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.

Yersinia enterocolitica detection in pork products: Evaluation of isolation protocols.

Conventional methods for Yersinia enterocolitica detection in food samples are generally considered inadequate. Problems arise from the presence of the so-called "background flora", coupled to the low contamination level of the pathogen. Since, data on the microbial ecology occurring in competitive microflora are still lacking, MALDI TOF MS was used for strains 'identification after enrichment in PSB or ITC broths, and after plating on selective CIN medium at different incubation times. SYBR Green Real time PCR was used for the Y. enterocolitica strains' detection (4/O:3, 1A/O:5) in experimentally contaminated foods, as well as in naturally contaminated samples. A higher number of different bacterial genera (10 on CIN and 18 on PCA) was recorded after enrichment in PSB, whilst enrichment in ITC led to recovery of 6 and 10 genera on CIN and PCA, respectively. Yersiniaceae was the dominant family on the first day of incubation, but on the second day the percentage of isolation considerably decreased. By testing experimentally contaminated samples, substantial difficulties were encountered. The biotype 1A was always detected, whereas strain 4/O:3 proved to be poorly competitive. Based on the data, the enrichment media PSB and ITC, currently proposed for Y. enterocolitica detection, need to be improved to promote a successful pathogen's recovery.

Assessment of microbial communities on freshly killed wild boar meat by MALDI-TOF MS and 16S rRNA amplicon sequencing.

Wild boars (Sus scrofa) are the most widely distributed large mammals and recent increase in consumption of wild boar meat urges the need of microbiological quality criteria. The aim of the study was to characterize the initial bacterial contamination on freshly-killed wild boar meat using a culture-dependent approach with ISO-methods combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification and 16S rRNA amplicon sequencing. Moreover, the presence of foodborne pathogens was examined using Real-Time-PCR and confirmed by classical isolation. Analysing 22 unrelated wild boar meat samples showed a higher bacterial contamination level compared to pork, with Salmonella present in almost one third of the samples. A great variability of the microbial contamination between the samples was recorded, as well as complementary results between culturing and 16S rRNA amplicon sequencing as frequently isolated genera were not always detected, and vice versa. Furthermore, the foodborne pathogen Salmonella was never detected with 16S rRNA amplicon sequencing, demonstrating the necessity for a cautious approach in the implementation of new analysis techniques in food safety. The present work determines that attention should be paid to the trade of non-inspected meat directly to retail or consumers.

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.

Detection of Human Bocavirus Species 2 and 3 in Bivalve Shellfish in Italy.

Human bocavirus (HBoV) has been shown to be a common cause of respiratory infections and gastroenteritis in children. Recently, HBoVs have been detected in sewage and river waters in Italy and worldwide. However, studies on their presence in other water environments and in bivalve mollusks are not yet available. In this study, 316 bivalve shellfish samples collected in three Italian regions over a 6-year period (2012 to 2017) were analyzed by nested PCR and sequencing using broad-range primer pairs targeting the capsid proteins VP1 and VP2 of HBoV. The virus was detected in 27 samples (8.5% of the total samples), and a statistically significant difference was found within the three regions. A further 13 samples, collected in geographic and temporal proximity to positive samples, were included in the study to assess the spread of HBoV in shellfish production areas at the time of contamination. Twelve of these additional samples were found to be positive for HBoV. All positive samples in this study were characterized as HBoV species 2 (17 samples; 8 different sequences) or species 3 (22 samples; 4 different sequences). This study reports the occurrence of HBoV in bivalve shellfish and shows evidence of considerable spatial spread of the virus throughout shellfish production areas. Further studies are needed to elucidate both the role of HBoV as an agent of gastroenteritis and the risk for foodborne transmission of this virus.IMPORTANCE Human bocavirus is recognized as an important cause of acute respiratory tract infections and has recently been considered an etiological agent of gastroenteritis in the pediatric population. Our findings document that HBoVs are detected in bivalve shellfish with a relevant prevalence and suggest that an assessment of the risk for foodborne transmission of these viruses should be undertaken.

First Detection of Hepatitis E Virus in Shellfish and in Seawater from Production Areas in Southern Italy.

Shellfish samples (n = 384) from production areas, water samples from the same areas (n = 39) and from nearby sewage discharge points (n = 29) were analyzed for hepatitis E virus (HEV) by real-time and nested RT-PCR. Ten shellfish samples (2.6%) and five seawater samples (12.8%) tested positive for HEV; all characterized strains were G3 and showed high degree of sequence identity. An integrated surveillance in seafood and waters is relevant to reduce the risk of shellfish-associated illnesses.

Detection of Norovirus GII.17 Kawasaki 2014 in Shellfish, Marine Water and Underwater Sewage Discharges in Italy.

Norovirus (NoV) is a major cause of non-bacterial acute gastroenteritis worldwide, and the variants of genotype GII.4 are currently the predominant human strains. Recently, a novel variant of NoV GII.17 (GII.P17_GII.17 NoV), termed Kawasaki 2014, has been reported as the cause of gastroenteritis outbreaks in Asia, replacing the pandemic strain GII.4 Sydney 2012. The GII.17 Kawasaki 2014 variant has also been reported sporadically in patients with gastroenteritis outside of Asia, including Italy. In this study, 384 shellfish samples were subjected to screening for human NoVs using real-time PCR and 259 (67.4%) tested positive for Genogroup II (GII) NoV. Of these, 52 samples, selected as representative of different areas and sampling dates, were further amplified by conventional PCR targeting the capsid gene, using broad-range primers. Forty shellfish samples were characterized by amplicon sequencing as GII.4 (n = 29), GII.2 (n = 4), GII.6 (n = 2), GII.12 (n = 2), and GII.17 (n = 3). Sixty-eight water samples (39 seawater samples from the corresponding shellfish production areas and 29 water samples from nearby underwater sewage discharge points) were also tested using the above broad-range assay: eight NoV-positive samples were characterized as GII.1 (n = 3), GII.2 (n = 1), GII.4 (n = 2), and GII.6 (n = 2). Based on full genome sequences available in public databases, a novel RT-PCR nested assay specific for GII.17 NoVs was designed and used to re-test the characterized shellfish (40) and water (8) samples. In this second screening, the RNA of GII.17 NoV was identified in 17 additional shellfish samples and in one water sample. Upon phylogenetic analysis, these GII.17 NoV isolates were closely related to the novel GII.17 Kawasaki 2014. Interestingly, our findings chronologically matched the emergence of the Kawasaki 2014 variant in the Italian population (early 2015), as reported by hospital-based NoV surveillance. These results, showing GII.17 NoV strains to be widespread in shellfish samples collected in 2015 in Italy, provide indirect evidence that this strain has started circulating in the Italian population. Notably, using a specific assay, we were able to detect many more samples positive for GII.17 NoV, indicating that, in food and water matrices, broad-range assays for NoV may grossly underestimate the prevalence of some, less common, NoVs. The detection of the GII.17 strain Kawasaki 2014 in clinical, water and food samples in Italy highlights the need for more systematic surveillance for future disease control and prevention.