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Background Until recently, metronidazole was the first-line treatment for Clostridioides difficile infection and it is still commonly used. Though resistance has been reported due to the plasmid pCD-METRO, this does not explain all cases. Objectives To identify factors that contribute to plasmid-independent metronidazole resistance of C. difficile. Methods Here, we investigate resistance to metronidazole in a collection of clinical isolates of C. difficile using a combination of antimicrobial susceptibility testing on different solid agar media and WGS of selected isolates. Results We find that nearly all isolates demonstrate a haem-dependent increase in the MIC of metronidazole, which in some cases leads to isolates qualifying as resistant (MIC &amp;gt;2 mg/L). Moreover, we find an SNP in the haem-responsive gene hsmA, which defines a metronidazole-resistant lineage of PCR ribotype 010/MLST ST15 isolates that also includes pCD-METRO-containing strains. Conclusions Our data demonstrate that haem is crucial for medium-dependent metronidazole resistance in C. difficile.

Haem is crucial for medium-dependent metronidazole resistance in clinical isolates of Clostridioides difficile