FIG 3.

Fecal toxin levels and amounts of C. difficile UK6 spores in mice orally immunized with NTCD, NTCD_mTcd138 spores, or PBS followed by infection with C. difficile UK6 spores. Feces were collected on postinfection days 0, 1, and 2 and then dissolved (0.1 g/ml) in PBS with proteinase inhibitors. TcdA (A) or TcdB (B) levels in feces were determined by ELISA (*, P < 0.05 versus PBS or NTCD). (C) Fecal samples were collected at day 4 postchallenge for bacterial enumeration. An equal amount of 95% ethanol was mixed with each fecal sample for 30 min. Ethanol was removed by centrifugation, and pellets were diluted and spread on BHI-supplemented (BHIS) agar plates containing 10% taurocholic acid in an anaerobic chamber. After incubation for 48 h, the colonies on each plate were counted. The tcdB gene was amplified to distinguish toxigenic C. difficile (UK6) and nontoxigenic C. difficile strains. Bars represent means ± SD. (*, P < 0.05 versus PBS; ns, P > 0.05, i.e., no significance versus PBS; P = 0.0687 between groups NTCD and NTCD_mTcd138). Student's unpaired t test was used for statistical significance.