ATP-binding cassette transporters mediate and regulate metronidazole resistance in Clostridioides difficile

Metronidazole (MTZ) resistance in Clostridioides difficile is associated with decreased efficacy of MTZ. We studied the MTZ-resistant C. difficile strain sh182IR (minimum inhibitory concentration [MIC] = 32 mg/L) to characterize the role of ATP-binding cassette (ABC) family transporters in MTZ resistance in C. difficile. The strain overexpresses ABC multidrug efflux pump components.

In strain sh182IR, ClosTron mutagenesis was applied to disrupt cd17530, a gene encoding the ABC transporter ATP-binding protein, and the effects on efflux capacity and MIC of MTZ were evaluated. The upstream regulatory gene cd17520 was also sequenced.

Disruption of cd17530 reduced the efflux capacity of fluorescent substrates and MIC of MTZ in C. difficile sh182IR. Repair of cd17530 successfully restored the MTZ MIC and efflux capacity of the strain. We also confirmed that the upstream TetR/AcrR family transcription factor gene cd17520 in sh182IR harbours a frameshift mutation. This mutation resulted in the loss of function of the peptide chain encoded by the gene and upregulation of cd17530-17540-17550.

The frameshift mutation in the transcriptional repressor cd17520, which results in the derepression of the cd17530-17540-17550 operon, contributes to ABC transporter-mediated MTZ resistance in C. difficile.