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The occurrence of antibiotic-resistant bacteria in foodstuff involves a human health risk. Edible insects are a precious resource; however, their consumption raises food safety issues. In this study, the occurrence of antibiotic resistant bacteria in laboratory-reared fresh mealworm larvae (Tenebrio molitor L.) and frass was assessed. Antibiotics were not used during the rearing. Enterobacteriaceae and enterococci were isolated from 17 larvae and eight frass samples. In total, 62 and 69 isolates presumed to belong to Enterobacteriaceae and Enterococcus spp., respectively, were obtained and tested for antibiotic susceptibility via disk diffusion. Based on the results, isolates were grouped, and representative resistant isolates were identified at species level through 16S rRNA gene sequencing. For enterococci resistance, percentages higher than 15% were observed for vancomycin and quinupristin-dalfopristin, whereas Enterobacteriaceae resistance higher than 25% was found against cefoxitin, ampicillin, and amoxicillin-clavulanic acid. Based on the species identification, the observed resistances seemed to be intrinsic both for enterococci and Enterobacteriaceae, except for some ß-lactams resistance in Shigella boydii (cefoxitin and aztreonam). These could be due to transferable genetic elements. This study suggests the need for further investigations to clarify the role of edible insects in the spreading of antibiotic resistance determinants through the food chain.
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In the circular economy framework, hydrosols, by-products of the essential oil industry, are gaining attention for their potential in waste reduction and resource reuse. This study analyzed hydrosols from six edible flowers, investigating their chemical composition (VOC-Hyd) and antibacterial properties alongside volatile organic compounds of fresh flowers (VOC-Fs) and essential oils (EOs). Antirrhinum majus exhibited ketones as major VOC-Fs (62.6%) and VOC-Hyd (41.4%), while apocarotenoids dominated its EOs (68.0%). Begonia cucullata showed alkanes (33.7%) and aldehydes (25.7%) as primary VOC-Fs, while alkanes were prevalent in both extracts (65.6% and 91.7% in VOC-Hyd and in EOs, respectively). Calandula officinalis had monoterpenoids in VOC-Fs and VOC-Hyd (89.3% and 49.7%, respectively), while its EOs were rich in sesquiterpenoids (59.7%). Dahlia hortensis displayed monoterpenoid richness in both VOC-Fs and extracts. Monocots species' VOC-Fs (Polianthes tuberosa, Tulbaghia cominsii) were esters-rich, replaced by monoterpenoids in VOC-Hyd. P. tuberosa EO maintained ester richness, while T. cominsii EOs contained a significant percentage of sulfur compounds (38.1%). Antibacterial assays indicated comparable minimum inhibitory concentration profiles across VOC-Hyd: B. calcullata and P. tuberosa against Staphylococcus aureus and Salmonella enterica ser. typhimurium, T. cominsii against Escherichia coli and S. enterica, A. majus and C. officinalis against S. aureus, and D. hortensis against S. enterica.
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This paper aims to assess the disinfecting capacity of a double-distilled ozonated water solution as an alternative to common antibiotic and antiseptic devices. Seventy-five dogs were subjected to the surgical procedures of scaling and dental extraction and included in three study groups: Group 1 subjected to antibiotic pre-treatment (association amoxicillin + clavulanic acid and ampicillin + metronidazole) and disinfection with chlorhexidine, and Group 2 and 3 devoid of preventive anti-microbial treatment in which disinfection was performed, respectively, with ozonated water and chlorhexidine. Sampling by bacteriological buffer was carried out to evaluate the bacterial count in the oral cavity. The analysis of the samples determined the total mesophilic bacterial count by seeding on the culture medium via the inclusion of PCA (Plate Count Agar). The results highlighted the anti-bacterial efficacy of intra-operative ozone as an alternative to pre-surgical antibiotic treatment.
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The study aims to identify antibacterial properties in unfertilized eggs of Scyliorhinus canicula and Scyliorhinus stellaris. Despite challenging marine conditions, these eggs retain their integrity for extended periods and remain unaffected by pathogens. The antibacterial activity was measured using minimum inhibitory and minimum bactericidal concentration analysis. The eggs of S. stellaris exhibited a slight inhibitory effect against Staphylococcus aureus and Listeria monocytogenes, whereas both species' eggs showed no activity against gram-negative microorganisms.
