Intestinal calcium and bile salts facilitate germination of Clostridium difficile spores
Cefoperazone-treated C57BL/6 mice (n = 8) were infected with 1500 spores of WT or Δ32980 by oral gavage. Colonization levels were assessed daily and are presented in total CFU per gram of feces. (a) Multiple t tests were performed and p = 0.23 for each time point tested (a). Ileal contents were collected from Cefoperazone treated C57BL/6 mice (n = 3) and ex vivo germination assays were performed. 1x103 spores of Cd630 or Δ32980 were incubated for one hour at 37°C in ileal contents, calcium depleted ileal contents or calcium depleted ileal contents treated with 15 mM CaCl2. Samples were then incubated at 65°C for 20 min and then plated on BHIS-Tc plates. Data are presented as % loss of Heat Resistance (b). Free calcium levels of ileal contents, calcium depleted ileal contents or calcium depleted ileal contents treated with 15 mM CaCl2 were measured using a calcium colorimetric assay. Levels of calcium (mM) were determined using a standard curve (c). Assays were performed in triplicate using ileal contents from three mice. Error bars are mean plus or minus SD. Statistical significance was calculated using Two-way ANOVA. (*) p<0.05 (****) p<0.0001.