Occurrence of virulence genes among Vibrio cholerae and Vibrio parahaemolyticus strains from treated wastewaters.

Pathogenic Vibrio species are an important cause of foodborne illnesses. The aim of this study was to describe the occurrence of potentially pathogenic Vibrio species in the final effluents of a wastewater treatment plant and the risk that they may pose to public health. During the 1-year monitoring, a total of 43 Vibrio strains were isolated: 23 Vibrio alginolyticus, 1 Vibrio cholerae, 4 Vibrio vulnificus, and 15 Vibrio parahaemolyticus. The PCR investigation of V. parahaemolyticus and V. cholerae virulence genes (tlh, trh, tdh, toxR, toxS, toxRS, toxT, zot, ctxAB, tcp, ace, vpi, nanH) revealed the presence of some of these genes in a significant number of strains. Intraspecies variability and genetic relationships among the environmental isolates were analyzed by random amplified polymorphic DNA-PCR (RAPD-PCR). We report the results of the first isolation and characterization of an environmental V. cholerae non-O1 non-O139 and of a toxigenic V. parahaemolyticus strain in Tunisia. We suggest that non-pathogenic Vibrio might represent a marine reservoir of virulence genes that can be transmitted between strains by horizontal transfer.

Development of a colony hybridization method for the enumeration of total and potentially enteropathogenic Vibrio parahaemolyticus in shellfish.

Vibrio parahaemolyticus is a marine microorganism, recognized as cause of gastroenteritis outbreaks associated with seafood consumption. In this study the development and the in-house validation of a colony hybridization method for the enumeration of total and potentially pathogenic V. parahaemolyticus is reported. The method included a set of three controls (process, hybridization and detection control) for the full monitoring of the analytical procedure. Four digoxigenin-labeled probes were designed for pathogenic strains enumeration (tdh1, tdh2, trh1 and trh2 probes) and one for total V. parahaemolyticus count (toxR probe). Probes were tested on a panel of 70 reference strains and 356 environmental, food and clinical isolates, determining the inclusivity (tdh: 96.7%, trh: 97.8%, toxR: 99.4%) and the exclusivity (100% for all probes). Accuracy and linearity of the enumeration were evaluated on pure and mixed cultures: slopes of the regression lines ranged from 0.957 to 1.058 depending on the target gene and R(2) was greater than or equal to 0.989 for all reactions. Evaluation was also carried on using four experimentally contaminated seafood matrices (shellfish, finfish, crustaceans and cephalopods) and the slopes of the curves varied from 0.895 (finfish) to 0.987 (cephalopods) for the counts of potentially pathogenic V. parahaemolyticus (R(2)≥0.965) and from 0.965 to 1.073 for total V. parahaemolyticus enumeration (R(2)≥0.981). Validation was performed on 104 naturally contaminated shellfish samples, analyzed in parallel by colony hybridization, ISO/TS 21872-1 and MPN enumeration. Colony hybridization and ISO method showed a relative accuracy of 86.7%, and a statistically significant correlation was present between colony hybridization enumeration and MPN results (r=0.744, p<0.001). The proposed colony hybridization can be a suitable alternative method for the enumeration of total and potentially pathogenic V. parahaemolyticus in seafood.

Detection and quantification of Vibrio parahaemolyticus in shellfish from Italian production areas.

