Detection of pathogenic Vibrio spp. in foods: polymerase chain reaction-based screening strategy to rapidly detect pathogenic Vibrio parahaemolyticus, Vibrio cholerae, and Vibrio vulnificus in bivalve mollusks and preliminary results.

The majority of human diseases attributed to seafood are caused by Vibrio spp., and the most commonly reported species are Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae. The conventional methods for the detection of Vibrio species involve the use of selective media, which are inexpensive and simple but time-consuming. The present work aimed to develop a rapid method based on the use of multiplex real-time polymerase chain reaction (PCR) to detect V. parahaemolyticus, V. vulnificus, and V. cholerae in bivalve mollusks. 30 aliquots of bivalve mollusks (Mytilus galloprovincialis) were experimentally inoculated with two levels of V. parahaemolyticus, V. vulnificus, and V. cholerae. ISO 21872-1:2017 was used in parallel for qualitative analysis. The limit of detection of 50% was 7.67 CFU/g for V. cholerae, 0.024 CFU/g for V. vulnificus, and 1.36 CFU/g for V. parahaemolyticus. For V. vulnificus and V. cholerae, the real-time PCR protocol was demonstrated to amplify the pathogens in samples seeded with the lowest and highest levels. The molecular method evaluated showed a concordance rate of 100% with the reference microbiological method. V. parahaemolyticus was never detected in samples contaminated with the lowest level, and it was detected in 14 samples (93.33%) seeded with the highest concentration. In conclusion, the developed multiplex real-time PCR proved to be reliable for V. vulnificus and V. cholerae. Results for V. parahaemolyticus are promising, but further analysis is needed. The proposed method could represent a quick monitoring tool and, if used, would allow the implementation of food safety.

First application of a droplet digital PCR to detect Toxoplasma gondii in mussels.

Toxoplasmosis, caused by the protozoan Toxoplasma gondii, is one of the main food-, water- and soil-borne zoonotic disease worldwide. Over the past 20 years many papers were published on the transmission of T. gondii by marine animals, including mollusks, which can concentrate the oocysts and release them. Sporulated oocysts may remain viable and infective for 18 months in seawater. Therefore, raw or undercooked bivalve mollusks pose a risk to humans. This study aimed to apply and validate for the first time a very sensitive digital droplet polymerase chain reaction (ddPCR) protocol to detect and quantify T. gondii DNA in mussels. Four concentration levels: 8000 genomic copies (gc)/µL, 800 gc/µL, 80 gc/µL, 8 gc/µL of a T. gondii reference DNA were tested. DNA was extracted from 80 pools of mussels (Mytilus galloprovincialis). Forty pools were contaminated with T. gondii reference DNA and used as positive controls, while 40 pools were used as negative controls. DdPCR reaction was prepared using a protocol, previously developed by the authors, for detection of T. gondii in meat. Amplification was obtained up 8 gc/µL. All infected replicates resulted positive, as well as no droplets were detected in negative controls. The droplets produced in the reaction ranged from 8,828 to 14,075 (average 12,627 droplets). The sensitivity and specificity of ddPCR were 100% (95%CI = 94.3-99.9). In addition, 100 pools of mussels collected in the Gulf of Naples were used to validate the protocol. Of these 16% were positive (95% CI = 9.7-25.0) for T. gondii. Samples were also tested by real-time PCR and no positive samples were found. Data obtained from ddPCR showed good identification of negative and positive samples with higher specificity and efficiency than real-time PCR. This tool could be very useful for a rapid sensitive detection of low DNA concentrations of T. gondii in mussels, reducing the risk of toxoplasmosis in humans.

The Sous Vide Cooking of Mediterranean Mussel (Mytilus galloprovincialis): Safety and Quality Assessment.

