Infections with Vibrio vulnificus resulting in septicemia and high mortality have been correlated with pre-existing liver disease and hemochromatosis. As these conditions are associated with impaired iron metabolism and as iron availability in the host has been implicated in the pathogenicity of a number of bacterial infections, the role of iron as a possible factor in the pathogenesis of V. vulnificus was examined. Injection of mice with iron resulted in a lowering of the 50% lethal dose from 106 to 1.1 cells and in a reduction in the time of death postinfection. Elevated serum iron levels were also produced by damaging livers with injections of CC14. The inoculum size required to kill these mice was directly correlated with serum iron levels. Since the portal of infection of this organism may be by ingestion of contaminated seafood, the effects of iron upon orally induced infection were also studied. The effects of adding iron, transferrin, or Desferal (an iron chelate) upon the growth of V. vulnificus in human and rabbit sera were also examined. Iron appeared to be the limiting factor in the ability of this organism to survive or grow in mammalian sera. These results, both in vitro and in vivo, provided strong evidence that iron may play a major role in the pathogenesis of V. vulnificus. Vibrio vulnificus, a halophilic bacterium, is perhaps the most invasive of vibrio species. Unlike other vibrios, it is associated with a high incidence of septicemia correlated with a high degree of mortality (2, 9). An experimental basis for this invasiveness was demonstrated in laboratory animals by Poole and Oliver (14) and by J. B. Dellinger (M.S. thesis, University of North Carolina, Charlotte, 1980) in histological examinations of ligated ileal loop infections. Blake et al. (2) have provided a comprehensive clinical presentation of infections produced by this bacterium. Seventy-five percent of infections resulting in septicemia were associated with underlying diseases that can result in iron overload. These diseases include hemochromatosis, thalassemia, and chronic cirrhosis. The importance of iron for microorganisms has long been recognized, and the availability of host iron has been proposed as a contributing factor in a number of experimental bacterial infections (1, 3, 18-20). Iron within the mammalian host is bound to various proteins and is, therefore, not readily available for bacterial acquisition (17). For example, the bactericidal or bacteriostatic properties of serum have been attributed to the sequestering of iron by transferrin. These properties can be abolished by increasing the saturation of transferrin with iron (3,5). In view of the high correlation of V. vulnificus infections with diseases involving increased iron status, the effects of iron and iron-chelating agents upon the pathogenicity and upon the ability of this organism to survive in serum were examined. MATERIALS AND METHODS Organisms. V. vulnificus (CDC strain C7184) was used for injections into 6to 8-week-old male mice (ICR) or for inoculation...

Of 38 isolates of Vibrio vulnificus examined, all avirulent strains produced only translucent colonies. All virulent strains, with the exception of biogroup 2 (eel pathogens), exhibited both opaque and translucent colonies. Isogenic morphotypes were examined for a variety of phenotypic and virulence traits. Only the ability to utilize transferrin-bound iron and the presence of a surface polysaccharide were found to correlate with colony opacity and virulence.

Translucent, avirulent spontaneous phase variants of Vibrio vulnificus M06-24 reverted back to the original opaque, encapsulated phenotype under both in vivo and in vitro conditions. Two translucent, acapsular mutants, which did not show phase variation, were constructed by using the transposon Tn5 ISSOL: :phoA (TnphoA). Loss of capsule was accompanied by decreases in virulence, hydrophilicity, and serum resistance. The ability to utilize transferrin-bound iron for growth was lost in only one of the two unencapsulated mutants.

Using plate counts, total cell counts, and direct viable counts, we examined the fate of cells of Vibrio vulnificus placed into natural estuarine waters during both winter and summer months. Cells inoculated into membrane diffusion chambers and placed into estuarine waters entered into a viable but nonculturable (VBNC) state in January and February, when the water temperatures were low (average, <15؇C). In contrast, when cells in the VBNC state were placed into the same waters in the warmer months of August through November (average water temperature of ca. 21؇C), the cells appeared to undergo a rapid (typically, within 24 h) resuscitation to the fully culturable state. These results were independent of whether the cells were in the logarithmic or stationary phase and whether they were encapsulated or not. This study indicates that the inability to isolate V. vulnificus from cold estuarine sites may be accounted for by entrance of the cells into a VBNC state and that recovery from this state in natural environments may result from a temperature upshift.

Previous studies in our laboratory, as well as clinical evidence, have suggested that increased iron levels in the host may be important in infections caused by the halophilic pathogen Vibrio vulnificus. To study iron acquisition, we induced siderophore production by growth in a low-iron medium, and biochemical testing indicated the production of both hydroxamateand phenolate-type siderophores. The siderophores were extracted from growth filtrates with ethyl acetate (for phenolates) and phenol-chloroform-ether (for hydroxamates). These extracts enhanced the growth of V. vulnificus when the bacterium was grown in ironlimited medium. The ability of these siderophores to stimulate the growth of