Protocol to study recurrence of Clostridioides difficile infection in murine model with built-in colonization and contamination controls

Abstract

Pre-colonization with endogenous murine Clostridioides difficile strains and cross-contamination between animals can compromise recurrent C. difficile infection (R-CDI) studies. We present a murine R-CDI protocol with built-in colonization and contamination controls. Mice receive an antibiotic cocktail and clindamycin before challenge with C. difficile spores, followed by vancomycin to induce recurrence. Selective cycloserine-cefoxitin-fructose agar (CCFA)/taurocholate-CCFA (TCCFA) fecal cultures quantify colonization, while PCR ribotyping identifies endogenous contaminants as a quality control. A two-handler workflow minimizes cross-contamination.