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Appl. Environ. Microbiol. doi:10.1128/AEM.02776-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Real-time RT-PCR for transcriptional expression analysis of virulence and housekeeping genes in VBNC Vibrio parahaemolyticus after recovery in culture

Laboratoire de Microbiologie, Département Environnement Microbiologie et Phycotoxines, IFREMER, 29280 Plouzané, France

^* To whom correspondence should be addressed. Email: dhervio{at}ifremer.fr.

	Abstract

A real-time reverse transcription (RT)-PCR was developed to determine whether viable but nonculturable (VBNC) Vibrio parahaemolyticus recovering culturability, induced expression of virulence genes coding for the thermostable direct hemolysin (TDH) and for the type III secretion system 2 (TTSS2). The culturability of a clinical strain Vp5 of V. parahaemolyticus in artificial seawater at 4°C was monitored and the VBNC state was obtained. One day after the VBNC state, temperature upshifts to 20°C and 37°C allowed recovery of culturability. Standard curves for relative quantification were established for the housekeeping genes rpoS, pvsA, fur, pvuA, and for tdh2 gene and for the TTSS2 genes (VPA1354, VPA1346, VPA1342). Expression of the pvsA and pvuA genes was stable and used to normalize expression. No transcriptional induction of the selected virulence genes was observed in the temperature conditions used to recover the culturability of the VBNC bacteria. The study demonstrates that recovery of culturability by VBNC cells of pathogenic V. parahaemolyticus is strictly restricted to regrowth, without correlation with induction of virulence gene expression. Disease induction would mainly depend on host-pathogen interactions that allow the expression of the virulence genes. It is the first time that the use of mRNA to detect viable cells was evaluated computing half-lives of multiple mRNA populations in conditions to obtain the VBNC state.