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The past decade witnessed the emergence in Shiga toxin-producing Escherichia coli (STEC) infections linked to the consumption of unpasteurized milk and raw milk cheese. The virulence of STEC is primarily attributed to the presence of Shiga toxin genes (stx1 and stx2) carried by Stx-converting bacteriophages, along with the intimin gene eae. Most of the available information pertains to the "Top 7" serotypes associated with STEC infections. The objectives of this study were to characterize and investigate the pathogenicity potential of E. coli UC4224, a STEC O174:H2 strain isolated from semi-hard raw milk cheese and to develop surrogate strains with reduced virulence for use in food-related studies. Complete genome sequence analysis of E. coli UC4224 unveiled the presence of a Stx1a bacteriophage, a Stx2a bacteriophage, the Locus of Adhesion and Autoaggregation (LAA) pathogenicity island, plasmid-encoded virulence genes, and other colonization facilitators. In the Galleria mellonella animal model, E. coli UC4224 demonstrated high pathogenicity potential with an LD50 of 6 CFU/10 & mu;L. Upon engineering E. coli UC4224 to generate single and double mutant derivatives by inactivating stx1a and/or stx2a genes, the LD50 increased by approximately 1 Log-dose in the single mutants and 2 Log-doses in the double mutants. However, infectivity was not completely abolished, suggesting the involvement of other virulence factors contributing to the pathogenicity of STEC O174:H2. Considering the possibility of raw milk cheese serving as a reservoir for STEC, cheesemaking model was developed to evaluate the survival of UC4224 and the adequacy of the respective mutants as reduced-virulence surrogates. All tested strains exhibited the ability to survive the curd cooking step at 48 & DEG;C and multiplied (3.4 Log CFU) in cheese within the subsequent 24 h. These findings indicate that genomic engineering did not exert any unintended effect on the double stx1-stx2 mutant behaviou

The presence of multi-drug resistant (MDR) bacteria in ready-to-eat foods comprises a threat for public health due to their ability to acquire and transfer antibiotic-resistant determinants that could settle in the microbiome of the human digestive tract. In this study, Enterococcus faecium UC7251 isolated from a fermented dry sausage was characterized phenotypically and genotypically to hold resistance to multiple antibiotics including aminoglycosides, macrolides, beta-lactams, and tetracyclines. We further investigated this strain following a hybrid sequencing and assembly approach (short and long reads) and determined the presence of various mobile genetic elements (MGEs) responsible of horizontal gene transfer (HGT). On the chromosome of UC7251, we found one integrative and conjugative element (ICE) and a conjugative transposon Tn916-carrying tetracycline resistance. UC7251 carries two plasmids: one small plasmid harboring a rolling circle replication and one MDR megaplasmid. The latter was identified as mobilizable and containing a putative integrative and conjugative element-like region, prophage sequences, insertion sequences, heavy-metal resistance genes, and several antimicrobial resistance (AMR) genes, confirming the phenotypic resistance characteristics. The transmissibility potential of AMR markers was observed through mating experiments, where Tn916-carried tetracycline resistance was transferred at intra- and inter-species levels. This work highlights the significance of constant monitoring of products of animal origin, especially RTE foodstuffs, to stimulate the development of novel strategies in the race for constraining the spread of antibiotic resistance.

Clostridium tyrobutyricum represents the main spoiling agent responsible for late blowing defects (LBD) in hard and semi-hard cheeses. Its spores are resistant to manufacturing procedures and can germinate during the long ripening process, causing the burst of the cheese paste with a consequent undesirable taste. The lower quality of blown cheeses leads to considerable financial losses for the producers. The early identification of spore contaminations in raw milk samples thus assumes a pivotal role in industrial quality control. Herein, we developed a point of care (POC) testing method for the sensitive detection of C. tyrobutyricum in milk samples, combining fast DNA extraction (with no purification steps) with a robust colorimetric loop-mediated isothermal amplification (LAMP) technique. Our approach allows for the sensitive and specific detection of C. tyrobutyricum spores (limit of detection, LoD: ~2 spores/mL), with the advantage of a clear naked-eye visualization of the results and a potential semi-quantitative discrimination of the contamination level. In addition, we demonstrated the feasibility of this strategy using a portable battery-operated device that allowed both DNA extraction and amplification steps, proving its potential for on-site quality control applications without the requirement of sophisticated instrumentation and trained personnel.

Enterococcus lactis and the heterotypic synonym Enterococcus xinjiangensis from dairy origin have recently been identified as a novel species based on 16S rRNA gene sequence analysis. Enterococcus faecium type strain NCTC 7171T was used as the reference genome for determining E. lactis and E. faecium to be separate species. However, this taxonomic classification did not consider the diverse lineages of E. faecium, and the double nature of hospital-associated (clade A) and community-associated (clade B) isolates. Here, we investigated the taxonomic relationship among isolates of E. faecium of different origins and E. lactis, using a genome-based approach. Additional to 16S rRNA gene sequence analysis, we estimated the relatedness among strains and species using phylogenomics based on the core pangenome, multilocus sequence typing, the average nucleotide identity and digital DNA–DNA hybridization. Moreover, following the available safety assessment schemes, we evaluated the virulence profile and the ampicillin resistance of E. lactis and E. faecium clade B strains. Our results confirmed the genetic and evolution-ary differences between clade A and the intertwined clade B and E. lactis group. We also confirmed the absence in these strains of virulence gene markers IS16, hylEfm and esp and the lack of the PBP5 allelic profile associated with ampicillin resistance. Taken together, our findings support the reassignment of the strains of E. faecium clade B as E. lactis.

