Analysis of Vibrio vulnificus from market oysters and septicemia cases for virulence markers

Abstract

Representative encapsulated strains of Vibrio vulnificus from market oysters and oyster-associated primary septicemia cases (25 isolates each) were tested in a blinded fashion for potential virulence markers that may distinguish strains from these two sources. These isolates were analyzed for plasmid content, for the presence of a 460-bp amplicon by randomly amplified polymorphic DNA PCR, and for virulence in subcutaneously (s.c.) inoculated, iron-dextran-treated mice. Similar percentages of market oyster and clinical isolates possessed detectable plasmids (24 and 36%, respectively), produced the 460-bp amplicon (45 and 50%, respectively), and were judged to be virulent in the mouse s.c. inoculation-iron-dextran model (88% for each). Therefore, it appears that nearly all V. vulnificus strains in oysters are virulent and that genetic tests for plasmids and specific PCR size amplicons cannot distinguish between fully virulent and less virulent strains or between clinical and environmental isolates. The inability of these methods to distinguish food and clinical V. vulnificus isolates demonstrates the need for alternative subtyping approaches and virulence assays.

FIG. 1.

Plasmid profiles of selected V. vulnificus strains. Lanes: A, E. coli 39R861; B, E. coli V517; C, V. cholerae 1075/25; D, V. vulnificus CDC 9344-95; E, V. vulnificus CDC 9149-95; F, V. vulnificus CDC 9030-95; G, V. vulnificus CDC 9031-96; H, V. vulnificus CDC 9075-96. Strains in lanes E to H contain a band of approximately 40 kb that is a relaxed form of the 29-kb plasmid, and lane H also contains a relaxed form of the 6.9-kb plasmid.

FIG. 2.

Summary of quantitative data from mouse infection studies. Iron-dextran-treated mice were inoculated s.c. with 10³ CFU of V. vulnificus. Data are means ± standard deviations of values for log CFU per gram of skin lesion (Lesion CFU), log CFU per gram of liver tissue (Liver CFU), and rectal temperature (Temp). For all groups, n = 25. *, P = 0.01 (Student t test); ^, P < 0.005 (Mann-Whitney U test).