Isolation and characterization of novel Staphylococcus aureus bacteriophage Hesat from dairy origin.

A novel temperate phage, named Hesat, was isolated by the incubation of a dairy strain of Staphylococcus aureus belonging to spa-type t127 with either bovine or ovine milk. Hesat represents a new species of temperate phage within the Phietavirus genus of the Azeredovirinae subfamily. Its genome has a length of 43,129 bp and a GC content of 35.11% and contains 75 predicted ORFs, some of which linked to virulence. This includes (i) a pathogenicity island (SaPln2), homologous to the type II toxin-antitoxin system PemK/MazF family toxin; (ii) a DUF3113 protein (gp30) that is putatively involved in the derepression of the global repressor Stl; and (iii) a cluster coding for a PVL. Genomic analysis of the host strain indicates Hesat is a resident prophage. Interestingly, its induction was obtained by exposing the bacterium to milk, while the conventional mitomycin C-based approach failed. The host range of phage Hesat appears to be broad, as it was able to lyse 24 out of 30 tested S. aureus isolates. Furthermore, when tested at high titer (108 PFU/ml), Hesat phage was also able to lyse a Staphylococcus muscae isolate, a coagulase-negative staphylococcal strain. KEY POINTS: • A new phage species was isolated from a Staphylococcus aureus bovine strain. • Pathogenicity island and PVL genes are encoded within phage genome. • The phage is active against most of S. aureus strains from both animal and human origins.

Food safety culture in food companies: evaluation of the perception of food safety culture in three Tuscan food companies.

The Commission Regulation (EU) No. 2021/382 (European Commission, 2021), amending the Regulation (EC) No. 852/2004 (European Commission, 2004), introduced the obligation for companies to establish and maintain a food safety culture (FSC). The methodology to evaluate, implement, and enhance the level of FSC is up to the individual companies. This study aimed to investigate the perception of FSC among the employees of 3 Tuscan medium-sized enterprises in the food sector, producing cured meat (A), dairy products (B), and frozen fish products (C). The survey was conducted through the development and administration of a questionnaire based on a 5 points Likert scale, referring to different aspects of FSC, organized in 6 sections with 5-6 statements each and subjected to a percentage of employees between 76 and 85%, classified also by the length of service (≤3 and >3 years). For all the companies, the minimum median and mode value for scores obtained by the different sections was 4, and the minimum median and mode value for the single statement was 3 (A, B; except for a bimodal value 2-4) and 4 (C). The section awareness and perception of risk showed the highest mean scores in all companies. As for the length of service, senior employees gave lower scores than junior ones in all sections in B and 3 sections in C. Overall, the results of the questionnaires showed a good perception of FSC, even though it was possible to identify some partial weaknesses.

Occurrence of Campylobacter in Faeces, Livers and Carcasses of Wild Boars Hunted in Tuscany (Italy) and Evaluation of MALDI-TOF MS for the Identification of Campylobacter Species.

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.

Antimicrobial and anti-biofilm activity of manuka essential oil against Listeria monocytogenes and Staphylococcus aureus of food origin.

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.

Coagulase negative staphylococci from ovine bulk-tank milk: Effects of the exposure to sub-inhibitory concentrations of disinfectants for teat-dipping.

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.

Determination of Prolactin in Canine Saliva: Is it Possible to Use a Commercial ELISA kit?

Prolactin has been reported to be a remarkable index of stress response, both acute and chronic, in several species. The use of biological matrixes other than blood is receiving increasing interest in the study of hormones, due to the lower invasiveness in collection. This research aimed to investigate the possibility of using a commercial ELISA (enzyme-linked immunosorbent assay) kit for measuring canine prolactin in blood for the quantification of canine prolactin in saliva. Study 1 consisted of a validation protocol, using saliva samples collected from lactating and non-lactating dogs. Study 2 was conducted to investigate a possible correlation between prolactin concentration in saliva and plasma in sheltered dogs by using the same kit. Prolactin values were reliably read only when they came from blood samples, not from saliva, but tended to be low in most of the cases. Study 1 showed that saliva had a matrix effect. In study 2, saliva prolactin levels were low and in 42.9% of cases, not readable. No correlation between prolactin values in plasma and saliva was found (ρ=0.482; p=0.274). These findings suggested that the determination of prolactin in dog saliva through an ELISA kit created for measuring prolactin in dog blood was unreliable.

