Abstract:Vibrio parahaemolyticus is an important enteropathogen in Japan, Taiwan and other coastal regions.The influence of the regulation of iron on the pathogenesis of this pathogen has not been well characterized. in Japan and Taiwan (1, 24). Thermostable direct hemolysin (TDH) and other TDH-related hemolysins are the major virulence factors in this pathogen (7-9, 19, 30). Karunasagar et al (11) demonstrated that the addition of lysed erythrocyte extract or ferric ammonium citrate to bacterial cultures significantly enhances the virulence of V parahaemolyticus. Wong and Lee (26) also showed that the in vivo growth of this pathogen is enhanced by the presence of supplementary iron in a peritoneally infected mouse model. Factors involved in the acquisition of iron have been described for V parahaemolyticus. In our previous in vitro study, production of siderophore, specific outer membrane proteins (OMPs) and TDH were all enhanced in V parahaemolyticus by limiting iron in the culture (3). One of the specific OMPs (77 kDa), characterized by a putative TonB box in its N-terminal amino acid sequence, may be involved in the acquisition of iron-containing compounds (15). TonB-dependent proteins usually act as receptors in the iron acquisition systems of Escherichia coli and V cholerae (2, 6). In another study, an 83 kDa OMP was recognized as being associated with the utilization of hemin and hemoglobin in V parahaemolyticus (27).Besides their role in the acquisition of iron, iron-regulated proteins could also be associated with the virulence of pathogens. Iron-regulated IrgA has been demonstrated to be a virulence factor for infection in an animal model with classical V cholerae, and homologous sequences were detected in non-Ol V cholerae, V. parahaemolyticus, V fluvialis and V alginolyticus (5).In this study, we obtained several spontaneous iron-utilizing mutant strains (mutants) of V parahaemolyticus which showed limited growth in iron-limited medium and did not produce iron-regulated OMPs in normal growth medium. The goal of this study was to elucidate the roles of iron acquisition in the pathogenesis of V.parahaemolyticus by examining the changes in the virulence of these iron-utilizing mutants by in vitro and in vivo assays. We also reported on the utilization of different iron sources by different strains of V parahaemolyticus. Materials and MethodsBacterial strains. Pathogenic and non-pathogenic

Vibrio parahaemolyticus is one of the most important food-borne pathogens in Taiwan, Japan, and other countries with long coastlines. This paper reports on the development of a new random amplified polymorphic DNA (RAPD) method for the molecular typing of this pathogen. The 10-mer primer 284 (5′-CAG GCG CAC A-3′) was selected to generate polymorphic amplification profiles of the genomic DNA at an annealing temperature of 38°C. A total of 308 clinical isolates of V. parahaemolyticus collected during food poisoning outbreaks in Taiwan, mostly occurring between 1993 and 1995, plus 11 environmental and clinical reference strains were analyzed by this RAPD method. A total of 41 polymorphic RAPD patterns were recognized, and these patterns were arbitrarily grouped into 16 types (A to P). Types A, B, C, D, and E were the major types, and subtypes C3, C5, E1, B1, D2, and A2 were the major patterns. The major types were phylogenetically more closely related to each other than to any of the minor types.