Plasmid-mediated metronidazole resistance in Clostridioides difficile
Authors: Boekhoud Ilse M. , Hornung Bastian V. H. , Sevilla Eloisa , Harmanus Céline , Bos-Sanders Ingrid M. J. G. , Terveer Elisabeth M. , Bolea Rosa , Corver Jeroen , Kuijper Ed J. , Smits Wiep Klaas 
Published: May 2019

AbstractBackgroundMetronidazole is used to treat mild- to moderate Clostridioides difficile infections (CDI). 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). Resistance is also seen in animal isolates, predominantly of RT010.MethodsSix metronidazole susceptible and 12 metronidazole resistant isolates from human and animal origin, including the patient isolates, were analyzed by whole genome sequence (WGS) analysis. 585 susceptible and resistant isolates collected in various international studies were tested for the presence of plasmid by PCR. Plasmid copy number was determined by quantitative PCR.FindingsStable metronidazole resistance correlated with the presence of a 7kb plasmid, pCD-METRO. pCD-METRO was not detected in 562 susceptible isolates, but was found in toxigenic and non-toxigenic metronidazole resistant strains from multiple countries (n=22). 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.InterpretationWe describe the first plasmid-mediated resistance to a clinically relevant antibiotic in C. difficile. 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.FundingNetherlands Organisation for Scientific Research; Netherlands Center for One Health; European Center for Disease Prevention and ControlResearch in contextEvidence before this studyOn October 19, 2017, a PubMed search was performed with the terms ‘metronidazole resistance’ and ‘clostridium OR clostridioides’, without language restrictions. A single relevant paper was found describing a strain displaying stable metronidazole resistance not obtained by serial passaging, but no mechanism was identified in this study. On the same day, a PubMed search using terms ‘plasmid’ and ‘resistance’ and ‘clostridium difficile OR clostridioides difficile’ did not yield relevant literature on plasmid-mediated resistance in C. difficile.Added value of this studyThis study is the first report of plasmid-mediated resistance in C. difficile, and more generally, the first to ascribe a clinically relevant function to a C. difficile plasmid. Specifically, we report the sequence and annotation of the plasmid pCD-METRO and show that it confers stable resistance to metronidazole, is detected in both toxigenic and non-toxigenic strains of human and animal origin (including epidemic types), is internationally disseminated, is maintained at a higher copy number than characterized C. difficile plasmids and can be acquired horizontally.Implications of all the available evidenceMetronidazole is widely used as a treatment for mild-to-moderate CDI, though treatment failure occurs in up to ∼30 % of patients. Our data show that carriage of pCD-METRO results in stable metronidazole resistance in C. difficile and suggest that pCD-METRO is mobilizable from an as-of-yet unknown bacterium. Our findings warrant a further investigation into the role of this plasmid in metronidazole treatment failure and the influence of metronidazole use on the international dissemination of pCD-METRO. It also offers an opportunity to improve treatment success and reduce the dissemination of antimicrobial resistance by screening C. difficile isolates or donor fecal material prior to fecal microbiota transplant.