Lactobacillus helveticus is a thermophilic lactic acid bacterium that is widely employed as a starter culture for manufacturing several Swiss and Italian hard-cooked cheeses. The sequencing of L. helveticus Lh 23, which consists of 2,100,230 bp with a GC content of 36.5%, reveals industrially useful traits and interesting metabolic pathways.

Staphylococcus aureus is one of the major pathogens responsible for intramammary infections in small ruminants, causing severe economic losses in dairy farms. In addition, S. aureus can contaminate milk and dairy products and produce staphylococcal enterotoxins, being responsible for staphylococcal food poisoning. Currently, data on the population structure and the virulence gene patterns of S. aureus strains isolated from goat milk is limited. Therefore, this study aimed at defining Ribosomal Spacer PCR (RS-PCR) genotypes, clonal complexes (CC), spa types, and virulence gene profiles of S. aureus isolated from goat milk samples from Lombardy region of Italy. A total of 295 S. aureus isolates from 65 goat bulk tank milk samples were genotyped by RS-PCR. spa typing and virulence gene patterns of a subgroup of 88 isolates were determined, and MLST was performed on a further subgroup of 39 isolates, representing all the spa types identified during the analysis. This study revealed 7 major genotypic clusters (CLR, CLAA, CLZ, CLAW, CLBW, CLS, and CLI), of which S. aureus CLR (19.8%) was the most common. A total of 26 different spa types were detected, the most prevalent types were t1773 (24%), t5428 (22.7%), and t2678 (12.5%). Overall, 44.3% of all isolates harbored at least one enterotoxin gene. The most prevalent was the combination of sec-sel genes (35.2%). Based on their MLST, isolates were assigned to 14 different CC, with majority grouped as CC133 (24%), CC130 (19.6%), and CC522 (19.6%). The caprine S. aureus population was depicted with a minimum spanning tree and an evolutionary analysis based on spa typing and MLST, respectively. Then, the variability of such strains was compared to that of bovine strains isolated in the same space-time span. Our results confirmed that S. aureus isolates from goats have wide genetic variability and differ from the bovine strains, supporting the idea that S. aureus from small ruminants may constitute a distinct population.

Lactobacillus helveticus is a homofermentative thermophilic lactic acid bacteria that is mainly used in the manufacture of Swiss type and long-ripened Italian hard cheeses. In this study, the presence of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) were analysed in 25 L. helveticus genomes and identified in 23 of these genomes. A total of 40 CRISPR loci were identified and classified into five main families based on CRISPR repeats: Ldbu1, Lsal1, Lhel1, Lhel2 and a new repeat family named Lhel3. Spacers had a size between 30 and 40 bp whereas repeats have an average size of 30 bp, with three longer repeats. The analysis displayed the presence of conserved spacers in 23 of the 40 CRISPR loci. A geographical distribution of L. helveticus isolates with similar CRISPR spacer array profiles were not observed. Based on the presence of the signature protein Cas3, all CRISPR loci belonged to Type I. This analysis demonstrated a great CRISPR array variability within L. helveticus, which could be a useful tool for genotypic strain differentiation. A next step will be to understand the possible role of CRISPR/Cas system for the resistance of L. helveticus to phage infection. Significance and Impact of the Study: Lactobacillus helveticus, a lactic acid bacteria species widely used as starter culture in the dairy industry has recently also gained importance as health-promoting culture in probiotic and nutraceutical food products. The CRISPR/Cas system, a well-known molecular mechanism that provides adaptive immunity against exogenous genetic elements such as bacteriophages and plasmids in bacteria, was recently found in this species. In this study, we investigated the presence and genetic heterogeneity of CRISPR loci in 25 L. helveticus genomes. The results presented here represent an important step on the way to manage phage resistance, plasmid uptake and genome editing in this species.

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 TaqManTM 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.

Staphylococcus aureus is regarded as a leading cause of mastitis in goats. However, few data are available on the presence of methicillin-resistant S. aureus (MRSA) in this species. The aim of this study was to assess the prevalence of S. aureus and MRSA in bulk tank milk samples from dairy goat farms in Northern Italy. Eighty-five out of 197 samples (43.1%) tested positive for S. aureus with counts ranging from 10 to more than 1.5×104 cfu/mL. The MRSA was screened by both direct plating followed by a disk diffusion test to evaluate methicillin resistance and a selective enrichment method. Methicillin-resistance was confirmed by mecA-specific PCR. Methicillin-resistant S. aureus was identified in 4 samples (2.0%) and multilocus sequence typing (MLST) showed the presence of livestock-associated MRSA belonging to lineages ST398 (n=3) and ST1 (n=1). In one case we demonstrated that the same MRSA strain was able to persist over time on the farm, being isolated from both bulk tank milk and the udder of 3 goats 1 yr after the first isolation. The high prevalence of S. aureus-positive herds detected in this study and the presence of MRSA strains belonging to livestock-associated genotypes is of concern, and represents a novel finding in the Italian dairy goat production system. The application of stringent measures for the control of S. aureus mastitis at the farm level seems appropriate to reduce the economic losses, and to minimize the risk of foodborne illness and the transmission of MRSA to humans by occupational exposure.