Detection and Virulence Characterization of Listeria monocytogenes Strains in Ready-to-Eat Products.

The public health risk posed by Listeria monocytogenes in ready-to-eat (RTE) foods depends on the effectiveness of its control at every stage of the production process and the strain involved. Analytical methods currently in use are limited to the identification/quantification of L. monocytogenes at the species level, without distinguishing virulent from hypovirulent strains. In these products, according to EU Regulation 2073/2005, L. monocytogenes is a mandatory criterion irrespective of strain virulence level. Indeed, this species encompasses a diversity of strains with various pathogenic potential, reflecting genetic heterogeneity of the species itself. Thus, the detection of specific L. monocytogenes virulence genes can be considered an important target in laboratory food analysis to assign different risk levels to foods contaminated by strains carrying different genes. In 2015-2016, a severe invasive listeriosis outbreak occurred in central Italy, leading to the intensification of routine surveillance and strain characterization for virulence genetic markers. A new multiplex real-time polymerase chain reaction targeting main virulence genes has been developed and validated against the enzyme-linked fluorescent assay (ELFA) culture-based method. Results of the improved surveillance program are now reported in this study.

Unveiling hákarl: A study of the microbiota of the traditional Icelandic fermented fish.

Hákarl is produced by curing of the Greenland shark (Somniosus microcephalus) flesh, which before fermentation is toxic due to the high content of trimethylamine (TMA) or trimethylamine N-oxide (TMAO). Despite its long history of consumption, little knowledge is available on the microbial consortia involved in the fermentation of this fish. In the present study, a polyphasic approach based on both culturing and DNA-based techniques was adopted to gain insight into the microbial species present in ready-to-eat hákarl. To this aim, samples of ready-to-eat hákarl were subjected to viable counting on different selective growth media. The DNA directly extracted from the samples was further subjected to Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) and 16S amplicon-based sequencing. Moreover, the presence of Shiga toxin-producing Escherichia coli (STEC) and Pseudomonas aeruginosa was assessed via qualitative real-time PCR assays. pH values measured in the analyzed samples ranged from between 8.07 ±â€¯0.06 and 8.76 ±â€¯0.00. Viable counts revealed the presence of total mesophilic aerobes, lactic acid bacteria and Pseudomonadaceae. Regarding bacteria, PCR-DGGE analysis highlighted the dominance of close relatives of Tissierella creatinophila. For amplicon sequencing, the main operational taxonomic units (OTUs) shared among the data set were Tissierella, Pseudomonas, Oceanobacillus, Abyssivirga and Lactococcus. The presence of Pseudomonas in the analyzed samples supports the hypothesis of a possible role of this microorganism on the detoxification of shark meat from TMAO or TMA during fermentation. Several minor OTUs (<1%) were also detected, including Alkalibacterium, Staphylococcus, Proteiniclasticum, Acinetobacter, Erysipelothrix, Anaerobacillus, Ochrobactrum, Listeria and Photobacterium. Analysis of the yeast and filamentous fungi community composition by PCR-DGGE revealed the presence of close relatives of Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida zeylanoides, Saccharomyces cerevisiae, Debaryomyces, Torulaspora, Yamadazyma, Sporobolomyces, Alternaria, Cladosporium tenuissimum, Moristroma quercinum and Phoma/Epicoccum, and some of these species probably play key roles in the development of the sensory qualities of the end product. Finally, qualitative real-time PCR assays revealed the absence of STEC and Pseudomonas aeruginosa in all of the analyzed samples.

Contamination of hospital food with Clostridium difficile in Central Italy.

This study investigates the contamination of foods with Clostridium difficile in 3 hospitals of Central Italy. We used real-time PCR for tpi gene to analyse 350 food samples, including 296 cooked meals and 54 foods to be eaten raw. The molecular screening test was positive for 3 samples, but toxigenic C. difficile was isolated only in two of them. The prevalence in cooked food was 0,3% (1/296), while in uncooked processed foods was 1,9% (1/54). Data support the potential risk of food as a source of toxigenic C. difficile for hospitalized patients, but further investigations are needed.

