Development of 16S rRNA targeted PCR methods for the detection and differentiation of Vibrio vulnificus in marine environments

Abstract

A PCR method for the detection and differentiation of Vibrio vulnificus strains was developed as an alternative to culture methods by using combined primers directed against the variable regions of 16S rRNA. Primers designed from two variable regions of the Vibrionaceae 16S rRNA (corresponding to nucleotide numbers 1006 to 1023 and 1278 to 1258 in Escherichia coli 16S rRNA) was found to be species-specific for V. vulnificus by PCR. Additionally, tri-primer PCR of 16S rRNA was evaluated for the differentiation of V. vulnificus strains. Although the third primer, which was derived from the variable region, positions 454 to 473, cannot discriminate V. vulnificus from other bacteria, it was used to avoid the detection of type B 16S rRNA of this organism in PCR. The resulting 825 bp fragment in the presence of the 273 bp fragment, which is specific to V. vulnificus, in tri-primer PCR clearly differentiated type A 16S rRNA strains from type B. Enumeration of V. vulnificus in the samples of oyster and environmental samples was done by most probable numbers' (MPN) method of five preenrichment tubes of alkaline peptone water supplemented with polymyxin B following up the confirmation of positive tubes by streaking the samples onto mCPC agar or by 16S rRNA gene amplification. Higher numbers of presumptive V. vulnificus confirmed by selective media compared with those confirmed by PCR method in MPN method suggested that there would be some bacteria that cannot be discriminated from V. vulnificus on mCPC agar in environmental samples. In the biotyping of the V. vulnificus isolates in oyster samples, the majority of the strains (92.5%) belonged to biotype 1, and 7.5% of the strains belonged to biotype 2. However, strains of 16S rRNA of V. vulnificus isolates in the marine environment determined by tri-primer PCR appeared to be 35% type A and 65% type B. These results implied that the marine environment can serve as reservoir of both V. vulnificus biotypes 1 and 2, and strains of 16S rRNA type B were more frequent than strains of type A in that environment.