Staphylococcal food poisoning outbreaks occurred in Sicily (Italy) from 2009 to 2016.

Notification of foodborne outbreaks has been mandatory in Europe since 2005, and surveillance is carried out along the entire food chain. Here we report the results obtained from laboratory investigations about four cases of foodborne outbreaks that occurred in Sicily between 2009 and 2016, deemed to be related to staphylococcal enterotoxins (SEs) and coagulase-positive Staphylococci (CPS) by the Local Public Health Authority. Primosale cheese samples were processed by culture methods for enumeration of CPS and immunoenzymatic assays for detection and differentiation of the SEs possibly contained in food samples. In all cases, the mistrusted foods were found to be contaminated by CPS at bacterial loads between 5 and 8 log CFU/g and contained SE type C (SEC). The reported data confirm the risk of staphylococcal food poisoning associated with the consumption of raw milk cheese. SEC is the most commonly occurring SE in goat milk and dairy products and the most represented enterotoxin in Sicilian dairy products. Our results highlighted the need for improving the current monitoring efficiency and implementing the available laboratory methods to collect more faithful epidemiological data on the current prevalence of staphylococcal toxins in the food chain, including SEs currently not detectable by validated analytical methods.

Phenotypic Antimicrobial Resistance Profiles of Human Campylobacter Species Isolated in Northwest Italy, 2020-2023.

The spread of antimicrobial resistant Campylobacter strains, linked to antimicrobials use and abuse in humans and food animals, has become a global public health problem. In this study, we determine the prevalence of antimicrobial resistance (AMR) in human Campylobacter isolates (n = 820) collected in Piedmont, Italy, from March 2020 to July 2023. The strains underwent susceptibility testing to determine the minimal inhibitory concentration for erythromycin, ciprofloxacin, gentamicin, streptomycin, and tetracycline: 80.1% of the strains showed resistance to at least one antibiotic. The highest prevalence of AMR was noted for ciprofloxacin and tetracycline (72.1% and 52.9%, respectively) and the lowest for erythromycin and aminoglycosides (streptomycin/gentamicin) (3.2% and 5.4%, respectively). The prevalence of co-resistance against fluoroquinolones and tetracyclines was 41.1%. The prevalence of multidrug resistant strains was 5.7%. Our data support evidence that AMR in human Campylobacter strains is common, particularly against ciprofloxacin and tetracycline, two medically important antimicrobials for humans.

Detection and Characterization of Zoonotic Pathogens in Game Meat Hunted in Northwestern Italy.

Wildlife can represent a reservoir of zoonotic pathogens and a public health problem. In the present study, we investigated the spread of zoonotic pathogens (Salmonella spp., Yersinia enterocolitica, Listeria monocytogenes, Shiga-toxin-producing Escherichia coli (STEC), and hepatitis E virus (HEV)) considering the presence of virulence and antibiotic resistance genes in game meat from animals hunted in northwest Italy. During two hunting seasons (2020 to 2022), samples of liver and/or muscle tissue were collected from chamois (n = 48), roe deer (n = 26), deer (n = 39), and wild boar (n = 35). Conventional microbiology and biomolecular methods were used for the detection, isolation, and characterization of the investigated pathogens. Two L. monocytogenes serotype IIa strains were isolated from wild boar liver; both presented fosfomycin resistance gene and a total of 22 virulence genes were detected and specified in the text. Eight Y. enterocolitica biotype 1A strains were isolated from chamois (2), wild boar (5), and deer (1) liver samples; all showed streptogramin and beta-lactam resistance genes; the virulence genes found were myfA (8/8 strains), ymoA (8/8), invA (8/8), ystB (8/8), and ail (4/8). Our data underscore the potential role of wildlife as a carrier of zoonotic and antibiotic-resistant pathogens in northwest Italy and a food safety risk for game meat consumers.

Performance Evaluation of a Commercial Real-Time PCR Method for the Detection of Lupin Traces in Food.

