INVESTIGADORES
LUCCHESI paula Maria Alejandra
congresos y reuniones científicas
Título:
Phage and verotoxin levels related to cytotoxicity titers in vt2-positive isolates
Autor/es:
GRANOBLES VELANDIA, C. V.; KRÜGER, A.; ARROYO, G. H.; PARMA, A. E.; LUCCHESI, P. M. A.
Reunión:
Simposio; 8th INTERNACIONAL SYMPOSIUM ON SHIGA TOXIN (VEROCYTOTOXIN) - PRODUCING Escherichia coli INFECTIONS; 2012
Resumen:
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 cytotoxicity
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 cultured
in LB broth under inducing (0.5 lg/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 performed
at least twice.
lg/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
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 evidenced
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 vt2 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 be
detected by EIA.