Therefore, it has to be considered that STEC O104:H4 produces only STX2, while STEC O157:H7 produces both STX1 and 2. Concordant with the quantification of shiga toxin contents by EIA, cytotoxicity assays on Vero cells showed that treatment of STEC O157:H7 with

0.25x or 1x MIC enhanced the STX-activity of supernatants more than 100-fold (Figure 3A). Treatment of STEC O157:H7 with the 4x MIC of buy APO866 ciprofloxacin still increased STX activity in the supernatants more than 10-fold compared to non-treated controls. In contrast, treatment of STEC O104:H4 with 0.25x or 1x MIC of ciprofloxacin increased STX activity about 10- or almost 100-fold, respectively, compared to untreated controls. Importantly, the 4x MIC of ciprofloxacin reduced the shiga toxin activity in supernatants of STEC O104:H4 up to 10-fold compared to untreated controls. Figure 3 Cytotoxic activity of supernatants of STEC strains O157:H7 and O104:H4 treated with various antibiotics. The cell free supernatants of STEC cultures described in Figure 2 were 10-fold serially diluted and added to semi-confluent monolayers of Vero cells in microtiter plates. After incubation for 24 h, XTT-labeling reagent was added and cultures were incubated for another 24 h before measuring the viability MK0683 nmr of the Vero cells as OD450 of the samples. The cytotoxic activity of the supernatants was calculated as described in Methods. For each antibiotic,

the cytotoxicity of the supernatants is plotted against MycoClean Mycoplasma Removal Kit the dilution of the supernatants in the upper part of the panel. In these plots, the effect of the antibiotics on the cytotoxicity of the supernatants was determined

as the increment of cytotoxicity in comparison to untreated controls, as indicated exemplarily for the 1x and 4x MIC of ciprofloxacin by green dashed lines and red dashed lines, respectively. In the lower part of each panel the increments of the cytotoxicity are plotted for the various MIC of the respective antibiotic. Shown are the means and standard errors of three independent experiments. Statistical significance is indicated by asterisks: * for p < 0.05; ** for p < 0.01. These data confirm reports that ciprofloxacin can induce the accumulation of STX activity in the supernatants of STEC O157:H7 [3, 4] and they show a similar response of STEC O104:H4 to low concentrations of ciprofloxacin. However, the dose–response of these two strains of STEC markedly varies in that 4x MIC reduces toxin activity in supernatants of O104:H4 below that of untreated controls, while the same concentration still enhances the toxin activity more than 10-fold in supernatants of strain O157:H7. Meropenem at 0.25x and higher MIC enhanced STX activity in supernatants of strain O157:H7 up to 10-fold (Figure 3B). In contrast, meropenem at concentrations up to 1x MIC did not affect the STX activity of supernatants of strain O104:H4 and 4x MIC reduced the STX activity. Strain O157:H7 responded to fosfomycin at concentrations of 0.