In accordance with U.S. FDA Foods Program Regulatory Science Steering Committee guidelines, with this study, we optimized and validated a commercial real-time PCR method for the detection of low amounts of lupin in four classes of food matrices: chocolate cookies, ragù, Olivier salad, and barley and rice flour. DNA extracted from blank (true negative) samples artificially contaminated with lupin (Lupinus albus) flour at 1000 ppm underwent dilutions with the DNA extracted from the true negative samples up to 0.5 ppm. The limit of detection for real-time PCR was 0.5 ppm in the complex matrices (range, Ct 26-34), making this a specific, robust, and rapid method for lupin allergen detection and labeling. Our validation data support the suitability of this commercially available real-time PCR method for this purpose.

In Vivo Evaluation of the Potential of Thyme and Lemon Hydrolates as Processing Aids to Reduce Norovirus Concentration during Oyster Depuration.

In this study, we evaluated the use of hydrolates, co-products of essential oil distillation, as processing aids to improve the depuration process of Pacific oysters (Crassostrea gigas) as a post-harvest method aimed at reducing the norovirus (NoV) viral load. Live oysters were kept in water to which hydrolates of Thymus serpyllum and Citrus limon at 1% were added for 24 h. The concentration of NoV was quantified using the ISO 15216-1 quantitative real-time RT-PCR method in the oyster digestive tissue both before and after the treatment. The results showed a significant reduction of 0.2 log in the NoV GII concentration after 24 h of treatment with 1% C. limon hydrolate. Conversely, treatment with T. serpyllum did not appear to reduce the concentration of NoV compared to the control. Additionally, a sensory analysis was conducted through a blind survey comparing untreated and treated oysters. No changes in the sensory and physical characteristics of the oysters were observed, except for a decrease in the marine flavour intensity, which was positively perceived by consumers. These results indicate that the addition of hydrolates of C. limon at 1% during depuration might represent a promising processing aid for enhancing both the safety and acceptability of live oysters.

First Report of Food Poisoning Due to Staphylococcal Enterotoxin Type B in Döner Kebab (Italy).

Staphylococcal food poisoning results from the consumption of food contaminated by staphylococcal enterotoxins. In July 2022, the Turin local health board was notified of a suspected foodborne outbreak involving six children who had consumed döner kebab purchased from a takeaway restaurant. The symptoms (vomiting and nausea) were observed 2-3 h later. A microbiological analysis of the food samples revealed high levels (1.5 × 107 CFU/g) of coagulase-positive staphylococci (CPS). The immunoassay detected a contamination with staphylococcal enterotoxins type B (SEB). The whole genome sequencing of isolates from the food matrix confirmed the staphylococcal enterotoxin genes encoding for type B, which was in line with the SEB detected in the food. This toxin is rarely reported in staphylococcal food poisoning, however, because there is no specific commercial method of detection. The involvement of enterotoxin type P (SEP) was not confirmed, though the corresponding gene (sep) was detected in the isolates. Nasal swabs from the restaurant food handlers tested positive for CPS, linking them to the likely source of the food contamination.

Food Safety Monitoring of Salmonella spp. in Northern Italy 2019-2021.

Salmonella is the second most frequent bacterial pathogen involved in human gastrointestinal outbreaks in the European Union; it can enter the food-production chain from animal or environmental sources or from asymptomatic food operators. European food legislation has established microbiological criteria to ensure consumer protection. Salmonella is listed under both process hygiene criteria and food safety criteria. Each EU member state designates an agency to organize or perform controls and other official activities. This paper describes the official control plans performed by competent authorities in Northern Italy in the three-year period 2019-2021. A total of 4413 food samples were delivered to the IZS Food Safety laboratories for Salmonella detection, of which 36 (0.8%) tested positive. Salmonella was most frequently detected in poultry meat samples (25/36 positive samples) followed by other meat products and pork products. The official controls for the protection of consumer health apply the EU's farm-to-fork approach: the samples were collected during production (food production plants), from products on the market, and from collective catering (restaurants, cafeterias, canteens). This manuscript will provide information about the presence of Salmonella in foodstuffs that can help competent authorities to set control plans based on risk assessments.

