Hepatitis A virus strains circulating in the Campania region (2015-2018) assessed through bivalve biomonitoring and environmental surveillance

The genetic diversity of Hepatitis A Virus (HAV) circulating in the Campania Region in years 2015-2018 was investigated through the monitoring of sentinel bivalve shellfish and water matrices. Overall, 463 water samples (71 sewage samples, 353 coastal discharge waters, and 39 seawaters samples), and 746 bivalve shellfish samples were analyzed. Positivity for HAV was detected in 20/71 sewage samples, 14/353 coastal discharge waters, 5/39 seawaters, and 102/746 bivalve shellfish. Sixty-one of the positive samples were successfully sequenced and were characterized as genotype IA (n = 50) and IB (n = 11). The prevalent strain circulating in 2015 in both bivalves and waters was the IA strain responsible for the outbreak occurring around the same time in the Naples area. This variant was no longer identified in subsequent years (2017-2018) when, instead, appeared two of the IA variants of the multistate outbreak affecting men who have sex with men (MSM), VRD_521_2016, and RIVM-HAV16-090, with the former prevailing in both shellfish and water environments. HAV IB isolates were detected over the years in shellfish and in water matrices, but not in clinical samples, suggesting that this genotype had been circulating silently. An integrated surveillance system (environment/food/clinical cases) can be a useful tool to monitor changes in viral variants in the population, as well as an early warning system

Evaluation of DNA preparation methods combined with different PCR-based assays for Coxiella burnetii detection in milk

Introduction - Coxiella burnetii (Cb) is the causative agent of Q fever, a zoonosis that occurs worldwide. Due to health concerns, unpasteurized cow's milk and a number of dairy products produced by unpasteurized milk may contain virulent Cb. PCR method is commonly employed for sensitive, specific and rapid test for Cb detection in biological samples including milk. Aim - In this study, six DNA purification methods for recovering Cb DNA from experimentally contaminated cow's milk were evaluated, together with three PCR-based assays targeting the IS 1111 Cb-repeated element. Materials and methods - For DNA extraction, the cetyltrimethylammonium bromide method was implemented and the following commercial kits were used: QIAamp DNA Mini kit; DNeasy Mericon Food kit; NucliSENS miniMAG; NucleoSpin Food; Wizard Genomic DNA Purification Kit. The three assays considered were standard PCR, TaqMan real-time PCR and SYBR Green combined with the evaluation of the melting temperature of the amplicon. Results and discussion - The best extraction methods, QIAamp DNA Mini kit, DNeasy Mericon Food kit and NucleoSpin Food, combined with the TaqMan real-time PCR assay, allowed us to detect the presence of 5 Cb cells per mu L of milk. Conclusion - The analysis of bulk milk seems to be a suitable means of monitoring the Q fever health condition in cows' herds, as long as efficient extraction methods and sensitive amplification assays are used

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

Comparison of droplet digital PCR vs real-time PCR for Yersinia enterocolitica detection in vegetables

: Yersiniosis - the 4th most commonly reported zoonosis in the European Union - is caused by the consumption of food contaminated with the bacterium Yersinia enterocolitica. The number of human cases and contaminated food samples is probably underestimated since conventional molecular methods currently proposed for Yersinia enterocolitica detection proved to have several limitations. Critical issues associated with the detection of Yersinia enterocolitica in meat and/or meat product has already been investigated, whereas data on the possible limits of the molecular methods for Yersinia enterocolitica detection in vegetables are still lacking. According to ISO method (ISO 18867:2015), real-time polymerase chain reaction (rtPCR) should be adopted for Yersinia enterocolitica detection, even if it proved to be affected by some biases. Recently, Droplet Digital PCR (ddPCR) has been introduced as a useful tool to detect and quantify different pathogenic bacteria in complex food matrices. However, its potential application for Yersinia enterocolitica detection in vegetables has never been investigated before. In the present study two molecular platforms (rtPCR and ddPCR) were used to evaluate the pathogen's behaviour in experimentally contaminated leafy greens (Lactuca sativa L.) and to assess the rate of detection achievable after the incubation for eleven days at different temperatures. By comparing, noticeable differences emerged between the two technical approaches: only ddPCR allowed the detection of the pathogen in leafy greens when contaminated at low levels. Moreover, results of the present work highlighted the importance of length and temperature of incubation on the survival and/or the growth of Yersinia enterocolitica in vegetables: at 18 and 25 °C the concentration of the pathogen considerably decreases along incubation. Based on data, the use of rtPCR leads to an underestimation of the true prevalence of pathogenic Y. enterocolitica in vegetables, while temperature and time currently proposed for Y. enterocolitica (25 °C for 24 h), allow optimizing detection. To conclude, ddPCR may be undoubtedly proposed as a reliable alternative strategy for the quick detection of the pathogen in food samples

