Use of Bioinformatics Tools to Improve Molecular Tools for Enumerating Pathogenic Vibrio vulnificus and Vibrio parahaemolyticus

Abstract:

Vibrio bacteria occur naturally in estuarine and marine environments. Two species, V. vulnificus and V. parahaemolyticus, are responsible for most reported Vibrio illnesses in the United States. These pathogens are often prevalent in Gulf of Mexico coastal waters when warm temperatures occur concomitantly with certain other abiotic factors. V. vulnificus infections result from either consumption of contaminated raw oysters or wound infections following exposure to contaminated seawater. Both V. vulnificus and V. parahaemolyticus can cause gastroenteritis or septicemia. Pilus formation is related to retention time in oyster tissues; and these pili are essential virulence factors due to their significant role in colonization and adherence to human epithelial cells. Genes involved in pilus formation include pilA, B, and C. Motility of V. vulnificus may also play a role in localization to the sites of infection; flagella serve as an adhesin or as a structure for invasion into host cells. A key gene involved in formation of this structure is flgE. Genes needed for pathogenicity by V. vulnificus or V. parahaemolyticus include ToxR, vvhA, rtxA and tdh, among others. Many current methods of Vibrio detection rely on microbiological assays, often leading to underestimates of viable-but-not-culturable Vibrio. To improve detection, and to better understand this species' persistence in oyster tissues, we amassed clinical isolates’ gene sequences, with the goal of improving existing quantitative PCR primer sets. We identified gene sequences from genomes of pathogenic clinical isolates of V. vulnificus and V. parahaemolyticus and performed multiple sequence alignments for each gene to identify regions of conservation among all pathogenic strains that were also different from sequences of environmental strains. New primer sequences are based on a minimum of 3 clinical isolates’ sequences (V. vulnificus, vvhA); and some multiple sequence alignments contain data 20 or more isolates from both species (V. vulnificus and parahaemolyticus, tdh) or a single species (V. vulnificus, vvhA). These improved tools to rapidly detect pathogenic Vibrio in coastal Gulf of Mexico and associated estuaries are essential for policy decisions with respect to human impacts in the face of anticipated ecosystem changes.