Potential synergistic activity of quercetin with antibiotics against multidrug-resistant clinical strains of Pseudomonas aeruginosa

Abstract

Development of drug resistance in opportunistic pathogens is one of the major healthcare challenges associated with infection management. Combination therapy has many advantages due to the simultaneous action of two drugs on two separate cellular targets. However, selection of the drugs should offer safety and synergistic interaction against most of the strains. Here, the efficacy of antibiotics in combination with quercetin, a natural flavonoid capable of targeting quorum sensing was tested against biofilm-forming Pseudomonas aeruginosa strains previously isolated from catheter associated urinary tract infection. Based on the antibiotic susceptibility pattern, synergistic effect of quercetin with selected antibiotics (levofloxacin, ceftriaxone, gentamycin, tobramycin and amikacin) was tested at the fractional concentrations of MIC by the checkerboard method and the fractional inhibitory concentration index (FICi) was calculated to estimate the synergistic effect. Effect of the synergistic combinations were further tested using time-kill assay, and against biofilm formation and biofilm cell viability. Cytotoxicity assays were performed using Human Embryonic Kidney 293T cells (HEK-293T) using the effective drug combinations with respective controls. The biofilm formation and biofilm cell viability were drastically affected with quercetin and selected antibiotics combinations with ≥80% inhibition. In vitro infection studies showed that all the strains could exert significant cell killing (68 to 85%) and the drug combinations decreased the infection rate significantly by reducing the cell killing effect of P. aeruginosa (p<0.05). The synergistic effect of quercetin is attributed to its quorum sensing inhibitory properties. These findings indicate that quercetin along with existing antibiotics can potentiate the treatment against P. aeruginosa infection and may reduce the selection pressure due to antibiotic overuse.

Fig 1

(a) Growth pattern and (b) biofilm formation of P. aeruginosa strains used in study.

Fig 2. Checkerboard showing effect of antibiotic-quercetin combinations against P. aeruginosa strains.

Pink color represents growth inhibition (additive/MIC), cream color indicates the synergistic effect with growth inhibition. Green shows no reduction in the growth (no effect) based on the visible turbidity. The P. aeruginosa strains were treated with fractional concentration of antibiotic with quercetin in 96-well MHB for 24 h. The growth based on visible turbidity was used for interpreting the results.

Fig 3. Combenefit mapped surface HSA plot.

The data based on the OD₆₀₀ values obtained for antibiotic-quercetin combination from the 24 h incubation was used to generate this heat map. Light blue to dark blue shows increased reduction (synergy) while yellow-red shows no reduction (non-synergy).

Fig 4. Time kill assay data showing the growth pattern of P. aeruginosa strains treated with antibiotic-quercetin combinations at lowest FICi for 24 h.

(a-e) represents five different strains. (f) shows the CFU/mL data obtained from the cultures of 24 h incubation by serial dilution plate method.

Fig 5. Effect of antibiotic-quercetin combinations at lowest FICi on biofilm.

Percentage effective combination of antibiotics with quercetin showing reduction in the biofilm. (a) Percentage of inhibition of biofilm formation (b) Biofilm cell viability of the treated pre-formed biofilms. (a) The bacteria were grown in 96-well plate in presence of antibiotic-quercetin combination and biofilm intensity was assessed by crystal violet staining. (b) Biofilm viability was tested after treating the pre-formed biofilms in 96-well plate with antibiotic-quercetin combinations.

Fig 6. Biofilm viability as visualized by live/dead staining for P. aeruginosa (YU-V10, YU-V28, YU-V11, YU-V15 PAO1) using CLSM.

(a) PAO1, YU-V10, YU-V28 and (b) YU-V11 and YU-V15. The biofilms were treated with quercetin and antibiotics at the lowest FICi combination and stained with Acridine Orange-Propidium Iodide staining. Green color indicates the live cells and yellow to red color indicates dead cells.

Fig 7. Cell killing effect of P. aeruginosa strains on HEK 293T cells.

The antibiotic-quercetin combinations at the lowest FIC_i showing attenuation of infection induced cell killing. Data are presented as mean ± SD (n = 3). Statistical differences are presented *p<0.05, **p<0.01 and ***p<0.001.