In this study, prevalence, biotechnological and safety profiles of 588 Enterococcus isolates isolated from raw milk and Istrian cheese during different stages of ripening were analyzed. Despite the low and variable presence of enterococci in milk ((3.65±2.93) log CFU/mL), highly comparable enterococcal populations were established after 30 days of cheese ripening ((7.96±0.80) log CFU/g), confirming Enterococcus spp. as a major part of the core microbiota of Istrian cheese. The dominant species were E. faecium (53.8%) and E. faecalis (42.4%), while minor groups, consisting of E. durans (2.84%) and E. casseliflavus (0.95%), also occurred. A pronounced intraspecies variability was noticed based on molecular fingerprinting, with 35 strains (genotypes) detected. Most of the genotypes were farm-specific with one third being shared between the farms. This genotype variability reflected particular differences of Istrian cheese production, mainly variable salt concentration, ripening temperature and air humidity as well as microclimatic or vegetation conditions. There was considerable variation between the strains of the same species regarding wide range of biotechnologically important traits as well as their ability to survive in simulated gastrointestinal conditions. A considerable number of strains were resistant to critically important antibiotics such as tetracycline (43.56%), erythromycin (35.79%) and vancomycin (23.48%). Polymerase chain reaction-based detection did not identify any of the common genetic determinants for vancomycin and erythromycin resistance; for tetracycline tetM gene was detected. The presence of virulence genes including agg, efaAfs, gelE, cylM, cylB, cylA, esp, efaAfm, cob and cpd was frequently recorded, especially among E. faecalis strains.
Keywords: Enterococcus spp.; antibiotic resistance; cheese; molecular fingerprinting; spontaneous fermentation; virulence genes.