PRESENCE OF ENTEROCOCCI IN RAW COW'S MILK AND “PUZZONE DI MOENA” CHEESE

Abstract

ABSTRACT Raw cow's milk and cheese samples collected from factories producing “Puzzone di Moena” located in the alpine province of Trento (northern Italy), were analyzed for their enterococcal content. Presumptive enterococci (Gram positive, catalase negative, coccal shaped and positive for growth on kanamycin aesculin azide) were isolated, verified for their growth in presence of 6.5% NaCl, 40% bile salts and esculin hydrolysis, and analyzed by a genetic polyphasic approach consisting of intergenic spacer (ITS)-polymerase chain reaction (PCR) between the 16S and 23S rRNA genes, denaturing gradient gel electrophoresis (DGGE) of the V3 region of the 16S rRNA gene and a sodA gene-based multiplex PCR. Results showed the presence of Enterococcus faecalis as the main species found in milk and cheese. Enterococcus casseliflavus and Enterococcus durans were also detected in raw milk. In order to exclude clonal relatedness, the isolates were subjected to randomly amplified polymorphic DNA-PCR (RAPD-PCR). Winter cheese was characterized by a higher biodiversity than summer production, both in terms of Enterococcus species and number of strains. No strain was found positive for vancomycin resistance as determined by vanA- and vanB-specific PCR. Two strains were shown to be characterized by an anti-Listeria behavior due to bacteriocin-like inhibitory substances (BLIS) production. BLIS producer strains persisted during the ripening of cheese. PRACTICAL APPLICATIONS Most of the traditional cheeses are made from raw milk, whose lactic acid bacterial population plays a major role during ripening. Enterococci from milk may survive the adverse hostile environment in cheese during ripening and be potentially transferred to consumers. Enterococcus strains isolated from milk involved in cheese production, and from ripened “Puzzone di Moena” cheeses, were shown not to host van genes, thus, they should not be involved in the process of antimicrobial resistance dissemination among intestinal microflora. Furthermore, it was found that bacteriocinogenic enterococci dominated over nonbacteriocin-like inhibitory substances producer Enterococcus strains during the cheese process, confirming previous studies showing that bacteriocin production may influence bacterial complexes. From an application standpoint, these results are basic in the selection of nonstarter lactic acid bacteria to be used as adjunct cultures in traditional cheese productions.