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Date of Graduation
Master of Science (MS)
Department of Biology
In this study 143 V. vulnificus isolates of clinical and environmental origin, were examined for growth on differential media, identified to species and tested for antibiotic resistance. A multiplex PCR was created and optimized, and phylogenetic analysis was conducted. The first objective was to compare phenotypic methods to identify V. vulnificus. Colony colors of confirmed V. vulnificus isolates on selective media (Vibrio vulnificus agar, thiosulfate citrate bilesalts sucrose agar, CHROMAgar Vibrio (CAV), and colistin polymyxin B cellobiose agar), mostly matched those characteristic of V. vulnificus. To test the ability of these media to select for V. vulnificus, new presumptive V. vulnificus isolates were collected and grown on the four media. Most of the tested media had very high false positive rates, with isolates not confirmed as V. vulnificus through PCR of the vvhA gene, growing with characteristic V. vulnificus colony colors. CAV was determined to be the most differential. The Biolog Microbial Identification System was used to identify V. vulnificus isolates and had a 96% correct identification rate. Antibiotic resistance of V. vulnificus isolates was assessed and compared with isolate origin. Almost all the V. vulnificus isolates were susceptible to all the antibiotics used. Only five isolates displayed any type of resistance and thus no relationship with origin could be established. A multiplex PCR protocol using amplification of vvhA and the 16S gene was developed, optimized, and tested to identify an isolate as V. vulnificus. Buffer optimization was simultaneously conducted and TBE was determined to be the most effective buffer for gel electrophoresis with the produced amplicons. Finally, a fragment of the vvhA gene from each isolate was sequenced. Analysis of the fragment resulted in phylogenetic trees with two distinct branches related to clinical or XI environmental isolates. Virulence factors associated with the constructed phylogeny but isolate origin did not. This study was conducted to further characterize a group of wellstudied V. vulnificus isolates and to find methods to differentiate clinical from environmental isolates. Continued analysis of these and additional isolates may further our knowledge of the species and reveal more characteristics indicative of pathogenicity.
Conrad, James, "Characterization of Vibrio vulnificus strains using phenotypic and genotypic assays" (2014). Masters Theses. 10.