Molecular mechanisms of antimicrobial resistance and virulence of Vibrio parahaemolyticus

Abstract

Vibrio parahaemolyticus is not only a clinically important foodborne pathogen worldwide but also a leading cause of foodborne illnesses in Hong Kong. My research is focused on understanding the molecular mechanisms of virulence and antimicrobial resistance in this pathogen. Through bioinformatics analysis, a novel adhesion protein that was referred to as VadF, was mined from the genomic sequence. Deletion of vadF gene in V. parahaemolyticus dramatically decreased its ability to attach to HeLa cells and its infectivity in mice model. Biochemical results showed that the N-terminal region closely linked to the transmembrane domain of VadF was responsible for the binding to N-terminal domains of fibronectin. The rest parts of VadF did not bind to fibronectin independently, but they stimulate VadF binding to fibronectin. In addition, a novel znuA homologue (vpa1307) that represents a novel subfamily of ZnuA was identified. It was shown that horizontal gene transfer is one of the most important factors that influence the virulence of V. parahaemolyticus. Phylogenetic analysis suggested that vpa1307 gene in V. parahaemolyticus is horizontally acquired. 40% of clinical isolates possessed this gene. The expression of vpa1307 gene was induced under zinc limitation. Loss of function and gain of function confirmed that Vpa1307 contributes to the growth of V. parahaemolyticus under zinc starvation condition. Moreover, Vpa1307 also contributed to cytotoxicity against HeLa cells as well as the pathogenesis in mice. These results suggest the acquired vpa1307 subfamily genes contribute to the fitness and virulence of Vibrio species. The second direction of my research is to understand the mechanisms of antimicrobial resistance in V. parahaemolyticus. Fluoroquinolones are the choices for the treatment of V. parahaemolyticus infections. It was showed that there is an increasing trend of fluoroquinolone resistance in V. parahaemolyticus. However, the underlying mechanisms for the fluoroquinolone resistance in this pathogen remain to be characterized. The present work showed that a single amino acid substitution in GyrA (Ser83Ile) and ParC (Ser85Leu) contributes to the primary mechanism of fluoroquinolone resistance. Four isolates were found to carry a plasmid with a novel fluoroquinolone resistance gene, qnrVC5. Efflux pumps have limited role in fluoroquinolones resistance among all strains. A 1.5 kb class I integron that may contribute to trimethoprim and rifampicin resistance by the dfrA27 and arr3 genes was also detected in these four isolates. In the ciprofloxacin resistant V. parahaemolyticus strain V1, a multiple drug resistance transferable plasmid, pVP1 (about 200 kb) was identified. pVP1 carried a novel quinolone resistance gene, qnrVC6. This gene conferred resistant to nalidixic acid and ciprofloxacin in E. coli. The genomic organizations showed that QnrVC6 is present within IS elements, which has never observed before, suggesting the qnrVC genes may be transferred among Vibrio spp. by divergent mobile genetic materials. Other drug resistance elements on pVP1 were blaPER-1, aacA3, catB2, aadA1 and dfrA1. Transmission of pVP1 among Vibrio species would cause huge threat to public health and needs close monitoring.