Factors Predicting the Intention of Eating an Insect-Based Product.

This study provides a framework of the factors predicting the intention of eating an insect-based product. As part of the study, a seminar was carried out to explore how the provision of information about ecological, health, and gastronomic aspects of entomophagy would modify consumer beliefs regarding insects as food. Before and after the informative seminar, two questionnaires about sociodemographic attributes and beliefs about the consumption of insects as food were given. Participants were then asked to carry out a sensory evaluation of two identical bread samples, but one was claimed to be supplemented with insect powder. Results showed that perceived behavioral control is the main predictor of the intention, followed by neophobia and personal insect food rejection. The disgust factor significantly decreased after the participants attended the informative seminar. Sensory scores highlighted that participants gave "insect-labelled" samples higher scores for flavor, texture, and overall liking, nevertheless, participants indicated that they were less likely to use the "insect-labelled" bread in the future. Our findings provide a better understanding of insect food rejection behavior and help to predict the willingness to try insect-based products based on some important individual traits and information.

Effect of an Italian propolis on the growth of Listeria monocytogenes, Staphylococcus aureus and Bacillus cereus in milk and whey cheese.

Propolis antimicrobial activity has been limitedly studied in food, particularly in dairy products. We studied the antimicrobial activity of an alcoholic extract of an Italian propolis in sterile skim milk, pasteurized cow's milk, and cow's and goat's whey cheese (ricotta). Following the determination of the minimal inhibitory concentration on Gram+ and Gram- bacteria, the extract was employed at 2 and 5% (P2, P5), using controls with the same ethanol concentrations (E2, E5) and without any addition. In milk trials, Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, and Pseudomonas fluorescens were tested. P2 and P5 samples registered significant decreases of Gram+ bacteria in skim milk. The same was true for P5 in cows' milk, but only with S. aureus for P2. Ricotta was inoculated with L. monocytogenes, S. aureus and B. cereus and stored at 8.5°C. In cow's milk ricotta, L. monocytogenes counts in P5 were always lower than control during the storage time, significantly so from the 14th day. In goat's ricotta, L. monocytogenes counts in P5 were at least one logarithm lower than E5, whereas the extract didn't show a significant effect on S. aureus and B. cereus. The antimicrobial activity of propolis, particularly on L. monocytogenes, could be employed in ready-to-eat refrigerated dairy products.

Plasma Tryptophan/Large Neutral Amino Acids Ratio in Domestic Dogs Is Affected by a Single Meal with High Carbohydrates Level.

Aim of this study was to evaluate the plasma ratio between l-tryptophan (TRP) and five large neutral amino acids (isoleucine + leucine + phenylalanine + tyrosine + valine) (5LNAAs) after a single meal with high carbohydrates level. Five female Labrador Retrievers were involved. Each dog was fed three different meals: M1 (a mix of puffed rice, minced meat and olive oil), M2 (puffed rice and olive oil) and M3 (commercial dry food usually consumed) once in the morning per day for 30 days. Blood was collected right before the first meal (t0) and after 2, 4, 6, 8, 10 and 24 h. Plasma amino acids’ concentrations were measured using an HPLC (High-performance liquid chromatography) method with fluorimetric detection. Plasmatic TRP concentrations showed no significant difference between M1, M2 and M3 samples at any sampling time. M2 led to a decrease in 5LNAAs levels and consequently led to a significant higher TRP/5LNAAs ratios in the 6 h period after the provision of carbohydrates, compared to both M1 and M3. In addition, the mean TRP/5LNAAs ratio was significantly higher in M2 than in M3 at t8 and t10. These results indicate that meal composition affects TRP/5LNAAs ratio and possibly, TRP bioavailability.

