Antifungal Azoles as Tetracycline Resistance Modifiers in Staphylococcus aureus

Abstract

Staphylococcus aureus has developed resistance to antimicrobials since their first use. The S. aureus major facilitator superfamily (MFS) efflux pump Tet(K) contributes to resistance to tetracyclines. The efflux pump diminishes antibiotic accumulation, and biofilm hampers the diffusion of antibiotics. None of the currently known compounds have been approved as efflux pump inhibitors (EPIs) for clinical use. In the current study, we screened clinically approved drugs for possible Tet(K) efflux pump inhibition. By performing in silico docking followed by in vitro checkerboard assays, we identified five azoles (the fungal ergosterol synthesis inhibitors) showing putative EPI-like potential with a fractional inhibitory concentration index of ≤0.5, indicating synergism. The functionality of the azoles was confirmed using ethidium bromide (EtBr) accumulation and efflux inhibition assays. In time-kill kinetics, the combination treatment with butoconazole engendered a marked increase in the bactericidal capacity of tetracycline. When assessing the off-target effects of the azoles, we observed no disruption of bacterial membrane permeability and polarization. Finally, the combination of azoles with tetracycline led to a significant eradication of preformed mature biofilms. This study demonstrates that azoles can be repurposed as putative Tet(K) EPIs and to reduce biofilm formation at clinically relevant concentrations. IMPORTANCE Staphylococcus aureus uses efflux pumps to transport antibiotics out of the cell and thus increases the dosage at which it endures antibiotics. Also, efflux pumps play a role in biofilm formation by the excretion of extracellular matrix molecules. One way to combat these pathogens may be to reduce the activity of efflux pumps and thereby increase pathogen sensitivity to existing antibiotics. We describe the in silico-based screen of clinically approved drugs that identified antifungal azoles inhibiting Tet(K), a pump that belongs to the major facilitator superfamily, and showed that these compounds bind to and block the activity of the Tet(K) pump. Azoles enhanced the susceptibility of tetracycline against S. aureus and its methicillin-resistant strains. The combination of azoles with tetracycline led to a significant reduction in preformed biofilms. Repurposing approved drugs may help solve the classical toxicity issues related to efflux pump inhibitors.

Keywords: Staphylococcus aureus; Tet(K) efflux protein; antifungal azoles; efflux pump inhibitors; repurposing.

FIG 1

Interaction of butoconazole (A), econazole (B), miconazole (C), tioconazole (D), and clotrimazole with the active site of Tet(K) (E). Cartoon (ball and stick) representation of Tet(K) showing azoles docked in the active site cleft (left). 3D electrostatic potential map (middle). Ligand interaction diagram in 2D representation showing several interactions involved in Tet(K)-azole binding (right).

FIG 2

(A) Growth kinetics of Staphylococcus aureus XU212 in CA-MHB under subinhibitory concentrations (1/4× MIC) of the azoles. OD₆₀₀ values are the means of three independent experiments. The control (untreated) set was included to monitor normal bacterial growth. (B) Tetracycline-azole synergy test using agar-well diffusion assay on S. aureus XU212. The agar wells containing azoles individually did not produce any zone of inhibition; however, tetracycline alone displayed a zone of inhibition of only 9 mm. The combination of tetracycline with all the azoles was bactericidal, as indicated by a significant increment in the zone of growth inhibition. The images are representatives of two independent experiments. Abbreviations: TET, Tetracycline; CLO, clotrimazole; TIO, tioconazole; MIC, miconazole; ECO, econazole; BUT, butoconazole. Shown are effects of subinhibitory concentrations (1/4× MIC) of clotrimazole (C), tioconazole (D), miconazole (E), econazole (F), and butoconazole (G) on the real-time accumulation of EtBr in a concentration-dependent manner; the positive-control reserpine (H) was included for comparison. The results presented correspond to the average of three independent assays ± SD.

FIG 3

Effect of subinhibitory concentrations (1/4× MIC) of clotrimazole (A), tioconazole (B), miconazole (C), econazole (D), and butoconazole (E) on the efflux inhibition of EtBr in the presence and absence of glucose; the positive-control reserpine (F) was included for comparison. The results presented correspond to the average of three independent assays ± SD.

FIG 4

(A to F) Efflux inhibition of tetracycline in S. aureus XU212, when the cells were treated with clotrimazole, tioconazole, miconazole, econazole, butoconazole, and reserpine at subinhibitory concentrations (1/4× MIC). The change in fluorescence was monitored in the presence and absence of glucose (4%). The tetracycline fluorescence was recorded at 535 nm by exciting at a wavelength of 405 nm. The results presented correspond to the average of two independent experiments with three repeats ± SD.

FIG 5

(A) Time-kill curves of S. aureus XU212 showing the bactericidal effect of tetracycline (256 μg/ml) in combination with butoconazole (12.5 μM). Each time point represents the mean log₁₀ CFU/ml ± SD of three readings. (B) Shows the postantibiotic effect induced by tetracycline (1× MIC) alone and in combination with butoconazole (1/4× MIC) for 2 h in S. aureus XU212. Time 0 h corresponds to the beginning of growth monitoring immediately after compound removal and cell resuspension in fresh CA-MHB. The value of PAE corresponds to the delay undergone by the treated culture with respect to the untreated control in reaching an OD value of one-half of the final OD. The assays were repeated three times independently, and results were presented as mean ± SD. (C) Bacterial membrane permeability is measured with fluorescent PI dye upon exposure to the azoles at 1× MIC. Increased fluorescence corresponds to cells with the permeabilized membranes. Paenibacillin (10 μM) was used as a positive control since it destabilizes the Gram-positive bacterial membrane. The drug-free control was included to monitor change in fluorescence. (D) The azoles do not depolarize the bacterial membrane. Shown is the change in fluorescence of S. aureus XU212 cells using a DiSC3(5) assay. Data are presented as a change in fluorescence before and after the addition of the azoles and valinomycin at 1× MIC. (E) S. aureus XU212 was exposed to the azoles at 1/4× MIC during 4 h. The ATP levels were quantified using a luciferin-luciferase bioluminescence detection assay. CCCP and valinomycin were included for comparison. The results presented correspond to the mean of three independent assays ± SD. Results were considered highly significant when the P value was <0.001 (***).

FIG 6

(A) Influence on the biofilm eradication ability of tetracycline (128 μg/ml) alone or in combination with the azoles at subinhibitory concentrations (1/4× MIC); the crystal violet staining assessed the biomass of S. aureus XU212 after exposure to tetracycline and the tetracycline-azole combination for 24 h. The percent values represent the amount of biofilm formation with respect to drug-free control. The experiments were carried out in three biological repeats, and results correspond to average ± SD. Results were considered significant when the P value was <0.05 (*) and highly significant when the P value was <0.001 (***). (B and C) Quantification of the effects of the azoles at subinhibitory concentrations (1/4× MIC) in combination with tetracycline at 1/2× MIC and synergistic MICs on mature biofilm; the viable bacterial cells were determined using the MTT assay after exposure for 24 h. The percent values represent the number of live cells with respect to the drug-free control. The experiments were carried out in three biological repeats, and results correspond to average ± SD. Results were considered significant when the P value was <0.01 (**) and highly significant when the P value was <0.001 (***). (D) Effect of tetracycline (128 μg/ml) alone or in combination with butoconazole (12.5 μM) on biofilm eradication assessed by confocal laser scanning microscopy (40×); static biofilms after exposure for 24 h were stained with SYTO9. S. aureus XU212 within biofilm on glass carriers display green fluorescence. The images are representatives of two independent experiments.