Vibrio parahaemolyticus is a marine microorganism, recognized as an important cause of foodborne illness particularly in Asia, South America and United States. Outbreaks are rarely reported in Europe, but they can occur unexpectedly in relation, among other reasons, to the spread of highly virulent strains. It is known that the risk is proportional to exposure levels to pathogenic V. parahaemolyticus (i.e. carrying the tdh and/or the trh genes) but currently there is a lack of occurrence data for pathogenic V. parahaemolyticus in shellfish production areas of the Member States. In this study a total of 147 samples of bivalve molluscs, from harvesting areas of two Italian regions (Sardinia and Veneto) were analyzed for Escherichia coli and salmonella, according to Reg 2073/2005, and for detection and enumeration of total and toxigenic V. parahaemolyticus strains using a new DNA colony hybridization method. Environmental parameters (water temperature and salinity) were also recorded. Results of E. coli were consistently in agreement with the legislation limits for the harvesting class of origin and Salmonella was detected only in one sample. The average contamination levels for total V. parahaemolyticus were 84 and 73 CFU/g respectively for Sardinia and Veneto, with the highest value reaching 8.7 × 10(3)CFU/g. Nineteen samples (12.9%) resulted positive for the presence of potentially pathogenic V. parahaemolyticus strains, with levels ranging between 10 and 120 CFU/g and most of the positive samples (n=17) showing values equal or below 20 CFU/g. A significant correlation (r=0.41) was found between water temperature and V. parahaemolyticus levels, as well as with isolation frequency. The data provided in this study on contamination levels of total and potentially pathogenic V. parahaemolyticus, seasonal distribution and correlation with water temperature, will help in defining appropriate monitoring programs and post-harvest policies for this hazard, improving the management of the harvesting areas and the safety of bivalve molluscs.

Qualitative and quantitative assessment of viral contamination in bivalve molluscs harvested in Italy.

Bivalve molluscs are a well documented source of viral infection. Further data on shellfish viral contamination are needed to implement European Regulations with sanitary measures more effective against viral pathogens. To this aim, 336 samples of bivalve molluscs (185 mussels, 66 clams, 23 oysters and 62 samples from other species) collected in harvesting areas of class A and B of four Italian Regions were analyzed for qualitative and quantitative determination of hepatitis A virus (HAV) and Norovirus (NoV) GI and GII, using real time RT-PCR. The results showed a wide diffusion of viral contamination in the shellfish production areas considered. HAV prevalence was low (0.9%) with contamination levels that varied from 5 to 7 × 10(2)copies/g. On the contrary, NoV showed a high prevalence (51.5%), with a large variability according to the group considered (e.g. 47.8% for Crassostrea in Veneto, 79.7% for Mytilus in Campania, 84.6% for Tapes in Sardinia). NoV contamination affected class A and class B production areas to a different extent, with a statistically significant difference in both contamination prevalence (22.1% vs. 66.3%; p<0.0001) and quantity (average contamination level of 3.1 × 10(2) vs. 1.9 × 10(3) copies/g; p<0.05). The different species analyzed from class B harvesting areas (Mytilus, Tapes/Ruditapes and Crassostrea) showed a NoV prevalence respectively of 70.3%, 66.0% and 47.8% but comparable NoV contamination levels (between 8.4 × 10(2) and 4.9 × 10(3)copies/g). Other two bivalve species considered in the study (Donax spp. and Solen spp.) showed a relevant NoV presence (40.0% and 34.4% of samples). Finally, samples analyzed before and after commercial purification treatment showed a decrease of contamination prevalence after the treatment, but inconsistent results were recorded on NoV levels. The data obtained, together with other quantitative information to estimate consumer exposure, in association with studies on dose-response and on the effectiveness of post-harvest treatments, will provide a useful tool for the definition of microbiological criteria related to the different shellfish species.

Noroviruses in seafood: a 9-year monitoring in Italy.

Norovirus (NoV) are increasingly important as etiological agents of gastrointestinal infections. Consumption of bivalve molluscs and ready-to-eat fishery products is one of the most common ways of acquiring NoV foodborne infections, and the rise of outbreaks of viral gastroenteritis represents an important health problem that is also responsible for economic losses. The aim of this work was to define the prevalence of NoV contamination in preserved fishery products and in shellfish commercialized in Italy, taking into account the results obtained during 9 years of survey (2003-2011) and paying special attention to the regions more involved in national production. A total of 4463 samples were examined (2310 mussels, 1517 clams, 510 oysters, 22 other shellfish species, 104 preserved seafood products) and the average positivity rate for NoV presence was 4.1% and ranged from 0.6% in 2007 to 9.8% in 2003 and from 1.9% in preserved seafood products to 4.7% in mussels. Genetic characterization of circulating strains showed a prevalence of genogroup II genotypes, including GII.b and GII.e polymerase types and different GII.4 variants. This information could contribute to the optimization of risk-based sampling strategies for NoV contamination in seafood, taking into account variability in different species and from year to year.

