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.In the United States, Vibrio vulnificus is the leading cause of death associated with consumption of seafood (12, 15). Consumption of raw Gulf Coast oysters from April to November is responsible for nearly all of the cases. Although V. vulnificus is abundant in oysters during that time of year, cases are rare even in the high-risk group (i.e., those with preexisting liver disease or who are immunocompromised) (7). A major obstacle in developing effective control strategies is the inability to identify in the oysters V. vulnificus strains that are capable of causing human illness.Two major research needs identified at a 1994 V. vulnificus workshop sponsored by the Food and Drug Administration (FDA) were to develop methods to distinguish virulent V. vulnificus strains from avirulent strains and to determine the infectious dose (22). Since human volunteer studies with V. vulnificus are not ethical, a consensus approach was proposed to determine the infectious dose by relating disease frequency with exposure. It was also suggested that a collection of strains from oysters and human septicemia cases associated with oyster consumption should be characterized in various assays in an attempt to determine traits that may be linked to virulence. The Centers for Disease Control and Prevention (CDC), FDA, and various state departments of health collected approximately 75 well-characterized clinical strains from human septicemia cases with known sources of oysters consumed, patient histories, etc. A recent study of the abundance of V. parahaemolyticus and V. vulnificus in retail oysters by the Interstate Shellfish Sanitation Conference and the FDA generated a large collection of V. vulnificus cultures that is seasonally and geographically diverse and well defined (3). Molecular characterization and virulence assays of representative V. vulnificus isolates from these tw...

Genetic Variation in the Vibrio vulnificus Group 1 Capsular Polysaccharide Operon

“…Potential virulence in V. cholerae and V. parahaemolyticus is clearly defined by toxinencoding genetic markers (ctx and tdh, respectively), but these distinctions are less evident in V. vulnificus because all clinical and environmental strains are equally virulent in animal and cell culture models of pathogenesis (9). Previous associations of allelic variation with clinical origin were noted for V. vulnificus loci, including the 16S rRNA gene (34), the viuB gene for siderophore biosynthesis (37), and another ORF with no homology to sequences in GenBank (44).…”

Vibrio vulnificus rtxA1 gene recombination generates toxin variants with altered potency during intestinal infection

“…The acd + strains were not tested because, as discussed earlier, data suggested ORL 8074 may not produce an active MARTX Vv toxin and DAL 6-5000 was found previously to be avirulent by s.c. infection (23). The six selected strains showed a range of virulence: high virulence for lineage I laboratory-passaged clinical isolates CMCP6 and MO6-24/O (0-10% survival); intermediate virulence for lineage I clinical isolates LOS 6966 and FLA 9509 (50-83% survival); and low virulence for lineage II strains ATL 71503 and ATL 9579 (100% survival) (Table S2).…”

“…1) (21, 22). However, strain DAL 6-5000 does not have any plasmids (23), so MARTX Vv D must represent a chromosomal copy. In support of this notion, CECT4999 recently was reported to have a second copy of the rtxA1 gene on the chromosome (Genbank accession no.…”

The Biology of Vibrio vulnificus

“…While the concentration of V. vulnificus in estuarine waters is typically quite low (<10 CFU/ml), it becomes concentrated in such molluscan shellfish as oysters and clams due to their efficient use of filter-feeding to obtain food. The resultant levels in shellfish can reach 10 5 CFU/g of tissue or more (3,4,5). While the infectious dose is not known, it has been estimated to be as few as 100 cells or less (6).…”