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Antibacterianos , Pruebas de Sensibilidad Microbiana , Óvulo , Staphylococcus aureus , Animales , Staphylococcus aureus/efectos de los fármacos , Antibacterianos/farmacología , Tiburones , Listeria monocytogenesRESUMEN
The application of artificial intelligence and machine learning (ML) methods is becoming increasingly popular in computational toxicology and drug design; it is considered as a promising solution for assessing the safety profile of compounds, particularly in lead optimization and ADMET studies, and to meet the principles of the 3Rs, which calls for the replacement, reduction, and refinement of animal testing. In this context, we herein present the development of VenomPred 2.0 (http://www.mmvsl.it/wp/venompred2/), the new and improved version of our free of charge web tool for toxicological predictions, which now represents a powerful web-based platform for multifaceted and human-interpretable in silico toxicity profiling of chemicals. VenomPred 2.0 presents an extended set of toxicity endpoints (androgenicity, skin irritation, eye irritation, and acute oral toxicity, in addition to the already available carcinogenicity, mutagenicity, hepatotoxicity, and estrogenicity) that can be evaluated through an exhaustive consensus prediction strategy based on multiple ML models. Moreover, we also implemented a new utility based on the Shapley Additive exPlanations (SHAP) method that allows human interpretable toxicological profiling of small molecules, highlighting the features that strongly contribute to the toxicological predictions in order to derive structural toxicophores.
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Inteligencia Artificial , Aprendizaje Automático , Animales , HumanosRESUMEN
Tenebrio molitor (mealworm) is one of the most promising insect species to produce sustainable feed and food with high nutritional value. Insects may harbour microorganisms both in the gut and on the exoskeleton originating from the rearing environment. Staphylococcus aureus is a pathogenic microorganism frequently involved in food poisoning due to its enterotoxin production. This study aimed to evaluate the S. aureus growth and enterotoxins production following an experimental inoculation into the T. molitor rearing substrate (about 7 log CFU/g). Analyses on the substrate and larvae were performed over a testing period of seven days. The microbial population dynamics were also evaluated through total viable count and lactic acid bacteria count. The effects of fasting, washing, and cooking on the microbial loads of mealworms were evaluated. The results highlighted that mealworms and substrates can maintain their microbial loads of S. aureus over the tested period. Moreover, fasting and washing were generally not able to significantly reduce (p-value > 0.05) S. aureus count in mealworms. On the other hand, cooking significantly reduced (p-value < 0.001) the microbial load in almost all cases. No production of enterotoxins was revealed during the trial. Therefore, microbiological risks can be reduced by a wise choice of substrate, appropriate control measures, and thermal treatment of larvae.
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The interest in edible insects is continuously increasing due to their environmental, nutritional, and productive features. The aim of this study was to evaluate the effects and survival of Listeria monocytogenes in Zophobas morio rearing, using two different bacterial loads (2 and 6 log CFU/g). We also considered the effect of washing, fasting, and cooking treatments on the larvae. During the experimental trial, no mortality was observed among the larvae. The L. monocytogenes loads decreased over time, and it was below the detection limit for crates inoculated with 2 log CFU/g, while the substrate inoculated with 6 log CFU/g reached loads of 4.26 (without larvae) and 2.83 log CFU/g (with larvae). Due to the absence of L. monocytogenes in the larvae on day seven, it was not possible to assess the fasting treatment or the washing and the cooking. However, when looking at the total microbial count, significant effects were revealed for all treatments. The unfasted larvae showed no effect of washing, while the total microbial counts decreased after washing in the fasted larvae. This proves that fasting is a good treatment in terms of hygiene assurance for the consumer.
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A total of 193 wild boars hunted in Tuscany, an Italian region with a high presence of wild ungulates, were examined to assess the occurrence of Campylobacter species in faeces, bile, liver and carcasses, with the aim of clarifying their contribution to human infection through the food chain. Campylobacter spp. were found in 44.56% of the animals, 42.62% of the faecal samples, 18.18% of the carcass samples, 4.81% of the liver tissues and 1.97% of the bile samples. The Campylobacter species genotypically identified were C. coli, C. lanienae, C. jejuni and C. hyointestinalis. The prevalent species transpired to be C. coli and C. lanienae, which were isolated from all the matrices; C. jejuni was present in faeces and liver, while C. hyointestinalis only in faeces. Identification was carried out by matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF MS) on 66 out of 100 isolates identified genotypically, and the technique yielded unsatisfactory results in the case of C. lanienae, which is responsible for sporadic human disease cases. The level of Campylobacter spp. contamination of meat and liver underlines the need to provide appropriate food safety information to hunters and consumers.
