Repurposing salicylanilide anthelmintic drugs to combat drug resistant Staphylococcus aureus

Abstract

Staphylococcus aureus is a Gram-positive bacterium that has become the leading cause of hospital acquired infections in the US. Repurposing Food and Drug Administration (FDA) approved drugs for antimicrobial therapy involves lower risks and costs compared to de novo development of novel antimicrobial agents. In this study, we examined the antimicrobial properties of two commercially available anthelmintic drugs. The FDA approved drug niclosamide and the veterinary drug oxyclozanide displayed strong in vivo and in vitro activity against methicillin resistant S. aureus (minimum inhibitory concentration (MIC): 0.125 and 0.5 μg/ml respectively; minimum effective concentration: ≤ 0.78 μg/ml for both drugs). The two drugs were also effective against another Gram-positive bacteria Enterococcus faecium (MIC 0.25 and 2 μg/ml respectively), but not against the Gram-negative species Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter aerogenes. The in vitro antimicrobial activity of niclosamide and oxyclozanide were determined against methicillin, vancomycin, linezolid or daptomycin resistant S. aureus clinical isolates, with MICs at 0.0625-0.5 and 0.125-2 μg/ml for niclosamide and oxyclozanide respectively. A time-kill study demonstrated that niclosamide is bacteriostatic, whereas oxyclozanide is bactericidal. Interestingly, oxyclozanide permeabilized the bacterial membrane but neither of the anthelmintic drugs exhibited demonstrable toxicity to sheep erythrocytes. Oxyclozanide was non-toxic to HepG2 human liver carcinoma cells within the range of its in vitro MICs but niclosamide displayed toxicity even at low concentrations. These data show that the salicylanilide anthelmintic drugs niclosamide and oxyclozanide are suitable candidates for mechanism of action studies and further clinical evaluation for treatment of staphylococcal infections.

Fig 1. Chemical structure of the salicylanilide anthelmintic drugs niclosamide and oxyclozanide.

Fig 2. Salicylanilide anthelmintic drugs prolong survival of C. elegans infected with MRSA.

A 384 well assay plate was co-inoculated with nematodes, bacteria and either DMSO (negative control), vancomycin (positive control) or the salicylanilide anthelmintic drugs. Serial dilutions of the drugs were tested to adjust the concentration between 0.78–50 μg/ml. (A) Sytox Orange stained and bright field images of assay wells containing a gradient of the tested drugs. Dead worms take up the vital dye Sytox Orange and fluoresce. (B) Percent survival of infected worms was calculated from intensity of fluorescence measured in each well of the assay plate.

Fig 3. Antimicrobial activity of niclosamide and oxyclozanide against the ESKAPE pathogens.

Disc diffusion assay to detect zone of growth inhibition due to bactericidal activity of niclosamide and oxyclozanide against E. faecium, S. aureus methicillin resistant strain (MW2), K. pneumoniae, A. baumannii, P. aeruginosa and E. aerogenes.

Fig 4. Comparative time-kill kinetics of niclosamide and oxyclozanide against MRSA.

The survival of MW2 in a broth culture treated with DMSO, niclosamide or oxyclozanide at a concentration of 4 times the MIC. (MIC values: niclosamide 0.125 μg/ml, oxyclozanide 0.5 μg/ml).

Fig 5. Permeabilization of MRSA cells by niclosamide and oxyclozanide.

Time course of membrane permeabilization by oxyclozanide (A) and niclosamide (B) was measured by following the increase in Sytox Green fluorescence (λexc = 485 nm; λem = 530 nm). The drugs were tested at serially diluted concentrations between 1 to 64 μg/ml.

Fig 6. Hemolytic activity of niclosamide and oxyclozanide.

Sheep erythrocytes were treated with serial dilutions of Triton X-100 (.001–1%), oxyclozanide and niclosamide (0.063–64 μg/ml)

Fig 7. Cytotoxicity of niclosamide and oxyclozanide to HepG2.

The viability of HepG2 cells was measured after treatment with serially diluted concentrations (1–64 μg/ml) of niclosamide, oxyclozanide or the mitochondrial toxin rotenone (positive control). Cell viability was measured spectrophotometrically by detecting degradation of WST-1 dye into formazan by viable cells, which produces an intense color.