Abstract
Enterococci represent a significant part of microbiota in home-made dairy products due to their remarkable intrinsic resistance to critical growth conditions and technological properties. In this study, the predominant coccal lactic acid bacteria (LAB) were isolated from traditional dairy products made and largely consumed in villages of Chahar-Mahalo Bakhtiyari province of Iran. Thirty-six samples of home-made dairy products were collected from scattered households, and a total of 61 gram-positive and catalase-negative cocci were analyzed. Genetic fingerprinting (repetitive extragenic palindromic PCR (REP-PCR) and 16S–23S intergenic spacer (ITS) amplification) and gene sequencing (16S rRNA gene and pheS gene) were carried out to obtain an unambiguous identification of the isolates at the species level and to highlight genetic biodiversity among wild enterococci. It was shown that 42 out of 61 isolates belonged to Enterococcus spp., and Enterococcus faecium was the most frequently isolated species. Intraspecific diversity of the E. faecium species was further investigated, targeting the genes hyl, esp, and msrC, related to virulence and antibiotic resistance, and the results obtained suggest that two strains could be further characterized for possible use as starter cultures. In conclusion, the present study confirms that application of diverse independent molecular techniques is required to clearly identify Enterococcus spp. isolates and to unveil wild strain biodiversity. Moreover, a preliminary molecular screening of selected genetic features represents a fast and feasible approach to select, among a wide collection of isolates, the most promising candidates for further exhaustive characterization of safety and technological traits.
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Ahmadova A, Dimov S, Ivanova I, Choiset Y, Chobert J-M, Kuliev A, Haertlé T (2011) Proteolytic activities and safety of use of Enterococci strains isolated from traditional Azerbaijani dairy products. Eur Food Res Technol 233:131–140
Baker G, Smith J, Cowan DA (2003) Review and re-analysis of domain-specific 16S primers. J Microbiol Methods 55:541–555
Carver TJ, Rutherford KM, Berriman M, Rajandream M-A, Barrell BG, Parkhill J (2005) ACT: the Artemis comparison tool. Bioinformatics 21:3422–3423
Devriese L, Vancanneyt M, Descheemaeker P, Baele M, Van Landuyt H, Gordts B, Butaye P, Swings J, Haesebrouck F (2002) Differentiation and identification of enterococcus durans E. hirae and E. villorum. J App Microbiol 92:821–827
Dinleyici EC, Dalgic N, Guven S, Ozen M, Kara A, Arica V, Metin-Timur O, Sancar M, Kurugol Z, Tanir G (2013) The effect of a multispecies synbiotic mixture on the duration of diarrhea and length of hospital stay in children with acute diarrhea in Turkey: single blinded randomized study. Eur J Pediatr 172:459–464
Drahovská H, Kocíncová D, Seman M, Turna J (2002) PCR-based methods for identification of Enterococcus species. Folia Microbiol 47:649–653
Eaton TJ, Gasson MJ (2002) A variant enterococcal surface protein Espfm in Enterococcus faecium; distribution among food, commensal, medical, and environmental isolates. FEMS Microbiol Lett 216:269–275
Edalatian MR, Najafi MBH, Mortazavi SA, Alegría Á, Nassiri MR, Bassami MR, Mayo B (2012) Microbial diversity of the traditional Iranian cheeses Lighvan and Koozeh, as revealed by polyphasic culturing and culture-independent approaches. Dairy Sci Tech 92:75–90
EFSA (2012) Guidance on the assessment of Enterococcus faecium in animal nutrition. EFSA J 10:2682
Giraffa G, Carminati D, Neviani E (1997) Enterococci isolated from dairy products: a review of risks and potential technological use. J Food Protect 60:732–737
Hadji-Sfaxi I, El-Ghaish S, Ahmadova A, Batdorj B, Le Blay-Laliberté G, Barbier G, Haertlé T, Chobert J-M (2011) Antimicrobial activity and safety of use of Enterococcus faecium PC4. 1 isolated from Mongol yogurt. Food Control 22:2020–2027
Jackson CR, Fedorka-Cray PJ, Barrett JB (2004) Use of a genus- and species-specific multiplex PCR for identification of enterococci. J Clin Microbiol 42:3558–3565
Jamet E, Akary E, Poisson M-A, Chamba J-F, Bertrand X, Serror P (2012) Prevalence and characterization of antibiotic resistant Enterococcus faecalis in French cheeses. Food Microbiol 31:191–198
Jensen MA, Webster JA, Straus N (1993) Rapid identification of bacteria on the basis of polymerase chain reaction-amplified ribosomal DNA spacer polymorphisms. Appl Environ Microbiol 59:945–952