This study involves an investigation of the effects of various cooking temperatures, freeze-thaw processes, and food preservatives on the quality and shelf-life of sous vide Mediterranean mussels. Cooking temperatures of 80 °C or above significantly improved the microbiological quality, with bacterial counts remaining within the acceptability range for human consumption even after 21 days of refrigerated storage. Fast freezing followed by slow thawing preserved the highest moisture content, potentially improving texture. Sensory analysis revealed that refrigerated sous vide mussels maintained a comparable taste to freshly cooked samples. Frozen samples reheated via microwaving exhibited more intense flavour than pan-reheated or fresh mussels. Food additives, including citric acid, potassium benzoate, and potassium sorbate, alone or in combination with grape seed oil, significantly reduced total volatile basic nitrogen and thiobarbituric acid-reactive substances during 28 days of storage, indicating decreased spoilage and lipid oxidation. Mussels with a combination of these additives registered a nitrogen content as low as 22 mg of N/100g after 28 days, well below the limit of acceptability (<35 mg of N/100g). Food additives also inhibited bacterial growth, with mesophilic bacteria count below 3.35 Log CFU/g after 28 days, compared with 5.37 Log CFU/g in control samples. This study provides valuable insights for developing optimal cooking and preservation methods for sous vide cooked seafood, underscoring the need for further research on optimal cooking and freeze-thaw protocols for various seafood types.

Antimicrobial Resistance and Genomic Characterization of Salmonella Infantis from Different Sources.

The epidemiology of Salmonella Infantis is complex in terms of its distribution and transmission. The continuous collection and analysis of updated data on the prevalence and antimicrobic resistance are essential. The present work aimed to investigate the antimicrobial resistance and the correlation among S. Infantis isolates from different sources through the multiple-locus variable-number of tandem repeat (VNTR) analysis (MLVA). A total of 562 Salmonella strains isolated from 2018 to 2020 from poultry, humans, swine, water buffalo, mussels, cattle, and wild boar were serotyped, and 185 S. Infantis strains (32.92%) were identified. S. Infantis was commonly isolated in poultry and, to a lesser extent, in other sources. The isolates were tested against 12 antimicrobials, and a high prevalence of resistant strains was recorded. S. Infantis showed high resistance against fluoroquinolones, ampicillin, and tetracycline, which are commonly used in human and veterinary medicine. From all S. Infantis isolates, five VNTR loci were amplified. The use of MLVA was not sufficient to understand the complexity of the epidemiological relationships between S. Infantis strains. In conclusion, an alternative methodology to investigate genetic similarities and differences among S. Infantis strains is needed.

Alginate Coating Charged by Hydroxyapatite Complexes with Lactoferrin and Quercetin Enhances the Pork Meat Shelf Life.

In this work, the effect of an alginate-based coating loaded with hydroxyapatite/lactoferrin/quercetin (HA/LACTO-QUE) complexes during the storage of pork meat was evaluated. FT-IR spectra of HA/LACTO-QUE complexes confirmed the adsorption of QUE and LACTO into HA crystals showing the characteristic peaks of both active compounds. The kinetic releases of QUE and LACTO from coatings in an aqueous medium pointed out a faster release of LACTO than QUE. The activated alginate-based coating showed a high capability to slow down the growth of total viable bacterial count, psychotropic bacteria count, Pseudomonas spp. and Enterobacteriaceae during 15 days at 4 °C, as well as the production of the total volatile basic nitrogen. Positive effects were found for maintaining the hardness and water-holding capacity of pork meat samples coated with the activated edible coatings. Sensory evaluation results demonstrated that the active alginate-based coating was effective to preserve the colour and odour of fresh pork meat with overall acceptability up to the end of storage time.

Occurrence and distribution of Salmonella serovars in carcasses and foods in southern Italy: Eleven-year monitoring (2011-2021).

