The contents of STX were determined with a commercial EIA specific for both STX1 and 2 in two-fold serial dilutions of the supernatants. check details For each antibiotic, in the upper part of the panel the OD of the STX-specific signal is click here plotted against the dilution of the supernatants. In the lower part of each panel, the STX-titers are

shown which were determined in the plots of the OD as indicated exemplarily for the 1x (green dashed lines) and 4x (red dashed lines) MIC of ciprofloxacin. Briefly, from the OD-value of the undiluted sample of the untreated culture a horizontal dashed line was drawn until it intersected the plot of a given MIC. From this intersection a vertical line was drawn to determine the dilution at which the OD-value of the respective supe rnatant equaled the OD-value of the untreated control. The inverse of this dilution was defined as the STX-titer of the sample. Shown are the means and standard errors of three independent experiments. Statistical significance is indicated by asterisks: * for p < 0.05; ** JNJ-26481585 for p < 0.01. Fosfomycin at subinhibitory

concentrations as well as at the 4x MIC increased the titers of STX of supernatants of strain O157:H7 up to 4-fold as compared to untreated controls, while fosfomycin did not significantly affect titers of STX2 in cultures of O104:H4 (Figure 2C). Fosfomycin has already been discussed as a risk factor increasing clinical symptoms in an outbreak of STEC O157:H7 among school children [9]. Our data document increased titers of shiga toxins in fosfomycin-treated cultures of STEC O157:H7 and, therefore, seem to support the conclusion not to treat patients infected with STEC O157:H7 with fosfomycin. However, fosfomycin does not induce the release of STX2 from STEC O104:H4 and treatment with 4x MIC even reduced STX2-titers. Thus, high doses of fosfomycin could be useful for the treatment of infections with STEC O104:H4. Gentamicin did not enhance the release of shiga toxin from either STEC O157:H7 or O104:H4 (Figure 2D). Rifampicin at gradually increasing concentrations in the range of 0.25x

to 4x MIC gradually increased the titers of STX released 4��8C by both STEC O157:H7 and O104:H4 up to 64-fold of untreated controls (Figure 2E). Chloramphenicol at 1x MIC in cultures of STEC O157:H7 increased titers about 4-fold, while a 4x MIC reduced titers below those of untreated controls (Figure 2F). In contrast, chloramphenicol at both the 1x and 4x MIC in cultures of STEC O104:H4 reduced the STX2 titers below those of untreated controls. The determination of STX2 by EIA does only reveal the amount of immunologically detectable STX2, which is not necessarily tantamount to intact and active toxin. Thus, in order to assess the impact of antibiotics, the release of active STX2 was determined in the supernatants of fluid phase cultures of STEC O157:H7 and O104:H4 by the classical cytotoxicity assay on Vero cells.