Fig. 1.

(a) Concentration of combined TcdA and TcdB detected in the supernatants of the ΔPaLoc model strains and wild-type controls, as assessed by ELISA. Data represent the mean ± SD of ≥4 replicate values. P=<0.0001 as determined by one-way ANOVA followed by Dunnett's multiple comparison test. The R² value determined by assaying known concentrations of combined TcdA and TcdB from 0 to 125 ng/ml was 0.9955 and the following equation was used to convert absorbance values to toxin concentrations (x-0.1015)/0.0205 after accounting for the initial dilutions. (b) Cytotoxicity assay using Vero cells treated with 24 h supernatants from the ΔPaLoc model strains and wild-type controls. The end point represents the greatest dilution at which >50% of the cells had rounded. Data represent the mean ± SD of 5 replicate values. (c) Cytotoxicity assay using Vero cells treated with 48 h supernatants from the ΔPaLoc model strains and wild-type controls. Data represent the mean ± SD of ≥4 replicate values. P = ***<0.0002, ****<0.0001 as determined by one-way ANOVA followed by Dunnett's multiple comparison test. (d) Representative image of a Vero cell monolayer treated with a 1 × 10-1 dilution of R20291ΔPaLoc supernatant. (e) Representative image of a Vero cell monolayer treated with a 1 × 10-1 dilution of R20291 supernatant.