Monitoring and Genotyping of Norovirus in Bivalve Molluscan Shellfish from Northern Italian Seas (2018-2020).

Norovirus (NoV) is an enteric virus with foodborne transmission. Bivalve shellfish are a main source of infections and outbreaks. In Italy a voluntary based monitoring plan to check the safety of bivalve shellfish was set up at provincial level. This study describes the occurrence and distribution of NoV in the Northern Adriatic Sea and in the Ligurian Sea. From October 2018 to September 2020, 807 bivalve shellfish samples (n = 205 oysters, n = 182 mussels, n = 348 clams, n = 72 other bivalve shellfish) were tested by One-Step Retrotranscription Real-time polymerase chain reaction for NoV GI and GII and quantified according to the ISO 15216-2:2013 and ISO 15216-1:2017. Positive samples were further analyzed to determine genotype by sequencing of the ORF1/ORF2 junction of the viral genome. A total of 126 samples were positive for NoV, mussels, and oysters had the highest probability of being positive and positive samples were found mainly in the colder season. Of these samples, 46% were NoV GII, 13% NoV GI, and 40% carried both genogroups. Thirty-seven samples were typeable (GI n = 12 and GII n = 25) with GI samples belonging to four genotypes and GII samples belonging to five genotypes. GII.3 genotype was the most prevalent, followed by GII.4, particularly Sydney 2012 subtype, a leading cause of infections worldwide, was found in three oysters' and three clams' samples. The phylogenetic analysis revealed a high heterogeneity among the species that are scattered in several clusters. Considering the low infectious dose the overall presence of NoV in edible shellfish, particular those to be eaten raw or undercooked, is moderately high. The presence of genotypes frequently involved in human infections strengthens the need for ongoing monitoring, which should be extended at national level.

Preparation of Powdered Infant Formula: Could Product's Safety Be Improved?

The recent outbreak of Salmonella Agona linked to the consumption of infant formula (powdered formula) has rekindled the attention about the correct procedures for preparation and use of these products. International guidelines have already been published so far, particularly in association with Cronobacter sakazakii in early 2000s. FAO/WHO suggested to reconstitute formula with water at no less than 70°C. We therefore contaminated powdered formula with low levels of Salmonella spp and C sakazakii to evaluate the pathogens inactivation during the formula preparation using water at 70°C. In these conditions we observed a survival of both pathogens, indicating that the suggested recommendations may be not enough to guarantee the safety of this product. Higher temperatures are needed to reduce the biological risk, even if it may be not easily realized in actual domestic conditions. Moreover, the impact on the nutritional value of reconstituted formulas should be evaluated.

Identification of a major Listeria monocytogenes outbreak clone linked to soft cheese in Northern Italy - 2009-2011.

BACKGROUND: Molecular subtyping and enhanced surveillance in Lombardy region identified a cluster of possibly related listeriosis cases from 2006 to 2010. This cluster grouped 31 isolates that belonged to serotype 1/2a and Sequence Type 38 (ST38) as defined by Multilocus Sequence Typing (MLST). METHODS: Our study expanded the previous investigation to include cases from 2011 to 2014 and used Multi-Virulence-Locus Sequence Typing (MVLST) on all ST38 isolates to better understand their epidemiology and possibly identify a common source outbreak. RESULTS: Out of 306 L. monocytogenes clinical isolates collected, 43 (14.1%) belonged to ST38 with cases occurring in nine out of twelve Lombardy provinces. The ST38 isolates were split by MVLST into two Virulence Types (VTs): VT80 (n = 12) and VT104 (n = 31). VT104 cases were concentrated between 2009 and 2011 in two provinces, Bergamo and Milan. An epidemiologic investigation was performed and in one case, a matching VT104 isolate was retrieved from a soft cheese sample from a patient's refrigerator. CONCLUSIONS: Our findings revealed a major listeriosis outbreak in Northern Italy linked to soft cheese in 2009-2011, which went undetected by local health authorities. Our study shows that integrating subtyping methods with conventional epidemiology can help identify the source of L. monocytogenes outbreak clones.

Waterborne norovirus outbreak during a summer excursion in Northern Italy.

In September 2011, an acute gastroenteritis outbreak affected 33 children in Northern Italy. Patients had drunk river water during an excursion. Identical GI.4 norovirus genomes were detected from one patient's stools and from the river water. Improper discharge of human sewage into the river may have caused this waterborne outbreak.

Noroviruses in seafood: a 9-year monitoring in Italy.

