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-citratebile 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 24AE4% 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.

Vibrio parahaemolyticus is one of the principal bacterial causes for seafood-borne gastroenteritis in the world. In the present study, three sites located on the French Atlantic coast were monitored monthly for environmental parameters over 1 year. The presence of total and pathogenic V. parahaemolyticus in sediment, water and mussel samples was detected following enrichment by culture and real-time PCR (toxR gene, tdh, trh1 and trh2 virulence genes). Using generalized linear models, we showed that the presence of V. parahaemolyticus in water could be explained by a combination of mean temperature over the 7 days before the day of sampling (P < 0.001) and turbidity (P = 0.058). In mussels, an effect of chlorophyll a (P = 0.005) was detected when an effect of the mean salinity over the 7 days before sampling was significant for the sediment (P < 0.001). We did not detect any significant effect of phytoplanktonic blooms or of the number of culturable bacteria on V. parahaemolyticus presence. No sample was revealed positive for tdh. The presence of trh1 and trh2 was positively influenced by the mean temperature during the 2 days before the day of sampling (P < 0.001 and P = 0.032). The importance of these ecological parameters is discussed in relation to the biology of V. parahaemolyticus.

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°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°C before inoculation in ASW at 4°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.

During a 2-year study, the presence of human pathogenic bacteria and noroviruses was investigated in shellfish, seawater and/or surface sediments collected from three French coastal shellfish-harvesting areas as well as in freshwaters from the corresponding upstream catchments. Bacteria isolated from these samples were further analyzed. Escherichia coli isolates classified into the phylogenetic groups B2, or D and enterococci from Enterococcus faecalis and E. faecium species were tested for the presence of virulence genes and for antimicrobial susceptibility. Salmonella members were serotyped and the most abundant serovars (Typhimurium and its monophasic variants and Mbandaka) were genetically characterized by high discriminative subtyping methods. Campylobacter and Vibrio were identified at the species level, and haemolysin-producing Vibrio parahaemolyticus were searched by tdh- and trh- gene detection. Main results showed a low prevalence of Salmonella in shellfish samples where only members of S. Mbandaka were found. Campylobacter were more frequently isolated than Salmonella and a different distribution of Campylobacter species was observed in shellfish compared to rivers, strongly suggesting possible additional inputs of bacteria. Statistical associations between enteric bacteria, human noroviruses (HuNoVs) and concentration of fecal indicator bacteria revealed that the presence of Salmonella was correlated with that of Campylobacter jejuni and/or C. coli as well as to E. coli concentration. A positive correlation was also found between the presence of C. lari and the detection of HuNoVs. This study highlights the importance of simultaneous detection and characterization of enteric and marine pathogenic bacteria and human noroviruses not only in shellfish but also in catchment waters for a hazard assessment associated with microbial contamination of shellfish.

The objective of this study was to explore the recovery of culturability of viable but nonculturable (VBNC) Vibrio parahaemolyticus after temperature upshift and to determine whether regrowth or resuscitation occurred. A clinical strain of V. parahaemolyticus Vp5 was rendered VBNC by exposure to artificial seawater (ASW) at 41C. Aliquots of the ASW suspension of cells (0.1, 1 and 10 ml) were subjected to increased temperatures of 201C and 371C. Culturability of the cells in the aliquots was monitored for colony formation on a rich medium and changes in morphology were measured by scanning (SEM) and transmission (TEM) electron microscopy. Samples of VBNC cells were fixed and examined by SEM, revealing a heterogeneous population comprising small cells and larger, flattened cells. Forty-eight hours after temperature upshift to 201C or 371C, both elongation and division by binary fission of the cells were observed, employing SEM and TEM, but only in the 10-ml aliquots. The results suggest that a portion of VBNC cells is able to undergo cell division. It is concluded that a portion of VBNC cells of V. parahaemolyticus subjected to cold temperatures remain viable. After temperature upshift, regrowth of those cells, rather than resuscitation of all bacteria of the initial inoculum, appears to be responsible for recovery of culturability of VBNC cells of V. parahaemolyticus. Nutrient in filtrates of VBNC cells is hypothesized to allow growth of the temperature-responsive cells, with cell division occurring via binary fission, but also including an atypical, asymmetric cell division.