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This study adds insight regarding the occurrence and spread of SARS-CoV-2 Variants of Concern (VOCs) and Var-iants of Interest (VOIs) in Italy in October and November 2022, by testing urban wastewater collected through-out the country. A total of 332 wastewater samples were collected from 20 Italian Regions/Autonomous Provinces (APs) within the framework of national SARS-CoV-2 environmental surveillance. Of these, 164 were collected in the first week of October and 168 in the first week of November. A similar to 1600 bp fragment of the spike protein was sequenced by Sanger (for individual samples) and long-read nanopore sequencing (for pooled Region/AP samples).In October, mutations characteristic of Omicron BA.4/BA.5 were detected in the vast majority (91 %) of the samples amplified by Sanger sequencing. A fraction of these sequences (9 %) also displayed the R346T mutation. Despite the low prevalence documented in clinical cases at the time of sampling, amino acid substitutions characteristic of sublineages BQ.1 or BQ.1.1 were detected in 5 % of sequenced samples from four Regions/APs. A significantly higher variability of sequences and variants was documented in November 2022, when the rate of se-quences harbouring mutations of lineages BQ.1 and BQ1.1 increased to 43 %, and the number of Regions/APs positive for the new Omicron subvariant more than tripled (n = 13) compared to October. Moreover, an increase in the number of sequences with the mutation package BA.4/BA.5 + R346T (18 %), as well as the detection of variants never observed before in wastewater in Italy, such as BA.2.75 and XBB.1 (the latter in a Region where no clinical cases asso-ciated with this variant had ever been documented) was recorded.The results suggest that, as predicted by the ECDC, BQ.1/BQ.1.1 is rapidly becoming dominant in late 2022. Environ-mental surveillance proves to be a powerful tool for tracking the spread of SARS-CoV-2 variants/subvariants in the population.

(1) Background: The surveillance of SARS-CoV-2 RNA in urban wastewaters allows one to monitor the presence of the virus in a population, including asymptomatic and symptomatic individuals, capturing the real circulation of this pathogen. The aim of this study was to evaluate the performance of different pre-analytical and analytical methods for identifying the presence of SARS-CoV-2 in untreated municipal wastewaters samples by conducting an inter-laboratory proficiency test. (2) Methods: three methods of concentration, namely, (A) Dextran and PEG-6000 two-phase separation, (B) PEG-8000 precipitation without a chloroform purification step and (C) PEG-8000 precipitation with a chloroform purification step were combined with three different protocols of RNA extraction by using commercial kits and were tested by using two primers/probe sets in three different master mixes. (3) Results: PEG-8000 precipitation without chloroform treatment showed the best performance in the SARS-CoV-2 recovery; no major differences were observed among the protocol of RNA extraction and the one-step real-time RT-PCR master mix kits. The highest analytic sensitivity was observed by using primers/probe sets targeting the N1/N3 fragments of SARS-CoV-2. (4) Conclusions: PEG-8000 precipitation in combination with real-time RT-PCR targeting the N gene (two fragments) was the best performing workflow for the detection of SARS-CoV-2 RNA in municipal wastewaters.

The SARS-CoV-2 Omicron variant emerged in South Africa in November 2021, and has later been identified worldwide, raising serious concerns. A real-time RT-PCR assay was designed for the rapid screening of the Omicron variant, targeting characteristic mutations of the spike gene. The assay was used to test 737 sewage samples collected throughout Italy (19/21 Regions) between 11 November and 25 December 2021, with the aim of assessing the spread of the Omicron variant in the country. Positive samples were also tested with a real-time RT-PCR developed by the European Commission, Joint Research Centre (JRC), and through nested RT-PCR followed by Sanger sequencing. Overall, 115 samples tested positive for Omicron SARS-CoV-2 variant. The first occurrence was detected on 7 December, in Veneto, North Italy. Later on, the variant spread extremely fast in three weeks, with prevalence of positive wastewater samples rising from 1.0% (1/104 samples) in the week 5-11 December, to 17.5% (25/143 samples) in the week 12-18, to 65.9% (89/135 samples) in the week 19-25, in line with the increase in cases of infection with the Omicron variant observed during December in Italy. Similarly, the number of Regions/Autonomous Provinces in which the variant was detected increased from one in the first week, to 11 in the second, and to 17 in the last one. The presence of the Omicron variant was confirmed by the JRC real-time RT-PCR in 79.1% (91/115) of the positive samples, and by Sanger sequencing in 66% (64/97) of PCR amplicons. In conclusion, we designed an RT-qPCR assay capable to detect the Omicron variant, which can be successfully used for the purpose of wastewater-based epidemiology. We also described the history of the introduction and diffusion of the Omicron variant in the Italian population and territory, confirming the effectiveness of sewage monitoring as a powerful surveillance tool.

