6. Casaus P, Nilsen T, Cintas LM, Nes IF, Hernández PE, Holo H (1997) Enterocin B, a new bacteriocin from Enterococcus faecium T136 which can act synergistically with enterocin A. Microbiology 143:2287–2294. CrossRefPubMedGoogle Scholar
  7. Christensen GD, Simpson WA, Younger JJ, Baddour LM, Barrett FF, Melton DM, Beachey EH (1985) Adherence of coagulase-negative staphylococci to plastic tissue culture plates: a quantitative model for the adherence of staphylococci to medical devices. J Clin Microbiol 22:996–1006PubMedPubMedCentralGoogle Scholar
  8. Cirkovik I, Bozic DD, Draganic V, Lozo J, Beric T, Kojic M, Arsic B, Garalejic E, Djukic S, Stankovic S (2016) Licheniocin 50.2 and bacteriocins from Lactococcus lactis subsp. lactis biovar. diacetylactis BGBU1-4 inhibit biofilms of coagulase negative staphylococci and Listeria monocytogenes clinical isolates. PLoS One 11:1–12. CrossRefGoogle Scholar
  9. Cocolin L, Rantsiou K, Iacumin L, Urso R, Cantoni C, Comi G (2004) Study of the ecology of fresh sausages and characterization of populations of lactic acid bacteria by molecular methods. Appl Environ Microbiol 70:1883–1894. CrossRefPubMedPubMedCentralGoogle Scholar
  10. Dezwaan DC, Mequio MJ, Littell JS, Allen JP, Rossbach S, Pybus V (2007) Purification and characterization of enterocin 62-6, a two-peptide bacteriocin produced by a vaginal strain Enterococcus faecium: potential significance in bacterial vaginosis. Microb Ecol Health Dis 19:241–250. CrossRefPubMedPubMedCentralGoogle Scholar
  11. Dias FS, Ramos CL, Schwan RF (2013) Characterization of spoilage bacteria in pork sausage by PCR-DGGE analysis. Food Sci Technol 33:468–474. CrossRefGoogle Scholar
  12. Djadouni F, Kihal M (2012) Antimicrobial activity of lactic acid bacteria and the spectrum of their biopeptides against spoiling germs in foods. Braz Arch Biol Technol 55:435–443CrossRefGoogle Scholar
  13. Eid R, El Jakee J, Rashidy A, Asfour H, Omara S, Kandil MM, Mahmood Z, Hahne J, Seida AA (2016) Potential antimicrobial activities of probiotic Lactobacillus strains isolated from raw milk. J Probiotics Health 4:1–8. CrossRefGoogle Scholar
  14. El-Deeb N, Sharaf MM, El-Adawi H (2015) Antibacterial and plasmid curing activity of lactic acid bacteria against multidrug resistant bacteria strains. Int J Pharmacol 11:114–121. CrossRefGoogle Scholar
  15. Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791CrossRefGoogle Scholar
  16. Fortina MG, Ricci G, Mora D, Manachini PL (2004) Molecular analysis of artisanal Italian cheeses reveals Enterococcus italicus sp. nov. Int J Syst Evol Microbiol 54:1717–1721. CrossRefPubMedGoogle Scholar
  17. Franciosi E, Carafa I, Nardin T, Schiavon S, Poznanski E, Cavazza A, Larcher R, Tuohy KM (2015) Biodiversity and γ-aminobutyric acid production by lactic acid bacteria isolated from traditional alpine raw cow’s milk cheeses. BioMed Res Int 2015:1–11. CrossRefGoogle Scholar
  18. Gaaloul N, Braiek OB, Berjeaud JM, Arthur T, Cavera VL, Chikindas ML, Hani K, Ghrairi T (2014) Evaluation of antimicrobial activity and safety aspect of Enterococcus italicus GGN 10 strain isolated from Tunisian bovine raw milk. J Food Saf 34:1–12. CrossRefGoogle Scholar
