Metronidazole resistance in Clostridioides difficile is conferred by a plasmid

Background. Metronidazole is utilized to treat mild- to moderate Clostridioides difficile infections. No clear mechanism for metronidazole resistance has been described for C. difficile. A patient treated in the Leiden University Medical Center suffered from recurrent CDI caused by a PCR ribotype (RT) 020 strain which developed resistance to metronidazole (MIC = 8 mg/L). We describe the first plasmid-mediated resistance to a clinically relevant antibiotic in C. difficile. Methods. Six metronidazole susceptible and 12 metronidazole resistant isolates from human and animal origin, including the patient isolates, were analyzed by whole genome sequence (WGS) analysis. 529 susceptible isolates collected in various international studies were tested for the presence of plasmid by PCR. Plasmid copy number was determined by quantitative PCR. Findings. Stable metronidazole resistance correlated with the presence of a 7kb plasmid, pCD-METRO. pCD-METRO was not detected in 529 susceptible isolates, but was found in toxigenic and non-toxigenic metronidazole resistant strains from multiple countries. The introduction of a pCD-METRO-derived vector into a susceptible strain led to a ~25 fold increase in the metronidazole MIC. The pCD-METRO replicon sustained a plasmid copy number of ~30, which is higher than currently known replicons for C. difficile. Interpretation. pCD-METRO is an internationally disseminated plasmid capable of conferring metronidazole resistance in C. difficile, including epidemic ribotypes. Our finding that pCD-METRO may be mobilizable can impact diagnostics and treatment of CDI. The assigned samples are: 18 genomic WGS samples of Clostridium difficile Of these are: - 12 resistant to Metronidazole - 6 sensitive - 7 from dogs (4 Spain, 3 Netherlands) - 11 from humans (all Netherlands) - 12 ribotype 010 - 5 ribotype 020 - 1 ribotype 078 6 of the 18 samples were obtained from 1 single patient. These include the 5 ribotype 20, of which the first in the treatment course was sensitive, and acquired the Metronidazole resistance plasmid in the course of the treatment. The ribotype 078 was also isolated from this patient within the treatment course, but did not have the resistance plasmid. The assigned genome assembly belongs to the last isolate in the treatment course of this patient, sample 026R. In addition, 4 more samples were added later: - 2 WGS samples of a single isolate, before and after acquiring the plasmid - 2 more WGS samples of resistant isolates