Probiotic characteristics of bacteriocin-producing Enterococcus faecium strains isolated from human milk and colostrum

  • Ufuk BagciEmail author
  • Sine Ozmen Togay
  • Ayhan Temiz
  • Mustafa Ay
Original Article


As potential probiotic traits of human milk-isolated bacteria have increasingly been recognized, this study aimed to evaluate the probiotic properties of bacteriocin-producing Enterococcus faecium strains isolated from human milk and colostrum. Among 118 human milk- and colostrum-isolated lactic cocci, only 29 were identified as Enterococcus. Of these, only four Enterococcus faecium isolates exhibited bacteriocigenic activity against several pathogenic Gram-positive bacteria, including Listeria monocytogenes. These isolates exhibited high acid (up to pH 3.0) and bile tolerance (0.5% oxgall) in simulated gastrointestinal conditions, demonstrating their ability to survive through the upper gastrointestinal tract. All of the E. faecium strains were shown to be sensitive to most of the antibiotics including vancomycin, tetracycline, rifampicin, and erythromycin, while they were resistant to kanamycin and chloramphenicol. None of the strains showed any virulence (gelE, agg2, clyA, clyB, clyM) and antibiotic resistance genes (vanA, vanB, ermB, tetM, and aac(6′)-le-aph(2″)-la). In addition, all the strains were able to assimilate cholesterol, ranging between 25.2–64.1% and they exhibited variable adherence (19–36%) to Caco-2 cells. Based on the overall results of this in vitro study, four of the E. faecium strains isolated from human milk and colostrum can be considered as promising probiotic candidates; however, further in vivo evaluations are required.


Funding information

Authors would like to thank the Hacettepe University Scientific Research Coordination Unit (Project number: 4753) for financial support to this research project.

Compliance with ethical standards

The human milk samples were collected from the volunteers at Hacettepe University Hospital. The study protocol was approved by the Committee on Ethical Practice of the Faculty of Medicine, Hacettepe University, Ankara, Turkey. Informed consent was obtained from all individual participants included in the study. This article does not contain any studies with animals performed by any of the authors.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

12223_2019_687_MOESM1_ESM.docx (704 kb)
ESM 1 (DOCX 703 kb)


  1. Abushelaibi A, Al-Mahadin S, El-Tarabily K, Shah NP, Ayyash M (2017) Characterization of potential probiotic lactic acid bacteria isolated from camel milk. LWT Food Sci Technol 79:316–325CrossRefGoogle Scholar
  2. Agerholm-Larsen L, Raben A, Haulrik N, Hansen A (2000) Effect of 8 week intake of probiotic milk products on risk factors for cardiovascular diseases. Eur J Clin Nutr 54:288–297CrossRefGoogle Scholar
  3. Albesharat R, Ehrmann MA, Korakli M, Yazaji S, Vogel RF (2011) Phenotypic and genotypic analyses of lactic acid bacteria in local fermented food, breast milk and faeces of mothers and their babies. Syst Appl Microbiol 34:148–155CrossRefGoogle Scholar
  4. Amenu D (2014) Probiotic properties of lactic acid bacteria from human milk. J Med Microbiol Diagn 3:2161–0703CrossRefGoogle Scholar
  5. Ayyash M, Abushelaibi A, Al-Mahadin S, Enan M, El-Tarabily K, Shah N (2018) In-vitro investigation into probiotic characterisation of Streptococcus and Enterococcus isolated from camel milk. LWT Food Sci Technol 87:478–487CrossRefGoogle Scholar