Presence of Escherichia coli O157, Salmonella spp., and Listeria monocytogenes in Raw Ovine Milk Destined for Cheese Production and Evaluation of the Equivalence Between the Analytical Methods Applied.

Italy is one of the main producers and exporters of cheese made from unpasteurized sheep milk. Since raw milk and its derived products are known sources of human infections, cheese produced from raw sheep milk could pose a microbiological threat to public health. Hence, the objectives of the study were: to characterize the potential risk of the presence of pathogens Escherichia coli O157, Listeria monocytogenes, and Salmonella in raw ovine milk destined for cheese production obtained from all the sheep farms (n = 24) in the Marches region (Central Italy) that directly transform raw milk into cheeses and to evaluate the equivalence between the analytical methods applied. A three-step molecular method (simultaneous culture enrichment, species-specific DNA magnetic isolation, and multiplex real-time polymerase chain reaction) was used for milk (n = 143) and cheese (n = 5) analysis over a 3-year period. L. monocytogenes was not detected on any of the farms, while E. coli O157 was found on three farms, although only using the molecular method. Four farms tested positive for Salmonella spp., and Salmonella enterica subsp. diarizonae serovar 61:k:1,5,7 was isolated in one of those cases. This information highlights the need to develop preventative measures to guarantee a high level of consumer safety for this specific product line, and the molecular method could be a time-saving and sensitive tool to be used in routine diagnosis.

Evaluation of the quality of foods for special diets produced in a school catering facility within a HACCP-based approach: a case study.

A study was carried out to verify the appropriateness of the Hazard Analysis and Critical Control Point (HACCP) plan adopted in a school catering facility. To that end, the microbiological quality of foods, the correct implementation of special diets (lactose- and gluten-free) and the nutritional value of foods were assessed. Thirty-six samples of lactose-free and 87 samples of gluten-free special diet food preparations were subjected to microbiological, chemical, and nutritional analyses. The data collected demonstrate the effectiveness of the HACCP plan in reducing the occurrence of microbial and chemical (lactose and gluten) cross-contamination. The data obtained from the nutritional analyses showed that the dietary intake provided by the meals under study was satisfactory.

Assessment of the Microbiological Safety and Hygiene of Raw and Thermally Treated Milk Cheeses Marketed in Central Italy between 2013 and 2020.

A profile of the microbial safety and hygiene of cheese in central Italy was defined based on an analysis of 1373 cheeses sampled under the Italian National Control Plan for Food Safety spanning the years 2013 to 2020 and tested according to Commission Regulation (EC) No. 2073/2005 (as amended). A total of 97.4% of cheese samples were assessed as being satisfactory for food safety criteria and 80.5% for process hygiene criteria. Staphylococcal enterotoxin was found in 2/414 samples, while Salmonella spp. and Listeria monocytogenes were detected in 15 samples out of 373 and 437, respectively. Escherichia coli and coagulase-positive staphylococci counts were found unsatisfactory in 12/61 and 17/88 cheese samples, respectively. The impact of milking species, milk thermal treatment, and cheese hardness category was considered. A statistically significant association (p < 0.05) was found between milk thermal treatment and the prevalence of coagulase-positive staphylococci and Listeria monocytogenes and between hardness and unsatisfactory levels of Escherichia coli. The data depict a contained public health risk associated with these products and confirm, at the same time, the importance of strict compliance with good hygiene practices during milk and cheese production. These results can assist in bolstering risk analysis and providing insights for food safety decision making.

Widespread Multidrug Resistance of Arcobacter butzleri Isolated from Clinical and Food Sources in Central Italy.

