In vitro selection, via serial passage, of Clostridium difficile mutants with reduced susceptibility to fidaxomicin or vancomycin.

Current treatments for Clostridium difficile infection include vancomycin, metronidazole and fidaxomicin. LFF571 is an experimental agent undergoing evaluation in humans for the treatment of moderate C. difficile infection. Reduced susceptibility of C. difficile to fidaxomicin or LFF571 in vitro can be mediated by single point mutations in genes encoding the targets, whereas the mechanism(s) mediating reduced susceptibility to vancomycin in vitro remains elusive. To further characterize mechanisms reducing susceptibility of C. difficile to vancomycin, fidaxomicin or LFF571 in vitro, selections via serial passage at low cell density were performed, followed by whole-genome sequencing. C. difficile strain ATCC 43255 and three clinical isolates were subjected to 10 passages on medium containing a range of concentrations of fidaxomicin, LFF571 or vancomycin. Genomic DNA from isolates with reduced susceptibility was sequenced using Illumina Whole Genome Sequencing. Clones exhibiting decreased susceptibility to fidaxomicin harboured mutations in rpoB and CD22120 (marR homologue). Clones exhibiting decreased susceptibility to vancomycin harboured mutations in rpoC and also in CD2725, CD3659 and sdaB, which encode a putative N-acetylglucosamine transferase, exonuclease and l-serine deaminase, respectively. All mutations resulted in non-synonymous substitutions. No clones with reduced susceptibility to LFF571 were selected in this study. Reduced susceptibility to fidaxomicin and vancomycin was associated with mutations mediating target modifications (RNA polymerase and cell wall, respectively), as well as with mutations that may contribute to reduced susceptibility via other mechanisms. The MIC of LFF571 was unaffected for those mutants with reduced susceptibility to fidaxomicin or vancomycin.