A pan-European ring trial to validate an International Standard for detection of Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus in seafoods.

Globally, vibrios represent an important and well-established group of bacterial foodborne pathogens. The European Commission (EC) mandated the Comite de European Normalisation (CEN) to undertake work to provide validation data for 15 methods in microbiology to support EC legislation. As part of this mandated work programme, merging of ISO/TS 21872-1:2007, which specifies a horizontal method for the detection of V. parahaemolyticus and V. cholerae, and ISO/TS 21872-2:2007, a similar horizontal method for the detection of potentially pathogenic vibrios other than V. cholerae and V. parahaemolyticus was proposed. Both parts of ISO/TS 21872 utilized classical culture-based isolation techniques coupled with biochemical confirmation steps. The work also considered simplification of the biochemical confirmation steps. In addition, because of advances in molecular based methods for identification of human pathogenic Vibrio spp. classical and real-time PCR options were also included within the scope of the validation. These considerations formed the basis of a multi-laboratory validation study with the aim of improving the precision of this ISO technical specification and providing a single ISO standard method to enable detection of these important foodborne Vibrio spp.. To achieve this aim, an international validation study involving 13 laboratories from 9 countries in Europe was conducted in 2013. The results of this validation have enabled integration of the two existing technical specifications targeting the detection of the major foodborne Vibrio spp., simplification of the suite of recommended biochemical identification tests and the introduction of molecular procedures that provide both species level identification and discrimination of putatively pathogenic strains of V. parahaemolyticus by the determination of the presence of theromostable direct and direct related haemolysins. The method performance characteristics generated in this have been included in revised international standard, ISO 21872:2017, published in July 2017.

Diversity of Vibrio spp. isolated at ambient environmental temperature in the Eastern English Channel as determined by pyrH sequencing.

AIMS: To describe the diversity of the culturable mesophilic and potentially pathogenic vibrios isolated at 22 and 37°C on TCBS medium, in September 2009 from seawater and surface sediments. METHODS AND RESULTS: q-PCR assays previously selected for the identification of bacterial strains isolated at 37°C were used in combination with the partial sequencing of two housekeeping genes, pyrH and toxR, to identify 315 strains isolated at 22°C. The great majority of the 37°C strains was identified by q-PCR assays, (five of the six species) with the predominance of Vibrio alginolyticus (85·9%) and V. harveyi (10·7%). The human pathogens V. parahaemolyticus and V. cholerae were rarely detected (two strains each). The 22°C strains were successfully identified by the phylogeny analysis of pyrH and toxR genes, revealing 20 Vibrio species, with the predominance of the clam pathogen V. celticus (36·8%). The Splendidus and the Harveyi groups represented the main Vibrio group at 22°C (80%) and 37°C (99·5%), respectively. CONCLUSIONS: The combination of q-PCR assays and the sequencing of pyrH and toxR genes highlighted two different Vibrio communities at 22 and 37°C both dominated by pathogenic species for marine organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: The sequencing of the pyrH gene revealed to be a valuable tool to identify environmental Vibrio spp. strains isolated at 22°C, as 92·3% of them were identified in this study.

Real-time PCR optimization to identify environmental Vibrio spp. strains.

AIMS: To identify Vibrio vulnificus, Vibrio cholerae and Vibrio alginolyticus using standardized DNA extraction method and real-time PCR assays, among a large number of bacterial strains isolated from marine environment. METHODS AND RESULTS: Methods for DNA extraction and real-time PCR were standardized to identify a large number of Vibrio spp. strains isolated through regular collection campaigns of environmental samples. Three real-time PCR assays were developed from a multiplex PCR, targeting V. vulnificus, V. cholerae and V. alginolyticus on the dnaJ gene. After testing their specificity, these systems were applied for the identification of 961 strains isolated at 22°C (446 strains) and 37°C (515 strains) in September 2009. The predominance of V. alginolyticus (82·6%) among the Vibrio spp. strains isolated at 37°C was shown. At 22°C, only 1·6% of the strains were identified by PCR and they were V. alginolyticus. CONCLUSIONS: Reproducible and specific real-time PCR assays combined to a DNA extraction method on microplates were used to constitute a large environmental Vibrio strains collection and to identify and detect potential human pathogenic Vibrio isolated at 37°C. For environmental strains isolated at 22°C, because of the higher species diversity, other approaches, like sequencing, should be chosen for identification. SIGNIFICANCE AND IMPACT OF THE STUDY: The protocol developed in this study provides an appropriate and rapid screening tool to identify a large number of bacterial strains routinely isolated from the environment in long-term studies.

Effect of oxygen and temperature on the dynamic of the dominant bacterial populations of pig manure and on the persistence of pig-associated genetic markers, assessed in river water microcosms.

AIMS: The aim is to evaluate the dynamic of Bacteroides-Prevotella and Bacillus-Streptococcus-Lactobacillus populations originating from pig manure and the persistence of pig-associated markers belonging to these groups according to temperature and oxygen. METHODS AND RESULTS: River water was inoculated with pig manure and incubated under microaerophilic and aerobic conditions, at 4 and 20°C over 43 days. The diversity of bacterial populations was analysed by capillary electrophoresis-single-strand conformation polymorphism. The persistence of the pig-associated markers was measured by real-time PCR and compared with the survival of Escherichia coli and enterococci. Decay was characterized by the estimation of the time needed to produce a 1-log reduction (T90). The greatest changes were observed at 20°C under aerobic conditions, leading to a reduction in the diversity of the bacterial populations and in the concentrations of the Pig-1-Bac, Pig-2-Bac and Lactobacillus amylovorus markers with a T90 of 10·5, 8·1 and 17·2 days, respectively. CONCLUSIONS: Oxygen and temperature were found to have a combined effect on the persistence of the pig-associated markers in river waters. SIGNIFICANCE AND IMPACT OF THE STUDY: The persistence profiles of the Pig-1-Bac, Pig-2-Bac and Lact. amylovorus markers in addition to their high specificity and sensitivity support their use as relevant markers to identify pig faecal contamination in river waters.