Lactobacillus plantarum and Streptococcus thermophilus as starter cultures for a donkey milk fermented beverage.

Donkey milk is recently gaining attention due to its nutraceutical properties. Its low casein content does not allow caseification, so the production of a fermented milk would represent an alternative way to increase donkey milk shelf life. The aim of this study was to investigate the possibility of employing selected Streptococcus thermophilus and Lactobacillus plantarum isolates for the production of a novel donkey milk fermented beverage. Lysozyme resistance and the ability to acidify donkey milk were chosen as main selection parameters. Different fermented beverages (C1-C9) were produced, each with a specific combination of isolates, and stored at refrigerated conditions for 35days. The pH values and viability of the isolates were weekly assessed. In addition, sensory analysis was performed. Both S. thermophilus and L.plantarum showed a high degree of resistance to lysozyme with a Minimum Bactericidal Concentration>6.4mg/mL for 100% of S. thermophilus and 96% of L. plantarum. S. thermophilus and L. plantarum showed the ability to acidify donkey milk in 24h at 37°C, with an average ΔpH value of 2.91±0.16 and 1.78±0.66, respectively. Four L. plantarum and two S. thermophilus were chosen for the production of fermented milks. Those containing the association S. thermophilus/L. plantarum (C1-C4) reached a pH lower than 4.5 after 18h of fermentation and showed microbial loads higher than 7.00logcfu/mL until the end of the storage period. Moreover, comparing the microbial loads of samples containing both species and those containing S. thermophilus alone (C5), we highlighted the ability of L. plantarum to stimulate S. thermophilus replication. This boosted replication of S. thermophilus allowed to reach an appropriate pH in a time frame fitting the production schedule. This was not observed for samples containing a single species (C5-C9). Thus, L. plantarum strains seem to be good candidates in the production of a novel type of fermented milk, not only for their probiotic potential, but also for the enhancing effect on S. thermophilus growth.

Prevalence, phenotypic and genetic diversity of Campylobacter in poultry fresh meat and poultry products on retail sale in Tuscany (Italy).

In this study, the prevalence of Campylobacter spp. in poultry fresh meat and ready­to­cook products was evaluated. Seventy­three samples were collected at retail level from supermarkets and discount stores, obtaining 61.6% positivity. Of 133 Campylobacter isolates, 86 strains (Campylobacter coli, 58.1% and Campylobacter jejuni, 41.9%) were selected for characterisation on the basis of their SmaI and kpnI pulsed field gel­electrophoresis (PFGE) profiles, to exclude clonal replicates. Campylobacters resulted highly resistant to tetracycline, ciprofloxacin, and nalidixic acid (79.1%, 72.1% and 65.1%, respectively); 50% of C. coli and 13.9% of C. jejuni were resistant to ciprofloxacin and erythromycin, the most important antimicrobials for human campylobacteriosis therapy. Five C. coli were resistant to 5/7 of the tested antimicrobials. HS4c was the prevailing C. jejuni serotype group (22.3%), whereas 8 other serotypes were identified in low percentages. SmaI and kpnI profiles showed a wide variability. The survey showed a high Campylobacter contamination of poultry meat and poultry products at retail level in Tuscany, Italy. A wide strains' heterogeneity and a remarkable level of strains' antimicrobial resistance have been reported, confirming the need for an improvement of specific preventive measures along the production chain.

Improving pork burgers quality using Zingiber officinale Roscoe powder (ginger).

Pork burgers were evaluated for physical-chemical characteristics, fatty acids profile, lipid oxidation, antioxidant capacity, microbiological growth and sensory evaluation during storage time of seven days at 4°C as function of three formulations as only meat (control, B) and meat added with ginger powder at the percentage of 1 and 2% (BG1 and BG2). BG1 and BG2 were less redness than control ones with incremented yellow hue. These modifications in color parameters did not modify sensory characteristics of burgers. PUFA were incremented (both PUFAω3 and PUFAω6) by the addition of ginger. Furthermore, BG1 and BG2 burgers showed to be less sensitive to lipid oxidation and to possess an increase in antioxidant capacity. Microbial growth evaluation of total aerobic count and Pseudomonas spp. showed that ginger powder delayed in time the bacterial contamination. Results highlighted that the presence of ginger led to an enhanced shelf life and health characteristics of burgers (increasing peroxidisability, ratio hypocholesterolemic/hypercholesterolemic and ratio ω3/ω6; reducing atherogenicity and thrombogenicity).