Salmonella is one of the most common agents of foodborne illness. The genus Salmonella includes two species (Salmonella bongori and S. enterica) and six subspecies (enterica I, salamae II, arizonae IIIa, diarizonae IIIb, houtenae IV, and indica VI), each of which contains multiple serotypes associated with animal and human infections. The aim of the study was to evaluate the presence of Salmonella spp. in carcasses of food-producing animals and foods in southern Italy and the serovar distribution among different sources. From 2011 to 2021, a total of 12,246 foods and 982 samples from animal carcasses were collected and analyzed. The overall percentage of positive samples was 5.84% (N = 773) and a significant increase in prevalence was observed by comparing the years 2011-2015 (257, 3.27%) and 2016-2021 (516, 9.61%; p < 0.05). The highest percentage of positive food samples was observed in "Meat and Meat Products" (N = 327/2,438, 13.41%) followed by "Fish and fishery products" (N = 115/1,915, 6.01%). In carcasses, the highest percentage of positive samples was reported from broilers (N = 42/81, 51.85%) followed by buffalo (N = 50/101, 49.50%) and pork (N = 140/380, 36.84%). After typing, the isolates were assigned to the species S. enterica and to the subspecies: enterica (N = 760, 98.32%), diarizonae (N = 8, 1.03%), salamae (N = 3, 0.39%) and houtenae (N = 2, 0.26%). S. Infantis was the most frequently detected (N = 177, 24.76%), followed by S. Derby (N = 77, 10.77%), monophasic S. Typhimurium (N = 63, 8.81%), S. Typhimurium (N = 54, 7.55%), and S. Rissen (N = 47, 6.57%). By comparing the sampling period 2011-2015 with that of 2016-2021, an increase in the prevalence of S. Infantis and monophasic S. Typhimurium and a decrease of S. Typhimurium were recorded (p < 0.05). Thus, present data suggest that, despite the implementation of national and European control strategies to protect against Salmonella, the prevalence of this pathogen in southern Italy is still increasing and a change of national control programs to protect against Salmonella are necessary.

Sponge Whirl-Pak Sampling Method and Droplet Digital RT-PCR Assay for Monitoring of SARS-CoV-2 on Surfaces in Public and Working Environments.

The SARS-CoV-2 can spread directly via saliva, respiratory aerosols and droplets, and indirectly by contact through contaminated objects and/or surfaces and by air. In the context of COVID-19 fomites can be an important vehicle of virus transmission and contribute to infection risk in public environments. The aim of the study was to analyze through surface sampling (sponge method) the presence of SARS-CoV-2 in public and working environments, in order to evaluate the risk for virus transmission. Seventy-seven environmental samples were taken using sterile sponges in 17 animal farms, 4 public transport buses, 1 supermarket and 1 hotel receptive structure. Furthermore, 246 and 93 swab samples were taken in the farms from animals and from workers, respectively. SARS-CoV-2 detection was conducted by real-time RT-PCR and by digital droplet RT-PCR (dd RT-PCR) using RdRp, gene E and gene N as targets. None of the human and animal swab samples were positive for SARS-CoV-2, while detection was achieved in 20 of the 77 sponge samples (26%) using dd RT-PCR. Traces of the RdRp gene, gene E and gene N were found in 17/77 samples (22%, average concentration 31.2 g.c./cm2, range 5.6 to 132 g.c./cm2), 8/77 samples (10%, average concentration 15.1 g.c./cm2, range 6 to 36 g.c./cm2), and in 1/77 (1%, concentration 7.2 g.c./cm2). Higher detection rates were associated with sampling in animal farms and on public transport buses (32% and 30%) compared to the supermarket (21%) and the hotel (no detection). The result of the study suggests that the risk of contamination of surfaces with SARS-CoV-2 increases in environments in which sanitation strategies are not suitable and/or in highly frequented locations, such as public transportation. Considering the analytical methods, the dd RT-PCR was the only approach achieving detection of SARS-CoV-2 traces in environmental samples. Thus, dd RT-PCR emerges as a reliable tool for sensitive SARS-CoV-2 detection.

Development of a droplet digital polymerase chain reaction tool for the detection of Toxoplasma gondii in meat samples.