Background SARS CoV2 pandemic has generated interest in research of virus spread sources, apart from human contagion. In particular different studies are performed to understand surfaces involvement; during the epidemic event, different types of studies on surfaces have been performed. However, there is little information regarding the presence of the virus in food; wastewater contamination and poor hygiene practices could be the sources of viral contamination in handled food products. The aim of this work was to analyze different types of food for Sars CoV 2 presence. Methods 390 samples of molluscs, 353 vegetables, 10 meat, 72 water, 169 berries, 16 seafood products and ready-to-eat dishes were analysed (2020-2021). Samples were selected from those provided for routine analysis during the pandemic period. The preparation was done based on ISO 15216-2 indications, followed by extraction with Nuclisense®Minimag® system; real-time PCR was applied to detect the Sars CoV2 virus Open Reading Frame 1ab, with two different master mixes. Results All samples tested were negative for ORF1ab detection. Many hypothesis can be made about negative Results: it could be possible that contamination levels in water, or on surfaces in contact with food, are too low to generate a detection signal; again, handling practices may be sufficient to avoid the transmission of the virus to food. Otherwise, the selected genetic target and the Real-time PCR performed, could have been not so efficient in detecting virus as expected. Conclusions In this preliminary work, food does not seem to be a substrate for Sars CoV2 contamination; other food categories or transformation/production processes could be investigated to confirm the obtained results. Also, different targets and amplification systems would have to be evaluated to verify the rule of foodstuffs as Sars CoV2 vehicles. Key messages Primary production and manual processed food don't seem to be at risk for SARS CoV2 contamination. Food has not been identified as a source of Sars CoV2 spreading.

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.

In the dairy industry, the late blowing defect in medium and long ripened cheeses causes important economic losses. This cheese defect, caused mainly by the anaerobic spore former Clostridium tyrobutyricum, is characterized by gas production resulting in holes formation in the paste, and in unpleasant aromas related to the butyrate production. Since longtime, the reference method used to detect the presence of total spore number in milk is the Most Probable Number (MPN), a semi-quantitative method which measures the bacterial growth and the gas production after heat treatment of the samples. This method often gives uncertain results, it requires long incubation periods and fails to differentiate between different species of spore-forming bacteria. A TaqMan real-time quantitative PCR targeting the phosphotransacetylase gene (pta) for C. tyrobutyricum was previously developed as an alternative technique to the traditional method, faster and more specific, and therefore of great interest to preventively determine the contamination of milk by C. tyrobutyricum. The aim of this work was the validation of this quantitative real-time PCR protocol for the C. tyrobutyricum detection in milk. Following the method validation, 120 samples of UHT and raw milk artificially contaminated with C. tyrobutyricum spores were analyzed in increasing concentrations, relating the results of the molecular method with those obtained by the MPN. Finally, 144 raw milk samples with a possible natural contamination were analyzed and demonstrated a positivity rate of 15.28% (CI95% 10.31–22.05%).

