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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. The 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.

Bivalves can concentrate biological and chemical pollutants, causing foodborne outbreaks whose occurrence is increasing, due to climatic and anthropic factors that are difficult to reverse, hence the need for improved surveillance. This study aimed to evaluate the hygienic qualities of bivalves sampled along the production and distribution chain in Sicily and collect useful data for consumer safety. Bacteriological and molecular analyses were performed on 254 samples of bivalves for the detection of enteropathogenic Vibrio, Arcobacter spp., Aeromonas spp., Salmonella spp., and beta-glucuronidase-positive Escherichia coli. A total of 96 out of 254 samples, collected in the production areas, were processed for algal biotoxins and heavy metals detection. Bacterial and algal contaminations were also assessed for 21 samples of water from aquaculture implants. Vibrio spp., Arcobacter spp., Aeromonas hydrophila, Salmonella spp., and Escherichia coli were detected in 106/254, 79/254, 12/254, 16/254, and 95/254 molluscs, respectively. A total of 10/96 bivalves tested positive for algal biotoxins, and metals were under the legal limit. V. alginolyticus, A. butzleri, and E. coli were detected in 5, 3, and 3 water samples, respectively. Alexandrium minutum, Dinophysis acuminata, Lingulodinium polyedra, and Pseudonitzschia spp. were detected in water samples collected with the biotoxin-containing molluscs. Traces of yessotoxins were detected in molluscs from water samples containing the corresponding producing algae. Despite the strict regulation by the European Commission over shellfish supply chain monitoring, our analyses highlighted the need for efficiency improvement.

Seafood can vehiculate foodborne illnesses from water to humans. Climate changes, increasing water contamination and coastlines anthropization, favor the global spread of Vibrio spp. and the occurrence of antibiotic-resistant isolates. The aim of this study was to evaluate the spread of potentially pathogenic Vibrio spp. in fishery products collected in Sicily and to assess their antibiotic resistance. Bacteriological and molecular methods were applied to 603 seafood samples to detect V. parahaemolyticus, V. cholerae, V. vulnificus, and Vibrio alginolyticus in order to assess their pathogenicity and antimicrobial resistance. About 30% of bivalves and 20% of other fishery products were contaminated by Vibrio spp.; V. parahaemolyticus accounted for 43/165 isolates, 3 of which were carrying either tdh or trh; V. cholerae accounted for 12/165 isolates, all of them non-O1 non-O139 and none carrying virulence genes; and V. vulnificus accounted for 5/165 isolates. The highest rates of resistance were observed for ampicillin, but we also detected strains resistant to antibiotics currently included among the most efficient against Vibrio spp. In spite of their current low incidence, their rise might pose further issues in treating infections; hence, these results stress the need for a continuous monitoring of antimicrobial resistance among fishery products and an effective risk assessment.

This study shows the frequency of seeds samples contaminated by Salmonella spp. collected randomly from local markets; on 30 black pepper sample no contaminated sample was found while Salmonella spp. was detected in 3 of 36 (8.3%) analyzed sesame samples; three different serotypes were identified: S. Montevideo, S. Stanleyville e S. Tilene. The efficacy of gamma irradiation to inactivate Salmonella Montevideo in black pepper and sesame irradiated between 1 and 5 kGy was evaluated. 3 kGy is sufficient to reduce of 3-4 log CFU/g; whereas 5 kGy have been need to reduce 5.5-6 log CFU/g for samples of black pepper and sesame. No statistically significant differences were found between black pepper and sesame.

Marine biotoxins can accumulate in filter-feeders bivalve molluscs, that may represent a source of potential health problems being vectors of toxins, that are transferred to humans through their consumption. Harmful Algal Blooms impact on aquaculture may give also economic losses due to temporary closures of contaminated shellfish harvest and marketing. The presence of toxic algae for Paralytic Shellfish Poisoning (PSP), with recurrent toxic blooms of dinoflagellates, such as several Alexandrium species, been known since 2000 in the waters of an Ionian bay of Sicily, the Syracuse harbour, where shellfish farms are located. Our previous works reported in this area the positivity for PSP toxin in mussels (Mytilus galloprovincialis) with saxitoxin concentrations above the limit of the law and the simultaneous presence of toxic species of the genus Alexandrium in the waters. This work reports new recent episodes of algal blooms of Alexandrium minutum in the waters of the Syracuse harbour and PSP toxin contamination in farmed mussels, with values beyond the limits established by law, with the consequent immediate closure of the production area. PSP toxicity was detected with the MBA (Mouse Bioassay) with the confirm carried out with Lawrence method to quantify the total saxitoxin equivalents and characterize the toxic profile. Regular application of the implemented health plan is very important in order to prevent any risk and protect consumer health.