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In recent years, essential oils (EOs) have received interest due to their antibacterial properties. Accordingly, the present study aimed to investigate the effectiveness of the EOs obtained from seven species of Salvia on three strains of Listeria monocytogenes (two serotyped wild strains and one ATCC strain), a bacterium able to contaminate food products and cause foodborne disease in humans. The Salvia species analysed in the present study were cultivated at the Botanic Garden and Museum of the University of Pisa, and their air-dried aerial parts were subjected to hydrodistillation using a Clevenger apparatus. The obtained EOs were analysed via gas chromatography coupled with mass spectrometry for the evaluation of their chemical composition, and they were tested for their inhibitory and bactericidal activities by means of MIC and MBC. The tested Eos showed promising results, and the best outcomes were reached by S. chamaedryoides EO, showing an MIC of 1:256 and an MBC of 1:64. The predominant compounds of this EO were the sesquiterpenes caryophyllene oxide and ß-caryophyllene, together with the monoterpenes bornyl acetate and borneol. These results suggest that these EOs may possibly be used in the food industry as preservatives of natural origins.
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The COVID-19 pandemic has further confirmed to the community that direct contact with contaminated surfaces and objects represents an important source of pathogen spreading among humans. Therefore, it is of paramount importance to design effective germicidal paints to ensure a rapid and potent disinfectant action of surfaces. In this work, we design novel germicide polymeric coatings by inserting quaternary ammonium and sugar groups on the macromolecular backbone, thus respectively endowing the polymer with germicide features and hydrophilicity to interact with the surfaces. An aliphatic polyketone was selected as a starting polymer matrix that was functionalized with primary amine derivatives via the Paal-Knorr reaction. The resulting polymers were deposited on cellulose filter papers and checkboard charts with excellent coating yield and substrate coverage as determined by scanning electron microscopy for cellulose. Remarkably, the substrates coated by the functional polymers bearing quaternary ammonium compounds showed excellent bactericide properties with antibacterial rate of 99% and logarithmic reduction of >3. Notably, the polymers with higher hydrophobicity showed better retention on the substrate after being treated with water at neutral pH.
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Lavandula angustifolia Mill., known as one of the best essential oil-bearing plants, is an aromatic plant that is well cultivated in many Mediterranean regions due to its adaptability to variations in climatic and edaphic conditions. Therefore, its essential oil (EO) composition and its antimicrobial activity change as a consequence of abiotic and biotic factors. The chemical composition of L. angustifolia EO collected during four consecutive years of growth was one of the aims of this work. The volatile profile evidenced the prevalence of linalool and linalool acetate even though they switched their positions according to age. Plants in their first year were characterized by a high amount of sesquiterpene compounds (22.1% of the identified fraction). This percentage decreased during plant growth, not representing more than 5.3% in the fourth year. It is interesting to note that both the third- and fourth-year plants showed a content of monoterpenes that exceeded 90% of the total identified constituents. The EO extracted from the oldest plants evidenced higher activity on the studied strains, with more sensitivity on the Gram-positive ones. Tuscan lavender EO, especially that obtained from the four-year-old plants, is of great interest for its potential industrial applications and constitutes an example for the valorization of marginal Tuscan land and good-quality production.
