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

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Abstract
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.