Microarray-based detection of genetic heterogeneity, antimicrobial resistance, and the viable but nonculturable state...

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 species/serogroup-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.