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.

Antimicrobial Resistance of Salmonella Strains Isolated from Human, Wild Boar, and Environmental Samples in 2018-2020 in the Northwest of Italy.

Antimicrobial resistance is one of the most challenging public health problems worldwide, and integrated surveillance is a key aspect in a One Health control strategy. Additionally, Salmonella is the second most common zoonosis in Europe. We aimed to investigate the circulation of Salmonella strains and their related antimicrobial resistance in human, environmental, and wild boar samples from the northwest of Italy, from 2018 to 2020, to obtain a more comprehensive epidemiological picture. Salmonella Typhimurium 1,4,[5],12:i:-, S. Veneziana and S. Newport were the most common serotypes occurring in humans, the environment, and wild boar, respectively. Antimicrobial resistance was rather common in Salmonella isolates, with those from human displaying the highest degree of resistance against sulfadiazine−sulfamerazine−sulfamethazine (>90% of resistance). Moreover, resistance against azithromycin were exclusively observed in environmental samples, while only 7.7% (95% CI = 1.6−20.8) of wild boar isolates experienced resistance against trimethoprim−sulfamethoxazole. Multidrug resistance concurrently involved up to seven antimicrobial classes in human isolates, including third-generation cephalosporins and fluoroquinolones. Salmonella Typhimurium in humans and serotypes Goldcoast and Rissen from environmental sources showed the highest levels of resistance. This study shows diverse antimicrobial resistance patterns in Salmonella strains isolated from different sources and gives a broad picture of antimicrobial resistance spread in wild animals, humans, and the environment.

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.

Evidence of Antimicrobial Resistance and Presence of Pathogenicity Genes in Yersinia enterocolitica Isolate from Wild Boars.

Yersinia enterocolitica (Ye) is a very important zoonosis andwild boars play a pivotal role in its transmission. In the last decade, the wild boar population has undergone a strong increase that haspushed them towards urbanized areas, facilitating the human-wildlife interface and the spread of infectious diseases from wildlife to domestic animals and humans. Therefore, it is important to know the serotype, antimicrobial resistance and presence of pathogenicity genes of Yersinia enterocolitica (Ye) isolated in species. From 2013 to 2018, we analyzed the liver of 4890 wild boars hunted in Liguria region; we isolated and serotyped 126 Ye positive samples. A decisive role in the pathogenicity is given by the presence of virulence genes; in Ye isolated we found ystB (~70%), ymoA (45.2%), ail (43.6%) and ystA (~20%). Moreover, we evaluated the susceptibility at various antimicrobic agents (Ampicillin, Chloramphenicol, Enrofloxacin, Gentamicin, Kanamycin, Trimethoprim-Sulfamethoxazole, Sulfisoxazole, Ceftiofur and Tetracycline). The antibiotic resistance was analyzed, and we found a time-dependent increase. It is important to shed light on the role of the wild boars as a reserve of potentially dangerous diseases for humans, and also on the antibiotic resistance that represents a public health problem.

Thermotolerant Campylobacter spp. in chicken and bovine meat in Italy: Prevalence, level of contamination and molecular characterization of isolates.

Campylobacter species are common foodborne pathogens associated with cases of human gastroenteritis worldwide. A detailed understanding of the prevalence, contamination levels and molecular characteristics of Campylobacter spp. in cattle and chicken, which are likely the most important sources of human contamination, is imperative. A collection of 1243 poultry meat samples (665 chicken breasts and 578 chicken thighs) and 1203 bovine meat samples (689 hamburgers and 514 knife-cut meat preparations) were collected at retail outlets, in randomly selected supermarkets located in different Italian regions during one year. Of these samples, 17.38% of the poultry meat and 0.58% of the bovine meat samples tested positive for Campylobacter, of which 131 were Campylobacter jejuni (57.96%) and 95 were Campylobacter coli (42.03%). Campylobacter isolates were genotyped with the aim of assessing the genetic diversity, population structure, source distribution and Campylobacter transmission route to humans. All isolates were molecularly characterized by pulse field gel electrophoresis (PFGE), and further genotyped using multilocus sequence typing (MLST) and fla-SVR sequencing to gain better insight into the population structure. Antibiotic resistance was also investigate. The highest levels of resistance among chicken strains were observed for ciprofloxacin (88.25%), nalidixic acid (81.45%) and tetracycline (75.6%). PFGE analysis revealed 73 pulsotypes for C. jejuni and 54 pulsotypes for C. coli, demonstrating the existance of different and specific clones circulating in Italy. MLST of C.jejuni isolates mainly clustered in the CC353, CC354, CC21, CC206 and CC443; while C.coli isolates clustered only in CC828. The most common flaA alleles were 287 for C. jejuni and 66 for C. coli. Our study confirms that poultry meat is the main source of Campylobacteriosis, whereas red meat had a low level of contamination suggesting a minor role in transmission. The high presence of Campylobacter in retail chicken meat, paired with its increased resistance to antimicrobials with several multidrug resistance profiles detected, is alarming and represents a persistent threat to public health.

