Para-cresol production by Clostridium difficile affects microbial diversity and membrane integrity of Gram-negative bacteria

Abstract

Clostridium difficile is a Gram-positive spore-forming anaerobe and a major cause of antibiotic-associated diarrhoea. Disruption of the commensal microbiota, such as through treatment with broad-spectrum antibiotics, is a critical precursor for colonisation by C. difficile and subsequent disease. Furthermore, failure of the gut microbiota to recover colonisation resistance can result in recurrence of infection. An unusual characteristic of C. difficile among gut bacteria is its ability to produce the bacteriostatic compound para-cresol ( p-cresol) through fermentation of tyrosine. Here, we demonstrate that the ability of C. difficile to produce p-cresol in vitro provides a competitive advantage over gut bacteria including Escherichia coli, Klebsiella oxytoca and Bacteroides thetaiotaomicron. Metabolic profiling of competitive co-cultures revealed that acetate, alanine, butyrate, isobutyrate, p-cresol and p-hydroxyphenylacetate were the main metabolites responsible for differentiating the parent strain C. difficile (630Δ erm) from a defined mutant deficient in p-cresol production. Moreover, we show that the p-cresol mutant displays a fitness defect in a mouse relapse model of C. difficile infection (CDI). Analysis of the microbiome from this mouse model of CDI demonstrates that colonisation by the p-cresol mutant results in a distinctly altered intestinal microbiota, and metabolic profile, with a greater representation of Gammaproteobacteria, including the Pseudomonales and Enterobacteriales. 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. In general, 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 the viability of intestinal bacteria as well as the major metabolites produced in vitro. These observations are upheld in a mouse model of CDI, in which p-cresol production affects the biodiversity of gut microbiota and faecal metabolite profiles, suggesting that p-cresol production contributes to C. difficile survival and pathogenesis.

Author summary

Clostridium difficile is a bacterium responsible for causing the majority of antibiotic associated diarrhoea outbreaks world-wide. In the United States of America, C. difficile infects half a million people annually. Antibiotics disrupt the natural protective gut microbiota, rendering people susceptible to C. difficile infection, which leads to potentially life-threatening disease and complications. C. difficile is transmitted by spores, which are able to survive in harsh environments for long periods of time. After initial treatment for C. difficile, up to 35% of patients develop the disease again, thus requiring additional and more successful treatment. Here, we use novel techniques to show that C. difficile produces a compound, p-cresol, which has detrimental effects on the natural protective gut bacteria. We show that p-cresol selectively targets certain bacteria in the gut and disrupts their ability to grow. By removing the ability of C. difficile to produce p-cresol, we show that it makes C. difficile less able to recolonise after an initial infection. This is linked to significant alterations in the natural healthy bacterial composition of the gut. Our study provides new insights into the effects of p-cresol production on the healthy gut microbiota and how it contributes to C. difficile survival and pathogenesis.