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Tn5-induced protease-deficient strains of Aeromonas hydrophila with reduced virulence for fish

Protease-deficient strains of Aeromonas hydrophila TF7 were induced by transposon Tn5 mutagenesis, with Escherichia coli 1830(pJB4JI) as the Tn5 donor. The parent strain has the cell surface characteristics associated with virulence for fish, and as it produces a single metalloprotease, mutants could be distinguished by direct plating on brain-heart infusion skim milk agar. Mutants Pd-7 and Pd-10 still produced metalloprotease, but at reduced levels and only after prolonged incubation. The activities of other exoenzymes and hemolysin were unaffected, and the mutants autoagglutinated in broth, indicating that the cell surface characteristics of A. hydrophila TF7 had been retained. Unlike the parent strain, the mutants did not produce lesions or mortalities in rainbow trout (Salmo gairneri) when 5 X 10(6) CFU were injected intramuscularly. The bacterial cells were completely cleared from the site of the injection and the organs within 7 days. For 60-g rainbow trout held at 10 degrees C, the 50% lethal dose of Pd-10 was greater than 10(7) CFU, compared with 8.1 X 10(5) CFU for the parent strain. The mutants were significantly more susceptible than the parent strain to the bactericidal effect of fresh normal trout serum in vitro. Mutants Pd-7 and Pd-10 grew as well as the parent on M9 salts-glucose medium but more slowly on heat-inactivated fish serum. Thus, protease appears to be able to contribute to the establishment of A. hydrophila infection in fish both by overcoming initial host defenses and by providing nutrients for cell proliferation.