Soy, Rice and Oat Drinks: Investigating Chemical and Biological Safety in Plant-Based Milk Alternatives.

During the last decades, plant-based milk has become very appreciated by consumers, becoming a staple ingredient, especially for alternative breakfasts. Milk contains lactose, which is a sugar hydrolysed by the lactase enzyme. Lactose intolerance and lactose malabsorption are very common food intolerances among individuals. However, a lot of consumers consider themselves as lactose intolerant on the basis of self-reported intolerance and start to avoid dairy products, ignoring that plant-based milk alternatives are not nutritionally comparable to animal milk, especially in terms of protein intake. The aim of this study is to grow folder knowledge of the security of plant-based drinks, helping competent authorities to issue a risk assessment and to apply national plans about consumer safety. Results show that proper sanitary practices, such as pasteurization, are necessary in plant-based milk alternatives as well as in dairy milk. Chemical analysis has highlighted that there are no pesticide risks for consumers.

Investigation of a Staphylococcus aureus sequence type 72 food poisoning outbreak associated with food-handler contamination in Italy.

On August 2019 a staphylococcal food poisoning outbreak occurred in an elderly home in Piedmont, Italy. The epidemiological investigation performed among the persons that consumed the meal identified chicken salad as the most likely source of the outbreak. Staphylococcus aureus was isolated from a total of seven samples, namely one vomit sample from a guest of the nursing home, two food samples (chicken salad with and without mayonnaise) and nasal swabs collected from a total of four persons working in the kitchen of the nursing home. The maximum likelihood tree obtained using single nucleotide polymorphisms analysis revealed that the isolates from the aforementioned samples clustered together. Multilocus sequence typing revealed that they belonged to Sequence Type 72. Fourier transform infrared spectroscopy (FTIR) was used in parallel to single nucleotide polymorphisms and whole genome sequencing for the determination of the degree of relatedness of the isolates. The results of the FTIR showed the same clustering obtained with single nucleotide polymorphisms and whole genome sequencing and revealed the source of infection. This study underlines the importance of both laboratory evidence and epidemiological data for outbreak investigation and further confirms that FTIR is a suitable support for the short-term epidemiological investigation on source attribution in case of a S. aureus infection.

Monitoring of Antimicrobial Resistance of Salmonella Serotypes Isolated from Humans in Northwest Italy, 2012-2021.

Salmonella enterica is among the most common causes of foodborne outbreaks in humans in Europe. The global emergence of resistance to antimicrobials calls for close monitoring of the spread and prevalence of resistant Salmonella strains. In this study, we investigated the occurrence of antimicrobial resistance of Salmonella serotypes isolated from humans between 2012 and 2021 in Piedmont, northwest Italy. A total of 4814 Salmonella strains (168 serotypes) were tested against six classes of antimicrobials. Many strains (83.3%) showed resistance to at least one antibiotic: tetracycline (85.1%), ampicillin (79.2%), quinolones (47.4%), and gentamicin (28.4%). Between the first (2012-2016) and the second study period (2017-2021), a decrease in antimicrobial resistance was noted for tetracycline (from 92.4% to 75.3%), ampicillin (from 85.3% to 71.3%), quinolones (from 49.4% to 44.6%), and cefotaxime (from 34.8% to 4.0%). Many multidrug resistant Salmonella strains (43.6%) belonged to S. ser. Typhimurium, S. ser. Infantis, and S. ser. Typhimurium 1,4,[5],12:i:-. Overall, multidrug resistance decreased from 60.7% to 26.4%, indicating a reduction in the antimicrobial resistance of Salmonella strains in Piedmont and in Europe and demonstrating the effectiveness of the measures that were put in place to reduce antimicrobial resistance.