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Lavandula , Aceites Volátiles , Antibacterianos/farmacología , Cromatografía de Gases y Espectrometría de Masas , Lavandula/química , Monoterpenos , Aceites Volátiles/química , Aceites Volátiles/farmacologíaRESUMEN
The activity of manuka (Leptospermum scoparium) essential oil (EO) on biofilms of foodborne Listeria monocytogenes and Staphylococcus aureus has been studied. Seven strains of L. monocytogenes and 7 of S. aureus (5 methicillin-resistant) were tested. EO minimal inhibitory concentration (MIC), EO minimal bactericidal concentration (MBC) and biofilm production quantification were determined for each strain by microtiter methods. Moreover, EO Minimum Biofilm Inhibitory Concentration (MBIC) and Minimum Biofilm Eradicating Concentration (MBEC) were determined on 2 L. monocytogenes and 3 S. aureus that showed the best biofilm production. Finally, on 4 strains out of 5 (2 L. monocytogenes and 2 S. aureus) EO Biofilm Reduction Percentage (BRP) vs. untreated controls was assessed after a treatment with EO subinhibitory concentrations. The chemical composition of manuka essential oil was determined by Gas Chromatography- Electron Impact Mass Spectrometry (GCEIMS). The manuka EO demonstrated good antimicrobial activity: L. monocytogenes MIC and MBC were 0.466 mg/ml and 0.933 mg/ml, respectively, whereas S. aureus MIC and MBC were 0.233 mg/ml and 0.466 mg/ml, respectively. Furthermore, L. monocytogenes showed a MBIC of 0.933 mg/ml and a MBEC in the range of 0.933-1.865 mg/ml, whereas S. aureus had a MBIC in the range of 7.461-14.922 mg/ml and a MBEC of 14.922 mg/ml. L. monocytogenes revealed no significant BRP after the treatment with manuka EO, whereas S. aureus showed a BRP higher than 50% with MIC/2 and MIC/4 EO concentrations. These results provide information for feasible manuka EO applications in food production systems.
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Tenebrio molitor (T. molitor) (mealworm) larvae are one of the most promising insects for feed-food purposes. Mealworms are rich in several macro and micro nutritional elements and can be practically reared on side stream substrates. In this study, the effects of seven different cooking techniques were tested on the nutritional value of mealworms focusing the attention on protein digestibility, fatty acid (FA) profile, and oxidative status. Uncooked larvae (UC) were used as control and compared to two combinations of temperature/time in oven cooking (70°C for 30 min, OC70-30, 150°C for 10 min, OC150-10), two methods of frying (mealworms fried in sunflower oil as deep fry, DF, or pan fry, PF), microwaving (MW), boiling (in plastic bag under vacuum, BO), and steaming (ST). Proximate composition, in vitro digestibility (gastric and duodenal), FA profile, and oxidative status (tocopherol and tocotrienol, carbonyl, and lipid oxidation) were then tested. Cooking technique affected all the tested parameters. As expected, cooking affected proximate composition in relation to the method applied (dry matter increased after oven cooking and frying; lipids increased by frying). In vitro digestion revealed the highest value for the OC70-30 method, followed by UC and ST. Deep frying revealed the worst digestibility percentage. FA profile was deeply affected by the cooking technique, with general decrease in SFA and MUFA. The highest modifications in FA profile were revealed in ST larvae with an increased percentage of linoleic acid linked to the lowering of SFA and MUFA contents. Furthermore, deep frying larvae in sunflower oil increased the relative abundance of PUFAs. Tocols values were higher in DF and MW groups than PF (about 6-fold more) and all other groups (7-fold more). Carbonyls increased with oven cooking (OC150-10 and OC70-30), whereas the values were lower with frying and similar to ST and UC. Lipid oxidation was highest as well in OC150-10 but similar to frying methods (DF and PF). Based on the obtained results, it can be concluded that mealworm larvae surely meet human nutritional requirements, but the cooking method must be carefully chosen to maintain a high nutritional value.
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In the present paper, we focused our attention on Cinnamomum camphora (L.) J. Presl. (Lauraceae), studied at three levels: (i) micromorphological, with the analysis of the secretory structures and a novel in-depth histochemical characterization of the secreted compounds; (ii) phytochemical, with the characterization of the essential oils from young stems, fruits, and leaves, subjected to different conservation procedures (fresh, dried, stored at -20 °C, stored at -80 °C) and collected in two different years; (iii) bioactive, consisting of a study of the potential antibacterial activity of the essential oils. The micromorphological investigation proved the presence of secretory cells characterized by a multi-layered wall in the young stems and leaves. They resulted in two different types: mucilage cells producing muco-polysaccharides and oil cells with an exclusive terpene production. The phytochemical investigations showed a predominance of monoterpenes over sesquiterpene derivatives; among them, the main components retrieved in all samples were 1,8-cineole followed by α-terpineol and sabinene. Conservation procedures seem to only influence the amounts of specific components, i.e., 1,8-cineole and α-terpineol, while analyses on each plant part revealed the presence of some peculiar secondary constituents for each of them. Finally, the evaluation of the antibacterial activity of the essential oil showed a promising activity against various microorganisms, as Listeria monocytogenes, Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa. In conclusion, we combined a micromorphological and phytochemical approach of the study on different plant parts of C. camphora, linking the occurrence of secretory cells to the production of essential oils. We compared, for the first time, the composition of essential oils derived from different plant matrices conserved with different procedures, allowing us to highlight a relation between the conservation technique and the main components of the profiles. Moreover, the preliminary antibacterial studies evidenced the potential activity of the essential oils against various microorganisms potentially dangerous for plants and humans.
