Repurposing of Ciclopirox to Overcome the Limitations of Zidovudine (Azidothymidine) against Multidrug-Resistant Gram-Negative Bacteria

Abstract

Multidrug-resistant (MDR) Gram-negative bacteria are the top-priority pathogens to be eradicated. Drug repurposing (e.g., the use of non-antibiotics to treat bacterial infections) may be helpful to overcome the limitations of current antibiotics. Zidovudine (azidothymidine, AZT), a licensed oral antiviral agent, is a leading repurposed drug against MDR Gram-negative bacterial infections. However, the rapid emergence of bacterial resistance due to long-term exposure, overuse, or misuse limits its application, making it necessary to develop new alternatives. In this study, we investigated the efficacy of ciclopirox (CPX) as an alternative to AZT. The minimum inhibitory concentrations of AZT and CPX against MDR Gram-negative bacteria were determined; CPX appeared more active against β-lactamase-producing Escherichia coli, whereas AZT displayed no selectivity for any antibiotic-resistant strain. Motility assays revealed that β-lactamase-producing Escherichia coli strains were less motile in nature and more strongly affected by CPX than a parental strain. Resistance against CPX was not observed in E. coli even after 25 days of growth, whereas AZT resistance was observed in less than 2 days. Moreover, CPX effectively killed AZT-resistant strains with different resistance mechanisms. Our findings indicate that CPX may be utilized as an alternative or supplement to AZT-based medications to treat opportunistic Gram-negative bacterial infections.

Keywords: ciclopirox; drug repurposing; motility; multidrug-resistant; zidovudine (azidothymidine).

Figure 1

Confirmation of Mcr-1 expression in E. coli ATCC 25922. (a) Confirmation of mcr-1 expression from the related plasmid. PCR amplification using pQE-60-specific primer sets was performed for KS7001 (pQE-60, lane 1) and KS8001 (pQE-60-mcr-1, lane 2), which overproduced a His-tagged Mcr-1 protein [45], on 1% agarose gel wis shown. M and asterisk (*) indicate the DNA size marker (GeneRuler 1kb Plus DNA ladder; Thermo Scientific, MA, USA) and a non-specific PCR product, respectively. (b) Detection of Mcr-1 protein. Mcr-1 protein from KS8001 was detected by Western blotting with an Anti-His tag Antibody (Sino Biological, Wayne, PA, USA). Image acquisition and quantitative analysis were performed by using ChemiDoc^TM MP Imaging System (Bio-Rad, Hercules, CA, USA) and Image Lab^TM Software (ver 5.2.1; Bio-Rad, Hercules, CA, USA).

Figure 2

Generation of resistance phenotypes by AZT or CPX. (a) Schematic representation of the resistance generation method. Bactericidal activity of (b) AZT or (c) CPX against E. coli ATCC 25922. Representative data from n = 3 experiments, as described in Section 3.4 are shown. Relative increase of resistance of ATCC 25922 to (d) AZT or (e) CPX. The relative increase of resistance as a fold change with respect to non-drug-treated ATCC 25922 or Keio-tdk cells (set to 1 for each set) was calculated based on MIC changes from (b,c). The values shown in the graph at the indicated days are averaged values from n = 3 experiments, with standard deviations (p < 0.05).

Figure 3

Bactericidal activity of CPX in AZT resistant strains. (a) PCR amplification of the tdk-encoding gene. Sequence information for ATCC 25922 was obtained from the NCBI database (accession No. CP009072.1). Sequence alignment of (b) the tdk gene. DNA sequencing results of tdk-encoding region from AZT-R and ATCC 25922, read using the primers Keio-tdk-F and -R, were aligned using Clustal Omega (ClustalW2, v2.1, http://www.clustal.org; accessed on 10 January 2022) [50]. (c) Bactericidal activity of CPX. The bactericidal activity of CPX in AZT-resistant E. coli cells (AZT-R or AZT-tdk-R) and control cells (ATCC 25922) was determined at the indicated concentrations. Data shown here are from one representative experiment from triplicate experiments. The LB agar plates were imaged with a digital camera (Samsung NX200, Suwon, Korea).