Mouse C. difficile associated disease model

Female C57 BL/6 mice that were 7 to 8 weeks old were obtained from Charles River Laboratory and housed in sterile caging for the in-life portion of each study. Animals were randomly organized into groups of 20 (n = 20) and placed on drinking water supplemented with a cocktail of antibiotics immediately upon arrival. These antibiotics and their concentrations were: Kanamycin (0.4 mg/mL), Colistin (850 units/mL), Gentamicin (0.035 mg/mL), Metronidazole (.215 mg/mL), Vancomycin (0.045 mg/mL) [23]. Animals were left on the antibiotic supplemented water for 5 days, and then switched to normal water for 24 h. Mice were orally inoculated with 1 × 10⁶ C. difficile spores, and clindamycin was administered subcutaneously at 10 mg/kg of body weight. Starting the day of infection, and each day after, approximately 0.1–0.2 g of feces was collected from cages to determine C. difficile counts and associated amounts of toxin A and B. Bedding was changed daily to ensure fresh feces were collected for analysis, and census of survivors were recorded daily for 14 days after infection. Feces were weighed before sterile 1x PBS was added to the recovered feces, this solution was then homogenized, and 1 mL was separated for each total CFU recovery, spore recovery, and toxin A and B expression. Viable cell counts, spore counts, and toxin expression were quantified as described in the Material and Methods. The homogenized solution separated for spore quantification was heated to 65 ± 2 °C for 1 h to facilitate the isolation of only spores, while the fecal matter separated for toxin expression was diluted approximately 100x - 500x for quantification. This allowed it to fall within detection range of the ELISA used to determine toxin concentration.