Clostridioides difficile virulence genes expression in an insert plate model

Clostridioides difficile is a Gram positive bacterium sporulant and requires strict anaerobiosis. The intestinal carriage of C. difficile can be asymptomatic, but in cases of infection it is associated with different clinical signs of disease that can vary from mild diarrhea to pseudomembranous colitis or even death. Expression of C. difficile virulence gene was studied in an in vitro insert plate model. The objective of this study was to identify the timing of expression of virulence gene of C. difficile. In compartment “in”, a nutritive matrix, salt biles and a quantity of C. difficile spores were placed. In compartment “out”, a nutritive matrix and salt biles were placed. Several plates were made to study a plate without feces, two with a variation of the concentration of spores, two with a variation of the concentration of feces concentration and four with a variation of the microflora. Total DNA and RNA were extracted and RT-qPCR targeting mldA (evolved in cell division), gluD (housekeeping genes), tcdB and tcdA (toxins genes) This method of culture allows the growth of C. difficile under different growth conditions. The plate without feces is the benchmark. The expression of genes varies depending on the growth conditions and some donors (D2 and D4) will promote the production of toxins. Among the donors, donors 4 gives us a pattern that is closer to the expression of the virulence genes of the insert without feces. The next step of our study will use metatranscriptomics to identify the expression of certain genes that have an impact on colonization.