Conventional, Gnotobiotic, and Humanized Microbiota Mouse Models of C. difficile Infection.

Abstract

Clostridioides difficile infection (CDI) remains a significant public health challenge, with murine models serving as a cornerstone for investigating host-pathogen interactions, microbiota dynamics, and therapeutic interventions. Although no single mouse model has become the standard, antibiotic-induced disruption of the gut microbiota remains the most common approach to facilitate C. difficile colonization. Here, we describe flexible and reproducible mouse models of CDI that can be adapted to diverse experimental goals, including studies of acute disease, long-term colonization, and microbiota-mediated resistance. This protocol includes detailed guidance on antibiotic pretreatment, C. difficile challenge, clinical monitoring, and microbial enumeration. We also outline procedures for microbial reconstitution in germ-free mice, enabling the study of defined or humanized microbiota. Practical considerations for minimizing environmental contamination and optimizing reproducibility are also discussed.