Iranian Journal of Microbiology 2017. 9(4):224-233.

Bacteriocinogenic potential and virulence traits of Enterococcus faecium and E. faecalis isolated from human milk
Soodabeh Khalkhali, Naheed Mojgani

Abstract


Background and Objectives: Human milk is a continuous supply of Lactic Acid bacteria (LAB), including enterococci with probiotic potentials. The aim of this study was to analyze two Enterococcus species, isolated from human milk for their probiotic potential, bacteriocin producing ability and virulence traits.


Materials and Methods: Enterococcus faecium TA0033 and E. faecalis TA102 were tested for acid and bile tolerance, survival in simulated gastric and intestinal conditions. The antibacterial spectrum of the isolates was tested by agar well diffusion assay. The antagonistic agent was characterized by physico-chemical methods. The enterocin structural genes, virulence determinants, vancomycin resistance and biogenic amine genes, such as hdc1, hdc2, tdc, ldc and odc were also determined.


Results: The tested isolates survived acidic conditions, high bile salt (1%), simulated gastric and intestinal conditions. The culture supernatant fluids of the two isolates inhibited the growth of Escherichia coli, Listeria monocytogenes, Salmonella typhi, Staphylococcus aureus, Shigella dysenteriae and Streptococcus agalactiae. The antagonistic activity was lost in the presence of proteolytic enzymes but tolerated the action of catalase, lysozyme and lipase. In contrast to enterocin TA102, enterocin TA0033 possessed bactericidal mode of action. Bacteriocin structural genes, entA and entB were present in the genome of the two isolates, while E. faecalis TA102 additionally harboured entP and bac31 genes. The phenotypic and genotypic virulence assessment studies indicated hyaluronidase (hyl) production and vancomycin resistance in E. faecalis TA102 while, none of the isolates harboured the biogenic amine genes.


Conclusion: The presence of virulence genes in E. faecalis TA102 calls for careful monitoring of Enterococcus isolates for their safety parameters.


Keywords


Enterococcus, Bacteriocins, Biogenic amines, Human milk, Vancomycin, Virulence genes

Full Text:

PDF

References


Eaton TJ, Gasson MJ. Molecular screening of Enterococcus virulence determinants and potential for genetic exchange between food and medical isolates. Appl Environ Microbiol 2001; 67: 1628-1635.

Franz CM, Huch M, Abriouel H, Holzapfel WH, Galvez A. Enterococci as probiotics and their implications in food safety. Int J Food Microbiol 2011; 151: 125-140.

Kozak K, Charbonneau D, Sanozky-Dawes R, Klaehammer T. Characterization of bacterial isolates from the microbiota of mothers' breast milk and their infants. Gut Microbes 2015; 6: 341-351.

Ishibashi N, Himeno K, Fujita K, Masuda Y, Perez RH, Zendo T, et al. Purification and characterization of multiple bacteriocins and an inducing peptide produced by E. faecium NKR-5-3 from Thai fermented fish. Biosci Biotechnol Biochem 2012; 76: 947-953.

Carlos A, Semedo-Lemsaddek T, Barreto-Crespo M, Tenreiro R. Transcriptional analysis of virulence-related genes in enterococci from distinct origins. J Appl Microbiol 2010; 108: 1563-1575.

Miele A, Bandera M, Goldstien BH. Use of primers selective for vancomycin resistance genes to determine van genotype in enterococci and to study gene organization in VanA isolates. Antimicrob Agents Chemoth 1995; 39: 1772-1778.

Elsanhoty RM, Ramadan MW. Genetic screening of biogenic amines production capacity from some lactic acid bacteria strains. Food Control 2016; 68: 220-228.

Laukova A, Szaboova R, Pleva P, Bunkova L, Chrastinova L. Decarboxylase-positive Enterococcus faecium strains isolated from rabbit meat and their sensitivity to enterocins. Food Sci Nutr 2017; 5: 31-37.

Khalkhali S, Mojgani N. Characterization of candidate probionts isolated from human breast milk. Cell Mol Biol 2017; 82-88.

Mojgani N, Vaseji N, Khalkhali S, Naz Baloch M. Biochemical and molecular analysis of the antilisterial peptides produced by Enterococcus hirae strains isolated from raw ewe milk. J Adv Biol Biotech 2017;11: 1-11.

Gopal PK, Prasad J, Smart J, Gill HS. In vitro adherence properties of L. rhamnosus DR20 and Bifidobacterium lactis DR10 strains and their antagonistic activity against an enterotoxigenic Escherichia coli. Int J Food Microbiol 2001; 67:207-216.

Mojgani N, Hussaini F, Vaseji N. Characterization of indigenous Lactobacillus strains for probiotic properties. Jundishapur J Microbiol 2015; 8(2): e17523.

Schillinger U, Lücke FK. Antibacterial activity of L. sake isolated from meat. Appl Environ Microbiol 1989; 55: 1901-1906.

