Typing ofVibrio vulnificusStrains by Variability in Their 16S-23S rRNA Intergenic Spacer Regions

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Abstract
Amplification of the 16S-23S rDNA spacer region (ISR1) is a simple and rapid procedure for subtyping bacteria, especially those with several ribosomal operons including Vibrio vulnificus. V. vulnificus contains nine ribosomal operons with four or five ISR1 classes that differ in size and sequence. In the present study, 47 V. vulnificus strains of both shellfish and clinical origin were subtyped by their ISR1 patterns using "universal" primers, which target conserved sequences located in the 16S and the 23S rRNA genes. Sixteen different ISR1 patterns were observed that were grouped into two major clusters. Most (21/27, 77.8%) clinical isolates examined in this study grouped into a single cluster containing ISR1 patterns I, V, XI, and XII and these were highly similar (75%). This cluster was restricted to strains carrying the type B 16S rDNA (rrs) sequence which has been associated with human illness in previous studies. The remaining cluster consisted primarily of shellfish isolates. The highest variability in the ISR1 patterns was observed among shellfish isolates. Sequence analysis of the ISR1 region of selected strains demonstrated that all of them possess five ISR1 classes, with two "conserved sequence blocks" at the 5' and 3' end of the ISR1. All of these strains carried at least one tRNA gene and different classes differed in their tRNA gene composition. Some of the same ISR1 classes differed in size mainly due to an insertion of 35 bp in either of the conserved sequence blocks. These results demonstrate the feasibility of the ISR1 technique for V. vulnificus subtyping and suggest that ISR1 patterns appear to be linked to rrs sequence types and perhaps with virulence.

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