Niclosamide as a Repurposing Drug against Corynebacterium striatum Multidrug-Resistant Infections

Abstract

Corynebacterium striatum (C. striatum) is an emerging multidrug-resistant (MDR) pathogen associated with nosocomial infections. In this scenario, we screened the antimicrobial activity of the anthelmintic drugs doramectin, moxidectin, selamectin and niclosamide against 20 C. striatum MDR clinical isolates. Among these, niclosamide was the best performing drug against C. striatum. Niclosamide cytotoxicity was evaluated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay on immortalized human keratinocyte cells (HaCaT). After 20 h of treatment, the recorded 50% cytotoxic concentration (CC₅₀) was 2.56 μg/mL. The antibacterial efficacy was determined via disc diffusion, broth microdilution method and time-killing. Against C. striatum, niclosamide induced a growth inhibitory area of 22 mm and the minimum inhibitory concentration that inhibits 90% of bacteria (MIC₉₀) was 0.39 μg/mL, exhibiting bactericidal action. The biofilm biomass eradicating action was investigated through crystal violet (CV), MTT and confocal laser scanning microscopy (CLSM). Niclosamide affected the biofilm viability in a dose-dependent manner and degraded biomass by 55 and 49% at 0.39 μg/mL and 0.19 μg/mL. CLSM images confirmed the biofilm biomass degradation, showing a drastic reduction in cell viability. This study could promote the drug-repurposing of the anthelmintic FDA-approved niclosamide as a therapeutic agent to counteract the C. striatum MDR infections.

Keywords: Corynebacterium striatum; drug-repurposing; multidrug-resistant pathogen.

Figure 1

Antibacterial screening study of niclosamide and conventional antibiotics: (A) inhibition halo recorded for C. striatum (S₁) after treatment with vancomycin (VA, 5 µg), linezolid (LDZ, 10 µg), ciprofloxacin (CIP, 5 µg), rifampicin (RP, 5 µg), benzylpenicillin (PG, 1 µg) and or clindamycin (CD, 2 µg); (B) diameter of the inhibition area exhibited by C. striatum (S₁) after exposure with niclosamide (N, 5 µg).

Figure 2

Antibacterial potential of anthelmintic drugs: (A) heatmap representation of bacterial inhibition after treatment of C. striatum S₁ with doramectin, moxidectin, selamectin and niclosamide for 20 h; (B) time-kill kinetics of niclosamide against C. striatum cells. Negative control (CTRL-): untreated cells; CTRL+: bacteria treated with vancomycin (5 μg/mL); The data represent the mean ± standard deviation (SD).

Figure 3

SEM analyses of C. striatum cells treated with niclosamide at 1 × MIC (A–C) and ½ × MIC (D–F). The negative CTRL was represented by bacteria treated with the solvent (G–I) and the positive CTRL was represented by the treatment with vancomycin (5 μg/mL) (J–L). The treatment was carried out for 20 h at 37 °C. The images were obtained (from left to right) at 10,000×, 20,000×, and 40,000× magnification.

Figure 4

The effect of niclosamide on the preformed biofilm of C. striatum: (A,B) degradation of the biofilm matrix quantified by CV-staining; (C) Evaluation of the cell viability in the degraded biofilm; CTRL-: untreated biofilm; CTRL+: bacteria treated with vancomycin (20 μg/mL); CTRL DMSO: biofilm treated with the solvent used to dissolve the drug; The data represent the mean ± SD. Dunnett’s multiple comparisons test: **** p < 0.0001; *** p < 0.0005; ** p < 0.0096; ns p > 0.05. Ordinary one-way ANOVA: p < 0.0001, R² = 0.9935; (D) confocal images of C. striatum biofilm treated with solvent (left) and niclosamide (right); red indicates dead cells, green points to live cells while yellow/orange is the result of the overlapping of dead and living cells; the images were acquisitions from three “randomly selected” areas.

Figure 5

HaCaT cell viability after 24 h of treatment with niclosamide. CTRL-: untreated cells; CTRL+: dimethyl sulfoxide (DMSO) used at toxic concentration; CTRL DMSO: solvent used to dissolve the drug. The data represent the mean ± SD. Dunnett’s multiple comparisons test: **** p < 0.0001; ** p < 0.0096; ns p > 0.05. Ordinary one-way ANOVA: p < 0.0001, R² = 0.9982.

Figure 6

Intracellular survival of C. striatum in the HaCaT cell line after treatment with niclosamide. CTRL-: untreated cells; CTRL DMSO: cells treated with the solvent used to dissolve the drug. The data represent the mean ± SD. Dunnett’s multiple comparisons test: *** p < 0.0002; * p < 0.03. Ordinary one-way ANOVA: p < 0.0001, R² = 0.9679.