Norovirus (NoV) are increasingly important as etiological agents of gastrointestinal infections. Consumption of bivalve molluscs and ready-to-eat fishery products is one of the most common ways of acquiring NoV foodborne infections, and the rise of outbreaks of viral gastroenteritis represents an important health problem that is also responsible for economic losses. The aim of this work was to define the prevalence of NoV contamination in preserved fishery products and in shellfish commercialized in Italy, taking into account the results obtained during 9 years of survey (2003-2011) and paying special attention to the regions more involved in national production. A total of 4463 samples were examined (2310 mussels, 1517 clams, 510 oysters, 22 other shellfish species, 104 preserved seafood products) and the average positivity rate for NoV presence was 4.1% and ranged from 0.6% in 2007 to 9.8% in 2003 and from 1.9% in preserved seafood products to 4.7% in mussels. Genetic characterization of circulating strains showed a prevalence of genogroup II genotypes, including GII.b and GII.e polymerase types and different GII.4 variants. This information could contribute to the optimization of risk-based sampling strategies for NoV contamination in seafood, taking into account variability in different species and from year to year.

Inactivation of Listeria monocytogenes and Salmonella spp. in Milano-Type Salami Made with Alternative Formulations to the Use of Synthetic Nitrates/Nitrites.

During the manufacture of Italian salami, a traditional meat product, a sequence of hurdles like meat fermentation, air-drying, and long ripening processes are generally sufficient to inhibit the growth of most pathogens. Furthermore, Italian salami are traditionally produced by adding synthetic nitrates/nitrites to raw meat with safety and technological aims, even if controversial opinions about their use still remain, particularly in relation to the consumer demand for natural food products. In this context, the aim of the study was to investigate the inactivation of Listeria monocytogenes and Salmonella spp. during the manufacturing process of Milano-type salami made with different formulations to evaluate the contribution of the hurdles and the vegetable or synthetic additives on the inactivation of pathogens. Thus, a challenge study was performed dividing ca. 400 kg of Milano-type salami batter into three batches: Batch (A) without nitrates/nitrites; Batch (B) with vegetable nitrates, and Batch (C) with synthetic nitrates/nitrites. The batches were separately inoculated with L. monocytogenes and Salmonella spp. and the pathogens' survival was evaluated during the fermentation, draining, and 70-day ripening of the Milano-type salami. The pathogen counts decreased in all tested conditions, even though the highest inactivation of L. monocytogenes and Salmonella spp. (p < 0.05) was observed when nitrates or nitrites were added to the batter. This study shows how the safety of these products cannot exclude the aspect of the hurdle technology during the process, which plays a major role in the reduction of pathogens, but additives like nitrates and nitrites allow for a greater margin of safety. Thus, further studies are needed to validate the use of natural compounds as alternatives to conventional preservatives in meat products. These results may provide new information to support food business operators in producing traditional foods with alternative preservatives and competent authorities in verifying the safety of the products made with natural compounds, and to control the process parameters responsible for the synergistic effect against pathogens such as L. monocytogenes and Salmonella spp.

Is SARS-CoV-2 a Concern for Food Safety? A Very Low Prevalence from a Food Survey during the COVID-19 Pandemic in Northern Italy.

In 2019, SARS-CoV-2 was identified as the cause of an easily transmissible disease that was declared as a world pandemic. Foodborne transmission was never reported. However, early studies suggested that food could be involved in SARS-CoV-2 entry in the human gastrointestinal tract leading to possible infection, and highlighting the importance of further studies to inspect possible issues linked to food consumption. In this perspective, this work aimed at monitoring SARS-CoV-2 presence in some food and mains water samples in Northern Italy during the COVID-19 pandemic (2020-2022). A total of 1806 foods, 112 mains water samples, and 580 swabs on meat and dairy product surfaces were analyzed for SARS-CoV-2 RNA detection by Real-time PCR. All the analyzed samples were negative to viral RNA detection with the exception of one vegetable sample. Even if data on foodborne coronavirus transmission suggested a limited importance of this pathway, the impact of the current pandemic in Northern Italy deserved a rigorous investigation to rule out such possibility. Indeed, gaining insight on all SARS-CoV-2 possible transmission pathways, including the foodborne route, seemed of interest to maintain consumers' confidence and trust in food safety, and for the effective management of the current, and future, possible pandemics.

Isothermal inactivation of Mycobacterium avium subsp. paratuberculosis in curd simulating the stretching phase in pasta-filata cheese process.

