C. difficile is the most common cause of nosocomial infection in the UK, causing in excess of 2,000 deaths each year in addition to enormous societal and healthcare burden. Worryingly, incidence has increased 34% since the start of the Covid-19 pandemic after a decade of relative stability. The glycopeptide vancomycin is one of a small number of antibiotics that are commonly used to treat C. difficile infection, and it has been the frontline therapeutic in the UK since 2021. Despite the importance of this antibiotic, resistance surveillance is rare and the capacity of C. difficile to evolve resistance is not known. To address this gap in our knowledge, we have combined highly parallel experimental evolution with genomics and molecular microbiology to identify and characterise routes to high-level vancomycin resistance. We have identified several distinct resistance mechanisms, although all were associated with fitness costs of varying severity. However, even in our short-term experimental evolution, there was evidence of emerging compensatory mutations that restored fitness.

Our findings suggest that while C. difficile is capable of evolving high-level vancomycin resistance, this outcome may be limited clinically due to pleiotropic effects on fitness. Given our reliance on vancomycin in the clinic however, it is likely inevitable that we will see widespread resistance before long. Indeed, there are already hints that resistant strains may be spreading under the radar.