Fig 4. Competition co-culture assays in the presence of endogenous p-cresol.

Relative fitness of C. difficile strains 630Δerm, hpdC::CT and the complement hpdC::CT::phpdC-A were grown in competitive co-culture for 24 hours with gut commensal species a) E.coli, b) K. oxytoca and c) B. thetaiotaomicron. The growth medium was supplemented with the intermediate in the p-cresol pathway, pHPA (at 0.2% v/v) and 50 ng/ml anhydrotetracycline to induce expression of hpdCA, encoded in trans. The relative representation of each strain was expressed as a percentage of the total CFU count. Error bars are representative of three independent replicates and show the variation across replicates and co-culture conditions. Regression analysis was used to determine significant differences in growth taking strain into consideration and marked ** p<0.01 and ***p<0.001. d) The concentration of p-cresol produced in the co-cultures of three independent replicates was quantified by HPLC and plotted in GraphPad Prism7. Statistical analysis was performed by Linear regression, significant differences in p-cresol production compared to 630Δerm are indicated *** p<0.001.