Different Resistance Mechanisms for Cadazolid and Linezolid in Clostridium difficile Found by Whole-Genome Sequencing Analysis

Cadazolid (CDZ) is a new antibiotic currently in clinical development for the treatment of Clostridium difficile infections. CDZ interferes with the bacterial protein synthesis machinery. The aim of the present study was to identify resistance mechanisms for CDZ and compare the results to those obtained for linezolid (LZD) in C. difficile by whole-genome sequencing (WGS) of strains generated by in vitro passages and to those obtained for LZD-resistant clinical isolates. Clones of C. difficile 630 selected with CDZ during 46 passages had a maximally 4-fold increase in CDZ MIC, while the LZD MIC for clones selected with LZD increased up to 16-fold. CDZ cross-resistance with LZD was maximally 4-fold, and no cross-resistance with other antibiotics tested was observed. Our data suggest that there are different resistance mechanisms for CDZ and LZD in C. difficile . Mutations after passages with CDZ were found in rplD (ribosomal protein L4) as well as in tra and rmt , whereas similar experiments with LZD showed mutations in rplC (ribosomal protein L3), reg , and tpr , indicating different resistance mechanisms. Although high degrees of variation between the sequenced genomes of the clinical isolates were observed, the same mutation in rplC was found in two clinical isolates with high LZD MICs. No mutations were found in the 23S rRNA genes, and attempts to isolate the cfr gene from resistant clinical isolates were unsuccessful. Analysis of 50% inhibitory concentrations (IC 50 s) determined in in vitro transcription/translation assays performed with C. difficile cell extracts from passaged clones correlated well with the MIC values for all antibiotics tested, indicating that the ribosomal mutations are causing the resistant phenotype.