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Brucellosis is a zoonosis caused by different Brucella species. Wild boar (Sus scrofa) could be infected by some species and represents an important reservoir, especially for B. suis biovar 2. This study aimed to investigate the prevalence of Brucella spp. by serological and molecular assays in wild boar hunted in Tuscany (Italy) during two hunting seasons. From 287 animals, sera, lymph nodes, livers, spleens, and reproductive system organs were collected. Within sera, 16 (5.74%) were positive to both rose bengal test (RBT) and complement fixation test (CFT), with titres ranging from 1:4 to 1:16 (corresponding to 20 and 80 ICFTU/mL, respectively). Brucella spp. DNA was detected in four lymph nodes (1.40%), five epididymides (1.74%), and one fetus pool (2.22%). All positive PCR samples belonged to Brucella suis biovar 2. The results of this investigation confirmed that wild boar represents a host for B.suis biovar. 2 and plays an important role in the epidemiology of brucellosis in central Italy. Additionally, epididymis localization confirms the possible venereal transmission.
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Leptospirosis is a re-emerging and worldwide diffused zoonosis. Recently, the high importance of their epidemiology was explained by the intermediate Leptospira strains. Among these strains, Leptospira fainei was the first intermediate strain detected in domestic and wild swine. Wild boars (Sus scrofa) are well known as a reservoir, as well as all swine, for pathogenic Leptospira, but very little information is available concerning intermediate Leptospira infection. The investigation aim was to evaluate if intermediate Leptospira can infect the reproductive systems of wild boars hunted in the Tuscany region (Italy), as previously demonstrated for pathogenic ones. The reproductive system tissue (testicles, epididymides, uteri), and placentas and fetuses, were collected from 200 regularly hunted animals. Bacteriological examination and real-time PCR were performed to detect intermediate Leptospira DNA. Unfortunately, no isolates were obtained. Using real-time PCR, in six (3%) male organs (both testicles and epididymis), intermediate Leptospira DNA was found. The amplification of the 16S rRNA gene identified that all DNA obtained belong to Leptospira fainei. The results of this investigation highlighted for the first time the localization of Leptospira fainei in the male wild boar reproductive system, opening up a new avenue to further investigate.
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Teat-dipping is one of the most effective methods to prevent mammary infections in ruminants, including sub-clinical mastitis caused by coagulase-negative staphylococci (CoNS). Improper disinfectant application could expose microorganisms to sub-inhibitory concentrations leading to phenotypic variations. In this study, 12 chlorhexidine-digluconate (CHDG)-tolerant (of which 4 qac positive) and 12 benzalkonium chloride (BC)-tolerant (of which 7 qac-positive) CoNS isolates from ovine milk were exposed to sub-inhibitory concentrations of CHDG and BC, respectively. Changes in disinfectant susceptibility against BC and CHDG, antibiotic resistance against 12 antibiotics and biofilm production were then assessed for both groups. After CHDG stress, 67 % and 83 % of the CHDG-stressed isolates doubled their MICs for BC and CHDG, respectively and 2 qac-negative isolates showed a four-fold increase of their MBCs for CHDG. After BC stress, MICs for BC and CHDG doubled in 58 % and 83 % of the BC-stressed isolates, respectively, while one qac-positive isolate increased four-fold the MIC for BC. Cross-resistance to antibiotics was assessed by disc diffusion method. Some qac-positive isolates varied their resistance profile, while a blaZ-positive isolate showed a resistant phenotype against ampicillin only after the exposure to the disinfectant. As for qac-positive isolates, one CHDG-stressed and 2 BC-stressed increased their resistance to kanamycin and cefoxitin, respectively. The Congo Red Agar test was carried out to assess the in vitro slime production: all isolates were negative after stress. In conclusion, sub-inhibitory exposure to disinfectants may affect disinfectant and antibiotic susceptibility, the latter in particular for qac-positive isolates and those hosting unexpressed antibiotic resistance genes.
