Microbiological and Toxicological Investigations on Bivalve Molluscs Farmed in Sicily.

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.

Prevalence and antimicrobial resistance profiles of Salmonella spp. in poultry meat.

The spread of multidrug resistant (MDR) Salmonella strains, along the poultry supply chain, can represent a relevant threat to human health. This study aimed to evaluate the prevalence and antimicrobial resistance of Salmonella spp. isolated from poultry meat for human consumption. Between 2019 and 2021, 145 samples were analyzed according to ISO 6579-1:2017. The strains isolated were identified by using biochemical-enzymatic assays and serotyping, according to the Kauffmann-White-Le Minor scheme. The antibiotic susceptibility tests were determined using the Kirby-Bauer method. Forty Salmonella spp. strains were isolated and serotyping showed Salmonella Infantis to be predominant. 80% of the isolated strains were MDR and identified as S. Infantis. This study confirms the circulation of MDR Salmonella isolated from poultry meat and highlights the predominance of the S. Infantis serovar, which represents an emerging risk factor under the One Health holistic approach.

Food risk associated with vegetable consumption, exposure to antimicrobial-resistant strains and pesticide residues.

This preliminary study aimed to detect biological and chemical contaminants in vegetables sold in Sicily for human consumption, assess the spread of antimicrobial-resistant (AMR) strains in these foods, and characterize their antimicrobial-resistance genes. A total of 29 fresh and ready-to-eat samples were analyzed. Microbiological analyses were performed for the detection of Salmonella spp. and the enumeration of Enterococci, Enterobacteriaceae, and Escherichia coli. Antimicrobial resistance was assessed by the Kirby-Bauer method, according to the Clinical and Laboratory Standards Institute guidelines. Pesticides were detected by high-performance liquid chromatography and gas chromatography coupled with mass spectrometry. No samples were contaminated by Salmonella spp., E. coli was detected in 1 sample of fresh lettuce at a low bacterial count (2 log cfu/g). 17.24% of vegetables were contaminated by Enterococci and 65.5% by Enterobacteriaceae (bacterial counts between 1.56 log cfu/g and 5.93 log cfu/g and between 1.6 log cfu/g and 5.48 log cfu/g respectively). From 86.2% of vegetables, 53 AMR strains were isolated, and 10/53 isolates were multidrug resistant. Molecular analysis showed that the blaTEM gene was detected in 12/38 ß-lactam-resistant/intermediate-resistant isolates. Genes conferring tetracycline resistance (tetA, tetB, tetC, tetD, tetW) were detected in 7/10 isolates. The qnrS gene was detected in 1/5 quinolone-resistant isolates, the sulI gene was detected in 1/4 sulfonamide- resistant/intermediate-resistant isolates and the sulIII gene was never detected. Pesticides were detected in 27.3% of samples, all of which were leafy vegetables. Despite the satisfactory hygienic status of samples, the high percentage of AMR bacteria detected stresses the need for an effective monitoring of these foods as well as adequate strategies to counteract the spread of AMR bacteria along the agricultural chain. Also, the chemical contamination of vegetables should not be underestimated, especially considering that leafy vegetables are commonly consumed raw and that no official guidelines about maximum residue limits of pesticides in ready-to-eat vegetables are available.

Food Is Reservoir of MDR Salmonella: Prevalence of ESBLs Profiles and Resistance Genes in Strains Isolated from Food.

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.

Occurrence and Molecular Characterization of Potentially Pathogenic Vibrio spp. in Seafood Collected in Sicily.

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.

Extra-Intestinal Fluoroquinolone-Resistant Escherichia coli Strains Isolated from Meat.

Extra-intestinal E. coli are emerging as a global threat due to their diffusion as opportunistic pathogens and, above all, to their wide set of antibiotic resistance determinants. There are still many gaps in our knowledge of their origin and spread pathways, although food animals have been adjudicated vehicles for passing mult-drug resistant bacteria to humans. This study analyzed 46 samples of meat purchased from retail stores in Palermo in order to obtain quinolone-resistant E. coli isolates. Strains were screened for their phylogenetic groups, ST131-associated single nucleotide polymorphisms (SNPs), and then typed by ERIC-PCR. Their set of virulence factors, namely, kpsMII, papA, sfaS, focG, iutA, papC, hlyD, and afa genes, were investigated and their fluoroquinolone-resistance determinants evaluated. The data obtained show a dramatically high prevalence of multidrug resistance patterns in the Palermo area, with 28% of the isolates having virulence factor genes typical of ExPEC strains. No B2 group or ST131 strains were detected. Moreover, 20% of our isolates showed positivity to all the plasmid-mediated quinolone resistance (PMQR) determinants, showing a potential to transfer these genes among other bacteria. Therefore, these data underline the possibility that food animals and, specifically, poultry in particular may be a significant source of resistant bacterial strains, posing a potential zoonotic risk.

Detection of Arcobacter spp. in food products collected from Sicilia region: A preliminary study.

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.

Assessment of the microbiological quality of fresh produce on sale in Sicily, Italy: preliminary results.

BACKGROUND: Fresh produce occupies an increasingly important place in the human food supply because of its health-promoting nutritional properties. Most fresh produce is eaten raw or after minimal processing and, consequently, pathogen contamination can represent a serious health risk. There has been an increase in foodborne outbreaks and cases associated with fresh produce, but literature data about the prevalence of pathogen contamination are inconsistent. This study was undertaken to assess the hygienic quality and the prevalence of the most common bacterial pathogens in fresh produce sold in retail markets in Sicily. A total of 125 samples of different types of vegetables were examined by standardized microbiological methods. RESULTS: The aerobic mesophilic count ranged between 2 log and 7 log cfu g(-1) and the Enterobacteriaceae counts between < 1 log and 6 log cfu g(-1), with statistically significant differences between unprocessed and minimally processed products (p < 0.05). Escherichia coli was detected only in leaf vegetables at a concentration of 2 log - 3 log cfu g(-1). Enterococci were found at a concentration of 2 log - 4 log cfu g(-1). Coagulase positive Staphylococci and sulphite-reducing Clostridia were not detected in any sample. Three samples tested positive for Listeria monocytogenes, Yersinia enterocolitica and Salmonella veneziana. CONCLUSION: Our study provides updated data on the microbiological quality of retail vegetables and confirms the need to implement strategies to increase microbial safety of fresh produce.

Staphylococcal food poisoning case and molecular analysis of toxin genes in Staphylococcus aureus strains isolated from food in Sicily, Italy.

A case of staphylococcal food poisoning was observed in two individuals of the same family after consumption of primosale, a semiripened sheep cheese produced in Sicily. Staphylococcus aureus isolated from the cheese produced enterotoxin C (SEC) and carried both the enterotoxin C (sec) and the toxic shock syndrome toxin (tsst-1) gene. Following this case, an extensive survey was conducted on 971 food samples (raw milk, cheese, meat, and food preparations). S. aureus was detected in 102 of 971 food samples, from all types of food with the exception of ricotta cheese. The tsst-1 gene was present in 42% of the strains, either alone or in combination with other toxin genes. The enterotoxin C gene was the most represented enterotoxin, but it was only found in dairy products. Six S. aureus isolates carried the sea gene alone, two isolates carried both sea and seb, and one isolate carried both sea and sec. A significant percentage (46%) of all isolates carried a toxin gene, creating significant concern that virulent S. aureus can be transmitted through food in Sicily.