The morbidity and mortality associated with Vibrio-mediated waterborne diseases necessitates the development of sensitive detection technologies that are able to elucidate the identity, potential pathogenicity, susceptibility, and viability of contaminating bacteria in a timely manner. For this purpose, we have designed a single multiplex PCR assay to simultaneously amplify 95 diagnostic regions (encompassing species͞serogroup-specific, antimicrobial resistance, and known toxin markers) and combined it with a long oligonucleotide microarray to create a platform capable of rapidly detecting and discriminating the major human pathogenic species from the genus Vibrio: V. cholerae, V. parahaemolyticus, V. vulnificus, and V. mimicus. We were able to validate this strategy by testing 100 geographically and temporally distributed isolates and observed an excellent concordance between species-and serotype-level microarray-based identification and traditional typing methods. In addition to accurate identification, the microarray simultaneously provided evidence of antibiotic resistance genes and mobile genetic elements, such as sulfamethoxazole-trimethoprim constins and class I integrons, and common toxin (ctxAB, rtxA, hap, hlyA, tl, tdh, trh, vvhA, vlly, and vmhA) and pathogenicity (tcpA, type III secretion system) genes that are associated with pathogenic Vibrio. The versatility of this method was further underscored by its ability to detect the expression of known toxin and virulence genes from potentially harmful viable but nonculturable organisms. The results suggest that this molecular identification method provides rapid and definitive information that would be of value in epidemiological, environmental, and health risk assessment surveillance.pathogen detection ͉ molecular diagnostics ͉ cholera M embers of the genus Vibrio are Gram-negative motile bacteria that are naturally occurring, free-living inhabitants of marine and estuarine environments throughout the world. Whereas the vast majority of Vibrio are nonpathogenic to humans, select strains from four species, V. cholerae, V. parahaemolyticus, V. vulnificus, and V. mimicus, are known to be important human pathogens that are predominantly associated with food and waterborne illness. V. cholerae serogroup O1 and V. cholerae serogroup O139, the most notable of the pathogenic Vibrio spp., are the etiologic agents of the severe diarrheal disease known as cholera (1, 2). The strains responsible for endemic, epidemic, and pandemic cholera are known to harbor two critical virulence factors, cholera toxin (CT) and the toxin coregulated pilus (2); however, the overall pathology of V. cholerae infections appears to be determined by the coordinated action of several virulence factors (3). Although closely related to V. cholerae (and each other), the diseases caused by V. parahaemolyticus (gastroenteritis, wound infections, septicemia), V. vulnificus (severe necrotizing wound infections, invasive fulminating septicemia), and V. mimicus (gastroenteritis) are associated wit...
