Clostridioides difficile infection (CDI) has become one of the emerging challenges in infectious and digestive diseases. The incidence of community-acquired CDI continues to increase even though the overall incidence of CDI did not change significantly [1]. Recurrent CDI is especially challenging for clinicians to prescribe relevant treatments [2]. Antimicrobial therapy remains the first option for the treatment of CDI according to the latest European and American guidelines [3,4]. Metronidazole (MTZ) and vancomycin were the primary antibiotics used to treat CDI until fidaxomicin was approved by the Food and Drug Administration in 2011 [5]. However, in the updated Infectious Diseases Society of America/Society for Healthcare Epidemiology of America and European Society of Clinical Microbiology and Infectious Diseases guidelines, MTZ is no longer recommended as the primary treatment option for adult patients with CDI due to poor efficacy [3,4]. Currently, MTZ is used only for intravenous administration in combination with vancomycin to treat fulminant CDIs [6,7].

MTZ treatment is inferior to vancomycin in the treatment of CDIs. In a pooled analysis of two multinational, randomized, controlled trials [8], MTZ showed lower clinical success rate than vancomycin (72.7% vs. 81.1%). In Quebec, the proportion of patients who failed to respond to MTZ treatment and added vancomycin or switched to vancomycin increased from 9.6% in 1991–2002 to 25.7% in 2003–2004 [9]. The reduced clinical efficacy may be related to decreased susceptibility to MTZ or the development of MTZ resistance in C. difficile [10].

Horizontal transfer of the pCD-METRO plasmid can mediate resistance to MTZ in C. difficile, although the exact mechanism remains unclear [11]. However, the majority of MTZ-resistant C. difficile strains do not carry pCD-METRO [12]. Limited data from proteomic and genomic studies have suggested that chromosomal resistance to MTZ in C. difficile may be related to genetic changes in pyruvate: ferredoxin oxidoreductase or other oxidoreductases, as well as increased expression of genes associated with in vivo oxidative stress tolerance [[13], [14], [15], [16]]. However, more data are required to confirm these mechanisms. In a previous study, we obtained an MTZ-resistant C. difficile strain sh182IR by in vitro induction, and no plasmid was identified in this strain. RNA-seq analysis revealed overexpression of pyruvate: ferredoxin oxidoreductase and efflux pumps in sh182IR, particularly the genes encoding transporters in the ATP-binding cassette (ABC) family. Subsequent efflux pump inhibition assays confirmed their contribution to MTZ resistance [17]. The overexpression of multidrug efflux pumps is a pivotal mechanism underlying drug resistance. The ABC family transporters are associated with the efflux of ciprofloxacin and vancomycin in C. difficile. The ABC transporter CprABC is associated with resistance to antimicrobial peptides [18]. After mutation of the ABC transporter CD2068 in Clostridium difficile, the half maximal inhibitory concentration (IC50) values for multiple antibacterial agents, including MTZ decreased [19], with the most notable reduction observed for cloxacillin, showing a 3.5-fold decrease in IC50. However, it is not clear whether ABC family transporters are involved in MTZ resistance in C. difficile. In this study, we address the scientific question of how ABC transporters contribute to MTZ resistance in C. difficile. We hypothesize that mutations in transcriptional regulatory elements lead to the elevated expression of the downstream ABC transporter, which enhances the efflux capacity of C. difficile and contributes to the development of MTZ resistance.

In the present study, we investigated the role of ABC transporters in MTZ resistance in C. difficile. ClosTron mutagenesis was used to disrupt genes that encode putative multidrug efflux pumps of the ABC family in the MTZ-resistant strain sh182IR [20]. We also analysed the upstream sequence of the efflux pump-encoding gene to characterize the regulatory mechanisms.