Abstract: Clostridium difficile is the major cause of antibiotic-associated diarrhoea world-wide. Disruption of the commensal microbiota, such as through use of broad-spectrum antibiotics, is key for colonisation by C. difficile and development of C. difficile disease (CDI). Furthermore, failure of the gut microbiota to recover diversity and re-establish a healthy microbiota, results in recurrence of infection. After an initial recurrent episode, patients are more likely to relapse multiple times. Understanding the mechanisms behind colonisation, relapse and virulence in C. difficile will provide an insight into development of novel treatments for C. difficile. Virulence factor of C. difficile, which contribute to disease, colonisation and transmission include ; spores, flagella, cell surface proteins, biofilm formation and p-cresol production. An unusual characteristic of C. difficile among gut bacteria is its ability to produce the bacteriostatic compound para-cresol through fermentation of tyrosine, via an intermediate para-hydroxyphenyl acetate (p-HPA). Here, we investigate the interaction of C. difficile with other commensal microbiota and the effect that p-cresol production by C. difficile has on their growth and microbial diversity. Using a gene inactivation mutant (hpdC::CT) of the decarboxylase responsible for the conversion of p-HPA to p-cresol, we demonstrate that p-cresol production provides a competitive advantage for C. difficile over commensal gut bacteria; E. coli, K. oxytoca and B. thetaiotaomicron. Metabolic profiling highlights the importance of acetate, butyrate, p-cresol and p-HPA in driving separation of wild-type C. difficile from a p-cresol mutant. Removing the ability to convert p-HPA to p-cresol leads to a fitness defect in a mouse relapse model of C. difficile infection. These mice have a distinctly altered gut microbiota composition, alongside altered faecal metabolite profiles. We demonstrate that Gammaproteobacteria are susceptible to exogenous p-cresol in-vitro and that there is a clear divide between bacterial Phyla and their susceptibility to p¬¬-cresol. The Gram-negative species were relatively sensitive to p-cresol, whereas Gram-positive species were more tolerant. This study demonstrates that production of p-cresol by C. difficile has an effect on relapse of CDI in a mouse model of infection, as well as the viability of Gram-negative commensal intestinal bacteria and metabolite production, suggesting that p-cresol production directly or indirectly contributes to C. difficile pathogenesis.