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Hepatitis E is an emerging disease in industrialized countries. The food-borne transmission of hepatitis E virus (HEV) is associated principally with products derived from the domestic pig, the wild boar, and deer; however, few quantitative data are available on HEV loads in animals used in food production. This study assessed HEV occurrence, viral load and genetic variability in wild boar hunted for domestic consumption in the district of Viterbo (Central Italy) where high anti-HEV IgG seroprevalence values are reported in humans. A total of 332 liver and 69 intestine samples were obtained from wild boar hunted between 2011 and 2014. The liver tissue in 54 of the animals (16.3%) was HEV-positive. Viral loads in quantifiable liver samples (n = 29) ranged between 3.2 × 102 and 3.8 × 105 genome copies (g.c.)/g with a mean value of 1.85 × 104 g.c./g. A statistically significant positive correlation was found between viral concentration in liver and intestinal tissue, though mean viral load in the intestines was lower (3.13 × 103 g.c./g). Twenty-six samples were characterized molecularly as genotype 3 (G3) and four subtypes (a, c, f and l) were detected. Finally, twelve samples with near identical sequences were identified as G3 but could not be assigned to any of the known subtypes, and could therefore represent a potentially new subtype.

Wild boar is an animal the population of which constantly increases in Europe. This animal plays an important role as a reservoir for several pathogens, including three of the most important zoonoses: salmonellosis, yersiniosis and listeriosis. The aim of this investigation was to evaluate the occurrence of antimicrobial-resistant and virulence factor genes of Salmonella spp., Yersinia enterocolitica and Listeria monocytogenes isolated from wild boar in Tuscany (Central Italy). During two consequent hunting seasons (2018/2019 and 2019/2020), rectal swabs, spleens and livers were collected from 287 hunted wild boar to isolate strains. Each isolate was tested to investigate its antimicrobial resistance and to detect virulence factor genes by PCR. Eighteen Salmonella strains (6.27%) were isolated. Of these, 66.7% were resistant to streptomycin, 13.4% to cephalothin, 6.67% to imipenem and one isolate (6.67%) was resistant simultaneously to five antimicrobials. Moreover, the most detected genes were sopE (73.4%), pipB (66.7%), sodCI (53.3%), spvR and spvC (46.7%). In total, 54 (17.8%) Yersinia enterocolitica were isolated, of them, 26 (48.1%), 9 (16.7%), 17 (31.5%), 1 (1.85%) and 1 (1.85%) belonged to biotypes 1, 2, 3, 4 and 5, respectively. All strains (100%) demonstrated resistance to cephalothin and 70.4% to amoxicillin-clavulanic acid, 55.6% to ampicillin, and 37.0% to cefoxitin. Additionally, the most detected genes were ystA (25.9%), inv (24.1%), ail (22.2%), ystB (18.5%) and virF (14.8%). Finally, only one Listeriamonocytogenes isolate (0.35%) was obtained, belonging to serogroup IVb, serovar 4b, and it was found to be resistant to cefoxitin, cefotaxime and nalidixic acid. The results highlighted the role of wild boar as a carrier for pathogenic and antimicrobial-resistant Salmonella spp., Yersinia enterocolitica and Listeria monocytogens, representing a possible reservoir for domestic animals and human pathogens.

Wild boar is an animal the population of which constantly increases in Europe. This animal plays an important role as a reservoir for several pathogens, including three of the most important zoonoses: salmonellosis, yersiniosis and listeriosis. The aim of this investigation was to evaluate the occurrence of antimicrobial-resistant and virulence factor genes of Salmonella spp., Yersinia enterocolitica and Listeria monocytogenes isolated from wild boar in Tuscany (Central Italy). During two consequent hunting seasons (2018/2019 and 2019/2020), rectal swabs, spleens and livers were collected from 287 hunted wild boar to isolate strains. Each isolate was tested to investigate its antimicrobial resistance and to detect virulence factor genes by PCR. Eighteen Salmonella strains (6.27%) were isolated. Of these, 66.7% were resistant to streptomycin, 13.4% to cephalothin, 6.67% to imipenem and one isolate (6.67%) was resistant simultaneously to five antimicrobials. Moreover, the most detected genes were sopE (73.4%), pipB (66.7%), sodCI (53.3%), spvR and spvC (46.7%). In total, 54 (17.8%) Yersinia enterocolitica were isolated; of them, 26 (48.1%), 9 (16.7%), 17 (31.5%), 1 (1.85%) and 1 (1.85%) belonged to biotypes 1, 2, 3, 4 and 5, respectively. All strains (100%) demonstrated resistance to cephalothin and 70.4% to amoxicillin-clavulanic acid, 55.6% to ampicillin, and 37.0% to cefoxitin. Additionally, the most detected genes were ystA (25.9%), inv (24.1%), ail (22.2%), ystB (18.5%) and virF (14.8%). Finally, only one Listeria monocytogenes isolate (0.35%) was obtained, belonging to serogroup IVb, serovar 4b, and it was found to be resistant to cefoxitin, cefotaxime and nalidixic acid. The results highlighted the role of wild boar as a carrier for pathogenic and antimicrobial-resistant Salmonella spp., Yersinia enterocolitica and Listeria monocytogens, representing a possible reservoir for domestic animals and human pathogens.