Evaluation of the efficacy of a training course in food safety addressed to food charity volunteers.

In Italy, the Banco Alimentare Onlus manages a network of 8,000 charitable organizations that distribute 67,000 tons of foodstuffs to 1.6 million needy persons. To provide their volunteers with the required food safety knowledge, the Banco Alimentare del Piemonte Onlus commissioned the Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta to hold training courses in food safety. Before and after each session, the participants completed a questionnaire to evaluate their knowledge on the topic of food safety. The responses were entered in a dedicated database and analyzed using STATA ver. 15.1. Comparison of the scores for each participant before and after training revealed a considerable discordance [ICC 0.06, 95% confidence interval (CI) 0.00-0.18]. Analysis of the post-training questionnaires showed that the number of questions left unanswered decreased and the number of correct answers increased. The difference between the percentage of correct and incorrect responses before and after the training course was statistically significant (P<0.001). Comparison of responses to the pre- and post-training questionnaires provided the data for statistical evaluation of the efficacy of the training course.

Outbreak of febrile gastroenteritis caused by Listeria monocytogenes 1/2a in sliced cold beef ham, Italy, May 2016.

In May 2016, two separate clusters of febrile gastroenteritis caused by Listeria monocytogenes were detected by the local health authority in Piedmont, in northern Italy. We carried out epidemiological, microbiological and traceback investigations to identify the source. The people affected were students and staff members from two different schools in two different villages located in the Province of Turin; five of them were hospitalised. The epidemiological investigation identified a cooked beef ham served at the school canteens as the source of the food-borne outbreak. L. monocytogenes was isolated from the food, the stools of the hospitalised pupils and the environment of the factory producing the cooked beef ham. All isolates except one were serotype 1/2a, shared an indistinguishable PFGE pattern and were 100% identical by whole genome sequencing (WGS). By combining a classical epidemiological approach with both molecular subtyping and WGS techniques, we were able to identify and confirm a Listeria gastroenteritis outbreak associated with consumption of sliced cold beef ham.

Draft Genome Sequences of Four Yersinia enterocolitica Strains, Isolated from Wild Ungulate Carcasses.

This study describes the draft genome sequences of four Yersinia enterocolitica strains, originally isolated from ungulate carcasses. These isolates were typed biochemically and two were determined to be highly virulent (biotype 1B). The draft genome sequences had a mean size of 4.77 Mb and a mean G+C content of 47.1%.

Food Safety and Sustainable Nutrition Workshops: Educational Experiences for Primary School Children in Turin, Italy.