Influence of Season and Food Type on Bacterial and Entero-Toxigenic Prevalence of Staphylococcus aureus.

Staphylococcus (S.) aureus is a coagulase-positive pathogen of interest for human health and food safety in particular. It can survive in a wide environmental temperature range (7-48 °C, optimum 37 °C). Its enterotoxins are thermostable, which increases the risk of potential contamination in a variety of food products. Here we investigated the influence of seasonality and food type on bacterial count and presence of S. aureus enterotoxins. To do this, we analyzed 3604 food samples collected over a 5-year period (2016-2020). Ordinal logistic regression showed an influence of both seasonality and food type on the bacterial count. Regarding bacterial counts, winter was found to be the season with the highest risk, while with regards to enterotoxin production, the highest risk was found in autumn, specifically in October. The risk of contamination with S. aureus was greatest for dairy products. Our findings may inform food epidemiologists about foodborne illness prevention and risk to human health.

Surveys on Exposure to Reptile-Associated Salmonellosis (RAS) in the Piedmont Region-Italy.

Reptile-associated salmonellosis (RAS), Salmonella infection in humans, is acquired through contact with reptiles. Reptiles have become popular pet animals, and RAS is likely to be an underestimated but growing problem. No epidemiological data about RAS are routinely collected in Italy. In order to estimate the occurrence of RAS in the Italian human population and to investigate the exposure, two epidemiological studies on patients with sporadic salmonellosis were carried out in the Piedmont region, along with an evaluation of human exposure in public places displaying reptiles and with a survey on people awareness. RAS appeared make up 7% of sporadic salmonellosis in the first study and 3% in the second, more extensive study. A prevalence of 11.7% and 5.7%, respectively, were calculated for the age range of 0-21 years. It was observed that in public places displaying reptiles, it was possible to easily come into contact with the animals and their environment. Some knowledge about RAS emerged from the interviews with the general population, but preventive measures are not completely applied by reptile owners. In conclusion, RAS in Italy is present and constitutes a proportion of the human salmonellosis cases in line with the percentages reported in other countries. Exposure to reptiles should always be considered as a risk factor, and people should be more informed about RAS and the related preventive measures.

Detection of Peanut Traces in Food by an Official Food Safety Laboratory.

Food safety laboratories rely on validated methods that detect hidden allergens in food to ensure the safety and health of allergic consumers. Here we present test results for the validation and accreditation of a real-time PCR assay for the detection of peanut traces in food products. The method was tested on five classes of food matrices: bakery and pastry products, meats, ready-to-eat and dairy products, and grains and milling products. Blank samples were spiked starting with the peanut samples (Arachis hypogaea) at a concentration of 1000 ppm. Serial dilutions were then prepared with the DNA extracted from the blank samples to a final concentration of 0.5 ppm. The limit of detection in grains and milling products, ready-to-eat, meats, bakery and pastry products was 0.5 ppm (range, Ct 27-34) and 2.5 ppm in dairy products (range, Ct 25-34). In order to determine the exclusivity parameter of the method, the ragù matrix was contaminated with Prunus dulcis (almonds), Glycine max (soy), Sinapis alba (mustard), Apium graveolens (celery), Allium cepa (onion), Pisum sativum (peas), Daucus carota (carrots), and Theobroma cacao (cocoa); no cross-reactions were observed. The method was rated satisfactory for sensitivity (98%), specificity (100%), robustness, and repeatability and it was fully validated and accredited.