Presence of pathogenic Vibrio parahaemolyticus in waters and seafood from the Tunisian Sea.

The occurrence of the hemolysin genes, tdh and trh, in Vibrio parahaemolyticus strains isolated from environmental samples collected from various exported seafood products comprising of fishes and shellfish (Mytilus edulis and Crassostrea gigas) or seawater, was studied. Eight strains were confirmed as V. parahaemolyticus by toxR -based polymerase chain reaction and only one strain out of these 8 strains was positive for tdh and trh genes. Toxigenic V. parahaemolyticus isolates are present in Tunisian coastal areas and they may also be present in Tunisian exported seafood products.

Norovirus monitoring in bivalve molluscs harvested and commercialized in southern Italy.

Norovirus (NoV) is the main cause of human nonbacterial gastroenteritis throughout the world. NoVs are classified into five genogroups: GI, GII, GIII, GIV, and GV. NoVs from GI and GII are the most commonly reported NoVs associated with human infections, and raw or undercooked shellfish have been identified as the main potential infection vehicle. European Commission Regulation 2073/2005 defines only bacteriological parameters for use as safety criteria for shellfish because reference methods for detection of viruses are lacking. From July 2007 to April 2010, 163 shellfish samples were collected in southern Italy from harvesting areas, authorized or nonauthorized retailers, and a restaurant after an outbreak of human gastroenteritis. The shellfish were analyzed for the presence of NoVs from GI and GII using the one-step real-time reverse transcription PCR protocol. A total of 94 shellfish samples (57.7%) were positive for the presence of NoV, and GII was the most frequently identified genogroup.

Duplex Real Time PCR for the detection of hepatitis A virus in shellfish using Feline Calicivirus as a process control.

The consumption of bivalve shellfish is a common cause of foodborne outbreaks of viral origin. The evaluation of the sanitary quality of these products, however, is still based on bacterial indicators of fecal contamination (Reg. (EC) No. 2073/2005 and No.1441/2007) even if it is known that they are not reliable indicators of viral contamination. In this study a duplex Real Time PCR method for quantitative detection of hepatitis A (HAV) in shellfish was developed. Feline Calicivirus (FCV) was used as a control for assessing the effectiveness of the concentration and extraction process and the ability to eliminate PCR inhibitors present in the food matrix. The specific primers and probes for detection of HAV and FCV, chosen respectively from the 5'-UTR region and in the ORF1 region, were labeled with two different dyes and detected simultaneously. The method was applied on spiked and non-spiked shellfish from a local market. The amplification of HAV in the presence of FCV showed good linearity (R(2)=0.994) and the sensitivity limit of the reaction was at least 5 x 10(2)TCID(50)g(-1) of an hepatopancreas extract.

Phylogenetic and evolutionary analysis of Vibrio parahaemolyticus and Vibrio alginolyticus isolates based on toxR gene sequence.

The Vibrio genus contains a large number of closely related bacterial species differing, in some cases, less than 1% in 16S rRNA gene sequence. The present study evaluated the usefulness of toxR gene for phylogenetic and evolution analysis on Vibrio isolates of environmental or clinical origin belonging to the two closely related species V. parahaemolyticus and V. alginolyticus. The phylogenetic analysis based on toxR gene, contrary to 16S rRNA gene, allowed a clear differentiation of the isolates belonging to the two species and showed the presence of two separate, statistically supported clusters in V. alginolyticus (subgroup A and B). Such division, partially reflected in the biochemical features of the isolates, could not be explained by spatial and/or temporal distance in the isolation, leading to the hypothesis of two distinct, co-existing clusters in the V. alginolyticus isolates analysed. The evolutionary analysis on the toxR sequence showed that while the substitutions inferred from the alignment of V. parahaemolyticus are best explained by the negative/neutral selection model, in V. alginolyticus--and particularly in subgroup B--is acting a positive evolutionary pressure. The site detected as under diversifying selection (P164L) could be related to conformational changes of ToxR protein.