The Arcobacter genus comprises a group of bacteria widely distributed in different habitats that can be spread throughout the food chain. Fluoroquinolones and aminoglycosides represent the most common antimicrobial agents used for the treatment of Arcobacter infections. However, the increasing trend of the antimicrobial resistance of this pathogen leads to treatment failures. Moreover, the test implementation and interpretation are hindered by the lack of reference protocols and standard interpretive criteria. The purpose of our study was to assess the antibiotic resistance pattern of 17 A. butzleri strains isolated in Central Italy from fresh vegetables, sushi, chicken breast, and clinical human samples to provide new and updated information about the antimicrobial resistance epidemiology of this species. Antimicrobial susceptibility testing was carried out by the European Committee on Antimicrobial Susceptibility Testing (EUCAST)'s disc diffusion method. All the strains were multidrug resistant, with 100% resistance to tetracyclines and cefotaxime (third generation cephalosporins). Some differences were noticed among the strains, according to the isolation source (clinical isolates, food of animal origin, or fresh vegetables), with a higher sensitivity to streptomycin detected only in the strains isolated from fresh vegetables. Our data, together with other epidemiological information at the national or European Union (EU) level, may contribute to developing homogeneous breakpoints. However, the high prevalence of resistance to a wide range of antimicrobial classes makes this microorganism a threat to human health and suggests that its monitoring should be considered by authorities designated for food safety.

Microbiological surveillance of a bovine raw milk farm through multiplex real-time PCR.

Raw milk is increasingly appreciated by consumers but can be contaminated by a variety of zoonotic pathogens. Therefore, preventive measures, such as on-farm hazard analysis critical control point (HACCP) programs, must be applied to protect consumers. The aim of the present study was the comparison of a multiplex real-time polymerase chain reaction (PCR) assay with a culture-based approach in an on-farm quality assurance program for the detection of Escherichia coli O157, Salmonella spp., and Listeria monocytogenes in bulk tank milk, in-line milk filters, manure, and feces. Results revealed that the real-time PCR was more sensitive in detecting E. coli O157 than the culture method in filters (48% vs. 4% positive), manure (93% vs. 7% positive) and feces (60% vs. 4% positive). The two methods were equally efficient in detecting L. monocytogenes (8% of filters), while Salmonella spp. was not detected in any sample. In conclusion, the real-time PCR, by reducing analysis time to two working days, can be proposed as a useful tool in the raw milk primary production setting as a rapid and user-friendly screening method.

Staphylococcus aureus artificially inoculated in mealworm larvae rearing chain for human consumption: Long-term investigation into survival and toxin production.

The present research reports the results of a long-term study (70 days) of the dynamics of Staphylococcus aureus artificially inoculated in a Tenebrio molitor rearing chain for human consumption. To this end, a rearing substrate consisting of organic wheat middlings was spiked with S. aureus to obtain three initial contamination levels, namely 1 (low level), 5 (medium level) and 7 (high level) Log colony forming unit per gram. Microbial viable counting coupled with metataxonomic analysis were performed to evaluate: i) the persistence and growth of S. aureus in the rearing substrate; ii) the colonization and growth of S. aureus in the insect larvae; iii) the occurrence and load of S. aureus in the frass (excrement from larvae mixed with substrate residues); iv) the presence of S. aureus enterotoxins in the rearing substrate, frass, and larvae. The results of the present study highlighted that wheat middlings contaminated with S. aureus do not represent a suitable environment for the multiplication of the pathogen, irrespective of the initial contamination level. Of note, frass originated from the larvae reared on contaminated wheat middlings might potentially represent a source of S. aureus, with cell loads depending on the initial contamination level. A complex resident microbiota was revealed by metataxonomic analysis. Interestingly, co-occurrence/co-exclusions analysis did not reveal associations between the target microorganism and the microbiota of wheat middlings, larvae, or frass. Considering safety aspects of larvae, the results overall collected suggested that, under the applied conditions, T. molitor represents an inhospitable or even hostile environment for S. aureus, with this latter showing counts below the detection limit in the larvae at the end of the 70-day rearing trial, irrespective of the initial contamination level. The results also suggested that a combination of bactericidal factors, including unfavorable environmental conditions (such as low aw of wheat middlings and frass), might have established in the rearing chain. Finally, the absence of staphylococcal toxins suggests that, even when S. aureus is present at high contamination levels, it is not able to produce toxins in wheat middlings, larvae, or frass.

