Aims: To characterize Lactococcus garvieae strains of dairy origin and to determine their technological properties and safety for their possible use in starter culture preparation. Methods and Results: Forty-seven L. garvieae isolates, recovered from two artisanal Italian cheeses were studied, in comparison with 12 fish isolates and the type strain of the species. Phenotypic typing revealed that the strains could be differentiated on the basis of their ecological niche of origin in lactose positive strains (all isolated from dairy sources) and lactose negative strains (all isolated from fish). Furthermore, the strains exhibited a high degree of physiological variability, showing the presence of 26 different biotypes. The strains possessed moderate acidifying and proteolytic activities and did not produce bacteriocins. A safety investigation revealed that all strains were sensitive to vancomycin and moderately resistant to kanamycin; some biotypes were tetracycline resistant. Production of biogenic amines or presence of genes encoding virulence determinants occurred in some isolates. Conclusions: The prevalence of L. garvieae in some artisanal Italian cheeses can be linked to the typicity of the products. Although in a few cases an antimicrobial resistance or a presence of virulence determinants may imply a potential hygienic risk, most of the strains showed positive properties for their possible adjunction in a starter culture preparation, to preserve the natural bacterial population responsible for the typical sensorial characteristics of the traditional raw milk cheeses. Significance and Impact of the Study: L. garvieae strains can be considered an important part of the microbial population associated with the natural fermentation of artisanal Italian cheeses. A deepened characterization of the strains may aid in understanding the functional and ecological significance of their presence in dairy products and in selecting new strains for the dairy industry.

Lactobacillus sanfranciscensisis a lactic acid bacterium that characterizes the sourdough environment. The genetic differences of 24 strains isolated in different years from sourdoughs, mostly collected in Italy, were examined and compared by PFGE and multilocus sequence typing (MLST). The MLST scheme, based on the analysis of six housekeeping genes (gdh,gyrA, mapA, nox, pgmAandpta) was developed for this study. PFGE with the restriction enzymeApaI proved to have higher discriminatory power, since it revealed 22 different pulsotypes, while 19 sequence types were recognized through MLST analysis. Notably, restriction profiles generated from three isolates collected from the same firm but in three consecutive years clustered in a single pulsotype and showed the same sequence type, emphasizing the fact that the main factors affecting the dominance of a strain are correlated with processing conditions and the manufacturing environment rather than the geographical area. All results indicated a limited recombination among genes and the presence of a clonal population inL. sanfranciscensis.The MLST scheme proposed in this work can be considered a useful tool for characterization of isolates and for in-depth examination of the strain diversity and evolution of this species.

A three year survey on the dominant yeast populations in samples of air, must and wine in different vineyards and cellars of two northern Italian vine-growing territories (six sites in Franciacorta and eight sites in Oltrepò Pavese areas) was carried out. A total of 505 isolates were ascribed to 31 different species by RFLP analysis of the ITS1–5.8SrRNA–ITS2 region and partial sequence analysis of the 26S rRNA gene. The most commonly found species were Saccharomyces cerevisiae (frequency, F′ = 58.7 %; incidence, I’ = 53.5 %), Hanseniaspora uvarum (F′ = 14.3 %; I′ = 5.3 %), Metschnikowia fructicola (F′ = 11.1 %; I’ = 5.0 %) and Torulaspora delbrueckii (F′ = 10.3 %; I’ = 3.8 %). Among 270 S. cerevisiae new isolates, 156 (57.8 %) revealed a different genetic pattern through polymorphism analysis of the interdelta regions by capillary electrophoresis, while 47 isolates (17.4 %) were clones of starter cultures. By considering the Shannon–Wiener index and results of principal component analysis (PCA) analyses, the year of isolation (vintage) proved to be a factor that significantly affected the biodiversity of the yeast species, whereas the geographical site (terroir) was not. Seventy-five per cent of S. cerevisiae isolates gathered in a unique cluster at a similarity level of 82 %, while the remaining 25 % were separated into minor groups without any evident relationship between δ-PCR profile and territory, year or source of isolation. However, in six cases a similar strain appeared at the harvesting time both in Franciacorta and Oltrepò Pavese areas, whereas surprisingly no strain was reisolated in the same vineyard or cellar for consecutive years.

Dairy products can harbor various microorganisms (e.g., Campylobacter spp., Salmonella spp., Listeria monocytogenes, verocytotoxin-producing Escherichia coli) arising from animal reservoirs, and which can become important sources of foodborne illness. Therefore, early detection of food pathogens is crucial to prevent diseases. We wished to develop an accurate quantitative protocol based on a droplet digital polymerase chain reaction (ddPCR) involving eight individual TaqMan™ reactions to detect simultaneously, without selective enrichment, Listeria spp., L. monocytogenes, Salmonella spp., verocytotoxin-producing E. coli and Campylobacter spp. in cheese. ddPCR (a “third-generation PCR”) provides absolute quantification of target DNAs without requirement of a standard curve, which simplifies experimentation and data comparability. The accuracy, specificity and sensitivity of the developed ddPCR system were assessed using purified DNA from 50 reference pathogenic and non-pathogenic strains from international or Italian collections and analyzing soft cheese samples artificially contaminated with serial dilutions (from 4×106 to 4×101 CFU/g) of pure cultures from the American Type Culture Collection. Finally, the performance of our ddPCR system was compared by parallel testing with quantitative PCR: it gave higher sensitivity (102 CFU/g for the Listeria spp. assay) without the necessity of a standard curve. In conclusion, this is the first ddPCR system developed for simultaneous detection of common foodborne pathogens in cheese using a single set of amplification conditions. As such, it could become a useful strategy for high-throughput screening of microorganisms to evaluate the quality and safety of food products.

Brettanomyces bruxellensis displays a high degree of genotypic and phenotypic polymorphism and is the main yeast species involved in wine spoilage. Th