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Applied and Environmental Microbiology, November 2002, p. 5641-5646, Vol. 68, No. 11
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.11.5641-5646.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Detection of Cytotoxin-Hemolysin mRNA in Nonculturable Populations of Environmental and Clinical Vibrio vulnificus Strains in Artificial Seawater

Marion Fischer-Le Saux,1,{dagger} Dominique Hervio-Heath,1* Solen Loaec,1 Rita R. Colwell,2 and Monique Pommepuy1

DEL-MP-Laboratoire de Microbiologie, Institut Français de Recherche pour l'Exploitation de la Mer, Plouzané, France,1 Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, Maryland2

Received 10 April 2002/ Accepted 1 August 2002

The objective of this study was to develop a molecular detection method that better estimates the potential risk associated with the presence of Vibrio vulnificus. For that purpose, we applied seminested reverse transcription-PCR (RT-PCR) to viable but nonculturable (VBNC) populations of V. vulnificus and targeted the cytotoxin-hemolysin virulence gene vvhA. Three strains, two environmental, IF Vv10 and IF Vv18, and one clinical, C7184, were used in this study. Artificial seawater, inoculated with mid-log-phase cells, was maintained at 4°C. VBNC cells resulted after 3, 6, and 14 days for C7184, IF Vv18, and IF Vv10, respectively. Our data indicate that seminested RT-PCR is sensitive for the detection of vvhA mRNA in artificial seawater when exclusively nonculturable bacteria are present. This is the first report of the expression of a toxin gene in VBNC V. vulnificus. Moreover, vvhA transcripts were shown to persist in nonculturable populations over a 4.5-month period, with a progressive decline of the signal over time. This result indicates that special attention should be given to the presence of potentially pathogenic VBNC cells in environmental samples when assessing public health risk.

* Corresponding author. Mailing address: DEL-MP-Laboratoire de Microbiologie, Ifremer, BP 70, Technopôle Brest-Iroise, 29280 Plouzané, France. Phone: 33 298 22 43 49. Fax: 33 298 22 45 94. E-mail: Dominique.Hervio.Heath{at}ifremer.fr.

{dagger} Present address: UMR de Pathologie Végétale, UFR Sciences—Université d’Angers, 49045 Angers cedex 01, France.

Applied and Environmental Microbiology, November 2002, p. 5641-5646, Vol. 68, No. 11
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.11.5641-5646.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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