Raw milk and dairy products are usually considered the major sources of Mycobacterium avium subsp. paratuberculosis (MAP) exposure for humans. During the production process of mozzarella cheese, as well as of other pasta-filata cheeses made with pasteurized or raw milk, curd is heated and stretched by addition of hot or boiling water. This step is the critical point for the inactivation of MAP during the production process, but, to our knowledge, no studies have been published about the thermal death time values of MAP in curd. The aim of this study was to determine the inactivation kinetics of MAP in curd used to produce pasta-filata cheese in six independent experiments. The milk was inoculated with a mix of MAP strains (field and registered strains) and, with the aim to simulate the thermal treatment of the curd during the stretching step, samples of 10 g of contaminated curd were vacuum packed and treated separately at six different temperatures from 60°C to 75°C in a water bath. MAP survival was then evaluated by plate count method and inactivation parameters were estimated for determining the thermal resistance of the pathogen directly in the curd. D-values increased from 0.15 min (D75-value) to 4.22 min (D60-value) and the calculated z-value was 10.2°C. These data aid: (i) to design food thermal process treatments defining acceptance limits of critical control points to ensure safety against MAP; (ii) to predict the time/temperature combinations needed to obtain a certain MAP log reduction during the curd stretching step; (iii) to optimize or validate pasta-filata cheese process.

Detection of Hepatitis A Virus and Norovirus in Different Food Categories: A 6-Year Survey in Italy.

To observe the prevalence of contamination by hepatitis A virus (HAV) and norovirus (NoV) in different food types, 9242 samples were analyzed over a 6-year period (January 2014-December 2019). Samples were from routine official activities by Competent Authorities (CAs) and Food Business Operators, according to Hazard Analysis and Critical Control Points plans. Analyses were performed in accordance with European and Italian regulations. Food types were obtained from different production areas of Italy, and ranged from mollusks, ready-to-eat (RTE) and packaged vegetables, frozen berries, tap water, fruit and RTE fruit salads, and processed and preserved foods. No risk management plans were set by the authors' laboratory, because they were still adopted by conferring customers. Analyses were conducted according to ISO/TS 15216-2:2013 (ISO in Part 2: Method for Qualitative Detection. International Organization for Standardization, Geneva, 2013). The data showed that 2.25% (95% CI: 2.0-2.6) of samples were contaminated by at least one virus type, and that the most detected pathogen was NoV GII (89.50% of all positives). Mollusks (filter-feeding animals) were the most contaminated category (92.31% of all positives) not only by NoV or HAV individually, but also by multiple HAV/NoV contaminations consisting of 22.59% of all positives. For NoV, there was a significant correlation between shellfish positivity and season, with the autumn-winter period being the most associated with risk. Conversely, berries, drinking water and RTE vegetables, previously linked to several outbreaks, showed a low rate of contamination. These results from data collection have implications for the improvement of sampling plans for HAV and NoV by Italian CAs, and by food-producing and distribution operators. Moreover, these findings obtained by a standardized qualitative method contribute the collection of data aimed at establishing new microbiological criteria not yet foreseen (but advocated) by current European rules.

Molecular characterization of noroviruses and rotaviruses involved in a large outbreak of gastroenteritis in Northern Italy.

Noroviruses and rotaviruses from a gastroenteritis outbreak affecting >300 people near Garda Lake (Northern Italy) in 2009 were investigated. Characterization of viruses from 40 patient stool samples and 5 environmental samples identified three distinct rotavirus and five norovirus genotypes; two of the latter were detected in both patient and environmental samples.

Comparison between two standardized cultural methods and 24 hour duplex SYBR green real-time PCR assay for Salmonella detectionin meat samples.

Food-borne diseases caused by Salmonella represent a worldwide public health problem. Salmonella must be absent in an established amount depending on the kind of the product and usually cultural methods have to be applied to evaluate the compliance of the products. ISO 6579:2002 in Europe and FSIS MLG 4.04.:2008 in the USA have usually been employed to detect Salmonella in meat, poultry and egg products. A Real Time PCR method using probes has recently been validated against the NMKL (Nordic Committee on Food Analysis) standard method. This method has been modified using the less expensive Sybr Green Real Time PCR approach and applied directly in the 18 hours preenrichment broth for the purpose of detecting Salmonella in meat products in less than 24 hours. The purpose of this study was to: - compare the effectiveness of ISO and FSIS cultural methods; - develop a new 24 hour duplex Sybr Green Real Time PCR-melting curve analysis; - evaluate the performance of Salmonella, Standard Method, Rapid Method, SYBR Green Real Time PCR. The equivalence between ISO and FSIS methods was demonstrated and the use of SYBR Green Real Time PCR as a screening tool for negative results seems appealing especially to evaluate compliance with the HACCP systems.