  19. Guillet C, Join-Lambert O, Le Monnier A, Leclercq A, Mechaї F, Mamzer-Bruneel MF, Bielecka MK, Scortti M, Disson O, Berche P, others (2010) Human listeriosis caused by Listeria ivanovii. Emerg Infect Dis 16:136–138. CrossRefPubMedPubMedCentralGoogle Scholar
  20. Guttiérrez J, Criado R, Martín M, Herranz C, Cintas LM, Hernández PE (2005) Production of enterocin P, an antilisterial pediocin-like bacteriocin from Enterococcus faecium P13, in Pichia pastoris. Antimicrob Agents Chemother 49:3004–3008. CrossRefGoogle Scholar
  21. H-Kittikun A, Biscola V, El-Ghaish S, Jaffrès E, Dousset X, Pillot G, Haertlé T, Chobert J-M, Hwanhlem N (2015) Bacteriocin-producing Enterococcus faecalis KT2W2G isolated from mangrove forests in southern Thailand: purification, characterization and safety evaluation. Food Control 54:126–134. CrossRefGoogle Scholar
  22. Hor YY, Liong MT (2014) Use of extracellular extracts of lactic acid bacteria and bifidobacteria for the inhibition of dermatological pathogen Staphylococcus aureus. Dermatologica Sinica 32:141–147. CrossRefGoogle Scholar
  23. Hwanhlem N, Biscola V, El-Ghaish S, Jaffrès E, Dousset X, Haertlé T, H-Kittikun A, Chobert JM (2013) Bacteriocin-producing lactic acid bacteria isolated from mangrove forests in southern Thailand as potential bio-control agents: purification and characterization of bacteriocin produced by Lactococcus lactis subsp. lactis KT2W2L. Probiotics Antimicro Prot 5:264–278. CrossRefGoogle Scholar
  24. Hwanhlem N, Chobert J-M, H-Kittikun A (2014) Bacteriocin-producing lactic acid bacteria isolated from mangrove forests in southern Thailand as potential bio-control agents in food: isolation, screening and optimization. Food Control 41:202–211. CrossRefGoogle Scholar
  25. Hwanhlem N, Jaffrès E, Dousset X, Pillot G, Choiset Y, Haertlé T, H-Kittikun A, Chobert J-M (2015) Application of a nisin Z-producing Lactococcus lactis subsp. lactis KT2W2L isolated from brackish water for biopreservation in cooked, peeled and ionized tropical shrimps during storage at 8 °C under modified atmosphere packaging. Eur Food Res Technol 240:1259–1269. CrossRefGoogle Scholar
  26. Kalschne DL, Womer R, Mattana A, Sarmento MP, Colla LM, Colla E (2015) Characterization of the spoilage lactic acid bacteria in «sliced vacuum-packed ham». Braz J Microbiol 64:173–181. CrossRefGoogle Scholar
  27. Kimura M (1980) A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120CrossRefGoogle Scholar
  28. Klewicka E, Libudzisz Z (2004) Antagonistic activity of Lactobacillus acidophilus bacteria towards selected food-contaminating bacteria. Pol J food Nutr Sci 13/54(2):169–174Google Scholar
  29. Lengkey HAW, Balia RL, Togoe I, Taşbac BA, Ludong M (2009) Isolation and identification of lactic acid bacteria from raw poultry meat. Biotechnol Anim Husb 25:1071–1077Google Scholar
  30. Lim H-J, Kim S-Y, Lee W-K (2004) Isolation of cholesterol-lowering lactic acid bacteria from human intestine for probiotic use. J Vet Sci 5(4):391–395CrossRefPubMedGoogle Scholar
  31. Line JE, Svetoch EA, Eruslanov BV, Perelygin VV, Mitsevich EV, Mitsevich IP, Levchuk VP, Svetoch OE, Seal BS, Siragusa GR and others (2008) Isolation and purification of enterocin E-760 with broad antimicrobial activity against gram-positive and gram-negative bacteria. Antimicrob Agents Chemother 52:1094–1100.