Prevalence, Molecular Characterization and Antimicrobial Susceptibility of Clostridioides difficile Isolated from Pig Carcasses and Pork Products in Central Italy.

In the last decade, the incidence and severity of Clostridioides difficile infections (CDIs) in humans have been increasing and community-associated infections have been described. For these reasons, the interest in C. difficile in food and in food animals has increased, suggesting other possible sources of C. difficile acquisition. This study evaluated the presence of C. difficile on pig carcasses at the slaughterhouse and in pork products in Central Italy. The contamination rate on pig carcasses was 4/179 (2.3%). Regarding food samples, a total of 216 pork products were tested (74 raw meat preparations and 142 ready-to-eat food samples made by cured raw meat). The real-time PCR screening was positive for 1/74 raw meat preparation (1.35%) and for 1/142 ready-to-eat food samples (0.7%) C. difficile was isolated only from the raw meat preparation (pork sausage). All the isolated strains were toxigenic and susceptible to all the tested antibiotics. Strains isolated from carcass samples displayed A+B+CDTa+CDTb+ profile, were toxinotype IV and belonged to the same ribotype arbitrary named TV93, while the one isolated from food samples displayed A+B+CDTa-CDTb- profile and it was not possible to determine ribotype and toxinotype, because it was lost after freeze storage. It was concluded that the prevalence of C. difficile in the pork supply chain is very low.

Whole-Genome Sequencing Characterization of Virulence Profiles of Listeria monocytogenes Food and Human Isolates and In Vitro Adhesion/Invasion Assessment.

Listeria monocytogenes (Lm) is the causative agent of human listeriosis. Lm strains have different virulence potential. For this reason, we preliminarily characterised via Whole-Genome Sequencing (WGS) some Lm strains for their key genomic features and virulence-associated determinants, assigning the clonal complex (CC). Moreover, the ability of the same strains to adhere to and invade human colon carcinoma cell line Caco-2, evaluating the possible correspondence with their genetic virulence profile, was also assessed. The clinical strains typed belonged to clonal complex (CC)1, CC31, and CC101 and showed a very low invasiveness. The Lm strains isolated from food were assigned to CC1, CC7, CC9, and CC121. All CC1 carried the hypervirulence pathogenicity island LIPI-3 in addition to LIPI-1. Premature stop codons in the inlA gene were found only in Lm of food origin belonging to CC9 and CC121. The presence of LIPI2_inlII was observed in all the CCs except CC1. The CC7 strain, belonging to an epidemic cluster, also carried the internalin genes inlG and inlL and showed the highest level of invasion. In contrast, the human CC31 strain lacked the lapB and vip genes and presented the lowest level of invasiveness. In Lm, the genetic determinants of hypo- or hypervirulence are not necessarily predictive of a cell adhesion and/or invasion ability in vitro. Moreover, since listeriosis results from the interplay between host and virulence features of the pathogen, even hypovirulent clones are able to cause infection in immunocompromised people.

Intensive Environmental Surveillance Plan for Listeria monocytogenes in Food Producing Plants and Retail Stores of Central Italy: Prevalence and Genetic Diversity.

Listeria monocytogenes (Lm) can persist in food processing environments (FPEs), surviving environmental stresses and disinfectants. We described an intensive environmental monitoring plan performed in Central Italy and involving food producing plants (FPPs) and retail grocery stores (RSs). The aim of the study was to provide a snapshot of the Lm circulation in different FPEs during a severe listeriosis outbreak, using whole genome sequencing (WGS) to investigate the genetic diversity of the Lm isolated, evaluating their virulence and stress resistance profiles. A total of 1217 samples were collected in 86 FPEs with 12.0% of positive surfaces at FPPs level and 7.5% at RSs level; 133 Lm isolates were typed by multilocus sequencing typing (MLST) and core genome MLST (cgMLST). Clonal complex (CC) 121 (25.6%), CC9 (22.6%), CC1 (11.3%), CC3 (10.5%), CC191 (4.5%), CC7 (4.5%) and CC31 (3.8%) were the most frequent MLST clones. Among the 26 cgMLST clusters obtained, 5 of them persisted after sanitization and were re-isolated during the follow-up sampling. All the CC121 harboured the Tn6188_qac gene for tolerance to benzalkonium chloride and the stress survival islet SSI-2. The CC3, CC7, CC9, CC31 and CC191 carried the SSI-1. All the CC9 and CC121 strains presented a premature stop codon in the inlA gene. In addition to the Lm Pathogenicity Island 1 (LIPI-1), CC1, CC3 and CC191 harboured the LIPI-3. The application of intensive environmental sampling plans for the detection and WGS analysis of Lm isolates could improve surveillance and early detection of outbreaks.

