Running Title: Mode of action and propensity of resistance development of cadazolid

25 Cadazolid is a new oxazolidinone-type antibiotic currently in clinical development 26 for the treatment of Clostridium difficile-associated diarrhea. Here we report 27 investigations on the mode of action and the propensity for spontaneous resistance 28 development in C. difficile strains. Macromolecular labeling experiments indicated that 29 cadazolid acts as a potent inhibitor of protein synthesis, while inhibition of DNA 30 synthesis was also observed, albeit only at substantially higher concentrations. Strong 31 inhibition of protein synthesis was also obtained in strains resistant to linezolid, in 32 agreement with low MICs against such strains. Inhibition of protein synthesis was 33 confirmed in coupled-transcription-translation assays using extracts from different C. 34 difficile strains, including strains resistant to linezolid, while inhibitory effects in DNA 35 topoisomerase assays were weak or not detectable under the assay conditions. 36 Spontaneous resistance frequencies of cadazolid were low in all strains tested 37 (generally < 10 at 2-4 x the MIC) and in multiple passages experiments (up to 13) 38 MICs did not significantly increase. Furthermore, no cross-resistance was observed as 39 cadazolid retained potent activity against strains resistant or non-susceptible to linezolid, 40 fluoroquinolones and the new antibiotic fidaxomicin. 41 In conclusion, the data presented here indicate that cadazolid acts primarily by 42 inhibition of protein synthesis with weak inhibition of DNA synthesis as potential second 43 mode of action and suggest a low potential for spontaneous resistance development. 44 45 on N ovem er 2, 2017 by gest httpaac.asm .rg/ D ow nladed fom