References 1. Giraffa G. 2002. Enterococci from foods. FEMS Microbiology Reviews, 744, 1-9. 2. Schirone M., Tofalo R., Visciano P., Corsetti A., Suzzi G. 2012. Biogenic amines in Italian Pecorino cheese. Food Microbiology, 3, 1-8. 3. Di Giacomo F., Casolani N, Del Signore A. 2014. Cheese making using pig rennet and calf rennet: microorganisms and volatile compounds in Farindola ewe cheese. Italian Journal of Food Science, 26, 153-162. 4. Schirone M., Tofalo R., Mazzone G., Corsetti A., Suzzi, G. 2011. Biogenic amine content and microbiological profile of Pecorino di Farindola cheese. Food Microbiology, 28, 128-136. 5. Izquierdo E., Marchioni E., Aoude-Werner D., Hasselmann C., Ennaha S. 2009. Smearing of soft cheese with Enterococcus faecium WHE 81, a multi-bacteriocin producer, against Listeria monocytogenes. Food Microbiology, 26 (1), 16-20. 6. Giraffa G. 2003. Functionality of enterococci in dairy products. International Journal of Food Microbiology, 88, 215-222. 7. NMKL (Nordic committee on food analysis). 2004. Enterococcus. Determination in foods and feeds No. 68. 4 th ed. Nordic Committee on Food Analysis, Oslo, Norway. 8. Kariyama R., Mitsuhata R., Chow J.W., Clewell D.B., Kumon H. 2000. Simple and reliable multiplex PCR assay for surveillance isolates of vancomycin-resistant enterococci. Journal of Clinical Microbiology, 38 (8), 3092-3095. 9. Rossetti L., Giraffa G. 2005. Rapid Identification of dairy lactic acid bacteria by M13-generated, RAPD-PCR fingerprinting databases. Journal of Microbiological methods, 63, 135-144. 10. De Garnica M.L., Valdezate S., Gonzalo C., Saez-Nieto J.A. 2013. Presence of the vanC1 gene in a vancomycin-resistant Enterococcus faecalis strain isolated from ewe bulk tank milk. Journal of Medical Microbiology, 62, 494-495. Results The concentration of Enterococci population during Pecorino di Farindola production and ripening is reported in Figure 1. The values are expressed in log 10 CFU/g or ml of sample. The value reported in Figure 1 at the sampling time 0 is the average value obtained by Enterococci enumeration on three milk samples and three curd samples. The other values are determined, for each ripening time, by average value of two cheese samples analyzed. The average values of plate counts were about 10 3 CFU/ml or g in raw milk and curd. The presence of Enterococci in Pecorino di Farindola ranged from 10 4 to 10 5 CFU/g at the beginning of ripening and reached 10 7 CFU/g in ripened cheeses (Table 1). Among Enterococcus populations, the following species were identified at different rates: Enterococcus faecalis (72.2%), Enterococcus faecium (13.3%), Enterococcus casseliflavus/gallinarum (11.1%) and Enterococcus hirae (3.4%) (Figure 2). Enterococcus species distribution during production and ripening is descripted in Figure 3. Multiplex PCR was performed to detect vancomycin-resistance genes (vanA, vanB, vanC1, and vanC2/C3). None of the isolates carried vanA and vanB genes. All E. gallinarum (nine isolates) showed vanC1 intrinsic resistance genotype; the only one E. casseliflavus isolated showed vanC2/C3 intrinsic resistance genotype. Conclusions In this work we reported high levels of Enterococci in Pecorino di Farindola cheese and this bacterial group can be considered among the most represented in ripened cheese. Since some Enterococcus strains have natural and acquired antibiotic resistance and efficient mechanisms for genetic material transfer we investigated the presence of vancomycin resistance genes vanA, vanB, vanC1, and vanC2/C3 (10). In this study all the isolates belonging to the species E. faecalis (72.2%), E. faecium (13.3%) and E. hirae (3.4%) showed no vancomycin-resistance genes. Remaining isolates (11.1%), belonging to the species E. gallinarum and E. casseliflavus, carried vanC resistance genes. The vanC intrinsic resistance genotype is associated with several enterococcal species: E. gallinarum (vanC1), E. casseliflavus (vanC2) and E. flavescens (vanC3) (10). The chromosomal location of vanC genes makes them presumably non-transferable, conferring an intermediate resistance level to vancomycin and sensitivity to teicoplanin. Our results confirm the intrinsic resistance to vancomycin of E. gallinarum and E. casseliflavus species, but further studies are necessary to evaluate resistance to other antimicrobial agents to exclude the possibility of horizontal transfer of resistance genes among Enterococci and other bacteria. Enterococci could also represent threat for human health due to expression of pathogenic factors, for example: aggregation factor, gelatinase, cytolysin, enterococcal surface protein. Moreover, as Enterococci may have different roles during cheese ripening, like production of volatile