  32. Mahdavi M, Jalali M, Kermanshahi RK (2007) The effect of nisin on biofilm formating foodborne bacteria using microtiter plate method. Res Pharm Sci 2:113–118Google Scholar
  33. Malini M, Savitha J (2012) Heat stable bacteriocin from Lactobacillus paracasei subsp. tolerans isolated from locally available cheese: an in vitro study. E3 J Biotechnol Pharm Res 3:28–41Google Scholar
  34. Merlich AG, Zhunko ID, Limanska NV, Ivanytsia VO (2017) Antagonistic activity of metabolic products of bacteria Lactobacillus plantarum and Enterococcus italicus with joint action against phytopathogenic bacteria. Microbiologiia i Biotechnologiia 3:45–54 (in Ukrainian. CrossRefGoogle Scholar
  35. Okuda K, Zendo T, Sugimoto S, Iwase T, Tajima A, Yamada S, Sonomoto K, Mizunoe Y (2013) Effects of bacteriocins on methicillin-resistant Staphylococcus aureus biofilm. Antimicrob Agents Chemother 57:5572–5579. CrossRefPubMedPubMedCentralGoogle Scholar
  36. Perez RH, Zendo T, Sonomoto K (2014) Novel bacteriocins from lactic acid bacteria (LAB): various structures and applications. Microb Cell Factories 13:1–8. CrossRefGoogle Scholar
  37. Pholsen S, Khota W, Pang H, Higgs D, Cai Y (2016) Characterization and application of lactic acid bacteria for tropical silage preparation. Anim Sci J 87:1202–1211. CrossRefPubMedGoogle Scholar
  38. Saad MA, Abdelsamei HM, Ibrahim EMA, Abdou AM, Sohaimy SAE (2015) Effect of pH, heat treatments and proteinase K enzyme on the activity of Lactobacillus acidophilus bacteriocin. Benha Vet Med J 28:210–215CrossRefGoogle Scholar
  39. Sadekuzzaman M, Yang S, Mizan MFR, Ha SD (2015) Current and recent advanced strategies for combating biofilms. Compr Rev Food Sci Food Saf 14:491–509. CrossRefGoogle Scholar
  40. Saeed S, Ahmad S, Rasool SA (2004) Antimicrobial spectrum, production and mode of action of staphylococcin 188 produced by Staphylococcus aureus 188. Pak J Pharm Sci 17:1–8PubMedGoogle Scholar
  41. Saelim K, Kaewsuwan S, Tani A, Maneerat S (2015) Physical, biochemical and genetic characterization of enterocin CE5-1 produced by Enterococcus faecium CE5-1 isolated from Thai indigenous chicken intestinal tract. Songklanakarin J Sci Technol 37:299–307Google Scholar
  42. Şahingil D, Işleroǧlu H, Yildirim Z, Akçelik M, Yildirim M (2011) Characterization of lactococcinBZ produced by Lactococcus lactis subsp. lactis BZ isolated from boza. Tukr J Biol 35:21–33. CrossRefGoogle Scholar
  43. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425. CrossRefPubMedGoogle Scholar
  44. Salman M, Shahid M, Jamil A, Ur Rahman S (2014) Effect of Lactobacillus acidophilus bacteriocin on Bacillus cereus biofilms. J Pure Appl Microbio 8:3721–3728Google Scholar
  45. Samelis J, Kakouri A, Rementzis J (2000) Selective effect of the product type and the packaging conditions on the species of lactic acid bacteria dominating the spoilage microbial association of cooked meats at 4 °C. Food Microbiol 17:329–340. CrossRefGoogle Scholar
  46. Satish Kumar R, Kanmani P, Yuvaraj N, Paari KA, Pattukumar V, Arul V (2011) Purification and characterization of enterocin MC13 produced by a potential aquaculture probiont Enterococcus faecium MC13 isolated from the gut of Mugil cephalus. Can J Microbiol 57:993–1001. CrossRefPubMedGoogle Scholar
  47. Sawa N, Wilaipun P, Kinoshita S, Zendo T, Leelawatcharamas V, Nakayama J, Sonomotoa K (2012) Isolation and characterization of Enterocin W, a novel two-peptide lantibiotic produced by Enterococcus faecalis NKR-4-1. Appl Environ Microbiol 78:900–903. CrossRefPubMedPubMedCentralGoogle Scholar