  6. Basson A, Flemming L, Chenia H (2008) Evaluation of adherence, hydrophobicity, aggregation, and biofilm development of Flavobacterium johnsoniae-like isolates. Microb Ecol 55:1–14CrossRefGoogle Scholar
  7. Bhardwaj A, Kaur G, Gupta H, Vij S, Malik RK (2011) Interspecies diversity, safety and probiotic potential of bacteriocinogenic Enterococcus faecium isolated from dairy food and human faeces. World J Microbiol Biotechnol 27:591–602CrossRefGoogle Scholar
  8. Boris S, Suarez J, Barbes C (1997) Characterization of the aggregation promoting factor from Lactobacillus gasseri, a vaginal isolate. J Appl Microbiol 83:413–420CrossRefGoogle Scholar
  9. Canzi E, Guglielmetti S, Mora D, Tamagnini I, Parini C (2005) Conditions affecting cell surface properties of human intestinal bifidobacteria. Antonie Van Leeuwenhoek 88:207–219CrossRefGoogle Scholar
  10. Cárdenas N, Arroyo R, Calzada J, Peirotén Á, Medina M, Rodríguez JM, Fernández L (2016) Evaluation of technological properties of Enterococcus faecium CECT 8849, a strain isolated from human milk, for the dairy industry. Appl Microbiol Biotechnol 100:7665–7677CrossRefGoogle Scholar
  11. Charteris WP, Kelly PM, Morelli L, Collins JK (1998) Antibiotic susceptibility of potentially probiotic Lactobacillus species. J Food Prot 61:1636–1643CrossRefGoogle Scholar
  12. Chou L-S, Weimer B (1999) Isolation and characterization of acid-and bile-tolerant isolates from strains of Lactobacillus acidophilus. J Dairy Sci 82:23–31CrossRefGoogle Scholar
  13. Collado MC, Meriluoto J, Salminen S (2008) Adhesion and aggregation properties of probiotic and pathogen strains. Eur Food Res Technol 226:1065–1073CrossRefGoogle Scholar
  14. Corcoran B, Stanton C, Fitzgerald G, Ross R (2005) Survival of probiotic lactobacilli in acidic environments is enhanced in the presence of metabolizable sugars. Appl Environ Microbiol 71:3060–3067CrossRefGoogle Scholar
  15. De Ambrosini VM, Gonzalez S, de Ruiz Holgado AP, Oliver G (1998) Study of the morphology of the cell walls of some strains of lactic acid bacteria and related species. J Food Prot 61:557–562CrossRefGoogle Scholar
  16. Deraz SF, Shehata MG, El-Banna AA, Khalil AA, El-Sahn MA (2013) A complementary “in vitro”study of bacteriocinogenic activity and probiotic characteristics of newly isolated Enterococcus faecium SFD. J Pure Appl Microbio 7:2673–2689Google Scholar
  17. Di Cesare A, Vignaroli C, Luna GM, Pasquaroli S, Biavasco F (2012) Antibiotic-resistant enterococci in seawater and sediments from a coastal fish farm. Microb Drug Resist 18:502–509CrossRefGoogle Scholar
  18. Di Cesare A, Luna GM, Vignaroli C, Pasquaroli S, Tota S, Paroncini P, Biavasco F (2013) Aquaculture can promote the presence and spread of antibiotic-resistant enterococci in marine sediments. PLoS One 8:e62838CrossRefGoogle Scholar
  19. Dressman JB, Berardi RR, Dermentzoglou LC, Russell TL, Schmaltz SP, Barnett JL, Jarvenpaa KM (1990) Upper gastrointestinal (GI) pH in young, healthy men and women. Pharm Res 7(7):756–761CrossRefGoogle Scholar
  20. 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. J Clin Microbiol 33:24–27Google Scholar
  21. Ebringer L, Ferenčík M, Krajčovič J (2008) Beneficial health effects of milk and fermented dairy products. Folia Microbiol 53:378–394CrossRefGoogle Scholar