Salmonella spp. are among the most frequent causes of foodborne diseases, and the increasing occurrence of MDR strains is an additional cause for concern. In the three-year period 2019–2021, we collected Salmonella spp. strains isolated from different food categories analysed in the context of Regulation (EC) No 2073/2005 in order to assess their antibiotic susceptibility profiles and ESBL production. To determine the susceptibility profiles and identify MDR strains, we used the Kirby–Bauer method to test 17 antibiotics. Double-disc and PCR testing then allowed us to assess the production of ESBLs and the presence of beta-lactamase resistance genes. Phenotypic tests showed that 36 out of 67 strains were MDR and 52.7% of these were ESBL producers. Finally, molecular investigations conducted on ESBL-producing strains revealed the presence of blaSHV, blaCTX-M and blaTEM genes. Our results confirmed the prevalence of S. Infantis, an MDR strain and ESBL producer, in chicken meat. This suggests that further research on the prevalence of antibiotic resistance genes (ARGs) in foodborne strains is needed, especially from a One Health perspective.

The spread of antibiotic resistance (AR) among zoonotic pathogens is a serious health problem, especially because in the last decade the massive use of antibiotics has favored the emergence of Multidrug Resistance (MDR) strains. Some species of the Salmonella genus are among the major causes of foodborne infections worldwide and could represent reservoirs of AR. For these reasons, the susceptibility to six antibiotic classes of 63 strains isolated from animals and food was determined to assess the presence of MDR strains. In addition, the detection of resistance genes was done for strains that resulted in MDR. A statistically significant difference was found when comparing the presence of Salmonella spp. MDR strains between strains isolated from animals and strains isolated from food. Our data seem to indicate that MDR occurs mostly in Salmonella strains isolated from food.

Salmonella spp. is among the leading causes of foodborne infections in humans and a large number of animals. Salmonella spp. is a pathogen involved in the dissemination of antimicrobial resistance because it can accumulate antibiotic resistance genes (ARGs). In this study, the antibiotic resistance profile to 15 antibiotics, belonging to six different classes, of 60 strains of Salmonella spp. collected from pets, farm animals, wildlife, and food in Sicily (Italy) was investigated by the Kirby-Bauer method. Given that almost 33.3% of the Salmonella spp. strains were resistant to tetracycline, Real-Time PCR analysis was applied on all the 60 strains to detect the presence of eight selected tet resistance genes. Besides, the presence of the int1 gene, related to the horizontal gene transfer among bacteria, was also investigated in all the strains by Real-Time PCR analysis. Our data showed that 56% of the isolated strains harbored one or more tet resistance genes and that these strains were most frequently isolated from animals living in close contact with humans. Concerning int1, 17 strains (28.3%) harbored this genetic element and eight of these simultaneously contained tet genes. The results of this study highlight the importance of using a molecular approach to detect resistance genetic determinants, whose spread can increase the diffusion of multidrug-resistant strains. Besides, the study of zoonotic bacteria such as Salmonella spp. which significantly contribute to ARGs dissemination should always follow a One Health approach that considers the health of humans, animals, and the environment to be closely related.

The traditional cultural method (PCR and Real-Time PCR) for Salmonella spp. detection and identification is laborious and time-consuming. A qualitative LAMP method detecting Salmonella spp. was validated in compliance with ISO 16140:2016. The results show a relative accuracy, sensitivity, and specificity of 100% in comparison with the reference method ISO 6579-1:2017; the LOD50 was set as 0.4 CFU/g. Additionally, a field study was carried out comparing the LAMP kit, a commercially available Real-Time PCR kit (FoodProof Salmonella, Biotecon Diagnostics), and the reference cultural method. The Salmonella spp. LAMP kit was suitable for reliable detection of Salmonella spp., simplifying and reducing the extent and the steps of the analytical process. A total of 105 samples of raw poultry meat were screened for the presence of Salmonella spp. according to three methods: the LAMP kit Salmonella spp. (Enbiotech), the Real-Time PCR kit FoodProof Salmonella (Biotecon), and the reference cultural method. Using these three methods, only one sample out of the 105 (0.95%) tested was positive for Salmonella spp. This sample was further investigated using the reference method described in ISO 6579-3:2014, in order to characterise the Salmonella strain. Following this further biochemical identification and serological typing, the isolate was characterised as Salmonella Infantis.