  48. Schägger H, von Jagow G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368–379CrossRefPubMedGoogle Scholar
  49. Schillinger U, Lüke F-K (1989) Antibacterial activity of Lactobacillus sake isolated from meat. Appl Environ Microbiol 55(8):1901–1906PubMedPubMedCentralGoogle Scholar
  50. Sifour M, Ouled-Haddar H, Idoui T, Aissaoui S, Namous H (2014) Screening of some factors affecting bacteriocin production from Lactobacillus curvatus G6 using Plackett-Burman design res rev. Biosci 8:386–393Google Scholar
  51. Simões LC, Simões M, Vieira MJ (2011) The effects of metabolite molecules produced by drinking water-isolated bacteria on their single and multispecies biofilms. Biofouling 27:685–699. CrossRefPubMedGoogle Scholar
  52. Song D-F, Zhu M-Y, Gu Q (2014) Purification and characterization of plantaricin ZJ5, a new bacteriocin produced by Lactobacillus plantarum ZJ5. PLoS one 9:1–8. CrossRefGoogle Scholar
  53. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729. CrossRefPubMedPubMedCentralGoogle Scholar
  54. Tiwari A, Singh NP, Sharma G, Dang S, Gupta S, Gabrani R (2015) Isolation and screening of lactic acid bacteria producing bacteriocin like inhibitory substance from soil. Int J Pharmacol Pharm Sci 2:32–36Google Scholar
  55. Trias R, Bañeras L, Montesinos E, Badosa E (2008) Lactic acid bacteria from fresh fruit and vegetables as biocontrol agents of phytopathogenic bacteria and fungi. Int Microbiol 11:231–236. CrossRefPubMedGoogle Scholar
  56. Vázquez-Boland JA, Kuhn M, Berche P, Chakraborty T, Domínguez-Bernal G, Goebel W, González-Zorn B, Wehland J, Kreft J (2001) Listeria pathogenesis and molecular virulence determinants. Clin Microbiol Rev 14:584–640. CrossRefPubMedPubMedCentralGoogle Scholar
  57. Visser R, Holzapfel WH (1992) Lactic acid bacteria in the control of plant pathogens. In: Wood BJB (ed) The lactic acid bacteria, vol 1. Elsevier Applied Science, London and New York, pp 193–210Google Scholar
  58. Wang T, Liu M (2016) The effect of bacteriocins derived from lactic acid bacteria on growth and biofilm formation of clinical pathogenic strains. Int J Clin Exp Med 9:7343–7348Google Scholar
  59. Weisburg WG, Barns SM, Pelletier DA, Lane DJ (1991) 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173:697–703CrossRefPubMedPubMedCentralGoogle Scholar
  60. Winkelströter LK, Gomes BC, Thomaz MRS, Souza VM, De Martinis ECP (2011) Lactobacillus sakei 1 and its bacteriocin influence adhesion of Listeria monocytogenes on stainless steel surface. Food Control 22:1404–1407. CrossRefGoogle Scholar
  61. Zmantar T, Slama RB, Fahila K, Kouidhi B, Bakhrouf A, Chaieb K (2016) Modulation of drug resistance and biofilm formation of Staphylococcus aureus isolated from the oral cavity of Tunisian children. Braz J Infect Dis 21:27–34. CrossRefPubMedGoogle Scholar

Copyright information

© Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2019

Authors and Affiliations

  1. 1.Microbiology, Virology and Biotechnology Chair, Biology DepartmentOdessa National I.I. Mechnikov UniversityOdessaUkraine
  2. 2.UR 1268 INRA Biopolymères Interactions AssemblagesNantes Cedex 3France
  3. 3.Department of Animal Nutrition and Feed Management, ulPoznan University of Life SciencesPoznanPoland
  4. 4.Institute of Biochemistry and BiophysicsUniversity of TehranTehranIran

Personalised recommendations