Verocytotoxicity in basal and induced conditions in relation to type and number of vt variants

KRÜGER, ALEJANDRA; LUCCHESI, PAULA M. A.; ARROYO, GUILLERMO H.; PARMA, ALBERTO E.

Buenos Aires

Simposio; 7th INTERNATIONAL SYMPOSIUM ON SHIGA TOXIN (VEROCYTOTOXIN) - PRODUCING Escherichia coli INFECTIONS.; 2009

Asociación Argentina de Microbiología y comité internacional

Verocytotoxins (VTs), are the major virulence factors of verocytotoxigenic Escherichia coli (VTEC). Several toxin variants have been identified and VTEC with different vt alleles differ markedly in their association with HUS. Previous studies also indicate that VT-encoding phages are highly variable. However, the level of expression of VT variants and their ability to be induced are scarcely documented. The objective of this study was to evaluate verocytotoxicity, with and without mitomycin C induction (induced and basal conditions, respectively), of VTEC strains carrying different vt variants or combinations of them. We selected 54 VTEC, belonging to several serotypes, isolated from cattle and derived foods in Argentina. Briefly, strains were grown in Penassay broth for 5 h at 37°C with shaking. Then, the cultures were divided into two tubes and mitomycin C was added to one of them. The bacteria were grown overnight. Twofold serial dilutions of bacterial supernatants were done in 96-well -well plates and freshly trypsinized Vero cells (4 x 105 Vero cells/ ml) were added. The toxin titer was expressed as the reciprocal of the highest sample dilution that caused cytotoxicity in 50% of the Vero cells after 48 h of incubation. We observed a high variability in the basal cytotoxic effect among isolates with only one vt variant. Moreover, isolates carrying different vt variants were also differentially affected during growth with mitomycin C. The isolates carrying vt2EDL933 had the highest basal titers, which increased on average 1,000 fold following mitomycin C treatment. The isolates carrying vt1EDL933 showed similar basal titers than those carrying vt2EDL933 but the increase was significantly lower after mitomycin C treatment (on average, 300 fold). The isolates carrying vt2vha or vt2hb variants had variable cytotoxic effects with and without mitomycin C treatment. However, the titers increased dramatically after induction (average: 7,700 and 9,300 fold, respectively). vt2g-positive strains showed low cytotoxic basal effect and minimal response to induction. Presence of more than one vt gene variant in the same isolate was not reflected in higher titers, and generally, they were lower than those from strains with only one gene variant. In conclusion, both basal and induced cytotoxic effects were associated with the vt variant more than the serotype or origin of the isolate. Besides, our results reinforce the idea that the presence of two or more phages within the same isolate could alter the expression of the vt genes.