Listeria monocytogenes(Lm) is a ubiquitous bacterium that causes the foodborne illness, listeriosis. Clonal complexes (CC), such as CC121, are overrepresented in the food production industry, and are rarely reported in animals and the environment. Working within a European-wide project, we investigated the routes by which strains are transmitted from environments and animals to food and the food production environment (FPE). In this context, we report, for the first time, the occurrence of a ST121 (CC121) strain isolated from a dolphin brain. The genome was compared with the genomes of 376 CC121 strains. Genomic comparisons showed that 16 strains isolated from food were the closest to the dolphin strain. Like most of the food strains analyzed here, the dolphin strain included genomic features (transposon Tn6188, plasmid pLM6179), both described as being associated with the strain’s adaptation to the FPE. Like all 376 strains, the dolphin strain contained a truncatedactAgene andinlAgene, both described as being associated with attenuated virulence. Despite this fact, the strain was able to cross blood-brain barrier in immunosuppressed dolphin exposed polychlorinated biphenyl and invaded by parasites. Our data suggest that the dolphin was infected by a food-related strain released into the Mediterranean Sea.View Full-Text LISTADAPT

Listeria monocytogenes (Lm) is a ubiquitous bacterium that causes the foodborne illness, listeriosis. Clonal complexes (CC), such as CC121, are overrepresented in the food production industry, and are rarely reported in animals and the environment. Working within a European-wide project, we investigated the routes by which strains are transmitted from environments and animals to food and the food production environment (FPE). In this context, we report, for the first time, the occurrence of a ST121 (CC121) strain isolated from a dolphin brain. The genome was compared with the genomes of 376 CC121 strains. Genomic comparisons showed that 16 strains isolated from food were the closest to the dolphin strain. Like most of the food strains analyzed here, the dolphin strain included genomic features (transposon Tn6188, plasmid pLM6179), both described as being associated with the strain&rsquo, s adaptation to the FPE. Like all 376 strains, the dolphin strain contained a truncated actA gene and inlA gene, both described as being associated with attenuated virulence. Despite this fact, the strain was able to cross blood-brain barrier in immunosuppressed dolphin exposed polychlorinated biphenyl and invaded by parasites. Our data suggest that the dolphin was infected by a food-related strain released into the Mediterranean Sea.

Salmonella enterica subspecies enterica serotype Hessarek (Salmonella Hessarek) is considered a serovar with high host specificity and is an uncommon cause of disease in humans; no cases of S. Hessarek bacteremia have been reported in humans to date. On 16 July 2019, a young male presented abdominal pain, vomit, diarrhea, and fever up to 41 °C, a few hours after a kebab meal containing goat meat; he went to the Emergency Room, where a Film Array® GI Panel (BioFire, Biomerieux Company, Marcy-L´Étoile, France) was performed on his feces and results were positive for Salmonella. The culture of the feces was negative, but the blood culture was positive for Salmonella spp., which was identified as Salmonella Hessarek by seroagglutination assays. The patient was treated with ceftriaxone 2 g intravenously qd for 8 days; he was discharged in good general conditions, and ciprofloxacin 500 mg per os bid for 7 more days was prescribed, after exclusion of endocarditis and of clinical signs of complicated bacteremia. This case of Salmonella Hessarek gastroenteritis with bacteremia is probably the first case of bloodstream human infection due to this agent ever described. Further studies are needed to ascertain the global burden of S. Hessarek disease in humans.

