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

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 >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.