The presence of Salmonella spp. in marine animals is a consequence of contamination from terrestrial sources (human activities and animals). Bacteria present in marine environments, including Salmonella spp., can be antibiotic resistant or harbor resistance genes. In this study, Salmonella spp. detection was performed on 176 marine animals stranded in the Sicilian coasts (south Italy). Antibiotic susceptibility, by disk diffusion method and MIC determination, and antibiotic resistance genes, by molecular methods (PCR) of the Salmonella spp. strains, were evaluated. We isolated Salmonella spp. in three animals, though no pathological signs were detected. Our results showed a low prevalence of Salmonella spp. (1.7%) and a low incidence of phenotypic resistance in three Salmonella spp. strains isolated. Indeed, of the three strains, only Salmonella subsp. enterica serovar Typhimurium from S. coeruleoalba and M. mobular showed phenotypic resistance: the first to ampicillin, tetracycline, and sulphamethoxazole, while the latter only to sulphamethoxazole. However, all strains harbored resistance genes (blaTEM, blaOXA, tet(A), tet(D), tet(E), sulI, and sulII). Although the low prevalence of Salmonella spp. found in this study does not represent a relevant health issue, our data contribute to the collection of information on the spread of ARGs, elements involved in antibiotic resistance, now considered a zoonosis in a One Health approach.

Bartonella henselae is a slow growing and facultative intracellular pathogen mainly transmitted by arthropod vectors adapted to domestic and wild mammalian reservoir hosts. Since cats are the major source of the B. henselae infection, this study aimed to evaluate the seroprevalence and the DNA presence in randomly sampled stray cats. Blood samples of 429 cats were collected from shelter of Palermo (Southern Italy) and sera and whole blood were analyzed for the presence of antibodies against B. henselae by indirect immunofluorescence assay (IFA) and by real-time polymerase chain reaction (PCR), respectively. Two hundred and three sera (47.3%) were positive to IFA and 148 blood samples (34.5%) to real-time PCR. Based on serological results, the evaluation of the potential risk factors (sex, age, coat color) was carried out. The multivariate analysis indicated that cats more than 12 months old were more likely to be seropositive to B. henselae than cats B. henselae in stray cats in Southern Italy.

Arcobacter spp. are emerging waterborne and foodborne zoonotic pathogens responsible for gastroenteritis in humans. In this work, we evaluated the occurrence and the antimicrobial resistance profile of Arcobacter isolates recovered from different aquatic sources. Besides, we searched for Arcobacter spp. in seaweeds and the corresponding seawater samples. Bacteriological and molecular methods applied to 100 samples led to the isolation of 28 Arcobacter isolates from 27 samples. The highest prevalence was detected in rivers followed by artificial ponds, streams, well waters, and spring waters. Seaweeds contained a higher percentage of Arcobacter than the corresponding seawater samples. The isolates were identified as Arcobacter butzleri (96.4%) and Arcobacter cryaerophilus (3.6%). All the isolates showed a multi-drug resistance profile, being resistant to at least three different classes of antibiotics. Molecular analysis of genetic determinants responsible for tetracycline resistance in nine randomly chosen isolates revealed the presence of tetO and/or tetW. This work confirms the occurrence and the continuous emergence of antibiotic-resistant Arcobacter strains in environmental samples; also, the presence of quinolone-resistant Arcobacter spp. in aquatic sources used for water supply and irrigation represents a potential risk for human health.

