Cytopathic effects of broth-culture filtrates from eight clinical isolates and one reference strain of on three cultured mammalian cell lines were investigated. All the strains, including NCTC 11637, produced cytotoxic factors that caused intracellular vacuolation on these cell lines. AGS and SflEp cells were more sensitive than HEp-2 cells. To examine the role of urease in the cytotoxic effect, a urease-negative mutant was produced. Filtrates from both wild-type and mutant strains produced similar vacuolation on Sfl Ep cells in the absence of urea, suggesting that produces a cytotoxic substance other than urease. In contrast, ammonia alone, or jack bean urease with urea, also induced rounding and detachment of Sfl Ep cells, whereas ammonium salts induced the production of small vacuoles. The combination of the broth filtrate of the wild-type strain and urea induced vacuolation followed by rounding and detachment of Sfl Ep cells. Evidence is presented that the latter changes are due to ammonia produced during incubation. Nevertheless, the amounts produced were less than that needed to induce cytopathic effects by itself. These results suggest that the cytotoxic substance induces intracellular vacuolation, and that the vacuolated cells are more susceptible to killing by ammonia. Thus both the cytotoxic substance and urease may contribute to the lethal cytotoxicity of


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