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

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.艰难梭菌（Clostridium difficile）是一种革兰氏阳性产芽孢厌氧菌，亦是抗生素相关性腹泻的主要致病菌。广谱抗生素治疗等手段对共生菌群的破坏，是艰难梭菌定植并引发后续疾病的关键前驱条件；此外，肠道菌群未能恢复定植抗性可导致感染复发。在肠道菌群中，艰难梭菌的一项特殊特性在于其可通过酪氨酸发酵生成抑菌性化合物对甲酚（para-cresol，p-甲酚）。本研究证实，艰难梭菌在体外生成p-甲酚的能力，可使其在与大肠杆菌（Escherichia coli）、产酸克雷伯菌（Klebsiella oxytoca）及卵形拟杆菌（Bacteroides thetaiotaomicron）等肠道菌群的竞争中获得竞争优势。对竞争性共培养体系的代谢组分析显示，乙酸、丙氨酸、丁酸、异丁酸、p-甲酚及对羟基苯乙酸，是区分亲本菌株艰难梭菌630Δerm与p-甲酚生成缺陷突变株的核心差异代谢物。此外，本研究发现p-甲酚生成缺陷突变株在艰难梭菌感染（CDI）小鼠复发模型中表现出定植适合度缺陷。对该CDI小鼠模型的菌群组分析显示，p-甲酚生成缺陷突变株的定植会显著改变肠道菌群组成与代谢谱，使伽马变形菌纲（Gammaproteobacteria）的相对丰度显著升高，该纲包含假单胞菌目（Pseudomonales）与肠杆菌目（Enterobacteriales）。本研究证实，伽马变形菌纲在体外对外部施加的p-甲酚具有易感性，且不同细菌门对p-甲酚的易感性存在明确分界。总体而言，革兰氏阴性菌对p-甲酚的敏感性相对较高，而革兰氏阳性菌则耐受性更强。本研究证实，艰难梭菌生成p-甲酚的能力会影响肠道细菌的存活能力，以及体外培养条件下的主要代谢产物谱。上述研究结果在CDI小鼠模型中得到了验证：p-甲酚的生成会改变肠道菌群的生物多样性与粪便代谢谱，提示p-甲酚的生成有助于艰难梭菌的定植存活与致病过程。