Toxoplasmosis is a zoonotic disease caused by the protozoan parasite Toxoplasma gondii. Infection in humans has usually been related to the consumption of raw, undercooked or cured meat. The aim of this study was to develop a droplet digital polymerase chain reaction (ddPCR)-based assay for the detection and quantification of T. gondii in meat samples. To optimize the ddPCR, T.gondii reference DNA aliquots at five known concentrations: 8000 cg/µl, 800 cg/µl, 80 cg/µl, 8 cg/µl were used. Moreover, results obtained by ddPCR and quantitative PCR (qPCR) were compared using 80 known samples (40 positive and 40 negative), as well as 171 unknown diaphragm tissue samples collected at slaughterhouses. The ddPCR showed a sensitivity of 97.5% and a specificity of 100%, with a detection limit of 8 genomic copy/µl of T. gondii. A nearly perfect agreement (κ = 0.85) was found between results obtained by ddPCR and qPCR for both positive and negative known samples analysed. On the 171 diaphragm tissue samples from field, 7.6% resulted positive by ddPCR and only 1.2% by qPCR. Therefore, this innovative method could be very useful for the detection of T. gondii in meat samples, aiming to prevent human infections.

Detection of SARS-CoV-2 RNA in Bivalve Mollusks by Droplet Digital RT-PCR (dd RT-PCR).

Bivalve shellfish are readily contaminated by human pathogens present in waters impacted by municipal sewage, and the detection of SARS-CoV-2 in feces of infected patients and in wastewater has drawn attention to the possible presence of the virus in bivalves. The aim of this study was to collect data on SARS-CoV-2 prevalence in bivalve mollusks from harvesting areas of Campania region. A total of 179 samples were collected between September 2019 and April 2021 and were tested using droplet digital RT-PCR (dd RT-PCR) and real-time RT-PCR. Combining results obtained with different assays, SARS-CoV-2 presence was detected in 27/179 (15.1%) of samples. A median viral concentration of 1.1 × 102 and 1.4 × 102 g.c./g was obtained using either Orf1b nsp14 or RdRp/gene E, respectively. Positive results were unevenly distributed among harvesting areas and over time, positive samples being more frequent after January 2021. Partial sequencing of the spike region was achieved for five samples, one of which displaying mutations characteristic of the Alpha variant (lineage B.1.1.7). This study confirms that bivalve mollusks may bioaccumulate SARS-CoV-2 to detectable levels and that they may represent a valuable approach to track SARS-CoV-2 in water bodies and to monitor outbreak trends and viral diversity.

SARS-CoV-2 detection in nasopharyngeal swabs: Performance characteristics of a real-time RT-qPCR and a droplet digital RT-PCR assay based on the exonuclease region (ORF1b, nsp 14).

The emergence and spread of SARS-CoV-2 has led to a compelling request for accurate diagnostic tests. The aim of this study was assessing the performance of a real-time RT-qPCR (rt RT-qPCR) assay and of a droplet digital RT-PCR (dd RT-PCR) targeting the nsp14 genome region for the detection of SARS-CoV-2 in nasopharyngeal swabs. A total of 258 nasopharyngeal swabs were analyzed with the nsp14 assays and, for comparison, with a reference assay targeting the RdRp and E genes. Conflicting results were further investigated by two additional protocols, the Centers for Disease Control and Prevention (CDC) real-time targeting N1/N2, and a nested RT-PCR for the spike region. Agreement of results was achieved on 226 samples (156 positive and 70 negative), 8 samples were positive in the reference assay and in the nsp14 rt RT-qPCR but negative with the dd RT-PCR, and 24 samples provided different combinations of results with the three assays. Sensitivity, specificity and accuracy (95 %C.I.) of the nsp14 assays were: 100.0 % (97.4-100.0), 98.7 % (92.1-100.0), and 99.6 % (97.5-100.0) for the rt RT-qPCR; 92.4 % (87.4-95.6), 100.0 % (94.2-100.0), and 94.7 % (91.1-97.0) for the dd RT-PCR. The results of the study support the use of the nsp14 real-time RT-qPCR and ddPCR for the detection of SARS-CoV-2 in nasopharyngeal swabs.

Impact of Freezing on the Microbiological Quality and Physical Characteristics of Buffalo Mozzarella Cheese.