European control and prevention policies are focused to guarantee a high level of protection of consumers' health. Food-borne diseases as obesity, diabetes, food allergy, and food-borne outbreaks are increasing. To prevent food-borne diseases, it is fundamental to involve consumers, in particular children, in educational experiences aimed to learn the proper behaviours to be applied. In this context, we designed and performed 5 educational workshops about food safety, hidden allergens in food and nutrition aimed to involve children attending primary and summer school. These experiences let us collect observations about children knowledge and behaviours. From May to October 2015, a total of 1708 children aged 6 to 11 years joined our workshops. Children were involved in listening activities, laboratory experiments, handling games and sensory experiences. All participants were familiar with food allergy and were interested to know how to behave with allergic people. Children showed great curiosity in discovering that many foods normally contain live bacteria. Less than 25% of children reported to skip breakfast, to have it watching TV or to spend few minutes for it. Many of them (>75%) thought that fruits and vegetables are all year-round available and are not related to a specific period. Very few participants (<25%) knew that freezing is the treatment to be applied to make fresh fish safe from parasites. Children involved in food safety and nutrition educational experiences have the opportunity to increase their awareness about the correct behaviours to prevent food-borne diseases and to improve their own critical thinking about food consumption.

Molecular Typing of Staphylococcus Aureus Isolate Responsible for Staphylococcal Poisoning Incident in Homemade Food.

In October 2012, two persons fell ill with symptoms consistent with staphylococcal food poisoning after eating home-canned tuna fish and tomatoes. Laboratory investigation detected the enterotoxins in the home-canned tuna and molecular analysis of the isolated Staphylococcus aureus confirmed it carried toxin genes. Qualitative enzyme-linked immunosorbent assay and enzime linked fluorescent assay methods and quantitative assay identified the enterotoxins in the food leftovers, specifically staphylococcal enterotoxins type A (SEA) and D (SED), respectively 0.49 and 2.04 ng/g. The laboratory results are discussed considering the relation to the fish in oil, survival and heat resistance of S. aureus, and presumptive microbial contamination due to improper handling during home-canning procedures. This is the first reported cluster of foodborne illnesses due to staphylococcal enterotoxins in tuna in Italy. In this study, we reported cases described and analysed for their spa-type. Showing a high heterogeneity of isolates, spa-type t13252 is correlated in a node of the minimum spanning tree and it has never been reported as responsible for foodborne outbreak. This case underlines the importance of risk communication and dissemination of home-canning guidelines to reduce the incidence of foodborne outbreaks caused by homemade conserves.

Canine adenovirus type 1 and Pasteurella pneumotropica co­infection in a puppy.

In 2008, a 2 months-old male German shepherd was presented with fever, depression, and evident organic wasting. The puppy died within 48 hours after the onset of clinical signs. A complete necropsy was performed. Bacteriological examination of samples from the brain, lung, liver, spleen, and bone marrow tested positive for Pasteurella pneumotropica. Histopathology demonstrated inflammatory and vascular lesions in the central nervous system and internal organs. Canine adenovirus type 1 nucleic acid was detected by polymerase chain reaction in the frozen brain but not in the formalin-fixed, paraffin-embedded liver and lung samples. The positive PCR was subsequently confirmed by indirect fluorescent antibody testing of the paraffin-embedded brain and liver sections. Although the liver is the primary site of viral damage, these laboratory findings suggest that Canine adenovirus type 1 infection should be included in the differential diagnosis of neuropathological diseases in dogs and that adenoviral infections could promote septicaemia caused by opportunistic pathogens.

Egg and Milk Proteins as Hidden Allergens in Food: 5-Year (2010 to 2014) Results of Food Allergen Monitoring in Piedmont, Italy.

Cow's milk and egg allergies are two of the most common food allergies. Manufacturers of food products containing milk or eggs or their derivatives as an ingredient are required by European Union regulations to list their presence on the ingredient label. Under European Union legislation, member states are mandated to carry out food safety monitoring programs to verify compliance with food labeling requirements. Through the Regional Integrated Plan for Food Safety, the Piedmont (Italy) regional authority carries out an annual program to determine the presence of undeclared allergens in foods. In the 5-year period from 2010 to 2014, a total of 1,566 food samples were analyzed for the presence of hidden egg and milk proteins. The average positive percentage was 2.8% (3.6% egg and 2% milk proteins). Comparison between the allergen concentration and the published eliciting dose (ED) for egg proteins (0.03 mg) and for total milk proteins (0.1 mg) indicated a high risk of allergen exposure for sensitized consumers. The calculated exposure was up to 135× (for milk) the ED01 reported in the literature. Food manufacturers will need to improve their allergen control programs to reduce allergen exposure and risk.