  22. Edwards U, Rogall T, Blöcker H, Emde M, Böttger EC (1989) Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. Nucleic Acids Res 17:7843–7853CrossRefGoogle Scholar
  23. Fernández L, Langa S, Martín V, Maldonado A, Jiménez E, Martín R, Rodríguez JM (2013) The human milk microbiota: origin and potential roles in health and disease. Pharmacol Res 69:1–10CrossRefGoogle Scholar
  24. Fernández M, Hudson JA, Korpela R, de los Reyes-Gavilán CG (2015) Impact on human health of microorganisms present in fermented dairy products: an overview. Biomed Res Int 2015:1–13Google Scholar
  25. Foulquié Moreno M, 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
  26. Franz CM, Specht I, Haberer P, Holzapfel WH (2001) Bile salt hydrolase activity of enterococci isolated from food: screening and quantitative determination. J Food Prot 64:725–729CrossRefGoogle Scholar
  27. Franz CM, Huch M, Abriouel H, Holzapfel W, Gálvez A (2011) Enterococci as probiotics and their implications in food safety. Int J Food Microbiol 151:125–140CrossRefGoogle Scholar
  28. Giraffa G (2003) Functionality of enterococci in dairy products. Int J Food Microbiol 88:215–222CrossRefGoogle Scholar
  29. Goldin BR, Gorbach SL, Saxelin M, Barakat S, Gualtieri L, Salminen S (1992) Survival of Lactobacillus species (strain GG) in human gastrointestinal tract. Dig Dis Sci 37(1):121–128CrossRefGoogle Scholar
  30. Guo L, Li T, Tang Y, Yang L, Huo G (2015) Probiotic properties of Enterococcus strains isolated from traditional naturally fermented cream in China. Microb Biotechnol 9(6):737–745CrossRefGoogle Scholar
  31. Heikkilä M, Saris P (2003) Inhibition of Staphylococcus aureus by the commensal bacteria of human milk. J Appl Microbiol 95:471–478CrossRefGoogle Scholar
  32. Herranz C, Casaus P, Mukhopadhyay S, Martínez JM, Rodríguez JM, Nes IF, Hernández PE, Cintas LM (2001) Enterococcus faecium P21: a strain occurring naturally in dry-fermented sausages producing the class II bacteriocins enterocin a and enterocin B. Food Microbiol 18:115–131CrossRefGoogle Scholar
  33. Iranmanesh M, Ezzatpanah H, Mojgani N (2014) Antibacterial activity and cholesterol assimilation of lactic acid bacteria isolated from traditional Iranian dairy products. LWT Food Sci Technol 58:355–359CrossRefGoogle Scholar
  34. Ishimwe N, Daliri EB, Lee BH, Fang F, Du G (2015) The perspective on cholesterol-lowering mechanisms of probiotics. Mol Nutr Food Res 59:94–105CrossRefGoogle Scholar
  35. Jiménez E, Delgado S, Fernández L, García N, Albújar M, Gómez A, Rodríguez JM (2008) Assessment of the bacterial diversity of human colostrum and screening of staphylococcal and enterococcal populations for potential virulence factors. Res Microbiol 159:595–601CrossRefGoogle Scholar
  36. Jiménez E, Ladero V, Chico I, Maldonado-Barragán A, López M, Martín V, Fernández L, Fernández M, Álvarez MA, Torres C, Rodríguez JM (2013) Antibiotic resistance, virulence determinants and production of biogenic amines among enterococci from ovine, feline, canine, porcine and human milk. BMC Microbiol 13:288CrossRefGoogle Scholar
  37. Jost T, Lacroix C, Braegger C, Chassard C (2013) Assessment of bacterial diversity in breast milk using culture-dependent and culture-independent approaches. Br J Nutr 110:1253–1262CrossRefGoogle Scholar
