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.

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.

Use of commercial protective cultures in portioned sheep milk cheeses to control Listeria monocytogenes.

The main objective of this study was to innovate soft and semi-cooked sheep milk cheese production processes with the use of a commercial protective culture able to control Listeria monocytogenes growth. A freeze-dried commercial culture of Lactobacillus plantarum was tested in DS cheese and PS cheese, two types of pasteurized sheep milk, raw-paste cheeses aged for no less than 20 and 30 days respectively. In the first step, in vitro tests were conducted to identify the most suitable matrix for the growth of L. plantarum in order to create a subculture that could be used at industrial cheese-making plants. During the second phase of the study, L. plantarum culture was introduced in the manufacturing process of the cheeses in a production plant. Finally, a challenge test was conducted on portioned DS and PS cheeses in order to evaluate the activity of the protective culture against L. monocytogenes: the cheeses were portioned, experimentally contaminated with L. monocytogenes strains, vacuum packed and stored at +4°C (correct storage conditions) and at +10°C (thermal abuse). Cheeses were analysed at the end of the shelf-life to evaluate the presence and growth of L. monocytogenes, to enumerate lactic acid bacteria and determine chemicalphysical features. The results confirmed that protective cultures are a useful technological innovation to control L. monocytogenes growth during cheese storage without altering composition, microflora and chemical- physical characteristics of the product. However, the use of protective cultures should be applied as an integration of risk control measures and not as a substitute for preventive actions.

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.

Selection of commercial protective cultures to be added in Sardinian fermented sausage to control Listeria monocytogenes.

Sardinian fermented sausage "Salsiccia Sarda" is a Mediterranean-style, semi-dry, fermented, RTE product, representing the main pork meat product in Sardinia (Italy). The high variability that characterizes the technological processes applied in different production plants results in sausages with different chemico-physical features sometimes permissive for the growth of Listeria monocytogenes. In order to guarantee the hygienic-sanitary quality of the final product and to innovate the manufacturing process, the main objective of this study was to evaluate the use of different commercial protective cultures to control L. monocytogenes growth in the Sardinian fermented sausage. In the first step, in vitro tests were carried out to evaluate the effectiveness of five freeze-dried bioprotective cultures availabe on the market in limiting the growth of L. monocytogenes. The two protective cultures that showed the best in vitro results were selected for a challenge test on artificially contaminated Sardinian fermented sausages. Moreover, the protective culture that showed the best results in inhibiting the growth of L. monocytogenes according to in vitro and challenge test experiments, was included into real production settings and validated in three producing plants. As a result, it was observed that protective cultures represent an important technological innovation for the Sardinian fermented sausage processing plants as they allow to control L. monocytogenes growth without altering the composition, the microflora and the chemical-physical characteristics of the product, thus ensuring safety and quality. Protective cultures also showed to reduce Enterobacteriaceae mean levels at the end of ripening and not to affect the natural concentration of lactic acid bacteria and coagulase-negative staphylococci.