Background Shiga toxin-producing Escherichia coli (STEC) are an important public health concern as they cause severe conditions (bloody diarrhea and hemolytic uremic syndrome - HUS) and have epidemic potential. Cattle are the main reservoir for the highly virulent STEC O157 strain, and other HUS-associated non-O157 strains. This study aimed to characterize strains from animals and humans and to compare their molecular profiles. Animal isolates were tested for the presence of virulence factors correlated to their pathogenic potential. Methods 74 STEC human isolates from 43 clinical cases (sporadic or epidemic) and 270 animal isolates from feces from 62 cattle farms were collected. Epidemiological investigation to collect environmental and suspected food samples was carried out for 22 cases. All isolates were typed with PFGE and their serotype was defined by Real-Time PCR. Animal isolates were also tested for the presence of subAB, saa, tia, cfk, adfO, hlyA, efaI1-lifA5’3’, and toxB virulence genes. Results O157 was detected in 44% of human cases, other relevant serogroups O26, O111, O103, O145 in 21, 9, 5 and 5% of cases, respectively. The source of infection was identified in one case (cheese contaminated by a O157 strain). Among animal isolates 2, 0.7, 0.7 and 0.3% were identified as O157, O11, O113, and O145, respectively. PFGE highlighted a high heterogeneicity among animal strains, however no pulsotype common to cattle and clinical isolates was found. adfO, cfk, efaI1-lifA5’3’ and toxB were found significantly correlated to eae (intimin). Conclusions Human and cattle strains were not correlated, however STEC diversity in cattle was very high and included some strains potentially pathogenic to humans. For this reason, upholding an integrated surveillance is very important. Key messages Serotypes relevant to human health were found in cattle in a small but not negligible frequence. No direct correlation was found between animal and clinical isolates.

Background Norovirus (NoV) is the leading cause of acute gastroenteritis in developed countries. Transmission is through direct contact, unsanitary food handling or ingestion of contaminated water or food. Shellfish bioaccumulate NoV and current post-harvest depuration is not effective for its removal. Materials and Methods A monitoring programme for NoV in bivalve shellfish harvested from Northern Adriatic Sea was initiated in 2016. From January 2016 to March 2019, 418 samples including 257 Manila clams, 73 mussels, 46 striped clams, and 42 oysters, were examined. Mollusc samples were tested for NoV genogroups I (NoV GI) and II (NoV GII) contamination by RealTime RT-PCR according to ISO 15216-2. Results Ninety-three out of 418 tested samples (22.2%) were contaminated by at least one NoV genogroup, the simultaneous presence of the two genogroups was detected in 19/418 of the cases. Positive samples were distributed among the tested species as follow: 29/73 (39.7%) mussels, 51/257 (19.5%) Manila clams, 8/42 (19.0%) oysters, 5/46 (10.9%) striped clam. In 2016 (n = 135), 2017 (n = 122), 2018 (n = 132) and in the first trimester of 2019 (n = 12), prevalence of NoV was 6.7%, 24.6%, 31.8% and 41.4%, respectively. NoV GII was largely predominant being detected, alone or in association with GI, in 98.9% of the contaminated samples. On the other hand, GI prevalence increased from 1.5% (2016) to 12.1% in 2018, maybe reflecting a higher circulation of this genogroup in production environments. The vast majority of positive samples (91.4%) were detected during the cold season (November to March). Conclusions Overall positive samples have increased over the years (from 6.7% in 2016 to 31.8% in 2018). NoV GII was the most frequently detected genogroup, but NoV GI prevalence significantly raised in 2018. The routine application of quantitative RT-PCR (ISO 15216-1) to determine the viral load in bivalve molluscs would expand knowledge on potential for foodborne transmission. Key messages Overall positive samples have increased over the years. The routine application of quantitative PCR to determine the viral load in bivalve molluscs would expand knowledge on potential for foodborne transmission.