Salmonellosis is a major cause of bacterial foodborne infection. Since 2016, an increased number of cases of gastroenteritis caused by Salmonella enterica serovar Enteritidis linked to eggs produced in Poland has been reported in Europe. In Italy, S. Enteritidis is one of the three most commonly reported serotypes, associated mainly with the consumption of contaminated eggs and derived products. In our work, we analysed 61 strains of S. Enteritidis obtained from humans and farms in the Abruzzi region, Italy, in 2018. We used Multiple-Loci Variable-Number Tandem Repeat (VNTR) analysis (MLVA)-based typing and Whole-Genome Sequencing (WGS) tools to identify closely related strains and perform cluster analysis. We found two clusters of genetically similar strains. The first one was present in the local farms and isolated from human cases and had single-linkage distance of no more than two core genes and less than five Single-Nucleotide Polymorphisms (SNPs). The second cluster contained strains isolated from humans and from a dessert (tiramis&ugrave, ) sample that shared identical core genome and were assigned the same SNP address. Cluster 2 isolates were found to be genetically similar to an S. Enteritidis strain from a multi-country outbreak linked to Polish eggs.

In the framework of a European Union (EU) Coordinated Monitoring Programme, different types of ready-to eat (RTE) products, including soft and semi-soft cheese (n = 398) and cooked meat products (n = 403), were collected at retail in Italy and tested for detection and enumeration of Listeria monocytogenes. An Integrative Survey of 2696 samples, including soft and semi-soft cheese (n = 894) and cooked meat products (n = 1802) was carried out to have statistically representative results at the national level. Considering the results obtained both from the EU and the national Integrative Survey, prevalence of contamination of meat products was 1.66% (95% CL: 1.02–2.73%) at the arrival of the samples at the laboratory and 1.92% (95% CL: 1.31%–2.82%) at the end of shelf-life. Spalla cotta was the most frequently contaminated meat product. Prevalence of contamination in cheese was 2.13% (95% CL: 1.37%–3.3%) at the arrival at the laboratory and 1.01% (95% CL: 0.41%–2.55%) at the end of shelf-life. To get information about differences between cheese rinds and pastes, these two parts were separately analysed in the Integrative Survey samples. L. monocytogenes was detected in 4.02% (95% CL: 2.60%–6.19%) of cheese rinds (n = 473), whereas only the 0.34% (95% CL: 0.12%–0.98%) of cheese pastes (n = 894) were contaminated. This difference was statistically significant (χ2 = 10.026, P

Non-typhoidal Salmonella enterica infection is a significant public health problem worldwide. The aim of this study was to characterize Salmonella enterica strains isolated from human specimens in central and southern Italy, for epidemiological studies. One hundred and fifty S. enterica strains were serotyped. Isolates were tested for their antimicrobial susceptibility, by disk diffusion method. The molecular characterizations, based on PCR, were carried out for the detection of invA gene and other virulence elements and phage marker genes. Eighteen different Salmonella serotypes were identified. The most common serotypes detected were S. Typhimurium, S. Enteritidis, the monophasic variant of S. Typhimurium (S. 4,[5],12:i:-), and S. Napoli. High resistance rates were recorded for tetracycline (64%), streptomycin (62%), sulphonamide (57%), and ampicillin (56%). The ASSuT R-type, also associated to resistance to other antibiotics, was highly prevalent in S. 4,[5],12:i:- (97%) and S. Typhimurium (55%), while the ACSSuT Rtype, also associated to other antibiotics, was observed prevalently in S. Typhimurium (20.4%). The genes of more common detection were invA (100%), sspH2 (86.6%), gtgB (84.6%), g8 (80%), sodC1 (77.3%), gipA (52.6%), sspH1 (52.6%).

Non-typhoidal Salmonella enterica infection is a significant public health problem worldwide. The aim of this study was to characterize Salmonella enterica strains isolated from human specimens in central and southern Italy, for epidemiological studies. One hundred and fifty S. enterica strains were serotyped. Isolates were tested for their antimicrobial susceptibility, by disk diffusion method. The molecular characterizations, based on PCR, were carried out for the detection of invA gene and other virulence elements and phage marker genes. Eighteen different Salmonella serotypes were identified. The most common serotypes detected were S. Typhimurium, S. Enteritidis, the monophasic variant of S. Typhimurium (S. 4,[5],12:i:-), and S. Napoli. High resistance rates were recorded for tetracycline (64%), streptomycin (62%), sulphonamide (57%), and ampicillin (56%). The ASSuT R-type, also associated to resistance to other antibiotics, was highly prevalent in S. 4,[5],12:i:- (97%) and S. Typhimurium (55%), while the ACSSuT R-type, also associated to other antibiotics, was observed prevalently in S. Typhimurium (20.4%). The genes of more common detection were invA (100%), sspH2 (86.6%), gtgB (84.6%), g8 (80%), sodC1 (77.3%), gipA (52.6%), sspH1 (52.6%).