Goat Paratuberculosis: Experimental Model for the Evaluation of Mycobacterium Persistence in Raw Milk Cheese.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of chronic proliferative enteritis found in ruminants, known as paratuberculosis (PTB). The spread of PTB is increasing in countries with advanced animal husbandry practices, leading to significant economic losses. Moreover, a supposed zoonotic role of MAP in Crohn's disease (CD) in humans has been discussed by the scientific community; however, although the association between MAP and CD has generally been accepted, it is still up for debate if MAP is the main cause of CD, a contributing factor, or merely a commensal organism for the development of CD. The aim of this study was to assess the survival of MAP during the entire production process of a traditional Italian goat's raw milk fresh cheese, the "Robiola di Roccaverano", assessing the survival rate and persistence of MAP in the final product. A mix of MAP field isolates from goats of the Roccaverano area and a reference ATCC strain were used to carry out milk in experimental inoculation. Samples of milk, curd and cheese were taken in two consecutive batches of production. Microbiological challenge tests, evaluated by f57-qPCR, showed a significant decrease in MAP charge during the cheesemaking process for both batches, suggesting the productive process has an impact on MAP survival.

Prevalence and Antimicrobial Resistances of Salmonella spp. Isolated from Wild Boars in Liguria Region, Italy.

Salmonella spp. is an important zoonotic agent. Wild boars might host this pathogen in the intestinal tract and might represent a risk for Salmonella spp. transmission to humans. Wild boars are widely spread in Liguria, due to the environmental characteristics of the region. The aim of the study was the isolation, typing, and investigation of antimicrobial susceptibility of the isolated strains of Salmonella spp. During the 2013-2017 hunting seasons, 4335 livers of wild boars were collected and analyzed for the presence of Salmonella spp. A total of 260 strains of Salmonella spp. were isolated and characterized, with a prevalence of 6%. The isolated strains belonged to all six Salmonella enterica subspecies. Most of them were identified as Salmonella enterica subs. enterica of which 31 different serotypes were identified. The dominating serotype identified was S. Enteritidis. The antimicrobial resistance profiles of the isolated strains were analyzed against sixteen molecules. Of the isolated strains, 94.6% were resistant to at least one of the tested antimicrobials. This study showed the circulation of resistant Salmonella spp. strains in the wild boar population living in this area of Italy, underling the potential risk for these animals to disseminate this pathogen and its antimicrobial resistances.

Antibiotics and Non-Targeted Metabolite Residues Detection as a Comprehensive Approach toward Food Safety in Raw Milk.

Antibiotic residues in milk are a serious health and technological problem in dairy processes. This study aims to verify the absence of administered antimicrobials after therapeutic treatments, taking into consideration the withdrawal period, and to evaluate the reliability of screening tests under field conditions after confirmatory HPLC-HRMS (High Performance Liquid Chromatography-High-Resolution Mass Spectrometry) Orbitrap analysis. Moreover, the presence of expected or non-targeted metabolites was investigated using the new Compound Discoverer approach. The presence of antimicrobial drugs was shown in 29% of the samples, and also sometimes their metabolites (for enrofloxacin and lincomycin), despite the fact that samples were collected at the seventh milking. Moreover, in 9% of the samples, undeclared treatments were revealed due to the presence of both parent drugs and metabolites. Lastly, the putative identification of two new enrofloxacin metabolites, ENRO-N-methylacetamide and ENRO-ornithine, was proposed. In the light of this evidence, it must be borne in mind that metabolites, some of which are pharmacologically active, may also pose a risk to consumers and for the entire processing of milk in the cheese industries.

Comparison of Clostridioides difficile strains from animals and humans: First results after introduction of C. difficile molecular typing and characterization at the Istituto Zooprofilattico Sperimentale of Piemonte, Liguria e Valle d'Aosta, Italy.

PCR ribotypes (RTs027 and 078) are known causes of Clostridioides difficile infection (CDI) in humans. Molecular typing and characterization of 39 C. difficile strains isolated from samples from humas and animals in 2016-2018 indicated an overlap of RTs between community-acquired patients (CA-CDI) and domestic animals from the same geographical area; 14 RTs were identified: 12 RTs were positive for toxins A/B; RT078, RT080 and RT126 were also positive for binary toxin (CDT). Most of the RTs from the animals (RTs020, 078, 106, 126) were also detected in the samples from humans. Strains grouped into three clusters: cluster I included prevalently human strains, mainly RT 018; clusters II and III included strains from humans and animals, mainly RT078 and RT020. The CA-CDI strains suggested animals as a reservoir of C. difficile isolated together with other microorganisms from animals, highlighting the association of enteric pathogens as a cause of infection and death.

