Multidrug Resistant Clostridioides difficile: The Presence of Antimicrobial Resistance Determinants in Historical and Contemporaneous Isolates, and the Impact Of Fluoroquinolone Resistance Development on PCR Ribotype 027 Fitness

Clostridioides difficile is the major cause of infectious antibiotic-associated diarrhoea, imparting a substantial clinical and financial burden on healthcare facilities. Resistance development, particularly to fluoroquinolones, has been implicated in major, international epidemics, predominantly associated with the hyper-virulent ribotype 027. The development of multiple antimicrobial resistance may contribute significantly to the considerable clinical challenges associated with this organism. In this study, optimised germination environments, antimicrobial susceptibility testing and next generation sequencing were utilised in the recovery and characterisation of an historical C. difficile collection (1980-86). Epidemiological comparisons of ribotype distribution and susceptibility patterns with modern surveillance data (2012-2016) sought to reveal antimicrobial resistance variance between two distinct periods. By correlating phenotypic resistance and genetic determinants, the dissemination of resistance genes was evaluated. Contributions of bacterial mutability to resistance propagation were investigated in response to fluoroquinolone exposure amongst seven prevalent, clinical ribotypes; (n=44). Through in vitro batch and continuous co-culture modelling, the impact of resistance-conferring gyrA and gyrB mutations on the fitness of ribotype 027 (n=7) was assessed. The majority of test antimicrobials (n=8/9) were less active against modern vs historical isolates. This is potentially due to increased antimicrobial exposure and subsequent selection/expansion of resistant strains. Moxifloxacin testing demonstrated the largest increase in resistant populations, reinforcing the notion of reduced susceptibility to modern fluoroquinolones as a potential contributory factor in disease. Phylogenetic analyses highlighted the complexity of molecular clock predictions, with 69% of historical genomes correlating with a 95% prediction interval. Elevated mutability was observed amongst ribotype 027, suggesting greater propensity for resistance evolution in this type. Common fluoroquinolone resistance-conferring substitutions revealed advantages to bacterial fitness. Continuous, competitive co-culture modelling of a Thr82>Ile mutant, 027 strain emphasised the fitness benefits of this polymorphism, retained in the absence of fluoroquinolone pressure. These findings indicate a potential contribution to the success of this ribotype.