Occurrence of Listeria monocytogenes in salami manufactured in the Marche Region (Central Italy).

The aim of the present study was to investigate the occurrence of Listeria monocytogenes in salami samples collected from production plants of the Marche Region, and to assess the end-product acceptability based on the former Italian regulations and European Commission (EC) Regulation No 2073/2005. Based on the limits specified in the former Italian regulations, the percentage of non-acceptable samples was 34.3%, whereas based on the limits specified in EC Regulation N degrees 2073/2005, a lower percentage (17.1%) was seen. A similar trend was seen also when only the Ciauscolo salami were considered, with 45.2 and 27.4% of non-acceptable samples, respectively. No correlations were identified between occurrence of L. monocytogenes and the main parameters or the manufacturing processes.

Characterization of A Staphylococcal Food Poisoning Outbreak in A Workplace Canteen during the Post-Earthquake Reconstruction of Central Italy.

In summer 2017, a foodborne outbreak occurred in Central Italy, involving 26 workers employed in the post-earthquake reconstruction. After eating a meal provided by a catering service, they manifested gastrointestinal symptoms; 23 of them were hospitalized. The retrospective cohort study indicated the pasta salad as the most likely vehicle of poisoning. Foods, environmental samples, and food handlers' nasal swabs were collected. Bacillus cereus (Bc) and coagulase-positive staphylococci (CPS) including S. aureus, together with their toxins, were the targets of the analysis. CPS, detected in all the leftovers, exceeded 105 CFU/g in the pasta salad, in which we found Staphylococcal Enterotoxins (SEs) (0.033 ng SEA/g; 0.052 ng SED/g). None of the environmental and human swabs showed contamination. We characterized 23 S. aureus from foods. They all belonged to the human biotype, showed the same toxigenic profile (sea, sed, sej, and ser genes), and had the same Pulsed Field Gel Electrophoresis (PFGE) pattern; none of them harbored mecA or mupA genes. We also detected Bc contamination in the pasta salad but none of the isolates harbored the ces gene for the emetic toxin cereulide. The EU Reference Laboratory for CPS confirmed the case as a strong-evidence outbreak caused by the ingestion of SEs produced by a single strain of S. aureus carried by the same human source. This outbreak was successfully investigated despite the emergency situation in which it occurred.

Distribution of Transferable Antibiotic Resistance Genes in Laboratory-Reared Edible Mealworms (Tenebrio molitor L.).

In the present study, the distribution of antibiotic resistance genes in laboratory-reared fresh mealworm larvae (Tenebrio molitor L.), their feeding substrates (carrots and wheatmeal), and frass was assessed. Microbial counts on selective media added with antibiotics highlighted the presence of lactic acid bacteria resistant to ampicillin and vancomycin and, more specifically, enterococci resistant to the latter antibiotic. Moreover, staphylococci resistant to gentamicin, erythromycin, tetracycline, and vancomycin were detected. Enterobacteriaceae resistant to ampicillin and gentamicin were also found, together with Pseudomonadaceae resistant to gentamicin. Some of the genes coding for resistance to macrolide-lincosamide-streptogramin B (MLSB) [erm(A), erm(C)], vancomycin [vanA, vanB], tetracycline [tet(O)], and ß-lactams [mecA and blaZ] were absent in all of the samples. For the feeding substrates, organic wheatmeal was positive for tet(S) and tet(K), whereas no AR genes were detected in organic carrots. The genes tet(M), tet(K), and tet(S) were detected in both mealworms and frass, whereas gene aac-aph, coding for resistance to amynoglicosides was exclusively detected in frass. No residues for any of the 64 antibiotics belonging to 10 different drug classes were found in either the organic wheatmeal or carrots. Based on the overall results, the contribution of feed to the occurrence of antibiotic resistance (AR) genes and/or antibiotic-resistant microorganisms in mealworm larvae was hypothesized together with vertical transmission via insect egg smearing.

