Effect of fluoroquinolone resistance mutation Thr-82→Ile on Clostridioides difficile fitness.

Objectives:Fluoroquinolone resistance is common among epidemic Clostridioides difficile PCR ribotype (RT) 027 and may have contributed to outbreaks of C. difficile infection (CDI). We investigated the impact of fluoroquinolone mutations on the bacterial fitness (BF) of C. difficile RT027 isolates. Methods:The BF of seven RT027 mutants with reduced susceptibility to moxifloxacin (moxifloxacin MIC 4-32 mg/L) was compared with their susceptible (moxifloxacin MIC 1-2 mg/L) progenitor strains in competitive batch culture (CBC), cell cytotoxicity and maximal growth rate assays. Comparative fitness dynamics of one gyrA Thr-82→Ile-harbouring isolate (CD3079M) versus the parent strain (CD3079) were also investigated in a continuous co-culture (CC) chemostat model. Mutant and parent strain populations were assessed every 24 h over 8 days using selective and non-selective agars. Sequencing was performed using NEBNext® Ultra™ chemistry and Illumina® HiSeq 3000 technologies. Results:BF was significantly increased in all Thr-82→Ile isolates (w = 1.08-1.22) in CBC assays (P = 0.002). Gly-429→Val and Gln-434→Lys (gyrB) also showed no burden to fitness (w = 1.24 and 1.18, respectively), but Asp-71→Tyr conferred reduced fitness (w = 0.80). CC results for strains CD3079 and CD3079M (Thr-82→Ile) supported CBC findings; mutant-to-parent ratios differed significantly by 96 h (x¯=1.80, P = 0.025). Conclusions:The absence of a fitness cost associated with the most prevalent fluoroquinolone resistance mutations may have contributed to the success of RT027. Furthermore, a demonstrable in vitro advantage over fluoroquinolone-susceptible parent strains in CC may contribute to the maintenance of RT027, even in the absence of fluoroquinolone selection pressure.