Effect of emergence of fluoroquinolone resistance on intrinsic expression of P-glycoprotein phenotype in corneal epithelial cells

Abstract

Purpose: Multidrug resistance (MDR) represents a major obstacle to the success of antimicrobial fluoroquinolone (FQ) therapy. MDR-associated efflux protein pumps antimicrobial agents out of the corneal cells, leading to suboptimal eradication of microbes. This article examines whether long-term FQ (levofloxacin, ofloxacin, and gatifloxacin) therapy can modify the MDR phenotype (P-glycoprotein [P-gp]) on corneal epithelial cells (Statens Seruminstitut Rabbit Cornea [SIRC]).

Methods: To study the effect of FQ, SIRC cells without any exposure to FQ (control) were compared with the cells exposed to ofloxacin, levofloxacin, and gatifloxacin at a concentration of 10 μg/mL for 3 weeks. Efflux activity of P-gp was assessed by in vitro uptake studies (fluorescent and radioactive), flow cytometry, and quantitative real-time polymerase chain reaction (qRT-PCR).

Results: In the presence of FQ, elevated P-gp expression was noted with uptake, flow cytometry, and qRT-PCR analyses. This study confirms that long-term exposure to antibiotics, particularly FQ, can induce overexpression of P-gp efflux transporter present on the corneal cells. P-gp overexpression is commonly noticed in anticancer drug resistance cell lines; however, for the first time, this report describes overexpression of P-gp due to FQ exposure.

Conclusions: Based on this result, it is suggested that strategies should be developed and implemented not only to overcome resistance to ocular pathogen but also to FQ-induced cellular resistance.

FIG. 1.

Radioactive uptake of [¹⁴C]Erythromycin by control and fluoroquinolone-treated SIRC cells. Each point represents mean SD of 3 determinations. *Statistically significant from control at a P value of <0.05. SIRC, Statens Seruminstitut Rabbit Cornea; SD, standard deviation; S-OFX, ofloxacin-treated SIRC cells; S-GFX, gatifloxacin-treated SIRC cells; S-LFX, levofloxacin-treated SIRC cell.

FIG. 2.

(A) Overall comparison of Rho-123 uptake with and without exposure of fluoroquinolones in SIRC cells. Each point represents mean SD of 4 determinations. *Statistically significant from control at a P value of <0.05. (B) Standard curve of Rho-123. Rho-123, Rhodamine-123; S-LFX, levofloxacin-treated SIRC cells.

FIG. 3.

Functional assay for the P-gp efflux pump on SIRC cells by flow cytometry analysis. P-gp, P-glycoprotein.

FIG. 4.

Relative fold induction of P-gp in SIRC cells treated with 3 fluoroquinolones. Data represent relative fold induction (n=3) of 3 different experiments. *Statistically significant from control at a P value of <0.05.