Revealing the microbiota of marketed edible insects through PCR-DGGE, metagenomic sequencing and real-time PCR.

The present study aimed to identify the microbiota present in six species of processed edible insects produced in Thailand and marketed worldwide via the internet, namely, giant water bugs (Belostoma lutarium), black ants (Polyrhachis), winged termites (alates, Termitoidae), rhino beetles (Hyboschema contractum), mole crickets (Gryllotalpidae), and silkworm pupae (Bombyx mori). For each species, two samples of boiled, dried and salted insects were purchased. The microbial DNA was extracted from the insect samples and subjected to polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), high-throughput sequencing and qualitative real-time PCR assays. The microbiota of the analyzed samples were widely characterized by the presence of spore-forming bacteria mainly represented by the genera Bacillus and Clostridium. Moreover, the genera Anaerobacillus, Paenibacillus, Geobacillus, Pseudomonas, Stenotrophomonas, Massilia, Delftia, Lactobacillus, Staphylococcus, Streptococcus, Vagococcus, and Vibrio were also detected. Real-time PCR allowed for ascertainment of the absence of Coxiella burnetii, Shiga toxin-producing E. coli (STEC), and Pseudomonas aeruginosa in all samples. The results of this study confirm the importance of combining different molecular techniques to characterize the biodiversity of complex ecosystems such as edible insects. The presence of potential human pathogens suggests the need for a careful application of good manufacturing practices during insect processing. This study provides further data that will be useful in risk analyses of edible insects as a novel food source.

The bacterial biota of laboratory-reared edible mealworms (Tenebrio molitor L.): From feed to frass.

Tenebrio molitor represents one of the most popular species used for the large-scale conversion of plant biomass into protein and is characterized by high nutritional value. In the present laboratory study, the bacterial biota characterizing a pilot production chain of fresh T. molitor larvae was investigated. To this end, different batches of fresh mealworm larvae, their feeding substrate (wheatmeal) and frass were analyzed by viable microbial counts, PCR-DGGE and Illumina sequencing. Moreover, the occurrence of Coxiella burnetii, Pseudomonas aeruginosa and Shiga toxin-producing E. coli (STEC) was assessed through qualitative real-time PCR assays. Microbial viable counts highlighted low microbial contamination of the wheatmeal, whereas larvae and frass were characterized by high loads of Enterobacteriaceae, lactic acid bacteria, and several species of mesophilic aerobes. Spore-forming bacteria were detected to a lesser extent in all the samples. The combined molecular approach used to profile the microbiota confirmed the low microbial contamination of wheatmeal and allowed the detection of Enterobacter spp., Erwinia spp., Enterococcus spp. and Lactococcus spp. as dominant genera in both larvae and frass. Moreover, Klebsiella spp., Pantoea spp., and Xenorhabdus spp. were found to be in the minority. Entomoplasmatales (including Spiroplasma spp.) constituted a major fraction of the microbiota of one batch of larvae. From the real-time PCR assays, no sample was positive for either C. burnetii or STEC, whereas P. aeruginosa was detected in one sample of frass. Based on the overall results, two sources of microbial contamination were hypothesized, namely feeding with wheatmeal and vertical transmission of microorganisms from mother to offspring. Since mealworms are expected to be eaten as a whole, the overall outcomes collected in this laboratory study discourage the consumption of fresh mealworm larvae. Moreover, microbial loads and the absence of potential pathogens known to be associated with this insect species should be carefully assessed in order to reduce the minimum risk for consumers, by identifying the most opportune processing methods (e.g., boiling, frying, drying, etc.).