Assessment of human enteric viruses in shellfish from the northern Adriatic sea.

Incidence and circulation of different strains of hepatitis A and Norovirus in shellfish were studied on 235 samples (Tapes philippinarum, Mytilus galloprovincialis, Ostrea spp. and Chlamys spp.) obtained from different sites, representing the shellfish production areas of the northern Adriatic sea. Shellfish were harvested in the period of one year and, after depuration, were examined for bacterial (Escherichia coli and Salmonella) and viral (HAV and NoV) contamination. Viral contamination was present on average in 22% of samples: specifically, 6% of samples tested positive for HAV, 14% for NoV and 2% for both viruses. None of the samples revealed the presence of Salmonella, and in most of them (93%) the number of E. coli was below the European legislation limit of 230 MPN/100 g. T. philippinarum was the species most often contaminated, as well as being the only species in which the legal limit for E. coli was, in some cases, exceeded. Both HAV and NoV contamination were detected throughout the year; NoV detection was slightly more frequent during winter months, but positive samples were also present in summer. The sequencing of the PCR products showed the circulation of only one HAV genotype (IA) and four different NoV genotypes (Hawaii, Melksham, Lordsdale and GGIIb) with a prevalence of the GGIIb genotype in the second period of the monitoring.

Evaluation of different polymerase chain reaction methods for the identification of Vibrio parahaemolyticus strains isolated by cultural methods.

Control of contamination by Vibrio parahaemolyticus in fishery products is often hampered by the lack of standardized methods and by the uncertainty associated with biochemical identification of the isolates. In this study, 5 polymerase chain reaction (PCR) methods for the identification of V. parahaemolyticus to the species level were evaluated by using 25 Vibrio reference strains and 163 isolates from fishery products, environmental sources, and clinical samples. Sequence targets of the methods were toxR, gyrB, and tlh genes (tested with 2 protocols), and the fragment pR72H. Isolate identification was confirmed by sequencing of the 16S rRNA gene and by PCR protocols for the identification of other Vibrio species. The PCR assay targeting the toxR gene achieved the highest performance (100% inclusivity and exclusivity). The 2 PCR protocols based on tlh gene detection, although showing the same inclusivity (100%), differed in the exclusivity (50 and 91%, respectively). Finally, the results provided by the PCR assays targeting the gyrB gene and pR72H fragment were less reliable and, in some cases, difficult to assess. According to the results of this study, the characteristics of accuracy expressed by the toxR identification method make it a suitable candidate as a reference method for the molecular identification of V. parahaemolyticus strains.

Resistance of hepatitis A virus in mussels subjected to different domestic cookings.

Hepatitis A is a worldwide infectious disease. Shellfish consumption has always been one of the major risk factors for hepatitis A infection, especially when these products are eaten raw or slightly cooked. Moreover, the cooking does not always guarantee the harmlessness of shellfish. The aim of the present study was to evaluate the hepatitis A virus (HAV) resistance in experimentally contaminated mussels, subjected to domestic cooking. Three different domestic preparations (mussel hors-d'oevre, mussel au gratin, mussels with tomato sauce) were performed according to the traditional Italian cookery using different time and temperature conditions. To detect HAV-RNA, RT-nested-PCR was used; the presence of the infectious virus in the positive samples was confirmed by an integrated cell culture-RT-PCR method. The infectious virus was completely inactivated only in "mussels in tomato sauce", while it was still present, even if not quantitatively determinable, in the other preparations. The study confirmed that some factors can influence the HAV sensitivity to thermal inactivation preventing a complete decontamination of the product.

Round-robin comparison of methods for the detection of human enteric viruses in lettuce.

