Morphological, cultural, biochemical, and serological characteristics of 79 strains of Vibrio parahemolyticus isolated from patients suffering from gastroenteric disease in Japan were compared with 17 suspected V. parahemolyticus cultures isolated from wound infections and 14 nonpathogenic vibrios isolated from an estuarine environment in the United States. These groups were differentiated on the basis of several key reactions which included: the range of growth temperature and salt tolerance; the production of catalase and acetoin; the hydrolysis of starch; the fermentation and utilization as single carbon source of sucrose, cellobiose, and arabinose; and the ability to swarm on 1 % agar. The separation of the groups on the basis of cultural and biochemical analyses was confirmed by means of slide agglutinations with specific anti-K antisera. The results of this study strongly suggest that the wound infection isolates are V. parahemolyticus species which are easily distinguished from the nonpathogenic estuarine vibrios. MATERIALS AND METHODS Cultures examined. A total of 110 cultures (Table 1) were used for the present studies. The first group of 79 V. parahemolyticus included 78 strains isolated in Japan from feces of patients suffering from gastroenteritis or from food implicated in food poisoning outbreaks. These strains represented all known K antigens with the exception of K-22 antigen. One strain, OY-G1-3, isolated from Puget Sound, was identified as V. parahemolyticus by specific bacteriophage susceptibility (1). The V. parahemolyticus group was obtained from the following donors:

Twenty-four randomly selected clinical and environmental Vibrio vulnificus isolates were tested for virulence in iron-overloaded mice (250 mg of iron dextran per kg of body weight). The log1o 50% lethal doses of 17 isolates were lower by >3.5 loglo units in iron-overloaded mice than in control mice. These isolates were classified as virulent. The 505% lethal doses of these virulent isolates were also lower in mice that were immunosuppressed by treatment with cyclophosphamide (150 mg/kg). Four of the seven isolates initially classified as avirulent were virulent in mice that were simultaneously iron overloaded and immunosuppressed. These isolates were classified as moderately virulent. The remaining three isolates were avirulent under all conditions. The incidence of virulent strains among clinical and environmental isolates did not differ. The virulent isolates produced high titers of hemolysin, were resistant to inactivation by serum complement, produced phenolate siderophore, and utilized transferrin-bound iron. The moderately virulent isolates differed from the virulent isolates only in their increased sensitivity to inactivation by serum complement. The avirulent isolates differed from those of the other two classes in their inability to either produce significant amounts of phenolate siderophore or utilize transferrin-bound iron. A modified agar plate diffusion method for transferrin-bound iron utilization was developed to differentiate the two classes of virulent isolates from the avirulent isolates in vitro.

The lethality of Listeria isolates was determined with normal adult mice and mice that were immunocompromised by treatment with 20 mg of carrageenan per kg. The mean 50% lethal doses (LD50s) of the pathogenic isolates were significantly lower (a = 0.05) in' the immunocompromised mice than in the untreated mice, with an average reduction of 5.8 1glo units. Ini contrast, the mean LD50s of the nonpathogenic isolates were lowèr in the immunocompromised mice by an average of only 0.4 loglo unit, a differehce that was not significant (a = 0.05). Whén immunocompromised mice were used, the LD50s of pathogenic Listeria monocytogenes isolates were lower than those of nonpathogenic L. innocua and L. seeligeri isolates by '6 loglo units and lower than those of nonpathogenic L. ivanovii isolates by .-41glo units. Pathogenic L. monocytogenes isolates could be distinguished from nonpathogenic isolates by their ability to cause deaths in immunocompromised mice in 3 days at a dose of-104 CFU per mouse. An alternative procedure using iron-overloaded mice failed to effectively differentiate pathogenic Listeria isolates.

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Three of 36 raw milk isolates of Yersinia enterocolitica produced enterotoxin in milk at 25 degrees C, but not at 4 degrees C. No strain tested could survive pasteurization.