NAuRA: Genomic Tool to Identify Staphylococcal Enterotoxins in Staphylococcus aureus Strains Responsible for FoodBorne Outbreaks.

Food contamination by staphylococcal enterotoxins (SEs) is responsible for many food poisoning outbreaks (FPOs) each year, and they represent the third leading cause of FPOs in Europe. SEs constitute a protein family with 27 proteins. However, enzyme immunoassays can only detect directly in food the five classical SEs (SEA-SEE). Thus, molecular characterization methods of strains found in food are now used for FPO investigations. Here, we describe the development and implementation of a genomic analysis tool called NAuRA (Nice automatic Research of alleles) that can detect the presence of 27 SEs genes in just one analysis- and create a database of allelic data and protein variants for harmonizing analyses. This tool uses genome assembly data and the 27 protein sequences of SEs. To include the different divergence levels between SE-coding genes, parameters of coverage and identity were generated from 10,000 simulations and a dataset of 244 assembled genomes from strains responsible for outbreaks in Europe as well as the RefSeq reference database. Based on phylogenetic inference performed using maximum-likelihood on the core genomes of the strains in this collection, we demonstrated that strains responsible for FPOs are distributed throughout the phylogenetic tree. Moreover, 71 toxin profiles were obtained using the NAuRA pipeline and these profiles do not follow the evolutionary history of strains. This study presents a pioneering method to investigate strains isolated from food at the genomic level and to analyze the diversity of all 27 SE-coding genes together.

Hazard perception and possibility of simplifying food safety management systems in small businesses in Piedmont region, Italy.

Small food businesses, because of their limited resources, are hard pressed to comply with the requirements of a conventional food safety management system (FSMS). To overcome it, the European legislation provides some flexibility in the application of FSMS. With this study, we evaluated the change in the perception and awareness of hazards in food production and in the attitudes of food business operators (FBOs) after a regional regulation was introduced to allow flexibility and a campaign of training activities on the FSMS simplification opportunities. Training activities targeting various stakeholders have been carried out in Piedmont region since 2014. A total of 283 FBOs from the dairy and the meat supply chains were recruited for a two-round survey. Overall, the majority of the FBOs believed that application of an FSMS helps to overcome official controls, to produce healthy foods, to better manage the production process, and for staff training; its usefulness for enhancing customer trust was judged of limited value. FBO knowledge on the possibility of simplifying the FSMS activities increased significantly between surveys, suggesting the success of the information campaigns. Over time, simplification increased in the meat but not in the dairy supply chain, where it involved nearly 70% of FBOs. The cost of FSMS (mainly due to microbiological analysis) and the time it takes were the main reasons for FBO resistance to its application. Simplification of FSMS procedures were welcomed by the vast majority (>90%) of FBOs. The perception of hazards was low and generic, suggesting the need for targeted training activities.

An Italian survey of undeclared allergens in food over the years 2014-2018.

Large population studies estimated that the frequency of food allergies is increasing worldwide. In the last two decades, a 'second wave' of the allergy epidemic has emerged as a growing public health problem. EU regulation strengthened information to consumers about allergens in pre-packed food, since December 2014 it has been extended to non-prepacked foods by the Regulation (EU) No 1169/2011 of the European Commission. The present work aims to present a five-year survey on the presence of nine types of allergen in several foods, including food of animal origin and baby food. The concentration found for each irregular allergen is generally low, but some samples with no gluten indication contained a concentration above 10 mg kg-1 with the highest value of 138.5 mg kg-1 in a dietetic food, during the screening in 2017. These data underline the importance and the necessity of a complete food labelling to protect the majority of allergic individuals.