A severe outbreak of listeriosis in central Italy with a rare pulsotype associated with processed pork products.

PURPOSE: From May 2015 to March 2016, an outbreak due to Listeria monocytogenes serotype 1/2a and clinical pulsotype never previously isolated in Europe occurred in central Italy, involving 24 confirmed clinical cases. The article provides a description of the outbreak and the investigation carried out by a multidisciplinary network. METHODOLOGY: Epidemiological and microbiological surveillance was conducted to confirm the outbreak and to detect the food vehicle of infection. The origin and destination of the implicated food and its ingredients were investigated by tracing-back and -forward investigation. RESULTS: Next-generation sequencing confirmed the unique outbreak strain. On 4 January 2016, a L. monocytogenes strain with pulsotype indistinguishable from that isolated from clinical cases in the outbreak was detected in a sample of hog head cheese purchased from a retail supermarket by one of the patients. The hog head cheese was produced by a small meat processing plant in the Marche region, where microbiological investigation confirmed environmental and food contamination by the outbreak strain. Plant production was suspended and all contaminated batches of the hog head cheese were withdrawn from the market by 19 February by local health authority. We subsequently observed a sharp decline in clinical cases, the last being reported on 11 March 2016. CONCLUSION: The key factor in the timely conclusion of this investigation was intersectoral collaboration among epidemiologists, microbiologists, veterinarians, statisticians and health and food safety authorities at national, regional and local levels.

The microbial ecology of a typical Italian salami during its natural fermentation.

We have investigated the bacteria and yeast ecology of the typical Italian Ciauscolo salami that is produced in Central Italy using a polyphasic approach based on culture-dependent and -independent methods. The physico-chemical analyses showed a progressive drop in pH and water activity (aw) during ripening. The viable counts revealed a dominance of lactic acid bacteria (LAB) over coagulase negative cocci (CNC) and yeasts. From the molecular identification of the isolates, the prevalence of Lactobacillus curvatus, Lb. plantarum and Staphylococcus xylosus was shown among the bacteria, while Debaryomyces hansenii was the prevalent species among the yeasts, and it was isolated throughout the whole ripening process. Minority species, namely Rhodotorula mucillaginosa and Trichosporon brassicae, were also recovered from the meat batter. The total microbial community was profiled without cultivation by analyzing the DNA that was directly extracted from the salami samples. Moreover, the cultivable community was profiled by analyzing the DNA recovered from bulk cells that were obtained by harvesting the colonies from serial-dilution agar plates. The 16S rRNA gene V1 and V3 regions were used as targets in the denaturing gradient gel electrophoresis (DGGE) profiling of the LAB and CNC communities, respectively, while the diversity and dynamics of the yeast population were assessed by analyzing a portion of the 28S rRNA gene. Our findings suggest that the microbial diversity of fermented meat products can be successfully investigated by this polyphasic approach that is based on the assessment of both the total and the cultivable community diversity.

Effect of farm characteristics and practices on hygienic quality of ovine raw milk used for artisan cheese production in central Italy.

A survey on ovine dairy farms directly transforming own-produced milk, in the Italian Marche region, was carried out to assess flock and milking practices that may influence milk hygienic-sanitary conditions. A census survey established that 24 dairy farms were located in this region. Bulk milk samples were collected throughout the milking period in each dairy farm in 2013. Analyzed variables were: (i) chemical parameters such as fat, protein and lactose content, dry matter and pH; and (ii) total bacterial (TBC) and somatic cell counts (SCC). Chemical parameter values were in agreement with published data while, geometric mean (GM) log10 SCC was 5.91 and TBC GM was 57 978 colony forming units/mL, in compliance with Eropean Union criteria. A positive correlation was found between SCC and TBC when GMs of all farm data were considered (Spearman's rho = 0.7925; P = 0.0001). Statistical analysis did not show significant correlation between SCC or TBC GM and dairy farm principal characteristics. Although SCC levels detected in the present study should suggest the need to implement mastitis control programs, Marche's dairy sheep flocks revealed a good hygienic condition level. This is an important aspect in implementing safety for end users of the final product.