Chlorophyll a might structure a community of potentially pathogenic culturable Vibrionaceae. Insights from a one-year study of water and mussels surveyed on the French Atlantic coast.

The present study focused on the isolation of culturable bacteria from mussels and sea water to identify Vibrionaceae potentially pathogenic for humans. Three sites located on the French Atlantic coast were monitored monthly (twice each month during summer) for 1 year. Environmental parameters were surveyed (water temperature, salinity, turbidity, chlorophyll a) and bacteria were detected by culture and identified by API 20E(®) systems (BioMérieux) and PCR. A total of seven species were detected (Grimontia hollisae, Photobacterium damselae, Vibrio alginolyticus, V. cholerae, V. fluvialis, V. vulnificus and V. parahaemolyticus) and species diversity was higher at the end of summer. Surprisingly, V. cholerae non-O1/non-O139 was detected in spring. No site effect was detected. Using Sørensen similarity indices and statistical analyses, we showed that chlorophyll a had a significant influence on the bacterial community detected in mussels and assemblages were more similar to one another when chlorophyll a values were above 20 µg l(-1) . No significant effect of any parameter was found on the community detected in water samples. Such surveys are essential for the understanding of sanitary crises and detection of emerging pathogens.

mRNA detection by reverse transcription-PCR for monitoring viability and potential virulence in a pathogenic strain of Vibrio parahaemolyticus in viable but nonculturable state.

AIMS: This work investigates the maintenance of viability and potential virulence of Vibrio parahaemolyticus in a viable but nonculturable population (VBNC) state by reverse transcription-polymerase chain reaction (RT-PCR). METHODS AND RESULTS: Housekeeping genes, 16S-23S rDNA and rpoS, as well as virulence genes, tdh1 and tdh2, were selected and detected by PCR in a pathogenic strain of V. parahaemolyticus (Vp4). Their expression was then studied by RT-PCR in V. parahaemolyticus Vp4 cultivated in rich medium at 37 degrees C. The 16S-23S rDNA and rpoS, tdh1, tdh2 genes were transcripted at the mid-logarithmic, stationary and late stationary phases, corresponding to various physiological states. The expression of these genes was also studied by RT-PCR in a VBNC population of V. parahaemolyticus Vp4 in artificial seawater (ASW). The effect of temperature (washing of bacterial culture and microcosms) on the attaining VBNC bacteria was first considered. Washing of V. parahaemolyticus Vp4, collected at the mid-logarithmic phase, at 10 or 4 degrees C before inoculation in ASW at 4 degrees C allowed bacteria entered the VBNC state between 22 and 31 days. The 16S-23S rDNA and rpoS gene were expressed in the VBNC bacteria whereas no expression of the tdh1 and tdh2 genes was observed in the same populations. CONCLUSION: The two selected housekeeping genes, 16S-23S rDNA and rpoS, proved to be good viability markers for V. parahaemolyticus Vp4 in culturable and VBNC states. These first data indicated that the pathogenic strain Vp4 would not maintain the expression of the virulence genes, tdh1 and tdh2, in VBNC state. SIGNIFICANCE AND IMPACT OF THE STUDY: Use of RT-PCR for investigating the maintenance or not of viability and potential virulence in VBNC V. parahaemolyticus will facilitate further study to evaluate the potential risk presented by this pathogen in the environment.

Occurrence of pathogenic vibrios in coastal areas of France.

AIMS: This study was carried out to investigate the occurrence of potentially pathogenic species of Vibrio in French marine and estuarine environments. METHODS AND RESULTS: Samples of coastal waters and mussels collected between July and September 1999 were analysed by culture, using selective media including thiosulphate-citrate-bile salts-sucrose and modified cellobiose-polymixin B-colistin agar. Presumptive Vibrio colonies were isolated and identified using selected biochemical tests. Specific primers based on flanking sequences of the cytolysin, vvhA gene, pR72H DNA fragment and 16S-23S rRNA intergenic spacer region (ISR) were used in a polymerase chain reaction (PCR) to confirm the identification of Vibrio vulnificus, V. parahaemolyticus and V. cholerae, respectively. In this study, V. alginolyticus (99 of 189) was the predominant species, followed by V. parahaemolyticus (41 of 189), V. vulnificus (20 of 189) and non-O1/non-O139 V. cholerae (three of 189). All 20 V. vulnificus isolates showed PCR amplification of the vvhA gene, 16 of which had been isolated from estuarine water. The PCR amplification of the pR72H DNA fragment in 41 V. parahaemolyticus isolates generated two unique amplicons of 387 and 320 bp. The latter, present in 24.4% of these isolates, had not previously been found in V. parahaemolyticus strains examined to date. Amplification of the trh gene in two of the isolates suggested these to be virulent strains. Three strains identified as V. cholerae by amplification of the 16S-23S rRNA ISR were confirmed to be non-cholera (non-O1/non-O139) strains. CONCLUSIONS: The results of this study demonstrated the presence of pathogenic Vibrio species in French coastal waters. Furthermore, the PCR approach proved useful for the rapid and reliable confirmation of species identification. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings indicate the potential sanitary risk associated with the presence of pathogenic Vibrio spp. in cultivated mussels and in the aquatic environment. The PCR can be used to detect pathogenic vibrios directly in environmental samples.