CYTOTOXICITY OF HEMOLYTIC, CYTOTOXIC NECROTIZING FACTOR 1-POSITIVE AND 1-NEGATIVE ESCHERICHIA-COLI TO HUMAN T24 BLADDER CELLS

Approximately one-half of Escherichia call isolates from patients with cystitis or pyelonephritis produce the pore-forming cytotoxin hemolys in, a molecule with the capacity to lyse erythrocytes and a range of n ucleated cell types. A second toxin, cytotoxic necrotizing factor 1 (C NF1), is found in approximately 70% of hemolytic, but rarely in nonhem olytic, isolates. To evaluate the potential interplay of these two tox ins, we used epidemiological and molecular biologic techniques to comp are the cytotoxicity of hemolytic, CNF1(+), and CNF1(-) cystitis strai ns toward human T24 bladder epithelial cells in vitro. A total of 29 i solates from two collections of cystitis-associated E. coli were evalu ated by using methylene blue staining of bladder monolayers at l-h int ervals after inoculation with each strain. Most (20 of 29) isolates da maged or destroyed the T24 monolayer (less than 50% remaining) within 4 h after inoculation. As a group, CNF1(+) isolates from one collectio n (11 strains) were less cytotoxic at 4 h than the CNF1- strains in th at collection (P = 0.009), but this pattern was not observed among iso lates from the second collection (18 strains). To directly evaluate th e role of CNF1 in cytotoxicity of hemolytic E. coli without the variab les present in multiple clinical isolates, we constructed mutants defe ctive in production of CNF1. Compared to the CNF1(+) parental isolates , no change in cytotoxicity was detected in these cnf1 mutants, Our re sults indicate that CNF1 does not have a detectable effect on the abil ity of hemolytic. coli to damage human bladder cell monolayers in vitr o.