Title

Enumeration of Vibrio parahaemolyticus in the Viable But Nonculturable State Using Direct Plate Counts and Recognition of Individual Gene Fluorescence In Situ Hybridization

Document Type

Article

Publication Date

5-1-2011

Department

Coastal Sciences, Gulf Coast Research Laboratory

Abstract

Vibrio parahaemolyticus is a gram-negative, halophilic bacterium indigenous to marine and estuarine environments and it is capable of causing food and water-borne illness in humans. It can also cause disease in marine animals, including cultured species. Currently, culture-based techniques are used for quantification of V. parahaemolyticus in environmental samples; however, these can be misleading as they fail to detect V. parahaemolyticus in a viable but nonculturable (VBNC) state which leads to an underestimation of the population density. In this study, we used a novel fluorescence visualization technique, called recognition of individual gene fluorescence in situ hybridization (RING-FISH), which targets chromosomal DNA for enumeration. A polynucleotide probe labeled with Cyanine 3 (Cy3) was created corresponding to the ubiquitous V. parahaemolyticus gene that codes for thermolabile hemolysin (tlh). When coupled with the Kogure method to distinguish viable from dead cells, RING-FISH probes reliably enumerated total, viable V. parahaemolyticus. The probe was tested for sensitivity and specificity against a pure culture of tlh(+), tdh(-), trh(-) V. parahaemolyticus, pure cultures of Vibrio vulnificus. Vibrio harveyi, Vibrio alginolyticus and Vibrio fischeri, and a mixed environmental sample. This research will provide additional tools for a better understanding of the risk these environmental organisms pose to human health. (C) 2011 Elsevier B.V. All rights reserved.

Publication Title

Journal of Microbiological Methods

Volume

85

Issue

2

First Page

114

Last Page

118