Enumeration of Vibrio parahaemolyticus in the viable but nonculturable state using direct plate counts and recognition of individual gene fluorescence in situ hybridization

• Published in: Journal of microbiological methods Vol. 85; no. 2; pp. 114 - 118
• Main Authors: Griffitt, Kimberly J., Noriea, Nicholas F., Johnson, Crystal N., Grimes, D. Jay
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.05.2011; Elsevier
• Subjects: bacteria; Bacterial Proteins - genetics; Bacteriological methods and techniques used in bacteriology; Bacteriology; Biological and medical sciences; Brackish; Colony Count, Microbial; DNA; fluorescence; fluorescence in situ hybridization; Fundamental and applied biological sciences. Psychology; genes; hemolysins; human health; humans; In Situ Hybridization, Fluorescence - methods; Microbial Viability; Microbiology; plate count; population density; Recognition of individual gene fluorescence in situ hybridization; RING-FISH; risk; VBNC; Viable but nonculturable; Vibrio alginolyticus; Vibrio fischeri; Vibrio harveyi; Vibrio parahaemolyticus; Vibrio parahaemolyticus - genetics; Vibrio parahaemolyticus - growth & development; Vibrio parahaemolyticus - isolation & purification; Vibrio vulnificus; waterborne diseases; Viable but nonculturable; Vibrio parahaemolyticus; Recognition of individual gene fluorescence in situ hybridization; VBNC; RING-FISH; Molecular hybridization; In situ; Method; Recognition of individual gene fluorescence; Enumeration(counting); Gene; Fluorescence in situ hybridization; Bacteria; Vibrionaceae; VBNC form; in situ hybridization
• DOI: 10.1016/j.mimet.2011.02.006
• ISSN: 0167-7012

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.