Hygiene evaluation and microbiological hazards of hunted wild boar carcasses

Within the European Union, different legal requirements must be applied in relation to the circumstances in which wild boar meat is supplied for human consumption. The present study performed from October to December 2019 in the Campania region aimed to monitor microbial contamination on 36 wild boar carcasses eviscerated in premises registered according to the EU Regulation 852/04 (19 animals) and hunters' private houses (17 animals). From each carcass, four areas (ham, back, jowl, and belly), were swabbed using cellulose sponges and analysed for the enumeration of mesophilic bacteria, Enterobacteriaceae and E. coli using the respective culture ISO methods. Real-time PCR was used for the detection of Salmonella spp., Yersinia enterocolitica, Campylobacter spp., and pathogenic E. coli. The presence of pathogenic bacteria was also evaluated in 36 meat samples to better understand the public health risks related to its consumption. Additionally, the presence of Y. enterocolitica was assessed on 36 tonsil samples since, in swine, this pathogen is frequently isolated in this organ. According to the limits settled by the EU Regulation 2073/2005 for pork, carcasses collected from registered premises resulted in more satisfactory mesophilic counts ( 0.05). The overall percentage of wild boars positive per at least one of the enteric pathogens tested was 79.0% (15 out of 19 animals) in registered premises and 82.4% (14 out of 17 animals) in private houses. Pathogenic E. coli was detected in 27 carcasses (75.0%), suggesting that wild boar could play a role as reservoir host and that the meat can cause public health concerns. In conclusion, in the present study an overall high bacterial level was observed on wild boar carcasses, and therefore the need for better slaughter hygiene was demonstrated. Based on the data, the meat resulting from private domestic slaughter may be of lower hygienic quality. The absence of a significant difference observed in the present study may be due to limited sample size and therefore further research should be performed

Determination of the microbiological contamination in minced pork by culture dependent and 16S amplicon sequencing analysis

Routine evaluation of bacterial contamination in minced pork is still mainly performed by the enumeration of indicator bacteria, including total aerobic colony count and E. coli, using standardized isolation methods. However, the bacterial community structure as well as the effect of the storage time and temperature on the aerobic plate count are largely unknown for this matrix. The aim of the study was to characterize the microbial community in minced pork by 16S rRNA amplicon sequencing compared to classical isolation methods combined with identification by Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI TOF MS) and 16S rRNA gene sequencing. Analysis of 14 unrelated samples showed that total aerobic counts determined at 30 °C and 7 °C showed no significant difference, but the richness was higher on PCA at 30 °C for 7 samples, equal in 5, and higher at 7 °C for 2 samples. Members of the genus Pseudomonas, along with the genera Brochothrix and Carnobacterium were commonly identified among both the mesophilic and psychrotrophic population. Comparing to 16S rRNA amplicon sequencing, some contrasting data were obtained. Except for Brochothrix spp. and Pseudomonas spp., that were abundant and always detected, genera obtained with the two methods in the same sample were not always the same. Comparison of different sample preparation techniques and DNA extraction methods demonstrated also in this matrix that different results on the microbial composition and complexity are obtained. Present data illustrate that the combined isolation and identification of isolates using MALDI TOF MS and 16S gene sequencing and overall community profiling using 16S rRNA amplicon sequencing provides complementary results and yields important insights into the complex relationship between microorganisms in a food

Determination of the microbiological contamination in minced pork by culture dependent and 16S amplicon sequencing analysis

Routine evaluation of bacterial contamination in minced pork is still mainly performed by the enumeration of indicator bacteria, including total aerobic colony count and E. coli, using standardized isolation methods. However, the bacterial community structure as well as the effect of the storage time and temperature on the aerobic plate count are largely unknown for this matrix. The aim of the study was to characterize the microbial community in minced pork by 16S rRNA amplicon sequencing compared to classical isolation methods combined with identification by Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI TOF MS) and 16S rRNA gene sequencing. Analysis of 14 unrelated samples showed that total aerobic counts determined at 30 degrees C and 7 degrees C showed no significant difference, but the richness was higher on PCA at 30 degrees C for 7 samples, equal in 5, and higher at 7 degrees C for 2 samples. Members of the genus Pseudomonas, along with the genera Brochothrix and Carnobacterium were commonly identified among both the mesophilic and psychrotrophic population. Comparing to 16S rRNA amplicon sequencing, some contrasting data were obtained. Except for Brochothrix spp. and Pseudomonas spp., that were abundant and always detected, genera obtained with the two methods in the same sample were not always the same. Comparison of different sample preparation techniques and DNA extraction methods demonstrated also in this matrix that different results on the microbial composition and complexity are obtained. Present data illustrate that the combined isolation and identification of isolates using MALDI TOF MS and 16S gene sequencing and overall community profiling using 16S rRNA amplicon sequencing provides complementary results and yields important insights into the complex relationship between microorganisms in a food