The Study of Clostridioides difficile Spore Germination for the Development of a Pro-Germination Sporicidal Strategy for the Elimination of C. difficile Spores

Abstract

The highly resistant spores of C. difficile are key to the transmission of C. difficile infection and are capable of withstanding adverse environmental conditions including the antimicrobial activity of traditional biocides but reactivate through germination in response to specific environmental germinants and co-germinants. The research presented in this thesis utilised an optical density germination assay to explore the induction of C. difficile spore germination by germinants and co-germinants to further understand the regulation of C. difficile spore germination and utilise this knowledge in a pro-spore germination strategy to eliminate C. difficile spores from the environment. The bile salt, taurocholate was an efficient germinant, while cholate, glycocholate and deoxycholate exhibited divergent germination activity with spores of C. difficile NCTC 11204 and C. difficile reference strains representing ribotypes 001, 002 and 015. Only glycine and calcium had the capacity as co-germinant to induce germination of C. difficile spores but combining glycine or calcium with selected amino acids reduced or completely inhibited the germination response of C. difficile spores. A novel bile salt-derived compound, C109, developed to have dual germination and antimicrobial functionality with C. difficile spores successfully initiated germination and also demonstrated antimicrobial activity against C. difficile vegetative cells. To develop a C. difficile sporicidal smart surface a series of polymeric formulations incorporating C109 and additional novel compounds C114 and C119 were evaluated. Germination of C. difficile spores was successfully induced but polymer composition and processing conditions were found to influence germination activity. Evaluation of the antimicrobial activity of polymer formulations incorporating C109 demonstrated a statistically significant reduction in the recovery of viable C. difficile spores. The dual germination and antimicrobial activity of the polymer formulation incorporating C109 was therefore successfully demonstrated, providing proof of concept for a sporicidal polymeric biomaterial targeting C. difficile spores, deserving of further development and investigation.