Metronidazole-resistant Clostridioides difficile: genomic and transcriptomic traits acquired under in-vitro metronidazole induction

Decreased effectiveness of metronidazole for the treatment of Clostridioides difficile infection has been documented. One reason for this is that levels of metronidazole in the colon are generally low; therefore, a modest increase in the minimum inhibitory concentration of metronidazole for C. difficile may result in an insufficient therapeutic concentration. Due to the lack of efficient genetic manipulation tools for C. difficile strains, the resistance mechanism is largely unknown. In this study, a metronidazole-resistant strain (SH182IR) was acquired by in-vitro induction with metronidazole from a clinical metronidazole-heteroresistant strain (SH182), and the genomic and transcriptional changes were investigated through whole-genome sequencing and RNA-seq. The morphology of the two strains was studied by transmission electron microscopy, and the roles of drug efflux pumps in metronidazole resistance were determined by inhibition assay. Genomic analysis showed that the ferrous iron transporter feoB3 was truncated in SH182IR, indicating that feoB3 contributed to the metronidazole resistance of C. difficile. RNA-seq analysis showed that genes involved in peptidoglycan synthesis, efflux pumps and metronidazole reductive action were expressed differentially between the two strains. Further cell imaging confirmed that cell wall thickness was significantly greater in SH182IR. The efflux pump inhibitor test showed that addition of reserpine or cyanide 3-chlorophenylhydrazone reduced metronidazole resistance in SH182IR, thus proving the role of efflux pumps in metronidazole resistance. These results found an association between genomic variation and metronidazole resistance in C. difficile, and show that metronidazole resistance in C. difficile is multi-factorial, involving metronidazole metabolism, cell wall thickness and efflux pumps. These findings will help improve knowledge and understanding of metronidazole resistance of C. difficile.