P-058. Cloning and Partial Characterization of a Novel Vibrio tubiashii Hemolysin Gene and the Development of a PCR Based Detection for V. tubiashii

V. Sathyamoorthy, A. R. Datta, C. J. Lee, B. D. Tall, B. A. McCardell, M. H. Kothary;
US FDA, Laurel, MD.

Work from our laboratory has demonstrated that Vibrio tubiashii (Vt), a known molluscan pathogen, causes intestinal fluid accumulation in suckling mice indicating its potential to be a diarrheal pathogen in humans. The pathogen expresses virulence factors such as vulnificolysin-like hemolysin/cytolysin and metalloprotease, similar to those of other pathogenic Vibrios. In this study, we have cloned a hemolysin gene of Vt by digesting the genomic DNA with HindIII, cloning the fragments into pGem, and screening the resultant library in Escherichia coli JM109 for hemolytic activity using sheep blood agar. Out of about 2000 colonies, 3 colonies were found to produce zones of hemolysis on blood agar. Plasmids from these colonies were sequenced using M13 primers. The sequence showed a strong homology to ribA gene, required for riboflavin biosynthesis and has been reported to be responsible for hemolytic activity in Helicobacter pylori. Plasmid from one of the hemolysin clones, pGem:Hly1, when introduced in E. coli BSV18 (ribA:Tn5) was able to restore its growth on a medium without riboflavin and also produced hemolysis on blood agar. Four sets of primers were designed based on the hemolysin gene sequence and were tested using 21 different Vibrio strains representing 10 different species at an annealing temperature of 62.7°C. Except for Vt strain 19105 (Vt105), none of the other strains including Vt strain 19109 (Vt109) produced the expected size amplicons with these primers. At annealing temperature 43°C, only the primer set 1 yielded amplicons of the right size (~300bp) with both Vt105 and Vt109 but the ~300 bp amplicon was not produced with other Vibrio species. Our results indicate that we have cloned a ribA homologue of Vt, and primers based on this gene may be useful as a specific identification tool for V. tubiashii.