Five methods that detect human enteric virus contamination in lettuce were compared. To mimic multiple contaminations as observed after sewage contamination, artificial contamination was with human calicivirus and poliovirus and animal calicivirus strains at different concentrations. Nucleic acid extractions were done at the same time in the same laboratory to reduce assay-to-assay variability. Results showed that the two critical steps are the washing step and removal of inhibitors. The more reliable methods (sensitivity, simplicity, low cost) included an elution/concentration step and a commercial kit. Such development of sensitive methods for viral detection in foods other than shellfish is important to improve food safety.

Reverse transcription-booster PCR for detection of noroviruses in shellfish.

The methods commonly used for norovirus (NV) detection are based on reverse transcription-PCR (RT-PCR) followed by confirmation of the amplified sequence. To increase sensitivity, an RT-booster PCR was developed. The proposed method showed an increase in sensitivity at least 2 log units for all the NV strains tested compared with the standard RT-PCR method. Higher sensitivity was confirmed in tests on experimentally and naturally contaminated shellfish.

Multicenter validation of PCR-based method for detection of Salmonella in chicken and pig samples.

As part of a standardization project, an interlaboratory trial including 15 laboratories from 13 European countries was conducted to evaluate the performance of a noproprietary polymerase chain reaction (PCR)-based method for the detection of Salmonella on artificially contaminated chicken rinse and pig swab samples. The 3 levels were 1-10, 10-100, and 100-1000 colony-forming units (CFU)/100 mL. Sample preparations, including inoculation and pre-enrichment in buffered peptone water (BPW), were performed centrally in a German laboratory; the pre-PCR sample preparation (by a resin-based method) and PCR assay (gel electrophoresis detection) were performed by the receiving laboratories. Aliquots of BPW enrichment cultures were sent to the participants, who analyzed them using a thermal lysis procedure followed by a validated Salmonella-specific PCR assay. The results were reported as negative or positive. Outlier results caused, for example, by gross departures from the experimental protocol, were omitted from the analysis. For both the chicken rinse and the pig swab samples, the diagnostic sensitivity was 100%, with 100% accordance (repeatability) and concordance (reproducibility). The diagnostic specificity was 80.1% (with 85.7% accordance and 67.5% concordance) for chicken rinse, and 91.7% (with 100% accordance and 83.3% concordance) for pig swab. Thus, the interlaboratory variation due to personnel, reagents, thermal cyclers, etc., did not affect the performance of the method, which will be proposed as part of a developing international PCR standard.

Comparison of PCR, electrochemical enzyme-linked immunosorbent assays, and the standard culture method for detecting salmonella in meat products.

An electrochemical enzyme-linked immunosorbent assay (ELISA) coupled with flow injection analysis (ELISA-FIA) and a PCR-based method using ST11 and ST15 primers for detecting salmonellae in meat were evaluated in comparison with the International Organization for Standardization (ISO) culture method. The methods were applied to experimentally contaminated and naturally contaminated meat samples. The results showed that both ELISA-FIA and PCR allowed detection of salmonella in a product contaminated with a low number of the microorganisms (1 to 10 salmonellae/25 g) after only 5 h of incubation of preenrichment broth, and they were just as effective as the ISO method.

[Microbiological risk associated with seafood consumption]. / Rischio microbiologico associato al consumo di prodotti ittici.

Seafood has always been an important source for human nutrition. The increase of its consumption, however, and the epidemiological data, confirming the role of seafood, especially shellfish, as a carrier of foodborne toxinfections, has brought the need of a greater control of his microbiological characteristics. In this review we considered the main pathogenous microorganisms associated to such products, with a special attention towards the emerging pathogens not yet considered in the legislation (autochthonous pathogens, e.g. vibrios and enteric viruses). We examined the conditions of their presence and survival in the marine environment, the mechanisms through which they contaminate seafood and their resistance to the treatments endured by food before the commercialization and at the moment of the consumption.

Evaluation of DNA extraction methods for use in combination with SYBR green I real-time PCR to detect Salmonella enterica serotype enteritidis in poultry.