Prevalence data were collected from official microbiological records monitoring four selected foodborne pathogens (Salmonella spp., Listeria monocytogenes, Escherichia coli O157:H7 and Campylobacter jejuni) in raw milk sold by self-service vending machines in seven Italian Regions (n. 60907 samples from 1239 vending machines) during the years 2008 to 2011. Data of samples analyzed both by culture-based and real-time PCR methods were collected in one Region. A total of 100 raw milk consumers in four regions were interviewed while purchasing raw milk from vending machines. One hundred and seventy eight samples out of 60907 were positive for one of the four foodborne pathogens investigated; overall, 18 samples were positive for Salmonella spp., 83 for L. monocytogenes, 24 for E. coli O157:H7 and 53 for C. jejuni in the seven Regions investigated. There were no significant differences in prevalence among Regions, but a significant increase in C. jejuni prevalence was observed over the years. A comparison of the two different analysis methods showed that real-time PCR is from 2.71 to 9.40 times more sensitive than culture-based method. Data on consumer habits showed that some behaviors may enhance the risk of infection due to raw milk consumption: 37% of consumers do not boil milk before consumption, 93% never use an insulated bag to transport raw milk home, and raw milk is consumed by children under five years of age. The study emphasizes that end-product controls alone are not sufficient to guarantee an adequate level of consumer protection. The beta distribution of positive samples in this study and the data on raw milk consumer habits are useful and appropriate for the development of a National Quantitative Risk Assessment of Salmonella spp., L. monocytogenes, E. coli O157 and C. jejuni related to raw milk consumption.

A quantitative risk assessment (RA) model was developed to describe the risk of campylobacteriosis linked to consumption of raw milk sold in vending machines in Italy. Exposure assessment was based on the official microbiological records of raw milk samples from vending machines monitored by the regional Veterinary Authorities from 2008 to 2011, microbial growth during storage, destruction experiments, consumption frequency of raw milk, serving size, consumption preference and age of consumers. The differential risk considered milk handled under regulation conditions (4°C throughout all phases) and the worst time-temperature field handling conditions detected. Two separate RA models were developed, one for the consumption of boiled milk and the other for the consumption of raw milk, and two different dose-response (D-R) relationships were considered. The RA model predicted no human campylobacteriosis cases per year either in the best (4°C) storage conditions or in the case of thermal abuse in case of boiling raw milk, whereas in case of raw milk consumption the annual estimated campylobacteriosis cases depend on the dose-response relationships used in the model (D-R I or D-R II), the milk time-temperature storage conditions, consumer behaviour and age of consumers, namely young (with two cut-off values of ≤5 or ≤6 years old for the sensitive population) versus adult consumers. The annual estimated cases for young consumers using D-R II for the sensitive population (≤5 years old) ranged between 1013.7/100,000 population and 8110.3/100,000 population and for adult consumers using D-R I between 79.4/100,000 population and 333.1/100,000 population. Quantification of the risks associated with raw milk consumption is necessary from a public health perspective and the proposed RA model represents a useful and flexible tool to perform future RAs based on local consumer habits to support decision-making on safety policies. Further educational programmes for raw milk consumers or potential raw milk consumers are required to encourage consumers to boil milk to reduce the associated risk of illness.

A quantitative risk assessment (RA) was developed to estimate haemolytic-uremic syndrome (HUS) cases in paediatric population associated with the consumption of raw milk sold in vending machines in Italy. The historical national evolution of raw milk consumption phenomenon since 2008, when consumer interest started to grow, and after 7Âyears of marketing adjustment, is outlined. Exposure assessment was based on the official Shiga toxin-producing Escherichia coli O157:H7 (STEC) microbiological records of raw milk samples from vending machines monitored by the regional Veterinary Authorities from 2008 to 2014, microbial growth during storage, consumption frequency of raw milk, serving size, consumption preference and age of consumers. The differential risk considered milk handled under regulation conditions (4°C throughout all phases) and the worst time–temperature field handling conditions detected. In case of boiling milk before consumption, we assumed that the risk of HUS is fixed at zero. The model estimates clearly show that the public health significance of HUS cases due to raw milk STEC contamination depends on the current variability surrounding the risk profile of the food and the consumer behaviour has more impact than milk storage scenario. The estimated HUS cases predicted by our model are roughly in line with the effective STEC O157-associated HUS cases notified in Italy only when the proportion of consumers not boiling milk before consumption is assumed to be 1%. Raw milk consumption remains a source of E.Âcoli O157:H7 for humans, but its overall relevance is likely to have subsided and significant caution should be exerted for temporal, geographical and consumers behaviour analysis. Health education programmes and regulatory actions are required to educate people, primarily children, on other STEC sources.