Issue/problem EU Regulation regarding fresh poultry meat considers only S. enteritidis or S. typhimurium as Food Safety Criteria. On the contrary, the isolation of other Salmonella spp. in this food matrices is considered compliant. Several bibliographical sources indicate an increase in the isolation trend of S. infantis in the poultry industry and in derived products. Description of the problem Episodes of food poisoning caused by fresh chicken meat contaminated with S. infantis have been recently reported. According to CDC, last year in USA, an outbreak caused by a S. infantis strain, resistant to multiple antibiotics, ended with 129 sick people, 25 hospitalizations and a death. National Salmonellosis Control Plan for Poultry 2019-21 establishes in Italy the abatement conditions for poultry farms in case of positive isolation of S. enteritidis, S. typhimurium and S. infantis. Results In 2018, the National Health System disposed the analysis of batches of fresh chicken meat from Poland, sampled in a cold storage facility in Northern Italy. Food Control Dept. of IZSLER analysed 156 samples for a total of 780 sampling units (s.u.), for Salmonella spp.: 72 samples were positive (46,15%;corresponding to 222 s.u. - 28,46%). 42 strains were serotyped as S. enteritidis, 149 as S. infantis, 26 as S. newport, 4 as S. kentucky and 1 as S. mbandaka. According to EU Regulation, batches positive for S. enteritidis were indicated as not compliant, but batches positive to other Salmonella were considered regular. Lessons EU legislation allows the circulation of chicken meat contaminated with S. infantis. Chicken meat should be consumed after appropriate well cooking eliminating this hazard, but it does not protect the consumer from the risk of cross-contamination of tools and surfaces at the household level. It’s therefore fundamental the application of correct processing practices in order to prevent the spreading of Salmonella in home kitchens and limit dangerous episodes of infection. Key messages S. infantis trend of isolation in the poultry industry is increasing. application of GMPs in home cooking is fundamental to limit food outbreaks of infection.

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.

Mammalian orthoreoviruses (MRVs) are emerging infectious agents that may affect wild animals. MRVs are usually associated with asymptomatic or mild respiratory and enteric infections. However, severe clinical manifestations have been occasionally reported in human and animal hosts. An insight into their circulation is essential to minimize the risk of diffusion to farmed animals and possibly to humans. The aim of this study was to investigate the presence of likely zoonotic MRVs in wild ungulates. Liver samples were collected from wild boar, red deer, roe deer, and chamois. Samples originated from two areas (Sondrio and Parma provinces) in Northern Italy with different environmental characteristics. MRV detection was carried out by PCR, confirmation by sequencing and typing for MRV type 3, which has been frequently associated with disease in pigs, were carried out for positive samples. MRV prevalence was as high as 45.3% in wild boars and 40.6% in red deer in the Sondrio area, with lower prevalence in the Parma area (15.4% in wild boars). Our findings shed light on MRV occurrence and distribution in some wild species and posed the issue of their possible role as reservoir.

The classical microbiological method for detection of Listeria monocytogenes requires around 7 days for final confirmation, and due to perishable nature of RTE food products, there is a clear need for an alternative methodology for detection of this pathogen. This study presents an international (at European level) ISO 16140-based validation trial of a non-proprietary real-time PCR-based methodology that can generate final results in the following day of the analysis. This methodology is based on an ISO compatible enrichment coupled to a bacterial DNA extraction and a consolidated real-time PCR assay. Twelve laboratories from six European countries participated in this trial, and soft cheese was selected as food model since it can represent a difficult matrix for the bacterial DNA extraction and real-time PCR amplification. The limit of detection observed was down to 10 CFU per 25 of sample, showing excellent concordance and accordance values between samples and laboratories (>75%). In addition, excellent values were obtained for relative accuracy, specificity and sensitivity (82.75%, 96.70% and 97.62%, respectively) when the results obtained for the real-time PCR-based methods were compared to those of the ISO 11290-1 standard method. An interesting observation was that the L. monocytogenes detection by the real-time PCR method was less affected in the presence of Listeria innocua in the contaminated samples, proving therefore to be more reliable than the reference method. The results of this international trial demonstrate that the evaluated real-time PCR-based method represents an excellent alterative to the ISO standard since it shows a higher performance as well as reduce the extent of the analytical process, and can be easily implemented routinely by the competent authorities and food industry laboratories.

