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

Date Published: September 12, 2018

Publisher: Public Library of Science

Author(s): Ian J. Passmore, Marine P. M. Letertre, Mark D. Preston, Irene Bianconi, Mark A. Harrison, Fauzy Nasher, Harparkash Kaur, Huynh A. Hong, Simon D. Baines, Simon M. Cutting, Jonathan R. Swann, Brendan W. Wren, Lisa F. Dawson, Theresa M. Koehler.


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 (630Δerm) 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.

Partial Text

Clostridium difficile is a Gram-positive spore-forming enteric pathogen and the leading cause of antibiotic-associated diarrhoea worldwide[1]. C. difficile infection (CDI) ranges from self-limiting diarrhoea to severe and life threatening pseudomembranous colitis[2]. C. difficile spores are the aetiological agent of CDI transmission and are resistant to desiccation, environmental stress, disinfectants and heat[3, 4]. These spores, present in both hospitals and the environment are transmitted via the faecal-oral route, contributing to both nosocomial and community acquired CDI [3]. Infection with C. difficile is frequently preceded by treatment with broad-spectrum antibiotics, which eliminate discrete taxa of the commensal intestinal microbiota resulting in dysbiosis and permitting colonisation by C. difficile. Certain bacterial taxa have been highlighted as important in the prevention of C. difficile colonisation[5–7]. Since restoration of microbial diversity can resolve recurrent infections, faecal transplantation is viewed as an effective treatment strategy[8]. However, a greater understanding of how C. difficile is able to influence the gut microbiota and disrupt intestinal homeostasis is a current imperative.

The indigenous microbiota has been shown to form an ecological barrier that prevents the ingress of pathogenic bacteria such as C. difficile [32]. However, the specific components of the intestinal microbiota that facilitate colonisation resistance are only recently becoming clear [5–7, 25, 33–36]. Both the treatment with broad-spectrum antibiotics and the availability of specific metabolites has been shown to play a role in the expansion of particular bacterial species within the human microbiota [37, 38]. Here, we present compelling evidence that C. difficile may directly modify the intestinal microbiota through production of p-cresol. We demonstrate that C. difficile displays a greater degree of tolerance to p-cresol compared to other common intestinal species, including the Gammaproteobacteria: E. coli, K. oxytoca and P. mirabilis, as well as the Bacteroidetes, B. thethaiotaomicron. We show that these bacterial species are susceptible to the effects of both endogenous and exogenous p-cresol, which was reflected in reductions of viable counts when these intestinal microbiota species were grown in competitive co-culture with C. difficile. Using a plasmid based complementation system to restore the expression of the p-HPA decarboxylase, we have shown that p-cresol production by C. difficile must exceed 5 mM to elicit a significant alteration in competitive growth dynamics. We have shown that C. difficile is able to utilise all the available p-HPA supplemented in the growth medium, which results in the production of up to 25 ±0.04 mM p-cresol in vitro (Fig 3), which is 1000-fold more than the amount of p-cresol produced from tyrosine metabolism by other organisms cultured from the intestinal microbiota (range 0.06–1.95 μg/ml)[11]. There is evidence that p-HPA is present in the human colon and detected in healthy human stool samples at 19 μM[15], therefore C. difficile can potentially utilise free tyrosine and p-HPA to produce p-cresol in vivo.