Pathogenic profile and antimicrobial resistance of Escherichia coli, Escherichia marmotae and Escherichia ruysiae detected from hunted wild boars in Sardinia (Italy).

The objective of this study was to evaluate the occurrence of E. coli in hunted wild boars in Sardinia (Italy) and to further characterize the isolates with Whole Genome Sequencing to assess the genetic relatedness and the presence of virulence and antimicrobial resistance (AMR) genes. Samples were taken from 66 wild boars between 2020 and 2022 slaughtered in five hunting houses. A total of 181 samples were tested, including 66 samples from mesenteric lymph nodes, 66 samples from colon content and 49 samples from carcass surface. Isolates referable to Escherichia species were detected in all of the wild boars sampled. On a selection of 61 isolates, sequencing was conducted and antimicrobial susceptibility was tested. Among these, three isolates were confirmed to be two Escherichia marmotae (cryptic clade V) and one Escherichia ruysiae (cryptic clade III). E. coli pathotypes identified were UPEC (13 %), ExPEC-UPEC (5.6 %) and ETEC (3.7 %). Moreover, 3/6 E. marmotae isolates had typical ExPEC genes. Genetic similarity was observed in isolates collected from animals slaughtered in the same hunting house; this suggests epidemiological links deriving from the presence of animals infected with closely related strains or the result of cross-contamination. Antimicrobial resistance genes were detected in three non-pathogenic E. coli isolates: one isolate had sul2, tet(B), aph(6)-ld and aph(3″)-lb resistance genes and two had the fosA7 gene. This study confirmed that wild boars can act as reservoirs and spreaders of pathogenic Escherichia species and it provides information for future comparative genomic analysis in wildlife. Although isolates showed a limited resistome, the detection of resistance in non-pathogenic isolates underlines the need to monitor antimicrobial resistance in the wild boar population. To the best of our knowledge, this is the first detection of E. mamotae and E. ruysiae isolates in wild boars in Italy and the presence of this pathogen in wildlife and livestock need to be investigated further.

Comparison of Activity of Commercial Protective Cultures and Thermophilic Lactic Acid Bacteria against Listeria monocytogenes: A New Perspective to Improve the Safety of Sardinian PDO Cheeses.

Listeria monocytogenes contamination that occurs during and post-processing of dairy products is a serious concern for consumers, and bioprotective cultures can be applied to control the growth of the pathogen in sheep milk cheeses. However, to respect specifications provided for protected designation of origin (PDO) cheeses, only autochthonous microorganisms can be used as bioprotective cultures in these products. This in vitro study aimed to evaluate thermophilic lactic acid bacteria (LAB) isolated from sheep milk as bio-preservative agents to control L. monocytogenes growth in PDO cheese. Results were compared with those obtained with a commercial protective culture (cPC) composed of a Lactiplantibacillus plantarum bacteriocin producer designed to inhibit L. monocytogenes growth in cheese. The in vitro antilisterial activities of n.74 autochthonous LAB and a cPC were tested against 51 L. monocytogenes strains using an agar well diffusion assay. In addition, 16S rRNA sequencing of LAB isolates with antilisterial activity was conducted and strains of Lactobacillus helveticus, Lactobacillus delbrueckii subsp. indicus, Lactobacillus delbrueckii subsp. sunkii, Lactobacillus delbrueckii subsp. lactis and Enterococcus faecalis were identified. In this study, 33.6% (74/220) bacterial strains isolated from milk had characteristics compatible with thermophilic LAB, of which 17.6% (13/74) had in vitro antilisterial activity. These results demonstrate that raw sheep milk can be considered an important source of autochthonous thermophilic LAB that can be employed as protective cultures during the manufacturing of Sardinian PDO cheeses to improve their food safety. The use of bioprotective cultures should be seen as an additional procedure useful to improve cheese safety along with the correct application of good hygienic practices during manufacturing and the post-processing stages.

Effect of Commercial and Autochthonous Bioprotective Cultures for Controlling Listeria monocytogenes Contamination of Pecorino Sardo Dolce PDO Cheese.