Carnivores are potential carriers of agents that infect their prey species, even though they themselves are not susceptible, such as the lagovirus that causes European brown hare syndrome (EBHS), a severe disease of brown hares endemic in Europe. During our wildlife surveillance in Lombardy, we identified an EBHS outbreak in a protected area by both virological analyses (sandwich ELISA and RT-PCR) of the target organs from one dead hare and serological examinations (competitive ELISA) of captured animals. Since four red foxes were contemporarily hunted in the same area, we examined their organs by RT-PCR for the EBHS agent (EBHSv). The intestinal content of one fox tested positive, while the fox’ other organs (liver, spleen, and mesenteric lymph nodes), and all of the samples from the remaining three foxes, tested negative. Moreover, in the gastrointestinal content of the positive fox, we found food debris that was genetically identified as being of hare origin. The competitive ELISA test for EBHSv antibodies gave negative results in all of the fox sera. Genetic analyses of the EBHSv amplicons obtained by RT-PCR in the hare and the fox indicated a full homology (99.9 % nucleotide and 100 % amino acid identity). These results support the fact that red fox, as other predators, feeding on EBHSv infected hares may have genetic prints of the virus in their gut contents. Even if we did not prove that lagovirus particles remained infective in the excreted feces and, thus, contaminated the ground in the outbreak area, these eventualities cannot be excluded, and we could at least conclude that red foxes might assume a potential role in the indirect transmission of lagovirus, as EBHSv.

Listeria monocytogenes is an important food-borne pathogen causing meningitis, meningo-encephalitis and abortion. Both sporadic and epidemic human listeriosis cases are associated with the consumption of contaminated foods. To assess the potential risk to consumer health, the presence of L. monocytogenes was investigated using qualitative and quantitative methods in raw milk (bulk tank milk and milk for vending machine) collected from 2010 to 2013 in Northern Italy (Lombardy and Emilia-Romagna regions). Overall, L. monocytogenes was detected in 145 on 8716 of raw milk samples, with a prevalence of 1.66% (95% C.I. 1.4%–1.7%). The prevalence ranged from 0.52% (95% C.I. 0.3%–0.9%) in 2012 to 2.7% (95% C.I. 2.0%–3.8%) in 2013, but no trend of increase was observed in four-years of investigation. The pathogen was detected from 2.2% (95% C.I. 1.9%–2.6%) of bulk tank milk and from 0.5% (95% C.I. 0.3%–0.8%) of milk for vending machine. A significative difference (p

The aims of this study were to (1) study the optimal growth condition for two Lactococcus lactis strains to produce the anti-listeria compounds and (2) evaluate the effect of films activated with a twofold concentrated cell-free supernatant of La. lactis to inhibit Listeria monocytogenes. For the growth conditions, skimmed milk added to either Ml7 or MRS provided the highest levels of anti-listeria activity assayed (800 arbitrary unit per milliliter) for both La. lactis strains. A significant decrease of Li. monocytogenes counts was observed in cheese packed in films treated with an average decrease of 2.12 log colony-forming unit per gram after 15 days of storage. Practical Applications The antimicrobial film for food packaging can be used for the preservation of perishable foods such as the sliced cheese. This film developed in our study inhibits Listeria monocytogenes growth in sliced cheese, and does not have an adverse effect on the lactic microbiota, contributing to the preservation of the quality of the product. Thus, it could be a useful tool for the control of the development of pathogens in foods during storage of dairy products.

The aim of this study was to evaluate the behaviour of Escherichia coli, Salmonella Typhimurium and Listeria monocytogenes in an innovative semi-dry reduced fat Italian salami. The product is made from pork meat and it is characterized by less than 20% fat, lactose-, gluten- and milk protein-free. It is developed in Italy according to EC Regulation No. 1924/2006 “on nutrition and health claims made on foods”. Multi-strain cocktails of each pathogen were used to inoculate (5 log cfu g−1) separately the salami batter. During the manufacture and ripening, E. coli, S. Typhimurium, and L. monocytogenes decreased by 2.5, 1.65 and 0.5 log cfu g−1 respectively from the initial inoculated levels. Experimental data indicated that, during the shelf life in a condition of moderate thermal abuse (8–12 °C), the portioned and vacuum packed low fat salami are not able to support the growth of L. monocytogenes.

