Detection of Vibrio vulnificus Pathogenic Isolates Through Use of Primers for the Sigma-38 Gene and Hemolysin/Cytolysin Gene

Abstract

Vibrio vulnificus (V. vulnificus) is a halophilic, asporogenous Gram-negative mesophilic bacterium belonging to the Gammaproteobacteria. It lives in marine and estuarine waters and grows at high levels in the Gulf of Mexico between April and October. Activation of virulence genes in V. vulnificus is regulated by various environmental factors, such as temperature, salinity, and pH. Transcription of these genes is regulated by a specific sigma factor, RpoS, encoded by the gene which is present in most members of Proteobacteria and is highly conserved. RpoS is seen under stress, starvation and stationary phase, and controls expression of the virulence factor gene, vvhA, which produces a cytolysin that leads to hemolysis of erythrocytes and leucocytes, causing release of iron. Previous primers designed to amplify rpoS no longer worked to amplify a critical and highly conserved essential gene, and the problem arose on how to detect this locus in V. vulnificus. This study allowed increased accuracy for correctly detecting pathogenic V. vulnificus isolates by l) redesigning rpoS primers to perform conventional end point polymerase chain reaction (PCR) of 28 V. vulnificus isolates; 2) redesigning vvhA primers to perform conventional end point polymerase chain reaction (PCR) of 28 V. vulnificus isolates; and 3) developing a multiplex PCR assay to simultaneously detect both rpoS and vvhA genes.