Buffalo Mozzarella cheese from Campania is one of the most worldwide appreciated Italian dairy products. The increased demand for buffalo dairy products and the limited availability of the finest buffalo milk has prompted the diffusion of illicit practices, such as the use of milk, curd, or other products that are frozen or bought at low cost. The aim of this research was to provide preliminary results about the trend of the microbial communities of buffalo milk, curd and Buffalo Mozzarella cheese, during freezing storage of eleven months. At the same time, the alterations of physical properties and the presence of the molecular marker "γ4-casein", have been investigated. The results showed that freezing reduced the concentrations of the total bacterial count, Enterobacteriaceae, coliforms, Escherichia coli and yeasts in fresh milk and, the concentrations of the total bacterial count, coliforms, lactic acid bacteria and yeasts in mature curd. In the finished product, no notable decreases were observed, except for lactic acid bacteria. About the γ4-casein, no increase was observed in all matrices. These preliminary results allow us to conclude that the freezing process if properly carried out, does not compromise the microbiological quality and the physical properties of the Buffalo Mozzarella cheese.

Lactoferrin, Quercetin, and Hydroxyapatite Act Synergistically against Pseudomonas fluorescens.

Pseudomonas fluorescens is an opportunistic, psychotropic pathogen that can live in different environments, such as plant, soil, or water surfaces, and it is associated with food spoilage. Bioactive compounds can be used as antimicrobials and can be added into packaging systems. Quercetin and lactoferrin are the best candidates for the development of a complex of the two molecules absorbed on bio combability structure as hydroxyapatite. The minimum inhibiting concentration (MIC) of single components and of the complex dropped down the single MIC value against Pseudomonas fluorescens. Characterization analysis of the complex was performed by means SEM and zeta-potential analysis. Then, the synergistic activity (Csyn) of single components and the complex was calculated. Finally, the synergistic activity was confirmed, testing in vitro its anti-inflammatory activity on U937 macrophage-like human cell line. In conclusion, the peculiarity of our study consists of optimizing the specific propriety of each component: the affinity of lactoferrin for LPS; that of quercetin for the bacterial membrane. These proprieties make the complex a good candidate in food industry as antimicrobial compounds, and as functional food.

Towards an Integrated Approach for Monitoring Toxoplasmosis in Southern Italy.

Toxoplasmosis is a widespread worldwide zoonotic infection caused by the intracellular protozoan Toxoplasma gondii. This protozoan infection is considered one of the most important food-borne parasitic zoonoses globally. Beyond its impact on public health, toxoplasmosis has also important veterinary implications, because it causes miscarriage or congenital malformations in livestock with negative economic impacts. An integrated monitoring programme aimed to deepen the epidemiological data on toxoplasmosis and to identify the risk factors that may favour T. gondii infections in animals and humans was conducted in an endemic area of southern Italy. The monitoring activities were based on the following tasks: (i) parasitological analysis and risk factors for T. gondii in livestock (sheep, goat, cattle and water buffalo) farms; (ii) serological and molecular monitoring at slaughterhouse in meat-producing livestock; (iii) analysis of hospital discharge records (HDRs); (iv) outreach activities (information, dissemination and health education) to farmers, vet practitioners and school-age children. The present study confirmed a very high seroprevalence of T. gondii infection in livestock farms (e.g., up to 93.1% in sheep farms) in southern Italy and highlighted the potentially significant public health risk in this area.

Serotyping and Evaluation of Antimicrobial Resistance of Salmonella Strains Detected in Wildlife and Natural Environments in Southern Italy.

Wild animals are potential vectors of antibiotic-resistant bacteria in the environment. The present study aimed to investigate the occurrence of antimicrobial resistance among Salmonella serovars isolated from wildlife and the environment in Italy. A total of 164 Salmonella isolates were analyzed, and six different subspecies and 64 serovars were detected. High proportions of Salmonella isolates proved resistant to streptomycin (34.1%), followed by trimethoprim-sulfamethoxazole (23.2%), tetracycline (17.7%), ciprofloxacin (14.63%) and ampicillin (11.59%). By source, the lowest level of resistance was observed in Salmonella serovars isolated from a water environment, while antimicrobial resistance was frequent in strains collected from shellfish, reptiles and birds. Multidrug-resistant strains were recovered from seafood (n = 11), mammals (n = 3) and water (n = 1). Three S. Typhimurium monophasic variant strains showed asimultaneous resistance to ampicillin, streptomycin, tetracycline and trimethoprim-sulfamethoxazole, which represents a recognized alert resistance profile for this serovar. These data indicate the environmental dissemination of resistant strains due to anthropogenic activities, which, in southern Italy, probably have a higher impact on marine ecosystems than on terrestrial ones. Moreover, as most of the animals considered in the present study are usually consumed by humans, the presence of resistant bacteria in them is a matter of great concern.