compounds, bacteriocins and biogenic amines, our results suggest the need to further characterize enterococcal population in Pecorino di Farindola cheese. Introduction Enterococci are Gram-positive bacteria that usually inhabit gastrointestinal tracts of humans and animals; they are also widely distributed in a variety of environmental habitats, even when there is little or absent human and/or animal fecal contamination. Enterococci can be present in many types of food, such as vegetables, raw meat and cheese (1). Enterococci represent typical microbiota of ewe’s milk and consequently of sheep cheese; generally during ripening their numbers increase, while the level of lactococci decreases, so enterococcal population may become the most important microbial group in ripened sheep cheese (2). Enterococci can have an important role in maturation of sheep cheese during ripening and, producing volatile compounds, they can greatly contribute to flavor (3). The persistence and dominance of Enterococci during ripening has been attributed to their wide range of growth temperatures and their high tolerance to heat and salt (4). High levels of Enterococci in fermented food had been associated with biogenic amines formation. Furthermore Enterococci in fermented foods can produce bacteriocins, inhibiting multiplication of other pathogenic bacteria (such as Listeria monocytogenes) (5). Enterococci are typical opportunistic pathogens and can cause nosocomial infections such as gastroenteritis, endocarditis, bacteremia and urinary tract infections (6). Moreover some Enterococcus strains have natural and acquired antibiotic resistance and efficient mechanisms for genetic material transfer (1). The aim of this study was to characterize Enterococci population of Pecorino di Farindola, a traditional cheese of the eastern part of the Gran Sasso area, in the Abruzzo region, manufactured using raw ewes’ milk and pig rennet. In this work Enterococcus species have been identified throughout production and ripening process of Pecorino di Farindola and, in isolates, the presence of vancomycin genes encoding for the genotypes vanA, vanB and vanC has been investigated. Materials and methods Samples of raw milk, curd and Pecorino di Farindola cheese at different ripening stages (7, 14, 21, 35, 49, 63, 91, 121, 150 days) were analyzed. Enterococci enumeration and isolation were performed according to n°68 Nordic Committee on Food Analysis (2004): Enterococcus - Determination in foods and feeds (7). A total of 90 isolates of Enterococci were collected and subjected to biochemical identification by Vitek 2® bioMérieux system, an automated identification system that perform 43 biochemical tests for carbon source utilization, enzyme activities and antimicrobial resistance. To identify four clinically relevant species of Enterococci (E. faecalis, E. faecium, E. gallinarum, and E. casseliflavus) and to detect four glycopeptide resistance genotypes (vanA, vanB, vanC1 and vanC2/C3) a multiplex PCR using Qiagen Multiplex PCR kit was performed according to R. Kariyama et al. 2000 (8). PCR products were analyzed by capillary electrophoresis with QIAxcel Screen Gel software. Unidentified species isolates were subjected to sequencing of hypervariable region (first 500 bp) in the 5V end of the 16S rRNA gene (9). The sequence data were submitted to BLAST® (www.ncbi.nlm.nih.gov/BLAST) to identify Enterococci isolates. LXIX Convegno Perugia, 15-17 giugno 2015 Visciano P. 1 , Tofalo R. 1 , Santarelli G.A. 2 , Priore M. 2 , Alessiani A. 2 , Sacchini L. 2 , Di Marzio V. 2 , Schirone M. 1* , Suzzi G. 1 1 Faculty of BioScience and Technology for Food, Agriculture and Environment, University of Teramo, Teramo, Italy. 2 Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise ‘G. Caporale’, Teramo, Italy. * e-mail: mschirone@unite.it Identification, characterization and vancomycin-resistance of Enterococci in Pecorino di Farindola cheese Production/ ripening steps Log 10 CFU/g or ml Milk 2.62 Curd 2.70 7 5.17 14 5.43 21 4.08 35 4.12 49 4.93 63 4.84 91 5.19 121 7.45 150 7.22 Table 1. Enterococci enumeration data. Figure 2. Enterococci populations. 0 1 2 3 4 5 6 7 8 0 30 60 90 120 150 Log 10 CFU/g or ml Production/ ripening steps Figure 1. Concentration of Enterococci at different ripening stages in Log 10 CFU /ml or /g. Figure 3. Enterococci distribution during production and ripening. Enterococcus faecalis 72.2% Enterococcus faecium 13.3% Enterococcus gallinarum 10.0% Enterococcus casseliflavus 1.1% Enterococcus hirae 3.4% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Milk Curd 7 14 21 35 49 63 91 121 150 Enterococci distribution Production/ ripening steps E. hirae E. casseliflavus E. gallinarum E. faecium E. faecalis