  38. Jost T, Lacroix C, Braegger CP, Rochat F, Chassard C (2014) Vertical mother–neonate transfer of maternal gut bacteria via breastfeeding. Environ Microbiol 16:2891–2904CrossRefGoogle Scholar
  39. Kumar M, Tiwari SK, Srivastava S (2010) Purification and characterization of enterocin LR/6, a bacteriocin from Enterococcus faecium LR/6. Appl Biochem Biotechnol 160:40–49CrossRefGoogle Scholar
  40. Lei M, Dai X, Liu M (2015) Biological characteristics and safety examination of five enterococcal strains from probiotic products. J Food Saf 35:324–335CrossRefGoogle Scholar
  41. Lund B, Edlund C (2003) Bloodstream isolates of Enterococcus faecium enriched with the enterococcal surface protein gene, esp, show increased adhesion to eukaryotic cells. J Clin Microbiol 41:5183–5185CrossRefGoogle Scholar
  42. Lye H-S, Rahmat-Ali GR, Liong M-T (2010) Mechanisms of cholesterol removal by lactobacilli under conditions that mimic the human gastrointestinal tract. Int Dairy J 20:169–175CrossRefGoogle Scholar
  43. Mainville I, Arcand Y, Farnworth, ER (2005) A dynamic model that simulates the human upper gastrointestinal tract for the study of probiotics. Int J Food Microbiol 99(3):287–296CrossRefGoogle Scholar
  44. Maldonado-Barragán A, Caballero-Guerrero B, Jiménez E, Jiménez-Díaz R, Ruiz-Barba JL, Rodríguez JM (2009) Enterocin C, a class IIb bacteriocin produced by E. faecalis C901, a strain isolated from human colostrum. Int J Food Microbiol 133:105–112CrossRefGoogle Scholar
  45. Manero A, Blanch AR (2002) Identification of Enterococcus spp. based on specific hybridisation with 16S rDNA probes. J Microbiol Methods 50:115–121CrossRefGoogle Scholar
  46. Martín R, Langa S, Reviriego C, Jimínez E, Marín ML, Xaus J, Fernández L, Rodríguez JM (2003) Human milk is a source of lactic acid bacteria for the infant gut. J Pediatr 143:754–758CrossRefGoogle Scholar
  47. Martín V, Maldonado-Barragán A, Moles L, Rodriguez-Baños M, Campo R, Fernández L, Rodríguez JM, Jiménez E (2012) Sharing of bacterial strains between breast milk and infant feces. J Hum Lact 28:36–44CrossRefGoogle Scholar
  48. Mathers CD, Loncar D (2006) Projections of global mortality and burden of disease from 2002 to 2030. PLoS Med 3:e442CrossRefGoogle Scholar
  49. Morrissey JH (1981) Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem 117:307–310CrossRefGoogle Scholar
  50. Muyzer G, Teske A, Wirsen CO, Jannasch HW (1995) Phylogenetic relationships of Thiomicrospira species and their identification in deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresis of 16S rDNA fragments. Arch Microbiol 164:165–172CrossRefGoogle Scholar
  51. Naidu AS, Bidlack WR, Clemens RA (1999) Probiotic spectra of lactic acid bacteria (LAB). Crit Rev Food Sci Nutr 39(1):13–126CrossRefGoogle Scholar
  52. Ouwehand AC, Forssten S, Hibberd AA, Lyra A, Stahl B (2016) Probiotic approach to prevent antibiotic resistance. Ann Med 48:246–255CrossRefGoogle Scholar
  53. Özmen Toğay S, Celebi Keskin A, Açık L, Temiz A (2010) Virulence genes, antibiotic resistance and plasmid profiles of Enterococcus faecalis and Enterococcus faecium from naturally fermented Turkish foods. J Appl Microbiol 109:1084–1092CrossRefGoogle Scholar
  54. Pantev A, Kabadjova P, Dalgalarrondo M, Haertlé T, Ivanova I, Dousset X, Prévost H, Chobert JM (2002) Isolation and partial characterization of an antibacterial substance produced by Enterococcus faecium. Folia Microbiol 47:391–400CrossRefGoogle Scholar
