28Background. Metronidazole is used to treat mild-to moderate Clostridioides difficile infections (CDI). 29No clear mechanism for metronidazole resistance has been described for C. difficile. A patient 30 treated in the Leiden University Medical Center suffered from recurrent CDI caused by a PCR 31 ribotype (RT) 020 strain which developed resistance to metronidazole (MIC = 8 mg/L). Resistance is 32 also seen in animal isolates, predominantly of RT010. 33Methods. Six metronidazole susceptible and 12 metronidazole resistant isolates from human and 34 animal origin, including the patient isolates, were analyzed by whole genome sequence (WGS) 35 analysis. 585 susceptible and resistant isolates collected in various international studies were tested 36 for the presence of plasmid by PCR. Plasmid copy number was determined by quantitative PCR. 37Findings. Stable metronidazole resistance correlated with the presence of a 7kb plasmid, pCD-38 METRO. pCD-METRO was not detected in 562 susceptible isolates, but was found in toxigenic and 39 non-toxigenic metronidazole resistant strains from multiple countries (n=22). The introduction of a 40 pCD-METRO-derived vector into a susceptible strain led to a ~25 fold increase in the metronidazole 41 MIC. The pCD-METRO replicon sustained a plasmid copy number of ~30, which is higher than 42 currently known replicons for C. difficile. 43Interpretation. We describe the first plasmid-mediated resistance to a clinically relevant antibiotic in 44 C. difficile. pCD-METRO is an internationally disseminated plasmid capable of conferring 45 metronidazole resistance in C. difficile, including epidemic ribotypes. Our finding that pCD-METRO 46 may be mobilizable can impact diagnostics and treatment of CDI. 47
Research in context 51Evidence before this study. On October 19, 2017, a PubMed search was performed with the terms 52 'metronidazole resistance' and 'clostridium OR clostridioides', without language restrictions. A single 53 relevant paper was found describing a strain displaying stable metronidazole resistance not obtained 54 by serial passaging, but no mechanism was identified in this study. On the same day, a PubMed 55 search using terms 'plasmid' and 'resistance' and 'clostridium difficile OR clostridioides difficile' did 56 not yield relevant literature on plasmid-mediated resistance in C. difficile. 57Added value of this study. This study is the first report of plasmid-mediated resistance in C. difficile, 58 and more generally, the first to ascribe a clinically relevant function to a C. difficile plasmid. 59Specifically, we report the sequence and annotation of the plasmid pCD-METRO and show that it 60 confers stable resistance to metronidazole, is detected in both toxigenic and non-toxigenic strains of 61 human and animal origin (including epidemic types), is internationally disseminated, is maintained at 62 a higher copy number than characterized C. difficile plasmids and can be acquired horizontally. 63 Implications of all the available evidence. Metronidazole is widely used as a t...