Hepatitis E Virus (HEV) Spread and Genetic Diversity in Game Animals in Northern Italy.

Hepatitis E virus (HEV) is the causative agent of hepatitis E, an emerging public health infection which has an increasing incidence across Europe. Because of the apparent lack of species barriers, HEV was characterized as a zoonotic agent. Swine are recognized as the main reservoir, but HEV is also found in wild animals such as ungulates, lagomorphs, and bats. Our work aimed at detecting the HEV presence in wild fauna in two hunting areas of Northern Italy (Parma and Sondrio areas) with different environmental and anthropic characteristics to investigate its possible role as reservoir. Liver samples were collected from wild boars, red deer, roe deer and chamois, and viral identification was carried out by One-Step RT Real-time PCR. Positive samples were genotyped, and phylogenetic analysis was performed. The virus was found only in the wild boar population, with different prevalence and subtypes in the two areas (14% HEV3a and 1.2% close to HEV3f in Parma and Sondrio, respectively). Wild ruminants seem otherwise to pose a marginal risk. Given the high pig farm density in the Parma area, and expansion of the wild boar population, continuous monitoring of the strains circulating in wildlife is crucial.

One-Day Molecular Detection of Salmonella and Campylobacter in Chicken Meat: A Pilot Study.

Salmonella and Campylobacter ssp. are bacterial pathogens responsible for most foodborne infections in EU countries. Poultry serves as a reservoir for these pathogens, and its important role in the meat industry makes it essential to develop a rapid detection assay able to provide results in one day. Indeed, the rapid identification of foodborne pathogens is an important instrument for the monitoring and prevention of epidemic outbreaks. To date, Salmonella and Campylobacter screening is mainly conducted through molecular methods (PCR or real-time PCR) performed after 18-24 h long enrichments. In this study, we evaluated short enrichments (0, 2, 4, and 6 h) combined with a colorimetric loop-mediated isothermal AMPlification (LAMP) or real-time PCR to detect Salmonella and Campylobacter in poultry meat contaminated at different concentration levels (101, 103, and 105 CFU/g). Our results show that real-time PCR allows the detection of Salmonella and Campylobacter, even after shorter enrichment times than prescribed by ISO references; particularly, it detected Salmonella down to 101 CFU/g since T0 and Campylobacter from 103 CFU/g since T0. Detection with LAMP was comparable to real-time PCR without the requirement of a thermal cycler and with shorter execution times. These characteristics make colorimetric LAMP a valid alternative when one-day results are needed, improving the timely identification of positive meat batches, even in the absence of specialized instrumentation.

A Structural Study on the Listeria Monocytogenes Internalin A-Human E-cadherin Interaction: A Molecular Tool to Investigate the Effects of Missense Mutations.

Listeria monocytogenes is a widespread foodborne pathogen of high concern and internalin A is an important virulence factor that mediates cell invasion upon the interaction with the host protein E-cadherin. Nonsense mutations of internalin A are known to reduce virulence. Although missense mutations are largely overlooked, they need to be investigated in respect to their effects in cell invasion processes. This work presented a computational workflow to early characterize internalin A missense mutations. The method reliably estimated the effects of a set of engineered missense mutations in terms of their effects on internalin A-E-cadherin interaction. Then, the effects of mutations of an internalin A variant from a L. monocytogenes isolate were calculated. Mutations showed impairing effects on complex stability providing a mechanistic explanation of the low cells invasion capacity previously observed. Overall, our results provided a rational approach to explain the effects of internalin A missense mutations. Moreover, our findings highlighted that the strength of interaction may not directly relate to the cell invasion capacity reflecting the non-exclusive role of internalin A in determining the virulence of L. monocytogenes. The workflow could be extended to other virulence factors providing a promising platform to support a better molecular understanding of L. monocytogenes epidemiology.

Investigation and Follow-Up of a Staphylococcal Food Poisoning Outbreak Linked to the Consumption of Traditional Hand-Crafted Alm Cheese.