  55. Pasquaroli S, Di Cesare A, Vignaroli C, Conti G, Citterio B, Biavasco F (2014) Erythromycin-and copper-resistant Enterococcus hirae from marine sediment and co-transfer of erm (B) and tcrB to human Enterococcus faecalis. Diagn Microbiol Infect Dis 80:26–28CrossRefGoogle Scholar
  56. Pereira DI, Gibson GR (2002) Cholesterol assimilation by lactic acid bacteria and bifidobacteria isolated from the human gut. Appl Environ Microbiol 68:4689–4693CrossRefGoogle Scholar
  57. Pérez-Sánchez T, Balcázar JL, García Y, Halaihel N, Vendrell D, de Blas I, Merrifield DL, Ruiz-Zarzuela I (2011) Identification and characterization of lactic acid bacteria isolated from rainbow trout, Oncorhynchus mykiss (Walbaum), with inhibitory activity against Lactococcus garvieae. J Fish Dis 34:499–507CrossRefGoogle Scholar
  58. 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
  59. Rahman MM, Kim W-S, Kumura H, Shimazaki K-i (2008) Autoaggregation and surface hydrophobicity of bifidobacteria. World J Microbiol Biotechnol 24:1593–1598CrossRefGoogle Scholar
  60. Reis NA, Saraiva MAF, Duarte EAA, Carvalho E, Vieira BB, Evangelista-Barreto NS (2016) Probiotic properties of lactic acid bacteria isolated from human milk. J Appl Microbiol 121:811–820CrossRefGoogle Scholar
  61. Ren D, Li C, Qin Y, Yin R, du S, Ye F, Liu C, Liu H, Wang M, Li Y, Sun Y, Li X, Tian M, Jin N (2014) In vitro evaluation of the probiotic and functional potential of Lactobacillus strains isolated from fermented food and human intestine. Anaerobe 30:1–10CrossRefGoogle Scholar
  62. Reviriego C, Eaton T, Martín R, Jiménez E, Fernández L, Gasson MJ, Rodríguez JM (2005) Screening of virulence determinants in Enterococcus faecium strains isolated from breast milk. J Hum Lact 21:131–137CrossRefGoogle Scholar
  63. Rivas FP, Castro MP, Vallejo M, Marguet E, Campos CA (2012) Antibacterial potential of Enterococcus faecium strains isolated from ewes’ milk and cheese. LWT Food Sci Technol 46:428–436CrossRefGoogle Scholar
  64. Rossi EA, Vendramini RC, Carlos IZ, Pei YC, de Valdez GF (1999) Development of a novel fermented soymilk product with potential probiotic properties. Eur Food Res Technol 209:305–307CrossRefGoogle Scholar
  65. Saelim K, Sohsomboon N, Kaewsuwan S, Maneerat S (2012) Probiotic properties of Enterococcus faecium CE5-1 producing a bacteriocin-like substance and its antagonistic effect against antibiotic-resistant enterococci in vitro. Czech J Anim Sci 57:529–539CrossRefGoogle Scholar
  66. Semedo T, Santos MA, Martins P, Lopes MFS, Marques JJF, Tenreiro R, Crespo MTB (2003) Comparative study using type strains and clinical and food isolates to examine hemolytic activity and occurrence of the cyl operon in enterococci. J Clin Microbiol 41:2569–2576CrossRefGoogle Scholar
  67. Shagger H, von Jagow G (1987) Tricine-SDS-PAGE for the separation of proteins in the 1–100 kDa range. Anal Biochem 168:368–379CrossRefGoogle Scholar
  68. Shehata AA, Tarabees R, Basiouni S, Gamil M, Kamal AS, Krüger M (2017) Phenotypic and genotypic characterization of bacteriocinogenic Enterococci against Clostridium botulinum. Probiotics Antimicrob Proteins 9:182–188CrossRefGoogle Scholar
  69. Sica MG, Brugnoni LI, Marucci PL, Cubitto MA (2012) Characterization of probiotic properties of lactic acid bacteria isolated from an estuarine environment for application in rainbow trout (Oncorhynchus mykiss, Walbaum) farming. Antonie Van Leeuwenhoek 101(4):869–879CrossRefGoogle Scholar
