Phage and verotoxin levels related to cytotoxicity titers in vt2-positive isolates

GRANOBLES VELANDIA, CLAUDIA V.; KRÜGER, ALEJANDRA; ARROYO, GUILLERMO H.; PARMA, ALBERTO E.; LUCCHESI, PAULA M. A.

Amsterdam

Congreso; 8th INTERNATIONAL SYMPOSIUM ON SHIGA TOXIN (VEROCYTOTOXIN)-PRODUCING Escherichia coli; 2012

Comité Internacional

Introduction and Objectives: Verotoxins, the main virulence factor of VTEC, are encoded in temperate phages, being their expression and release generally related to the lytic cycle of the phage. Since VTEC strains collected in our laboratory present differences in cyto-toxicity titers for Vero cells, our objective was to evaluate the relationship between these differences with the level of production of phages and verotoxin. Material and Methods:We performed the analysis with selected vt2-positive isolates that belonged to either the serotypes O157:H7 or O145:H-, or carried the emergent vt2g-variant. Their verotoxicity titers were determined in a previous study. The isolates were cul-tured in LB broth under inducing (0.5 ug/mL mitomycin C) and non-inducing conditions. For phage quantification, supernatants were collected 3 h post induction and for verotoxin detection (by Ridascreen EIA), after ON incubation. The cultures were monitored spectrophotometrically every hour for the first 5 h post induction and growth/lysis curves were constructed. Each experiment was per-formed at least twice. Results: The bacterial growth curves in the absence of induction were similar for all VTEC isolates, however, the bacterial growth/lysis curves differed when cultures were exposed to mitomycin C. All the isolates, except for two which were vt2g-positive, clearly evi-denced bacteriolysis under this condition. By the double-layer agar method using E. coli DH5a as host strain, phages were detected in supernatants of all O157:H7 and O145:H- isolates, but among the vt2g-positive isolates only in those that evidenced bacteriolysis under induction. Phage plaques hybridized to a vt 2 probe, except those from one vt2g-positive isolate. Higher phage titers were detected when the cultures had been induced and, in general, phage levels were related to cytotoxic titers. All O157:H7 and O145:H- isolates and one vt2g -positive isolate (FB 62), gave strong positive results when verotoxin production was evaluated. The other three vt2g-positive isolates tested negative, even under induction. As no differences were detected neither between induced and non-induced cultures nor among different VT2-producing isolates, dilutions of the supernatants were tested. By using diluted supernatants, FB 62 strain could be identified as the the weakest verotoxin producer, under both uninduced and induced conditions. Conclusions: Phage and verotoxin titers in culture supernatants were closely associated with the cytotoxicity levels of the isolates, showing an increase under induction. Curiously, one vt2g-positive isolate with low verotoxicity titers rendered phage plaques that did not correspond to vt2-phages and no verotoxin production could bedetected by EIA.