Borrelia burgdorferi is a bacterial pathogen transmitted by Ixodes ticks and is responsible for Lyme disease in both humans and dogs. The aim of this work was to evaluate B. burgdorferi diffusion among stray dogs in Palermo (Sicily, Italy) by serological methods in order to study the risk factors associated with the infection. Serum and blood samples of 316 dogs were collected from a shelter in Palermo, and were analyzed for the presence of antibodies against B. burgdorferi by indirect immunofluorescence assay (IFA), and of the ospA gene by real-time PCR, respectively. Seventeen sera (5.4%) were positive for the antibodies via IFA and one blood (0.3%) for ospA via real time PCR. On the basis of serological results, the evaluation of the potential risk factors (sex, age, breed and coat color) was carried out. The multivariate analysis indicated that male sex is a factor significantly associated with B. burgdorferi seropositivity. This study confirms that male dogs have a higher risk of developing the disease than females, and represents the first investigation on the spread of B. burgdorferi among stray dogs in Sicily.

The aim of the study was to evaluate the occurrence of Arcobacter spp. in food samples collected from Sicilia region. A total of 91 food products of animal origin (41 meat, 17 fresh milk, 18 shellfish) and 15 samples of fresh vegetables, were examined by cultural method and confirmed by biochemical analysis and PCR methods. The detection of Arcobacter spp. was performed, after selective enrichment, on two selective agar plates: Arcobacter agar and mCCD (modified charcoal cefoperazone deoxycholate) agar supplemented with CAT (Cefoperazone, Amphotericin B and Teicoplanin). Arcobacter species were isolated using the membrane filtration technique. In 13 (14.3%) out of the 91 tested samples, the presence of Arcobacter spp. was found: the isolates were confirmed by multiplex PCR and identified as belonging to the species A. butzleri and A. cryaerophilus. The highest prevalence rate was observed in chicken meat (8.8%) followed by shellfish (3.3%). Negative results have been obtained for raw milks and vegetables samples. The preliminary study highlights the importance of this emerging pathogen and the need for further studies on its prevalence and distribution in different types of food for human consumption.

The present work was carried out to investigate the microbiological profile of Sicilian ewes’ ricotta cheeses during fifteen years of investigations (2002-2016). The samples were collected between those conferred to the Istituto Zooprofilattico Sperimentale della Sicilia (IZSSi) Adelmo Mirri, Palermo (Italy), by the competent authority during official control, by food business operator in HACCP systems and in research projects. Enterobacteriaceae, Escherichia coli and coagulase-positive staphylococci (CPS) were found only in some samples. Bacillus cereus was detected in c.a. 16% of samples but the level of contaminations did not reach the threshold that leads to significant toxin production. Pathogenic bacteria such as Listeria monocytogenes, Salmonella spp. and Brucella spp. were never detected. Furthermore, a total of 47 of lactic acid bacteria (LAB) strains were identified at species level by sequencing the 16S rRNA gene, resulting in the identification of 10 species belonging to four genera Enterococcus, Lactobacillus, Lactococcus and Leuconostoc, commonly employed as starter and non starter cultures in different traditional cheese. Results of this study highlighted an improvement of the hygienic conditions of the Sicilian ewes’ ricotta cheeses during the last ten years of investigation. This observation was confirmed from reduction of undesired microorganisms such as Enterobacteriaceae, E.coli and CPS, used to define the process hygiene criteria. However, in order to improve the final quality of this product are needed further strategy such as the dairy makers training, with the aim to apply a good hygienic practices during the production.

This study aimed at detecting the presence of antibiotic-resistant Gram-negatives in samples of meals delivered at the University General Hospital of Palermo, Italy. Antibiotic resistant Gram negatives were isolated in July—September 2007 ffrom cold dishes and food contact surfaces and utensils. Bacterial strains were submitted to susceptibility test and subtyped by random amplification of polymorphic DNA (RAPD). Forty-six of 55 (83.6%) food samples and 14 of 17 (82.3%) environmental swabs were culture positive for Gram negative bacilli resistant to at least one group of antibacterial drugs. A total of 134 antibiotic resistant strains, 51 fermenters and 83 non-fermenters, were recovered. Fermenters and non-fermenters showed frequencies as high as 97.8% of resistance to two or more groups of antibiotics and non fermenters were 28.9% resistant to more than three groups. Molecular typing detected 34 different profiles among the fermenters and 68 among the non-fermenters. Antibiotic resistance was very common among both fermenters and non-fermenters. However, the wide heterogeneity of RAPD patterns seems to support a prominent role of cross-contamination rather than a clonal expansion of a few resistant isolates. A contribution of commensal Gram negatives colonizing foods to a common bacterial resistance pool should not been overlooked.