Droplet Digital PCR (ddPCR) Analysis for the Detection and Quantification of Cow DNA in Buffalo Mozzarella Cheese.

Buffalo mozzarella cheese is one of the most appreciated traditional Italian products and it is certified as a Protected Designation of Origin (PDO) product under the European Commission Regulation No. 1151/2012. It is obtained exclusively from buffalo milk. If made from cow milk, or a mixture of buffalo and cow milk, buffalo mozzarella cheese does not qualify as a PDO product. In order to maximize their profits, some producers market buffalo mozzarella that also contains cow milk as a PDO product, thus defrauding consumers. New methods for revealing this fraud are therefore needed. One such method is the droplet digital Polymerase Chain Reaction (ddPCR). Thanks to its high precision and sensitivity, the ddPCR could prove an efficacious means for detecting the presence of cow milk in buffalo mozzarella cheese that is marketed as a PDO product. ddPCR has proved able to detect the DNA of cow and/or buffalo milk in 33 buffalo mozzarella cheeses labelled as PDO products, and experimental evidence could support its application in routine analyses.

Exposure to Bacillus cereus in Water Buffalo Mozzarella Cheese.

Bacillus cereus is a spoilage bacterium and is recognized as an agent of food poisoning. Two food-borne illnesses are caused by B. cereus: a diarrheal disease, associated with cytotoxin K, hemolysin BL, non-hemolytic enterotoxin and enterotoxin FM, and an emetic syndrome, associated with the cereulide toxin. Owing to the heat resistance of B. cereus and its ability to grow in milk, this organism should be considered potentially hazardous in dairy products. The present study assessed the risk of B. cereus poisoning due to the consumption of water buffalo mozzarella cheese. A total of 340 samples were analyzed to determine B. cereus counts (ISO 7932:2005); isolates underwent molecular characterization to detect the presence of genes encoding toxins. Eighty-nine (26.1%) samples harbored B. cereus strains, with values ranging from 2.2 × 102 to 2.6 × 106 CFU/g. Isolates showed eight different molecular profiles, and some displayed virulence characteristics. Bacterial counts and the toxin profiles of isolates were evaluated both separately and jointly to assess the risk of enteritis due to B. cereus following the consumption of buffalo mozzarella cheese. In conclusion, the results of the present study showed that the risk of poisoning by B. cereus following the consumption of this cheese was moderate.

Detection and quantification of Campylobacter in foods: New analytic approaches to detect and quantify Campylobacter spp. in food samples.

The aim of the present study was to develop rapid qualitative and quantitative methods based on the use of Real-Time PCR and Droplet Digital PCR (ddPCR), in order to have reliable techniques to detect and quantify Campylobacter spp. in food samples. The gene 16S-rRNA was used as specific target for Campylobacter spp. Real- Time PCR evaluation assay and a not competitive internal control was ushered in it. To investigate the selectivity of the method, 26 Campylobacter strains and 40 non-Campylobacter strains were tested and in order to verify the application of Real- Time PCR method, 5 pork meat samples were experimentally inoculated with a Campylobacter jejuni strain. Subsequently, dilutions with a bacterial load of Campylobacter jejuni within 10-106 CFU/mL were chosen for the optimization of the ddPCR assay. Lastly, a total of 54 naturally contaminated foods samples were analyzed through molecular (Real-Time PCR and ddPCR) and traditional methods. The Real-Time PCR protocol demonstrated to amplify only the Campylobacter spp. strains and when Campylobacter jejuni was experimentally inoculated in meat samples the pathogen was always detected. The ddPCRs assay allowed to quantify a level of contamination of 10 CFU/mL, but it was unable to quantify levels of 105 - 106 CFU/mL. Lastly, Campylobacter spp. was never detected in the 54 samples tested. In conclusion, the novel analytic approach proposed, based on an initial screening of the samples with Real-Time PCR and then on quantification of Campylobacter spp. with a ddPCR on those positive, represents a quick monitoring tool and, if used correctly, it would allow the implementation of food safety.