The demand for rapid methods for the quantification of pathogens is increasing. Among these methods, those based on nucleic acids amplification (quantitative PCRs) are the most widespread worldwide. Together with the qPCR, a new approach named digital PCR (dPCR), has rapidly gained importance. The aim of our study was to compare the results obtained using two different dPCR systems and one qPCR in the quantification of three different bacterial pathogens: Listeria monocytogenes, Francisella tularensis, and Mycobacterium avium subsp. paratuberculosis. For this purpose, three pre-existing qPCRs were used, while the same primers and probes, as well as PCR conditions, were transferred to two different dPCR systems: the QX200 (Bio-Rad) and the Quant Studio 3D (Applied Biosystems). The limits of detection and limits of quantification for all pathogens, and all PCR approaches applied, were determined using genomic pure DNAs. The quantification of unknown decimal suspensions of the three bacteria obtained by the three different PCR approaches was compared through the Linear Regression and Bland and Altman analyses. Our results suggest that, both dPCRs are able to quantify the same amount of bacteria, while the comparison among dPCRs and qPCRs, showed both over and under-estimation of the bacteria present in the unknown suspensions. Our results showed qPCR over-estimated the amount of M. avium subsp. paratuberculosis and F. tularensis cells. On the contrary, qPCR, compared to QX200 dPCR, under-estimated the amount of L. monocytogenes cells. However, the maximum difference among PCRs approaches was

Background and aims. Salmonellosis is one of the most common and widely distributed food-borne diseases. The increasing complexity and globalization of the food industry are causing an increase of some of these large-scale food-borne illnesses, thus there is a need for improvements in public health signal detection and communication streams between laboratories and regulatory agencies. The aim of this study is to show how the early reporting of salmonellosis cases directly from the Laboratory of Microbiology to the Local Health Service Infectious Diseases Office along with the prompt response of the ASL, and the rapid involvement of the Local Veterinary Prevention Department resulted in an improved individuation and investigation of a suspected food-borne outbreak with anomalous manifestation. Materials and methods. From August to November 2014 the early warning from the Laboratory of Microbiology regarding Salmonella spp. isolates with the identical serogroup and antibiotic resistance phenotype, allowed for prompt identification of a food-borne infection. Results and conclusions. The genotyping analysis suggested that over the period considered there was more than a single monophasic Salmonella typhimurium isolate: one responsible for the sporadic cases that occurred in September and October, and another in November.

Silter cheese is a traditional hard cheese, produced in Valcamonica, Brescia, Italy. A total of 426 lactic strains isolated from Silter were analyzed to determine their probiotic characteristics. 274 out of 426 strains were found to produce bacteriocins against at least one of eight different pathogens (Salmonella enterica, Listeria monocytogenes, Salmonella derby, Salmonella thyphimurium, Salmonella napoli, Staphylococcus aureus, E. coli O157:H7, Salmonella enteritidis). In addition, 211 of 274 bactericin-producer strains adhered to Caco-2 cells and were characterized by RiboPrinter, revealing predominance of Enterococcus faecalis (26%) and Enterococcus durans-faecium (22%). These findings suggest that Silter may qualify as an important source of feasible probiotic strains.

Hepatitis A virus (HAV) was detected in two samples of mixed frozen berries linked to Italian hepatitis A outbreak in April and September 2013. Both viruses were fully sequenced by next-generation sequencing and the genomes clustered with HAV complete genomes of sub-genotype IA with nucleotide identities of 95–97 %.

According to EC Regulation No 2073/2005, for food business operators that produce ready-to-eat (RTE) product, it is crucial to be able to demonstrate if the product supports the growth of Listeria monocytogenes. The objective of the study was therefore to evaluate the behaviour of L. monocytogenes in sliced RTE turkey bresaola (made by cured turkey breast 4.5% NaCl, 1% sodium lactate, sodium nitrite 150 ppm and flavouring) during the shelf life of the product, simulating a contamination during the slicing operation. Considering a shelf life of 90 days, as defined by manufacturer, the packages of sliced bresaola were stored at 5°C for 7 days and at 8°C for the remaining storage time (83 days). L. monocytogenes count decreased during storage test from 1.43/1.98 log cfu/g in the three batches tested to 1.03 log cfu/g in one batch and to undetectable levels in the other two batches. The results show that the investigated product is unable to support the growth of L. monocytogenes.