The composition and physicochemical characteristics of short-aged Pecorino Sardo PDO (Protected Designation of Origin) cheese makes it permissive to Listeria monocytogenes growth. The PDO product specification stipulates that this cheese is produced with whole sheep's milk inoculated with cultures from the area of origin. Therefore, the use of bioprotective cultures for the inhibition of pathogens in PDO cheeses is allowed only if autochthonous microorganisms are used. Furthermore, bioprotective cultures are generally used on the cheese surface to prevent the outgrowth of L. monocytogenes, the application of which can be time-consuming and require specialist technical knowledge. In this study, we examine the direct addition of bioprotective cultures to the cheese vat and compare the activity of a commercial bioprotective culture (Lactiplantibacillus plantarum) and an autochthonous lactic acid bacterium with bioprotective properties (Lactobacillus delbruekii sups. sunkii), for the inhibition of L. monocytogenes in Pecorino Sardo PDO cheese. Three types of Pecorino Sardo PDO cheese were made with bioprotective cultures added directly to the cheese milk along with the starter inoculum: PSA, with the commercial bioprotective culture; PSB, with the autochthonous bioprotective culture; and a CTRL cheese with no bioprotective culture. A challenge test was performed on each of these cheeses by artificially contaminating the cheese surface with L. monocytogenes (2 Log10 CFU/g). Three batches of each cheese type were analyzed to enumerate mesophilic and thermophilic lactic acid bacteria and to investigate the growth potential of L. monocytogenes during manufacturing, at the end of ripening, at the end of shelf-life, and after 180 days from cheese production. Both bioprotective cultures tested in this study showed inhibitory action against the pathogen with 0.3-1.8 Log10 CFU/g (colony-forming unit per gram) reduction levels. The autochthonous organism, L. sunkii, was as effective as the commercially supplied culture, and the addition of the bioprotective cultures to the cheese-making procedure offered protection against L. monocytogenes. The direct addition of bioprotective cultures to the making procedure of Pecorino Sardo PDO cheese is a potentially innovative strategy to improve the safety of this product.

Preliminary data on the microbial profile of dry and wet aged bovine meat obtained from different breeds in Sardinia.

This study aimed to evaluate the influence of dry and wet aging on microbial profile and physicochemical characteristics of bovine loins obtained from four animals of two different breeds, namely two Friesian cull cows and two Sardo-Bruna bovines. During dry and wet aging aerobic colony count, Enterobacteriaceae, mesophilic lactic acid bacteria, Pseudomonas, molds and yeasts, Salmonella enterica, Listeria monocytogenes and Yersinia enterocolitica, pH and water activity (aw) were determined in meat samples collected from the internal part of the loins. Moreover, the microbial profile was determined with sponge samples taken from the surface of the meat cuts. Samples obtained from Friesian cows were analyzed starting from the first day of the aging period and after 7, 14, and 21 days. Samples obtained from the Sardo Bruna bovines were also analyzed after 28 and 35 days. Wet aging allowed better control of Pseudomonas spp. during storage that showed statistically lower levels (P>0.05) in wet-aged meats with respect to dry-aged meats during aging and particularly at the end of the period (P>0.01) in both cattle breeds. At the end of the experiment (21 days), aerobic colony count and Pseudomonas in Fresian cows' dry-aged meats showed mean levels >8 log, while lactic acid bacteria mean counts >7 log were detected in wet-aged meats of both cattle breeds. In meats submitted to dry aging, pH was significantly higher (P<0.01) with respect to wet-aged meats at all analysis times and in both cattle breeds. Aw showed a stable trend during both dry and wet aging without significant differences. These preliminary results highlight the critical importance of the strict application of good hygiene practices during all stages of production of these particular cuts of meat intended for aging.

Hunted Wild Boars in Sardinia: Prevalence, Antimicrobial Resistance and Genomic Analysis of Salmonella and Yersinia enterocolitica.

The objective of this investigation was to evaluate Salmonella and Yersinia enterocolitica prevalence in wild boars hunted in Sardinia and further characterize the isolates and analyse antimicrobial resistance (AMR) patterns. In order to assess slaughtering hygiene, an evaluation of carcasses microbial contamination was also carried out. Between 2020 and 2022, samples were collected from 66 wild boars hunted during two hunting seasons from the area of two provinces in northern and central Sardinia (Italy). Samples collected included colon content samples, mesenteric lymph nodes samples and carcass surface samples. Salmonella and Y. enterocolitica detection was conducted on each sample; also, on carcass surface samples, total aerobic mesophilic count and Enterobacteriaceae count were evaluated. On Salmonella and Y. enterocolitica isolates, antimicrobial susceptibility was tested and whole genome sequencing was applied. Salmonella was identified in the colon content samples of 3/66 (4.5%) wild boars; isolates were S. enterica subs. salamae, S. ser. elomrane and S. enterica subs. enterica. Y. enterocolitica was detected from 20/66 (30.3%) wild boars: in 18/66 (27.3%) colon contents, in 3/66 (4.5%) mesenteric lymph nodes and in 3/49 (6.1%) carcass surface samples. In all, 24 Y. enterocolitica isolates were analysed and 20 different sequence types were detected, with the most common being ST860. Regarding AMR, no resistance was detected in Salmonella isolates, while expected resistance towards ß-lactams (blaA gene) and streptogramin (vatF gene) was observed in Y. enterocolitica isolates (91.7% and 4.2%, respectively). The low presence of AMR is probably due to the low anthropic impact in the wild areas. Regarding the surface contamination of carcasses, values (mean ± standard deviation log10 CFU/cm2) were 2.46 ± 0.97 for ACC and 1.07 ± 1.18 for Enterobacteriaceae. The results of our study confirm that wild boars can serve as reservoirs and spreaders of Salmonella and Y. enterocolitica; the finding of Y. enterocolitica presence on carcass surface highlights how meat may become superficially contaminated, especially considering that contamination is linked to the conditions related to the hunting, handling and processing of game animals.