Staphylococcal food poisoning (SFP) is one of the most important foodborne diseases. This work describes a SFP event linked to the consumption of alm cheese and involved three people belonging to the same family. Leftovers of the consumed cheese, samples from the grocery store and the producing alm were collected and tested for Coagulase positive staphylococci (CPS) enumeration and for the presence of staphylococcal enterotoxins (SEs). Isolates were typed with MLST, spa typing, and tested for SEs and methicillin resistance genes. An in vitro test evaluated SEs production in relation to bacterial growth. The presence of CPS and SEs was detected in all cheese samples and all isolates belonged to the same methicillin sensitive ST8/t13296 strain harbouring sed, ser and sej genes. The in vitro test showed the production of enterotoxins started from 105 CFU/mL. The farmer was prescribed with corrective actions that led to eradication of the contaminating strain.

Geographical restriction of Hepatitis E virus circulation in wild boars (Sus scrofa) in Emilia-Romagna region, Northern Italy.

Hepatitis E virus (HEV) is a singlestrand RNA virus that causes an acute viral hepatitis in humans. Among its eight recognized genotypes, HEV-3 and HEV-4 are zoonotic, infecting humans, pigs and wild boars. Recently, HEV-3 has been also detected in red deer, which represents another reservoir of HEV. Consumption of raw pork products (mainly liver sausages), undercooked wild boar meat, raw wild boar liver and deer meat has been responsible for foodborne HEV human worldwide. From November 2018 to March 2019, liver samples collected from 97 wild boars hunted in Emilia-Romagna region (Northern Italy) were tested for HEV RNA. The hunting area included two territories for an extension of 33 km2, named A (about 13 km2,natural park, deciduous wood) and B (about 20 km2, cultivated fields in proximity of a river) areas. Distance between the two areas ranged between 8 to 10 km. A total of 73 wild boars were hunted in area A, and 24 in area B. HEV RNA was detected by Real-time RT- PCR in 23/73 liver samples of wild boars living in area A only (31.5% - 95% CI: 22.0-42.8%). The HEV sequences (n=13) clustered within genotype 3. The majority of positives belonged to animals < 12 months (12/25; 48%), followed by subadults (13-24 months) (7/16; 43.8%) and adults (4/32; 12.5%). This difference was found to be statistically significant (p=0.0024). In absence of pig farms, the restriction of HEV-positive animals to a well-defined territory of 13 km2 (Boschi di Carrega Regional Park) could hypothetically be related to the presence of red deer (Cervus elaphus), which lived in area A at the beginning of the hunting season. Further studies are needed to confirm or deny our hypothesis.

Assessment of the Antibiotic Resistance Profile, Genetic Heterogeneity and Biofilm Production of Methicillin-Resistant Staphylococcus aureus (MRSA) Isolated from The Italian Swine Production Chain.

The main aim of the present study was to evaluate the level of antibiotic resistance, prevalence and virulence features of methicillin-resistant Staphylococcus aureus (MRSA) isolated from heavy swine at abattoir level and farming environments in Lombardy (Northern Italy). With this scope, 88 different heavy swine farms were surveyed, obtaining a total of n = 440 animal swabs and n = 150 environmental swabs. A total of n = 87 MRSA isolates were obtained, with an overall MRSA incidence of 17.50% (n = 77) among animal samples and a 6.67% (n = 10) among environmental. Molecular characterisation using multilocus sequence typing (MLST) plus spa-typing showed that sequence type ST398/t899 and ST398/t011 were the most commonly isolated genotypes, although other relevant sequence types such as ST1 or ST97 were also found. A lack of susceptibility to penicillins, tetracycline and ceftiofur was detected in >91.95, 85.05 and 48.28% of the isolates, respectively. Resistance to doxycycline (32.18%), enrofloxacin (27.59%) and gentamicin (25.29%) was also observed. Additionally, a remarkable level of antibiotic multiresistance (AMR) was observed representing a 77.01% (n = 67) of the obtained isolates. Genetic analysis revealed that 97.70% and 77.01% of the isolates harboured at least one antibiotic resistance or enterotoxin gene, respectively, pointing out a high isolate virulence potential. Lastly, 55.17% (n = 48) were able to produce measurable amounts of biofilm after 24 h. In spite of the current programmes for antibiotic reduction in intensively farming, a still on-going high level of AMR and virulence potential in MRSA was demonstrated, making this pathogen a serious risk in swine production chain, highlighting once more the need to develop efficient, pathogen-specific control strategies.