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Desinfectantes , Enfermedades de las Ovejas , Infecciones Estafilocócicas , Animales , Antibacterianos/farmacología , Coagulasa , Desinfectantes/farmacología , Farmacorresistencia Bacteriana , Femenino , Pruebas de Sensibilidad Microbiana/veterinaria , Leche , Ovinos , Infecciones Estafilocócicas/veterinaria , StaphylococcusRESUMEN
Domestic dogs (Canis lupus familiaris) used for wild boar (Sus scrofa) hunting may represent incidental hosts for several zoonotic pathogens. This investigation aimed to evaluate the presence of anti-Leptospira antibodies and the occurrence, antimicrobial resistance, and virulence of Salmonella spp., Yersinia enterocolitica, and Listeria monocytogenes in sera and rectal swabs collected from 42 domestic hunting dogs in the Tuscany region (Italy). Regarding Leptospira, 31 out of 42 serum samples (73.8%) were positive and serogroup Pomona was the most detected (71.4%) at titers between 1:100 and 1:400. Four Salmonella isolates (9.52%) were obtained, all belonging to serotype Infantis; two of them showed antimicrobial resistance to streptomycin, while pipB and sopE presence was assessed in all but one isolate. Concerning Yersinia enterocolitica, seven isolates (16.7%) were obtained, six belonging to biotype 1 and one to biotype 4. Resistance to amoxicillin-clavulanic acid, cephalothin, and ampicillin was detected. Biotype 4 presented three of the virulence genes searched (ystA, ystB, inv), while isolates of biotype 1 showed only one gene. No Listeria monocytogenes was isolated from dog rectal swabs. The results suggest that hunting dogs are exposed to different bacterial zoonotic agents, potentially linked to their work activity, and highlight the possible health risks for humans.
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Wild boar is an animal the population of which constantly increases in Europe. This animal plays an important role as a reservoir for several pathogens, including three of the most important zoonoses: salmonellosis, yersiniosis and listeriosis. The aim of this investigation was to evaluate the occurrence of antimicrobial-resistant and virulence factor genes of Salmonella spp., Yersinia enterocolitica and Listeria monocytogenes isolated from wild boar in Tuscany (Central Italy). During two consequent hunting seasons (2018/2019 and 2019/2020), rectal swabs, spleens and livers were collected from 287 hunted wild boar to isolate strains. Each isolate was tested to investigate its antimicrobial resistance and to detect virulence factor genes by PCR. Eighteen Salmonella strains (6.27%) were isolated. Of these, 66.7% were resistant to streptomycin, 13.4% to cephalothin, 6.67% to imipenem and one isolate (6.67%) was resistant simultaneously to five antimicrobials. Moreover, the most detected genes were sopE (73.4%), pipB (66.7%), sodCI (53.3%), spvR and spvC (46.7%). In total, 54 (17.8%) Yersinia enterocolitica were isolated; of them, 26 (48.1%), 9 (16.7%), 17 (31.5%), 1 (1.85%) and 1 (1.85%) belonged to biotypes 1, 2, 3, 4 and 5, respectively. All strains (100%) demonstrated resistance to cephalothin and 70.4% to amoxicillin-clavulanic acid, 55.6% to ampicillin, and 37.0% to cefoxitin. Additionally, the most detected genes were ystA (25.9%), inv (24.1%), ail (22.2%), ystB (18.5%) and virF (14.8%). Finally, only one Listeria monocytogenes isolate (0.35%) was obtained, belonging to serogroup IVb, serovar 4b, and it was found to be resistant to cefoxitin, cefotaxime and nalidixic acid. The results highlighted the role of wild boar as a carrier for pathogenic and antimicrobial-resistant Salmonella spp., Yersinia enterocolitica and Listeria monocytogens, representing a possible reservoir for domestic animals and human pathogens.