The study summarises the results obtained over the period 2002-2013 by the Italian IT-Enter-Vet network, aimed at collecting data on Salmonella isolates from non-human sources. A total of 42,491 Salmonella isolates were reported with a progressive decrease over the years. S. Typhimurium was the most frequent serovar up to 2011, but then, it was overtaken by S. 4,[5],12,:i:-, S. Derby, S. Livingstone and S. Enteritidis alternated as the third most commonly isolated serovars. With regard to the sources of isolation, S. Typhimurium was distributed ubiquitously among the animal species. On the contrary, S. 4,[5],12,:i:- and S. Derby were strictly associated with pigs, whereas S. Livingstone, S. Enteritidis and S. Infantis were clearly related to poultry. Intriguingly, when the frequency of serovar distribution along the food chain was considered, it was evident that S. Typhimurium and S. Derby tended to persist along the chain, as they were isolated even more frequently from foods than from animals. A similar distribution was found for S. Enteritidis and S. Hadar. Despite limitations related to non-mandatory participation of laboratories in the network, the data presented are valuable to obtain a picture of the evolution of Salmonella from non-human sources over time in Italy.

In recent years, the incidence of foodborne diseases caused by shiga toxin-producing Escherichia coli (STEC) has increased globally. For this reason, within the specific regional control plan for the detection of STEC in food products in Italy, the presence of STEC in unpasteurized milk cheeses was investigated. In total 203 samples obtained from March 2011 to December 2013 were analysed, with two standard methods (ISO 16654:2001 and ISO 13136:2012). Two strains of E. coli O157 were isolated (2/161, 1.2%) but did not carry any virulence-associated genes and 22 stx-positive samples (22/146, 15.1%) were detected in enrichment cultures, mostly from ovine cheeses. Only two strains isolated from different ovine cheeses carried stx gene and none of these was eae-positive. This study confirms the presence of stx-positive E. coli and suggests that this type of food cannot be excluded as a potential vehicle of STEC.

The presence of foodborne pathogens (Salmonella spp., Listeria monocytogenes, Escherichia coli O157:H7, thermotolerant Campylobacter, Yersinia enterocolitica and norovirus) in fresh leafy (FL) and ready-to-eat (RTE) vegetable products, sampled at random on the Italian market, was investigated to evaluate the level of risk to consumers. Nine regional laboratories, representing 18 of the 20 regions of Italy and in which 97.7% of the country's population resides, were involved in this study. All laboratories used the same sampling procedures and analytical methods. The vegetable samples were screened using validated real-time PCR (RT-PCR) methods and standardized reference ISO culturing methods. The results show that 3.7% of 1372 fresh leafy vegetable products and 1.8% of 1160 "fresh-cut" or "ready-to-eat" (RTE) vegetable retailed in supermarkets or farm markets, were contaminated with one or more foodborne pathogens harmful to human health.

Following a Listeria monocytogenes detection in a mozzarella cheese sampled at a dairy plant in Lazio Region, further investigations have been conducted both by the competent Authority and the food business operatordairy factory (as a part of dairy factory HACCP control). In total, 90 dairy products, 7 brine and 64 environmental samples have been tested. The prevalence of Listeria monocytogenes was 24.4% in mozzarella cheese, and 9.4% in environmental samples, while brines were all negatives. Forty-seven strains of L. monocytogenes have been isolated, all belonging to 4b/4e serotype. In 12 of these, the macrorestriction profile has been determined by means of pulsed field gel electrophoresis. The profiles obtained with AscI enzyme showed a 100% similarity while those obtained with ApaI a 96.78% similarity. These characteristics of the isolated strains jointly with the production process of mozzarella cheese has allowed to hypothesise an environmental contamination.