The objective of this study was to develop a rapid, reproducible, and robust method for detecting Salmonella enterica serotype Enteritidis in poultry samples. First, for the extraction and purification of DNA from the preenrichment culture, four methods (boiling, alkaline lysis, Nucleospin, and Dynabeads DNA Direct System I) were compared. The most effective method was then combined with a real-time PCR method based on the double-stranded DNA binding dye SYBR Green I used with the ABI Prism 7700 system. The specificity of the reaction was determined by the melting temperature (T(m)) of the amplicon obtained. The experiments were conducted both on samples of chicken experimentally contaminated with serotype Enteritidis and on commercially available poultry samples, which were also used for comparisons with the standard cultural method (i.e., ISO 6579/2001). The results of comparisons among the four DNA extraction methods showed significant differences except for the results from the boiling and Nucleospin methods (the two methods that produced the lowest threshold cycles). Boiling was selected as the preferred extraction method because it is the simplest and most rapid. This method was then combined with SYBR Green I real-time PCR, using primers SEFA-1 and SEFA-2. The specificity of the reaction was confirmed by the T(m), which was consistently specific for the amplicon obtained; the mean peak T(m) obtained with curves specific for serotype Enteritidis was 82.56 +/- 0.22 degrees C. The standard curve constructed using the mean threshold cycle and various concentrations of serotype Enteritidis (ranging from 10(3) to 10(8) CFU/ml) showed good linearity (R(2) = 0.9767) and a sensitivity limit of less than 10(3) CFU/ml. The results of this study demonstrate that the SYBR Green I real-time PCR constitutes an effective and easy-to-perform method for detecting serotype Enteritidis in poultry samples.

The survival of hepatitis A virus in fresh produce.

Fresh produce has been repeatedly implicated as the source of human viral infections, including infection with hepatitis A virus (HAV). The objective of the present study was to evaluate the HAV adsorption capacity of the surface of various fresh vegetables that are generally eaten raw and the persistence of the HAV. To this end, the authors experimentally contaminated samples of lettuce, fennel, and carrot by immersing them in sterile distilled water supplemented with an HAV suspension until reaching a concentration of 5 log tissue culture infectious dose (TCID50)/ml. After contamination, the samples were stored at 4 degrees C and analysed at 0, 2, 4, 7, and 9 days. To detect the HAV, RT-nested-PCR was used; positive samples were subjected to the quantitative determination using cell cultures. The three vegetables differed in terms of their adsorption capacity. The highest quantity of virus was consistently detected for lettuce, for which only a slight decrease was observed over time (HAV titre = 4.44 +/- 0.22 log TCID50/ml at day 0 vs. 2.46 +/- 0.17 log TCID50/ml at day 9, before washing). The virus remained vital through the last day of storage. For the other two vegetables, a greater decrease was observed, and complete inactivation had occurred at day 4 for carrot and at day 7 for fennel. For all three vegetables, washing does not guarantee a substantial reduction in the viral contamination.

Effects of depuration of molluscs experimentally contaminated with Escherichia coli, Vibrio cholerae 01 and Vibrio parahaemolyticus.

AIMS: The aim of the present study was to investigate the behaviour of two pathogenic vibrios (Vibrio cholerae O1 and Vibrio parahaemolyticus) during depuration and to compare it with that of Escherichia coli, used as an indicator of suitability for consumption. METHODS AND RESULTS: Samples of Mytilus galloprovincialis were experimentally contaminated with E. coli, V. cholerae O1 and V. parahaemolyticus, depurated in a pilot plant using ozone and analysed at selected intervals. Numbers of E. coli and vibrios were estimated using an MPN method. The presence of vibrios was confirmed by the use of PCR. The target genes used were ctx for V. cholerae O1 and the restriction fragment pR72H for V. parahaemolyticus. There was a substantially smaller reduction in the numbers of both vibrios (approximately 1 log) during the depuration process than of E. coli (approximately 3 log). CONCLUSIONS: The results confirm the inadequacy of E. coli as an indicator that molluscs have been cleansed of other microbiological agents. SIGNIFICANCE AND IMPACT OF THE STUDY: The study confirms the risk associated with the consumption of mussels and the need to correctly conserve and cook them prior to consumption.