Is Leptospira able to survive in raw milk? Study on the inactivation at different storage times and temperatures.

The consumption of raw milk is currently increasing due to several beneficial aspects, such as nutritional qualities, taste, and health benefits. However, some authors highlight the potential risk associated with raw milk consumption. In Italy, while the absence of some pathogen microorganisms is set by the regional regulation DGR 381/2007, for other microorganisms, such as Leptospira, no recommendations are provided. Leptospira is not ascribed among classical milk pathogens; however, it can potentially be present in raw milk. The aim of this study was to evaluate the survival in raw milk of six serovars of Leptospira after storage at different temperatures (4 °C ± 2 °C, 20 °C ± 2 °C, and 30 °C ± 2 °C) for different incubation times (20 min, 45 min, 1 h, and 1 h and 30 min), in order to determine the potential risk for consumers. Moreover, the immediate effect of bovine, goat, and donkey raw milk on tested Leptospira serovars was visually evaluated. After incubation, all samples were subcultured in EMJH and incubated aerobically at 30 °C for 3 months. All inoculated media were weekly examined by dark-field microscope in order to assess Leptospira survival. Extemporary observation of strains' behavior in milk allowed to detect an almost immediate motility loss, and no leptospires were detected by microscopic observations carried out weekly during the trial period. According to our results, it could be possible to exclude raw milk as a source of Leptospira infection for consumers.

Fishes and their parasites in the water district of Massaciuccoli (Tuscany, Central Italy).

This study has been conducted in the district of Massaciuccoli (lake, marsh and reclaimed areas with drainage channels) in Tuscany region (Central Italy). The aim of the research was to detect the presence of parasites in fishes, in particular of Opisthorchis felineus, which causes an important zoonosis. Between 2010-2012, the health status of 381 fishes was monitored, morphometric characteristics were determined, and parasites were searched for and identified. Of the 381 examined fishes, 189 were free of parasites while 192 were infected, among them 91 presented multiple infections. Opisthorchis felineus was not found in any of the examined fishes.

Serological survey of Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, and Ehrlichia canis infections in rural and urban dogs in Central Italy.

INTRODUCTION: Borrelia burgdorferi sensu lato (s.l.) and Anaplasma phagocytophilum are well known zoonotic pathogens, whereas Ehrlichia canis is usually considered to be of veterinary concern, although on the basis of recent reports it also seems to be able to infect humans. OBJECTIVE: The aim of the study was to determine the seroprevalence of B. burgdorferi s.l., A. phagocytophilum and E. canis in an Italian canine population, and to verify if there are differences between dogs living in urban areas and those from a rural environment. MATERIALS AND METHOD: Blood sera of 1,965 dogs, 1,235 from cities and 730 from rural areas, were tested by indirect immunofluorescent assay (IFAT). RESULTS: The overall seroprevalence was highest for E. canis (7.07%), followed by A. phagocytophilum (4.68%), and B. burgdorferi s.l. (1.47%). Rural dogs showed the highest seroprevalence to B. burgdorferi s.l. and A. phagocytophilum. No significant differences were observed between rural and urban E. canis-positive dogs. A low percentage (1.32%) of dogs with dual seropositivity was detected, and no triple positive reactions were observed. No significant differences were detected in the seroprevalence of the three agents in relationship to the age and gender of the dogs. Seroprevalence in the five years considered were not statistically different, except for the lowest rate for E. canis observed in 2012. CONCLUSIONS: The results confirm the presence of B. burgdorferi s.l., A. phagocytophilum and E. canis in Italian dogs in both urban and rural areas. Monitoring pet dogs, which share the same environment with their owners, is useful for identifying the presence of tick-borne disease agents of both veterinary and public health significance.