Genetic relatedness among environmental, clinical, and diseased-eel Vibrio vulnificus isolates from different geographic regions by ribotyping and randomly amplified polymorphic DNA PCR

Abstract

Genetic relationships among 132 strains of Vibrio vulnificus (clinical, environmental, and diseased-eel isolates from different geographic origins, as well as seawater and shellfish isolates from the western Mediterranean coast, including reference strains) were analyzed by random amplified polymorphic DNA (RAPD) PCR. Results were validated by ribotyping. For ribotyping, DNAs were digested with KpnI and hybridized with an oligonucleotide probe complementary to a highly conserved sequence in the 23S rRNA gene. Random amplification of DNA was performed with M13 and T3 universal primers. The comparison between ribotyping and RAPD PCR revealed an overall agreement regarding the high level of homogeneity of diseased-eel isolates in contrast to the genetic heterogeneity of Mediterranean isolates. The latter suggests the existence of autochthonous clones present in Mediterranean coastal waters. Both techniques have revealed a genetic proximity among Spanish fish farm isolates and a close relationship between four Spanish eel farm isolates and some Mediterranean isolates. Whereas the differentiation within diseased-eel isolates was only possible by ribotyping, RAPD PCR was able to differentiate phenotypically atypical isolates of V. vulnificus. On the basis of our results, RAPD PCR is proposed as a better technique than ribotyping for rapid typing in the routine analysis of new V. vulnificus isolates.

FIG. 1

Dendrogram based on UPGMA cluster analysis of the 34 different ribotypes obtained in this study. The scale measures the percentage of similarity. Numbers next to the dendrogram are ribotype designations, and numbers of strains sharing the same ribotype are shown in parentheses.

FIG. 2

RAPD PCR patterns of V. vulnificus. The dendrogram was derived from an UPGMA cluster analysis of the RAPD PCR profiles of 131 V. vulnificus strains. The tracks show the processed band patterns after conversion, normalization, and subtraction of the background.

FIG. 2

RAPD PCR patterns of V. vulnificus. The dendrogram was derived from an UPGMA cluster analysis of the RAPD PCR profiles of 131 V. vulnificus strains. The tracks show the processed band patterns after conversion, normalization, and subtraction of the background.