In temperate climates, a seasonal trend was observed in the incidence of human campylobacteriosis cases, with peaks reported in spring and autumn in some countries, or in summer in others; a similar trend was observed in Campylobacter spp. dairy cattle fecal shedding, suggesting that cattle may play a role in the seasonal peak of human infection. The objectives of this study were to assess if a seasonal trend in thermophilic Campylobacter spp. contamination of raw milk exists and to evaluate a possible relation between this and the increase of human campylobacteriosis incidence in summer months. The results showed a mean prevalence of 1.6% of milk samples positive for thermophilic Campylobacter spp. with a wide range in different months during the three years considered of 0.0-3.1%. The statistical analysis showed a significant difference (PCampylobacter spp. between warmer and cooler months (2.3% vs 0.6%). The evidence of a seasonal trend in thermophilic Campylobacter spp. contamination of raw milk sold for direct consumption, with an increase of the prevalence in warmer months, may represent one of the possible links between seasonal trend in cattle fecal shedding and seasonal trend in human campylobacteriosis.

Aims: The aim of the study was to evaluate the behaviour of Listeria monocytogenes in the conditioning liquid of packaged water buffalo mozzarella cheese (WBMC). Methods and Results: The conditioning liquid was contaminated with L. monocytogenes, and the contaminated samples were stored at four different storage temperatures: 5 and 10°C for 22 days; 20°C for 9 days; 20°C for 3 days and then at 5°C for 6 days. The results showed that L. monocytogenes concentration decreased when contaminated samples were stored at 5°C. When WBMC was stored at 20°C and at 10°C, L. monocytogenes started to grow after a lag phase of 3 and 10 days, respectively. When samples were stored at variable temperature conditions, L. monocytogenes numbers showed a lag phase of 5 days. Conclusions: Use of a conditioning liquid characterized by acidity and a correct storage temperature is able to counteract pathogen replication during shelf life. A high concentration of lactic acid bacteria was associated with effective control of L. monocytogenes but the role of lactic acid bacteria in WBMC conditioning liquid requires further investigation. Significance and Impact of the Study: According to European regulations, food producers should be able to justify decision-making on the shelf life assigned to their products, taking into account reasonable storage conditions and use by consumers. The results of the trial yielded information for producers of WBMC and similar cheeses for decision-making on product shelf life.

The classical microbiological method for detection of Salmonella spp. requires more than five days for final confirmation, and consequently there is a need for an alternative methodology for detection of this pathogen particularly in those food categories with a short shelf-life. This study presents an international (at European level) ISO 16140-based validation study of a non-proprietary Real-Time PCR-based method that can generate final results the day following sample analysis. It is based on an ISO compatible enrichment coupled to an easy and inexpensive DNA extraction and a consolidated Real-Time PCR assay. Thirteen laboratories from seven European Countries participated to this trial, and pork meat was selected as food model. The limit of detection observed was down to 10 CFU per 25 g of sample, showing excellent concordance and accordance values between samples and laboratories (100%). In addition, excellent values were obtained for relative accuracy, specificity and sensitivity (100%) when the results obtained for the Real-Time PCR-based methods were compared to those of the ISO 6579:2002 standard method. The results of this international trial demonstrate that the evaluated Real-Time PCR-based method represents an excellent alternative to the ISO standard. In fact, it shows an equal and solid performance as well as it reduces dramatically the extent of the analytical process, and can be easily implemented routinely by the Competent Authorities and Food Industry laboratories.

Vibrio parahaemolyticus is an autochthonous microorganism of the marine environments, frequently isolated from seafood, including bivalve mollusks, and a human pathogen responsible for gastroenteritis outbreaks and sporadic cases. The mechanism of pathogenicity has yet to be comprehensively determined, but two haemolysins (TDH and TRH) have been recognized as virulence factors. The aims of this work were: a) the evaluation of the …

