Experimental Evolution of Vancomycin Resistance in Clostridioides difficile: Pathways and Mechanistic Insights

Clostridioides difficile is the leading cause of hospital-associated diarrhoea, and presents an urgent threat to global health – owing, at least in part, to its ability to resist a wide array of antibiotics. Treatments for C. difficile infection are limited, and rely largely upon metronidazole, fidaxomicin, and vancomycin. Vancomycin is the current front-line drug of choice in the UK, and is one of the primary treatments for C. difficile worldwide. Perhaps surprisingly for such an adaptable organism, vancomycin resistance in C. difficile is not widespread, and knowledge of resistance pathways, beyond regulatory changes to the van operon, is lacking. However, isolated reports of resistance in clinic, coupled with anecdotal reports of treatment failure, suggest more effort should be placed on understanding the routes, mechanisms and costs of resistance. To bridge this gap, this work aimed to provide a thorough genetic and molecular characterisation of vancomycin resistance in C. difficile. Experimental evolution under increasing vancomycin dose demonstrated high-level vancomycin resistance could evolve rapidly, at the cost of pleiotropic phenotypic changes. The genetic basis of resistance was revealed through genome sequencing of resistant strains, highlighting multiple alternative routes to resistance acquisition, including mutations in van genes, comR, and the previously uncharacterised dacS. Through recapitulation of observed mutations in a clean genetic background, the mechanisms of vanS and dacS mediated resistance – alterations to the terminal D-Ala in nascent peptidoglycan – were determined, and synergistic interactions between these pathways leading to high-level vancomycin resistance were uncovered. Importantly, only two mutations were required for high-level resistance, a vital consideration for clinical monitoring. Overall, these mutational and mechanistic insights provide a solid foundation to guide future genomic surveillance of vancomycin resistance in C. difficile.