Jurkovič D, Križková L, Sojka M, Takáčová M, Dušinský R, Krajčovič J, Vandamme P, Vancanneyt M (2007) Genetic diversity of Enterococcus faecium isolated from Bryndza cheese. Int J Food Microbiol 116:82–87
Kim O-S, Cho Y-J, Lee K, Yoon S-H, Kim M, Na H, Park S-C, Jeon YS, Lee J-H, Yi H (2012) Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62:716–721
Martín-Platero AM, Valdivia E, Maqueda M, Martínez-Bueno M (2009) Characterization and safety evaluation of enterococci isolated from spanish goats’ milk cheeses. Int J Food Microbiol 132:24–32
Mourad K, Nour-Eddine K (2006) Physicochemical and microbiological study of “shmen”, a traditional butter made from camel milk in the Sahara (Algeria): isolation and identification of lactic acid bacteria and yeasts. Grasas y Aceites 57:198–204
Naser SM, Thompson FL, Hoste B, Gevers D, Dawyndt P, Vancanneyt M, Swings J (2005) Application of multilocus sequence analysis (MLSA) for rapid identification of Enterococcus species based on rpoA and pheS genes. Microbiology 151:2141–2150
Ogier J-C, Serror P (2008) Safety assessment of dairy microorganisms: the Enterococcus genus. Int J Food Microbiol 126:291–301
Oguntoyinbo FA, Okueso O (2013) Prevalence, distribution and antibiotic resistance pattern among enterococci species in two traditional fermented dairy foods. Ann Microbiol 63:1–7
Ortigosa M, Irigoyen A, Urdin M, García S, Ibanez F, Torre P (2008) Sources of enterococci in Idiazábal-type cheese. Int J Food Microbiol 125:146–152
Portillo A, Ruiz-Larrea F, Zarazaga M, Alonso A, Martinez JL, Torres C (2000) Macrolide resistance genes in Enterococcus spp. Antimicrob Agents Chemother 44:967–971
Saavedra L, MaPa T, Sesma F, de Valdez GF (2003) Homemade traditional cheeses for the isolation of probiotic Enterococcus faecium strains. Int J Food Microbiol 88:241–245
Snauwaert C, Naser S, Vandamme P, Vancanneyt M (2006) Molecular identification and diversity of enterococci isolated from Slovak Bryndza cheese. J Gen Appl Microbiol 52:329–337
Sun Z, Liu W, Gao W, Yang M, Zhang J, Wu L, Wang J, Menghe B, Sun T, Zhang H (2010) Identification and characterization of the dominant lactic acid bacteria from kurut: the naturally fermented yak milk in Qinghai, China. J Gen App Microbiol 56:1–10
Švec P, Vancanneyt M, Seman M, Snauwaert C, Lefebvre K, Sedláček I, Swings J (2005) Evaluation of (GTG)5-PCR for identification of Enterococcus spp. FEMS Microbiol Lett 247:59–63
Tabatabaee F, Alizadeh Behbahani B, Mohebbi M, Mortazavi SA, Ghaitaranpour A (2012) Identification of lactic acid bacteria isolated from Tarkhineh, a traditional Iranian fermented food. Sci J Microbiol 1:152–159
Tyrrell GJ, Bethune RN, Willey B, Low DE (1997) Species identification of enterococci via intergenic ribosomal PCR. J Clin Microbiol 35:1054–1060
van Schaik W, Top J, Riley DR, Boekhorst J, Vrijenhoek JE, Schapendonk CM, Hendrickx AP, Nijman IJ, Bonten MJ, Tettelin H (2010) Pyrosequencing-based comparative genome analysis of the nosocomial pathogen Enterococcus faecium and identification of a large transferable pathogenicity island. BMC Genomics 11:239–257
Vancanneyt M, Lombardi A, Andrighetto C, Knijff E, Torriani S, Björkroth KJ, Franz CM, Moreno MRF, Revets H, De Vuyst L (2002) Intraspecies genomic groups in Enterococcus faecium and their correlation with origin and pathogenicity. Appl Environ Microbiol 68:1381–1391
Vankerckhoven V, Van Autgaerden T, Vael C, Lammens C, Chapelle S, Rossi R, Jabes D, Goossens H (2004) 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 Enterococcus faecium. J Clin Microbiol 42:4473–4479
Verdier-Metz I, Michel V, Delbes C, Montel M-C (2009) Do milking practices influence the bacterial diversity of raw milk? Food Microbiol 26:305–310
Versalovic J, Schneider M, De Bruijn F, Lupski JR (1994) Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Method Mol Cell Biol 5:25–40
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Fatemeh Nejati, Veronica Gatto, Anna Castioni, Marta Tebaldi, Mehdi babae, Fabio Fracchetti, Giovanna E. Felis declare that they have no conflict of interest.
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Nejati, F., Gatto, V., Castioni, A. et al. Genetic diversity of enterococci from Iranian home-made artisanal dairy products. Dairy Sci. & Technol. 95, 151–165 (2015). https://doi.org/10.1007/s13594-014-0193-2
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DOI: https://doi.org/10.1007/s13594-014-0193-2