Enumeration of Vibrio parahaemolyticus in the Viable But Nonculturable State Using Direct Plate Counts and Recognition of Individual Gene Fluorescence In Situ Hybridization
Coastal Sciences, Gulf Coast Research Laboratory
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.
Journal of Microbiological Methods
Griffitt, K. J.,
Noriea, N. F.,
Johnson, C. N.,
Grimes, D. J.
(2011). Enumeration of Vibrio parahaemolyticus in the Viable But Nonculturable State Using Direct Plate Counts and Recognition of Individual Gene Fluorescence In Situ Hybridization. Journal of Microbiological Methods, 85(2), 114-118.
Available at: http://aquila.usm.edu/fac_pubs/404