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

Area of Science:

• Microbiology
• Infectious Diseases
• Animal Models

Background:

• Recurrent Clostridioides difficile infection (R-CDI) studies are often compromised by endogenous murine bacterial strains and cross-contamination between animals.
• Reliable experimental models are crucial for understanding R-CDI pathogenesis and testing therapeutics.

Purpose of the Study:

• To present a refined murine protocol for studying recurrent Clostridioides difficile infection (R-CDI).
• To incorporate built-in controls for endogenous colonization and experimental cross-contamination.

Main Methods:

• Mice were treated with an antibiotic cocktail and clindamycin, followed by challenge with Clostridioides difficile spores.
• Vancomycin administration was used to induce infection recurrence.
• Fecal cultures using selective cycloserine-cefoxitin-fructose agar (CCFA) and taurocholate-CCFA (TCCFA) quantified colonization.
• PCR ribotyping identified endogenous contaminants for quality control.
• A two-handler workflow was implemented to minimize cross-contamination.

Main Results:

• The protocol effectively controls for endogenous Clostridioides difficile colonization.
• The methodology successfully minimizes cross-contamination between experimental animals.
• Fecal cultures and PCR ribotyping provided reliable measures of colonization and contamination.

Conclusions:

• The presented murine R-CDI protocol offers robust controls for endogenous colonization and cross-contamination.
• This model enhances the reliability and reproducibility of recurrent Clostridioides difficile infection research.
• The protocol facilitates accurate investigation into R-CDI pathogenesis and therapeutic strategies.