title: Enumeration of Vibrio parahaemolyticus in the viable but nonculturable state using direct plate counts and recognition of individual gene fluorescence in situ hybridization.
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.
authors.affiliation: The University of Southern Mississippi, Gulf Coast Research Laboratory, Ocean Springs, MS 39564, USA. kimberly.griffitt@usm.edu
authors.forename: Kimberly J
authors.initials: KJ
authors.lastname: Griffitt
authors.forename: Nicholas F
authors.initials: NF
authors.lastname: Noriea
authors.forename: Crystal N
authors.initials: CN
authors.lastname: Johnson
authors.forename: D Jay
authors.initials: DJ
authors.lastname: Grimes
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D015169:Colony Count, Microbial; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D050296:Microbial Viability; D014736:Vibrio parahaemolyticus / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification
chemical_list: D001426:Bacterial Proteins