Vibrio vulnificus is the leading cause of reported deaths from infections related to consumption of seafood in the United States. Affected predisposed individuals frequently die rapidly from sepsis. Otherwise healthy people can experience severe wound infection, which can lead to sepsis and death. A question is why, with so many people consuming contaminated raw oysters, the incidence of severe V. vulnificus disease is low. Molecular typing systems have shown associations of V. vulnificus genotypes and the environmental or clinical source of the strains, suggesting that different genotypes possess different virulence potentials. We examined 69 V. vulnificus biotype 1 strains that were genotyped by several methods and evaluated them for virulence in a subcutaneously inoculated iron dextran-treated mouse model. By examining the relationships between skin infection, systemic liver infection, and presumptive death (a decrease in body temperature), we determined that liver infection is predicated on severe skin infection and that death requires significant liver infection. Although most strains caused severe skin infection, not every strain caused systemic infection and death. Strains with polymorphisms at multiple loci (rrn, vcg, housekeeping genes, and repetitive DNA) designated profile 2 were more likely to cause lethal systemic infection with more severe indicators of virulence than were profile 1 strains with different polymorphisms at these loci. However, some profile 1 strains were lethal and some profile 2 strains did not cause systemic infection. Therefore, current genotyping schemes cannot strictly predict the virulence of V. vulnificus strains and further investigation is needed to identify virulence genes as markers of virulence.Vibrio vulnificus is a halophilic bacterium that is naturally present in estuarine waters and contaminates oysters and other shellfish. It is an opportunistic pathogen of humans that causes primary septicemia and wound infection in susceptible hosts and is the leading cause of reported seafood-related deaths due to infection in the United States (for a review, see reference 17). V. vulnificus strains are divided into three biotypes, with human disease caused predominantly by biotype 1 and disease of eels caused predominantly by biotype 2. Biotype 3 was recently isolated from wound infections in Israel and represents an emerging form of this species (3). Several factors have been definitively shown to contribute to virulence of V. vulnificus, including capsular polysaccharide (60), the ability to acquire iron (38, 45, 61), type IV pili (46), flagella (37, 47), RTX toxin (30, 35, 39), and others (17, 27). To date, no single factor has been identified that can distinguish between naturally occurring virulent and less virulent isolates of V. vulnificus.In susceptible humans, V. vulnificus causes a rapid, fulminating disease process resulting in extensive tissue damage (reviewed in reference 17). Primary septicemia is characterized by high fever, chills, hypotension, and septic shock. ...

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...

SummaryVibrio vulnificus is Gram-negative bacterium that contaminates oysters, causing highly lethal sepsis after consumption of raw oysters and wound infection. We previously described two sets of V. vulnificus strains with different levels of virulence in subcutaneously inoculated iron dextran-treated mice. Both virulent, clinical strains and attenuated, environmental strains could be recovered in high numbers from skin lesions and livers; however, the attenuated environmental strains required significantly higher numbers of colony-forming units (cfu) in the inoculum to produce lethal infection. Using some of these strains and an additional clinical strain, we presently asked if the different abilities to cause infection between the clinical and environmental strains were due to differences in rates of growth or death of the bacteria in the mouse host. We therefore constructed a marker plasmid, pGTR902, that functions as a replicon only in the presence of arabinose, which is not present in significant levels in animal tissues. V. vulnificus strains containing pGTR902 were inoculated into iron dextran-treated and untreated mice. Measuring the proportion of bacteria that had maintained the marker plasmid recovered from mice enabled us to monitor the number of in vivo divisions, hence growth rate; whereas measuring the number of marker plasmid-containing bacteria recovered enabled the measurement of death of the vibrios in the mice. The numbers of bacterial divisions in vivo for all of the strains over a 12-15 h infection period were not significantly different in iron dextran-treated mice; however, the rate of death of one environmental strain was significantly higher compared with the clinical strains. Infection of non-iron dextran-treated mice with clinical strains demonstrated that the greatest effect of iron dextran-treatment was increased growth rate, while one clinical strain also experienced increased death in untreated mice. V. vulnificus inoculated into iron dextran-treated mice replicated extremely rapidly over the first 4 h of infection with doubling times of approximately 15-28 min. In contrast, one of the environmental strains exhibited a reduced early growth rate. These results demonstrate that differences in virulence among naturally occurring V. vulnificus can be explained by diverse abilities to replicate rapidly in or resist defences of the host. The marker plasmid pGTR902 should be useful for examining virulence of bacteria in terms of differentiating growth verses death in animal hosts for most Gram-negative bacteria.