Evaluation of Hygiene and Safety Criteria in the Production of a Traditional Piedmont Cheese.

Traditional products and related processes must be safe to protect consumers' health. The aim of this study was to evaluate microbiological criteria of a traditional Piedmont cheese, made by two different cheese producers (A and B). Three batches of each cheese were considered. The following seven samples of each batch were collected: raw milk, milk at 38°C, curd, cheese at 7, 30, 60, 90 days of ripening. During cheese making process, training activities dealing with food safety were conducted. Analyses regarding food safety and process hygiene criteria were set up according to the EC Regulation 2073/2005. Other microbiological and chemical-physical analyses [lactic streptococci, lactobacilli, pH and water activity (Aw)] were performed as well. Shiga-toxin Escherichia coli, aflatoxin M1 and antimicrobial substances were considered only for raw milk. All samples resulted negative for food safety criteria; Enterobacteriaceae, E.coli and coagulase-positive staphylococci (CPS) were high in the first phase of cheese production, however they decreased at the end of ripening. A high level of CPS in milk was found in producer A, likewise in some cheese samples a count of >5 Log CFU/g was reached; staphylococcal enterotoxins resulted negative. The pH and Aw values decreased during the cheese ripening period. The competition between lactic flora and potential pathogen microorganisms and decreasing of pH and Aw are considered positive factors in order to ensure safety of dairy products. Moreover, training activities play a crucial role to manage critical points and perform corrective action. Responsible application of good manufacturing practices are considered key factors to obtain a high hygienic level in dairy products.

Occurrence of Thermotolerant Campylobacter in Raw Poultry Meat, Environmental and Pigeon Stools Collected in Open-Air Markets.

Campylobacteriosis was the most commonly reported zoonosis for confirmed human cases in European Union during 2011. Poultry meat was very often implicated in Campylobacter infections in humans. In Italy commerce of raw poultry meat is common in open-air markets: these areas can be considered at high risk of bacterial contamination due to the high presence birds like pigeons. The aim of this study was to collect data about the contamination by thermotolerant Campylobacter of raw poultry meat commercialised in open-air markets, of work-surfaces in contact with poultry meat and of pigeon stools sampled in the market areas in Turin, Northern Italy. Between September 2011 and December 2012, 86 raw poultry meat samples, 86 environmental swabs and 108 animal samples were collected in 38 open-air markets. Analysis were carried out according to ISO10272-1:2006 standard. C.coli was detected in 2.3% (2/86) of raw poultry meat samples, whereas no swab (0/86) resulted positive. Of pigeon stool 28% (30/107) was positive for C.jejuni (83.3% C.jejuni subsp. jejuni and 16.7% C.jejuni subsp. doylei). C.jejuni subsp. jejuni was isolated from 1 dead pigeon. Our results showed lower rates of contamination than those reported at retail in Europe. Although samples were collected in areas at high risk of contamination, raw poultry meat and work surfaces reported a low level of presence of thermotolerant Campylobacter. The high percentage of C.jejuni isolated from pigeon stools showed the importance of a continuous application of preventive measures by the food business operators and the surveillance activity by the Competent Authority.

Food Allergens: State of the Art in Piedmont Region in the Period 2011-2012.

The US National Institutes of Allergy and Infectious Diseases defines food allergy as adverse health effect arising from a specific immune response that occurs reproducibly on exposure to a given food. Undeclared allergens in food label represent a risk for consumers, as there is no therapy for food allergies. According to Directive 2003/89/EC, declaration of all ingredients and derived substances in the label is mandatory. In 2011-2012, in Piedmont region (North-western Italy) 285 food samples were analysed for ß-lactoglobulin and 234 for egg proteins. The aim of this work was to analyse 2 years data in order to assess the presence of undeclared milk and egg allergenic proteins in food placed on the market checking the compliance of labeling of food allergens. Analyses were carried out with ELISA tests, both for the detection of the egg and milk proteins. ß-lactoglobulin was found in 2.8% (8/286) of samples, while egg proteins in 4.7% (11/234).