A microbiota-generated bile salt induces biofilm formation in Clostridium difficile

Effects of bile salts and deoxycholate on biofilm formation by C. difficile strain 630Δerm. a Bacteria were grown in BHISG supplemented with the indicated concentrations of bile salt extract or b deoxycholate. a, b Biofilm formation was evaluated at 72？h. In (b), representative image of a CV stained biofilm produced by cells cultured in the presence of increasing DOC concentrations. c Biofilm formation was evaluated after 48？h growth in the presence of 240？μM bile acids [cholic acid (CA), deoxycholic acid (DCA), lithocholic acid (LCA), chenodexycholic acid (CDCA)] or bile salts [cholate (CHO), deoxycholate (DOC) or chenodeoxycholate (CDOC)]. Asterisks indicate statistical significance determined with a Kruskal–Wallis test followed by an uncorrected Dunn’s test (*p？≤？0.05, **p？≤？0.001, ***p？≤？0.001 vs BHISG with 0？mg/mL bile salts or 0？μM DOC). d Biofilm formation kinetics in the presence or absence of 240？μM DOC. Biofilm formation (a–d) was measured using a crystal violet assay that included two PBS washing before staining (see the Methods section). e Kinetics of CFU grown in the presence or absence of 240？μM DOC. The CFU counts were performed with unwashed biofilms (see the Methods section). Asterisks indicate statistical significance determined with a two-way ANOVA followed by a Fisher LSD test (***p？≤？0.001, ****p？≤？0.0001 vs 24？h). Data shown indicate the mean and the error bars represent the standard error of the mean of at least five experiments performed on different day