  70. Strompfová V, Lauková A (2007) In vitro study on bacteriocin production of enterococci associated with chickens. Anaerobe 13:228–237CrossRefGoogle Scholar
  71. Strompfova V, Laukova A (2009) Enterococci from piglets—probiotic properties and responsiveness to natural antibacterial substances. Folia Microbiol 54:538–544CrossRefGoogle Scholar
  72. Šušković J, Kos B, Matošić S, Besendorfer V (2000) The effect of bile salts on survival and morphology of a potential probiotic strain Lactobacillus acidophilus M92. World J Microbiol Biotechnol 16:673–678CrossRefGoogle Scholar
  73. Taheri P, Samadi N, Ehsani MR, Khoshayand MR, Jamalifar H (2012) An evaluation and partial characterization of a bacteriocin produced by Lactococcus lactis subsp lactis ST1 isolated from goat milk. Braz J Microbiol 43:1452–1462CrossRefGoogle Scholar
  74. Todorov SD, Dicks LM (2004) Characterization of mesentericin ST99, a bacteriocin produced by Leuconostoc mesenteroides subsp. dextranicum ST99 isolated from boza. J Ind Microbiol Biotechnol 31:323–329CrossRefGoogle Scholar
  75. Todorov S et al (2008) Boza, a natural source of probiotic lactic acid bacteria. J Appl Microbiol 104:465–477Google Scholar
  76. Toğay SÖ, Temiz A, Celebi A, Acik L, Yalçin SS (2014) Investigation of potential virulence genes and antibiotic resistance characteristics of Enterococcus faecalis isolates from human milk and colostrum samples. Turk J Biol 38:357–364CrossRefGoogle Scholar
  77. Tok E, Aslim B (2010) Cholesterol removal by some lactic acid bacteria that can be used as probiotic. Microbiol Immunol 54:257–264Google Scholar
  78. Valenzuela AS, Benomar N, Abriouel H, Cañamero MM, Gálvez A (2010) Isolation and identification of Enterococcus faecium from seafoods: antimicrobial resistance and production of bacteriocin-like substances. Food Microbiol 27:955–961CrossRefGoogle Scholar
  79. Vankerckhoven V, Huys G, Vancanneyt M, Snauwaert C, Swings J, Klare I, Witte W, van Autgaerden T, Chapelle S, Lammens C, Goossens H (2008) Genotypic diversity, antimicrobial resistance, and virulence factors of human isolates and probiotic cultures constituting two intraspecific groups of Enterococcus faecium isolates. Appl Environ Microbiol 74:4247–4255CrossRefGoogle Scholar
  80. Vidhyasagar V, Jeevaratnam K (2013) Evaluation of Pediococcus pentosaceus strains isolated from idly batter for probiotic properties in vitro. J Funct Foods 5:235–243CrossRefGoogle Scholar
  81. Wardal E et al (2014) Molecular analysis of vanA outbreak of Enterococcus faecium in two Warsaw hospitals: the importance of mobile genetic elements. Biomed Res Int:2014Google Scholar
  82. Zhang F, Qiu L, Xu X, Liu Z, Zhan H, Tao X, Shah NP, Wei H (2017) Beneficial effects of probiotic cholesterol-lowering strain of Enterococcus faecium WEFA23 from infants on diet-induced metabolic syndrome in rats. J Dairy Sci 100:1618–1628CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Department of Food Engineering, Faculty of EngineeringTrakya UniversityEdirneTurkey
  2. 2.Department of Food EngineeringUludag UniversityBursaTurkey
  3. 3.Department of Food EngineeringHacettepe UniversityAnkaraTurkey
  4. 4.Department of Food TechnologyÇanakkale Onsekiz Mart UniversityÇanakkaleTurkey

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