Biofilm Assay for <em>Clostridioides difficile</em> with Applications for Drug Discovery

Clostridioides difficile is a gastrointestinal bacterial pathogen able to take advantage of a dysbiotic microbiota environment to proliferate, secrete toxins, and damage the intestinal epithelium. A subset of C. difficile infection (CDI) patients will experience antibiotic (15%-30%) or fecal microbiota transplant (FMT) (<10%) treatment failure. Therefore, the development of additional therapeutic interventions is of critical importance. The role of C. difficile biofilms in recurrence is unclear. However, biofilms in other organisms are responsible for chronic and relapsing disease, suggesting this could also be the case in recurrent CDI. We hypothesize that biofilms of C. difficile present a valuable therapeutic target. The goal of the protocol presented here is to adapt a biofilm formation assay for the identification of repositionable compounds with activity against established C. difficile biofilms. The protocol refines a robust and reproducible assay for forming biofilms, couples it to a metabolic assay, and applies it to drug discovery. This protocol outlines the biofilm formation assay, biomass and metabolic activity readouts, drug susceptibility testing, drug screening of a repositioning library, and representative results.