Identification and Antimicrobial Activity of Medium-Sized and Short Peptides from Yellowfin Tuna (Thunnus albacares) Simulated Gastrointestinal Digestion.

Due to the rapidly increasing resistance to conventional antibiotics, antimicrobial peptides are emerging as promising novel drug candidates. In this study, peptide fragments were obtained from yellowfin tuna muscle by simulated gastrointestinal digestion, and their antimicrobial activity towards Gram-positive and Gram-negative bacteria was investigated. In particular, the antimicrobial activity of both medium- and short-sized peptides was investigated by using two dedicated approaches. Medium-sized peptides were purified by solid phase extraction on C18, while short peptides were purified thanks to a graphitized carbon black sorbent. For medium-sized peptide characterization, a peptidomic strategy based on shotgun proteomics analysis was employed, and identification was achieved by matching protein sequence database by homology, as yellowfin tuna is a non-model organism, leading to the identification of 403 peptides. As for short peptide sequences, an untargeted suspect screening approach was carried out by means of an inclusion list presenting the exact mass to charge ratios (m/z) values for all di-, tri- and tetrapeptides. In total, 572 short sequences were identified thanks to a customized workflow dedicated to short peptide analysis implemented on Compound Discoverer software.

Antimicrobial Susceptibility Testing for Salmonella Serovars Isolated from Food Samples: Five-Year Monitoring (2015-2019).

The continuous collection and analysis of updated data on the antimicrobic resistance among bacterial strains represent the essential core for the surveillance of this problem. The present work aimed to investigate the occurrence of antimicrobial resistance among Salmonella serovars isolated in foods in 2015-2019. A total of 178 Salmonella strains belonging to 39 serovars were tested against 10 antimicrobials. High proportions of Salmonella isolates were resistant to tetracycline (n = 53.9%), ciprofloxacin (n = 47.2%), ampicillin (n = 44.4%), nalidixic acid (n = 42.7%), and trimethoprim-sulfamethoxazole (n = 38.8%). Different resistance rates were recorded among the different serotypes of Salmonella, and S. Infantis, exhibited the highest resistance to antibiotics. A high percentage of strains isolated from poultry, pork, and bovine were resistant to at least one or two antimicrobials. Resistant and multidrug-resistant (MDR) strains were also recorded among the isolates from molluscan shellfish; however, the occurrence of resistant Salmonella strains isolated from this source was significantly lower compared with those reported for poultry, pork, and bovine. The high levels of resistance reported in the present study indicate a potential public health risk. Consequently, additional hygiene and antibiotic stewardship practices should be considered for the food industry to prevent the prevalence of Salmonella in foods.

Occurrence of HEV-RNA in Italian Regional Pork and Wild Boar Food Products.

Hepatitis E is an emerging threat in industrialized countries. The foodborne transmission linked to consumption of pork and game meat is considered the main source of autochthonous infection. In Europe, small outbreaks have been reported linked to the consumption of pork liver sausages and wild boar meat. Based on previous findings and on increasing evidence of pork and game meat as a vehicle for HEV infections, the present study investigated the occurrence of HEV in 99 pork and 63 wild boar sausages and salami sold in Southern Italy. The HEV genome was detected in four wild boar sausages. Sequencing from 2 wild boar sausages confirmed that the HEV strains detected belonged to HEV-3 genotype, not assigned to any defined subtype. Data obtained confirmed the possible occurrence of HEV in pork products and in game. Although the detection rate is low, these products are frequently consumed raw after curing, whose effect on virus viability is still unknown.