Lactate dehydrogenase release assay from Vero cells to distinguish verotoxin producing Escherichia coli from non-verotoxin producing strains

The Vero cell assay presently used for virulence testing of verotoxigenic E scherichia coli (VTEC) requires at least 48-96 h where cytotoxicity effects are examined under a microscope. Here, a complimentary rapid assay was dev eloped that measures endogenous lactate dehydrogenase (LDH) release from Ve ro or HEp-2 cells as an indicator of cytotoxicity. Toxin preparations from 24 VTEC strains induced 36-89% LDH from Vero cells and 15-62% LDH from HEp- 2 cells in 12-16 h. A verotoxin-positive but enterohemolysin negative strai n also showed a similar cytotoxicity effect. In contrast, three VT-negative strains caused only 13-16% LDH from Vero cells and 1-7% LDH from HEp-2 cel ls. Five presumptive E. coli isolates from naturally contaminated food and clinical sources did not induce significant LDH release from either cell li nes. PCR analysis confirmed the presence of vt1 or vt2 genes in E. coli sho wing positive LDH values. Similarly, RiboPrinter(R) analysis confirmed and identified the test strains as E. coli except for two meat isolates, which were identified as Hafnia alvei. Cytopathic effects of toxin preparations f rom VTEC revealed severe lysis, vacuole formation and death in Vero cells a nd multiple vacuoles and cell elongation in HEp-2 cells. The colorimetric c ytotoxicity assay described here can provide quantitative data for determin ing the virulence potential of verotoxigenic E. coli in 12-16 h. (C) 2001 E lsevier Science B.V. All rights reserved.