Aymerich T, Holo H, Håvarstein LS, Hugas M, Garriga M, Nes IF. Biochemical and genetic characterization of enterocin A from E. faecium, a new antilisterial bacteriocin in the pediocin family of bacteriocins. Appl Environ Microbiol 1996; 62: 1676-1682.

Nami Y, Haghshenas B, Haghshenas M, Khosroushahi AD, Rosli R, Khosroushahi Y. Antimicrobial activity and the presence of virulence factors and bacteriocin structural genes in E. faecium CM33 isolated from ewe colostrum. Frontiers Microbiol 2015; 6:1-10.

de Azeredo LA, Leite SG, Freire DM, Benchetrit LC, Coelho R. Proteases from actinomycetes interfere in solid media plate assays of hyaluronidase activity. J Microbiol Methods 2001; 45: 207-212.

Bover-Cid S, Holzapfel WH. Improved screening procedure for biogenic amine production by lactic acid bacteria. Int J Food Microbiol 1999; 53: 33-41.

Bauer A, Kirby WM, Sheris JKT. Antimicrobial susceptibility testing by a standard single disc method. Am J Clin Pathol 1996; 45: 493-496.

Vankerckhoven V, Autgaerden T, Vael C, Lammens C, Chapelle S, Rossi R, Jabes D, Goossens H. Development of a multiplex PCR for the detection of asa1, gelE, cylA, esp, and hyl genes in enterococci and survey for virulence determinants among European hospital isolates of E. faecium. J Clin Microbiol 2004; 42: 4473-4479.

Reviriego C, Eaton T, Martín R, Jiménez E, Fernández L, Gasson MJ, Rodríguez JM. Screening of virulence determinants in E. faecium strains isolated from breast milk. J Hum Lact 2005; 21: 131-138

Semedo MA, Santos P, Martins MFS, Lopes JJ, Figueiredo Marques R, Tenreiro MT, et al. 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 2003; 41: 2569-2576.

Satake S, Clark N, Rimland D, Nolte FS, Tenover FC. Detection of vancomycin-resistant Enterococci in fecal samples by PCR. J Clin Microbiol 1997; 35:2325-2330.

Landete JM, de Las Rivas B, Marcobal A, Muñoz R. Molecular methods for the detection of biogenic amine-producing bacteria on foods. Int J Food Microbiol 2007; 117(3):258-269.

Khalkhali S, Mojgani N. Enterococcus faecium; a suitable probiotic candidate for modulation of immune responses against pathogens. Int J Basic Sci Med 2017;2:77-82.

Ghrairi T, Frere J, Berjeaud JM, Manai M. Purification and characterization of bacteriocins produced by E. faecium from Tunisian Rigouta cheese. Food Control 2008; 19:162-169.

Borzenkov V, Surovtsev V, Dyatlov I. Obtaining bacteriocins by chromatographic methods. Adv Biosci Biotechnol 2014; 5: 446-451.

Chen YS, Yanagida F, Srionnual S. Characteristics of bacteriocin-like inhibitory substances from dochi-isolated E. faecium D081821 and D081833. Lett Appl Microbiol 2007; 44: 320-325.

Zendo T, Eungruttanagorn N, Fujioka S, Tashiro Y, Nomura K, et al. Identification and production of a bacteriocin from E. mundtii QU 2 isolated from soybean. J Appl Microbiol 2005; 99: 1181-1190.

Montel Mendoza G, Ale CE, Nader-Macías MEF, Pasteris SE. Characterization of a bacteriocin produced by E. gallinarum CRL 1826 isolated from captive bullfrog: evaluation of its mode of action against L. monocytogenes and Gram-Negatives. J Bioprocess Biotechnol 2015; 5: 250-256.

Casaus P, Nilsen T, Cintas L, Nes IF, Hernández PE, Holo H. Enterocin B, a new bacteriocin from E. faecium T136 which can act synergistically with enterocin A. Microbiol 1997; 143: 2287-2294.

Trivedi K, Radmila S, Renata K. Bacteriocin activity of enterococci and presence of genes related to pathogenesis. Chez J food Scis 2012; 30: 330-335.

Garbutt JM, Ventrapragada M, Littenberg B, Mundy LM. Association between resistance to vancomycin and death in cases of E. faecium bacteremia. Clin Infect Dis 2000; 30: 466-472.

Ogier JC, Serror P. Safety assessment of dairy microorganisms: the Enterococcus genus. Int J Food Microbiol 2008; 126: 291-301.

Linares DM, del Rio B, Ladero V, Martinez N, Fernandez M, Martin MC. Factors influencing biogenic amines accumulation in dairy products. Frontiers Microbiol 2012; 3: 180.


Refbacks

  • There are currently no refbacks.


Creative Commons Attribution-NonCommercial 3.0

This work is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly.