Reversion of heme-dependent metronidazole resistance in Clostridioides difficile

Since the emergence of epidemic Clostridioides difficile in the early 2000s, reduced susceptibility to metronidazole (MTZ), once a frontline antibiotic for treating C. difficile infection, has emerged. Consequently, MTZ is no longer a guideline-recommended antibiotic in many countries. Heme and Nim proteins have been shown to co-mediate MTZ non-susceptibility by increasing the MTZ minimum inhibitory concentration (MIC). This study aimed to identify drugs that reverse heme-dependent MTZ non-susceptibility in C. difficile. Drugs known to interfere with heme were screened for their ability to inhibit MTZ non-susceptible C. difficile. Broth and agar dilution methods were used to assess the reversion of MTZ non-susceptibility in diverse clinical isolates, and checkerboard assay was used to determine drug-MTZ interactions. Gene expression analysis was performed using quantitative PCR and reporter assays. We uncovered artemisinin (ART) as an adjuvant capable of resensitizing heme-dependent MTZ non-susceptible C. difficile. ART reduced elevated MTZ MIC (1-8 µg/mL) in non-susceptible strains to the susceptible level (≤0.5 µg/mL) despite the presence of heme. Additionally, we demonstrated that ART reverses nim-mediated MTZ resistance in Bacteroides fragilis. The findings showed that ART significantly reduced intracellular hemin levels in C. difficile, even in the presence of exogenous hemin. This study identifies an adjuvant that repotentiates MTZ activity in non-susceptible C. difficile, where resistance is co-mediated by heme and the nim gene, offering a novel approach to restore MTZ susceptibility in anaerobes.

Metronidazole (MTZ) is a widely used antibiotic for treating anaerobic bacterial infections, including Clostridioides difficile infection. However, the increasing emergence of MTZ resistance among anaerobic pathogens poses a significant threat to its continued clinical effectiveness. Heme-dependent MTZ resistance, one of the mechanisms of resistance, has been associated with poorer treatment outcomes in patients. In this study, we identified artemisinin (ART) and demonstrated that it can reverse heme-dependent MTZ resistance in diverse clinical isolates of C. difficile. Furthermore, ART effectively reversed resistance mediated by the nim genes in both C. difficile and Bacteroides fragilis. These findings open a possible avenue for restoring the efficacy of MTZ against resistant anaerobic bacteria, particularly those with the Nim-mediated resistance mechanism.