Influence of microbiota on the growth and gene expression of Clostridioides difficile in an in vitro coculture model

Abstract

Clostridioides difficile is an anaerobic, spore-forming, Gram-positive pathogenic bacterium. This study aimed to analyze the effect of two samples of healthy fecal microbiota on C. difficile gene expression and growth using an in vitro coculture model. The inner compartment was cocultured with spores of the C. difficile polymerase chain reaction (PCR)-ribotype 078, while the outer compartment contained fecal samples from donors to mimic the microbiota (FD1 and FD2). A fecal-free plate served as a control (CT). RNA-Seq and quantitative PCR confirmation were performed on the inner compartment sample. Similarities in gene expression were observed in the presence of the microbiota. After 12 h, the expression of genes associated with germination, sporulation, toxin production, and growth was downregulated in the presence of the microbiota. At 24 h, in an iron-deficient environment, C. difficile activated several genes to counteract iron deficiency. The expression of genes associated with germination and sporulation was upregulated at 24 h compared with 12 h in the presence of microbiota from donor 1 (FD1). This study confirmed previous findings that C. difficile can use ethanolamine as a primary nutrient source. To further investigate this interaction, future studies will use a simplified coculture model with an artificial bacterial consortium instead of fecal samples.

Keywords: Clostridioides difficile; gastrointestinal microbiome; gene expression; growth; in vitro technique; metabolism.