Behavior of Listeria innocua during the manufacturing and pit-ripening of Formaggio di Fossa di Sogliano PDO cheese.

Formaggio di Fossa di Sogliano is a traditional Italian Protected Designation of Origin (PDO) cheese ripened for a minimum of 5 months, with the feature of a ripening of at least 80 to at most 100 days in pits, digged into tuffaceous rocks according to medieval tradition of Italy. In this study, a challenge test using Listeria innocua as a surrogate of Listeria monocytogenes was performed, with the aim of increasing knowledge concerning the impact of the Fossa cheese process, and especially of the traditional ripening process of this PDO, on the behaviour of L. monocytogenes. Pasteurized milk was experimentally inoculated with 4.5 log CFU/mL cocktail by three L. innocua strains, and L. innocua and Mesophilic Lactic Acid Bacteria (LAB) counts as well as the evolution of temperatures, pH and aw values were monitored throughout the manufacturing and ripening processes. Throughout the ripening in maturation room a constant temperature of 8°C was observed reaching a temperature between 10 and 15.5°C during ripening into pit. In the final products data for LAB concentration, pH and aw values were roughly in accordance with literature, even if some differences were, probably due to variability of artisanal cheese productions. The numbers of L. innocua showed a slight decrease but remained stable until the end of ripening in maturation room, whereas a significant reduction of the microorganism was observed in the final product, at the end of the ripening into the pit. The findings give scientific evidence that the process of this PDO prevented the L. innocua growth, allowing us to speculate a similar behaviour of L. monocytogenes. Based on this study, the recommendation to extend as much as possible the ripening into pit (from 80 to 100 days) was provided to food business operators as a risk mitigation strategy to be implemented.

Assessment of human enteric viruses in shellfish from the northern Adriatic sea.

Incidence and circulation of different strains of hepatitis A and Norovirus in shellfish were studied on 235 samples (Tapes philippinarum, Mytilus galloprovincialis, Ostrea spp. and Chlamys spp.) obtained from different sites, representing the shellfish production areas of the northern Adriatic sea. Shellfish were harvested in the period of one year and, after depuration, were examined for bacterial (Escherichia coli and Salmonella) and viral (HAV and NoV) contamination. Viral contamination was present on average in 22% of samples: specifically, 6% of samples tested positive for HAV, 14% for NoV and 2% for both viruses. None of the samples revealed the presence of Salmonella, and in most of them (93%) the number of E. coli was below the European legislation limit of 230 MPN/100 g. T. philippinarum was the species most often contaminated, as well as being the only species in which the legal limit for E. coli was, in some cases, exceeded. Both HAV and NoV contamination were detected throughout the year; NoV detection was slightly more frequent during winter months, but positive samples were also present in summer. The sequencing of the PCR products showed the circulation of only one HAV genotype (IA) and four different NoV genotypes (Hawaii, Melksham, Lordsdale and GGIIb) with a prevalence of the GGIIb genotype in the second period of the monitoring.

Diversity and persistence of Listeria monocytogenes within the Gorgonzola PDO production chain and comparison with clinical isolates from the same area.

Listeria monocytogenes causes invasive syndromes with high fatality rates in specific population groups. Cheeses have been commonly implicated in outbreaks worldwide. Gorgonzola is a cheese only produced in Northwestern Italy (it is the third Italian cheese in terms of production and export) and L. monocytogenes is frequently isolated from the production chain. The aims of this study were to assess the distribution of L. monocytogenes Virulence Types (VTs) in isolates collected in Gorgonzola processing plants and to determine the presence of Epidemic Clones (ECs). Fifty-Six L. monocytogenes strains collected between 2004 and 2016 from cheese and environmental samples were subtyped with Multi-Virulence-Locus Sequence Typing (MVLST) and compared to previously typed strains. Most isolates (n=50) belonged to two new VTs (VT113 and VT114). The remaining isolates belonged to previously identified VTs: VT14-ECVIII (milk chocolate outbreak, 1994, USA) and VT80 (ricotta salata outbreak, 2012, USA). VT14, VT80 and VT113 were shared with isolates from apparently sporadic human cases in the same geographical area and temporal period (Piedmont and Lombardy, 2005-2016). The overall L. monocytogenes population appears to be homogeneous and may be characteristic of Gorgonzola production. Nevertheless, the detection in cheese and environmental samples of VTs observed in clinical isolates or outbreak related strains (VT80, VT14) contributed to better describe the current scenario and pointed out the need for increased surveillance.