Hepatitis E virus (HEV) is an important public health concern in many developing countries and it occurs in sporadic forms in industrialized areas. With the discovery of swine HEV in pigs, which is genetically closely related to human HEV, hepatitis E is considered to be a zoonotic disease. To investigate the circulation of HEV within a distinct area of Lombardy region (Northern Italy), 17 pig farms were subjected to monitoring study by collection of fresh stool samples each represented by ground-pooled specimens. In particular, three distinct types of breeding farms were focused, represented by farrow to weaning, farrow to finish and fattening farms, respectively. Epidemiological data confirm that in Europe the seroprevalence in pigs, more than 9 month of age, ranges from 51.4 to 75%, while in 3-9 months fatteners is about 38%. In France and Italy, the positivity among farms is respectively 30 and 97.4% and the seroprevalence in Italy is 50.2%. Since HEV viremia was typically observed in the early period of life in swine, faeces were collected in boxes containing weaning pigs. For the study, 183 stool samples were collected and amplifications were performed with universal primers specific for the ORF2 region of genome. Twentyeight samples resulted positive to HEV RNA and genotyping demonstrated that they were closely related to HEV strains belonging to genotype 3 and circulating in Europe. Comparison with reference strains from GenBank excluded their similarity to genotype 1, 2 or 4 confirming that genotype 3 strains are circulating in Europe. Since it was demonstrated that swine act as a reservoir for HEV, and since many strains into HEV genotype 3 share a strong molecular similarity to human HEV, it was important to detect the presence of HEV in a restricted area with a very high density of pigs.

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.

Consumption of raw or insufficiently cooked mussels contaminated with hepatitis A virus (HAV) is a major cause of infection to humans. The origin of mussels commonly used for the preparation of marinated seafood salads is often unknown, since different producers worldwide undergo a precooking treatment at the original collection site with methods and parameters not always indicated. These treatments could be insufficient for the inactivation of HAV, which is characterized by a high temperature resistance. Both high hydrostatic pressure (HHP) and marinade treatments have been shown to affect HAV vitality. In this study, two treatments (HHP and marinating) were combined in order to assess a potential synergistic effect on the virus vitality. A kinetic test was conducted by subjecting the experimentally-contaminated mussels (HAV titre: 10(6)/ml TCID50) to marinating, and to different HHP treatment (4,000; 5,000; and 6,000 bar for 1, 5, and 9 min). Virus post-treatment vitality was assessed by its ability to grow on cell cultures and by quantitative real-time RT-PCR to evaluate virus resistance under such conditions. Marinating treatment alone (final pH 4.3, and NaCl 2 %) did not inactivate the virus. On the other hand, the use of HHP treatment alone on non-marinated HAV-contaminated mussels was effective only above 5,000 bar for 5 min. The results of the present study elucidate the synergistic effect of a combination between marination and HHP treatments on the inactivation of the virus.

In the last years, consequently to EC Regulation no. 1924/2006 on nutrition and health claims made on foods, some Italian food businnes operators (FBOs) leaders in the meat sector, invested in research to develop innovative products such as low fat salami, containing up to 30% less fat than the traditional one. For FBOs it is essential to demonstrate for each production process whether the substrate allows the growth of L. monocytogenes and whether L. monocytogenes could reach or exceed the limit of 100 cfu g–1 at the end of the shelf life, as stated by EC Regulation no. 2073/2005. In the present study, the growth potential of L. monocytogenes during the shelf life of low fat salami packed in modified atmosphere was evaluated. The results show that the product is unable to support the growth of pathogen, even if the storage temperature is between 8 and 12°C.

Formagelle di capra is a raw goat cheese produced from whole chilled goat milk; traditional technology involving unpasteurised milk and indigenous lactic starter cultures is employed for its production in Italy. The purpose of this study was to assess the behaviour of Escherichia coli O157:H7 during the manufacturing and ripening of this raw goat milk cheese. Raw milk was experimentally inoculated with E. coli O157:H7 in a laboratory scale plant and the count was monitored during production and 30 days of ripening required for this cheese. Results showed that E. coli O157:H7 count increased to more than 1.5 Log cfu g–1 during cheese production and remained constant until the end of ripening. The evidence that E. coli O157:H7 is able to survive during the manufacturing and ripening process suggests that the 30-day ripening period alone is insufficient to eliminate levels of viable E. coli O157:H7 in Formaggelle di capra cheese and that the presence of low numbers of E. coli O157:H7 in milk destined for the production of raw goat milk cheeses could represent a potential source of infection for humans and a threat for consumers.