Development of a Broad-Spectrum Antimicrobial Combination for the Treatment of Staphylococcus aureus and Pseudomonas aeruginosa Corneal Infections

Abstract

Staphylococcus aureus and Pseudomonas aeruginosa are two of the most common causes of bacterial keratitis and corresponding corneal blindness. Accordingly, such infections are predominantly treated with broad-spectrum fluoroquinolones, such as moxifloxacin. Yet, the rising fluoroquinolone resistance has necessitated the development of alternative therapeutic options. Herein, we describe the development of a polymyxin B-trimethoprim (PT) ophthalmic formulation containing the antibiotic rifampin, which exhibits synergistic antimicrobial activity toward a panel of contemporary ocular clinical S. aureus and P. aeruginosa isolates, low spontaneous resistance frequency, and in vitro bactericidal kinetics and antibiofilm activities equaling or exceeding the antimicrobial properties of moxifloxacin. The PT plus rifampin combination also demonstrated increased efficacy in comparison to those of either commercial PT or moxifloxacin in a murine keratitis model of infection, resulting in bacterial clearance of 70% in the animals treated. These results suggest that the combination of PT and rifampin may represent a novel antimicrobial agent in the treatment of bacterial keratitis.

Keywords: Pseudomonas aeruginosa; Staphylococcus aureus; keratitis; polymyxin; rifampin; trimethoprim.

FIG 1

Antibiofilm activity of polymyxin B-trimethoprim with/without rifampin and polymyxin B-trimethoprim with/without tobramycin. Biofilms established for 48 to 72 h were treated with increasing concentrations of indicated drug concentration as derived by fold-increase over MIC planktonic cells. (A) Activity of rifampin with/without PT against S. aureus UAMS-1. (B) Activity of rifampin with/without PT against P. aeruginosa PAO1. (C) Activity of tobramycin with/without PT against S. aureus UAMS-1. (D) Activity of tobramycin with/without PT against P. aeruginosa PAO1. Performance of moxifloxacin and standard deviations shown (n = 3).

FIG 2

Bactericidal kinetics of polymyxin B-trimethoprim with/without rifampin. Bacterial cultures were treated with 2× MIC of the indicated drug and resulting CFU were plotted over time. (A) Activity of rifampin with/without PT against S. aureus UAMS-1. (B) Activity of rifampin with/without PT against P. aeruginosa PAO1. Performance of moxifloxacin and standard deviations shown (n = 3).

FIG 3

Antimicrobial resistance. Shown are optical signal measures of P. aeruginosa PAO1 cells (A) or S. aureus UAMS-1 cells (B) in medium supplemented with 1× MIC of polymyxin B-trimethoprim with/without rifampin or moxifloxacin. Plots are representative data from one of six independent assays.

FIG 4

Antimicrobial efficacy of polymyxin B-trimethoprim plus rifampin in a murine model of S. aureus keratitis. (A) Representative grade 0 (i), grade 1 (ii), grade 2 (iii), and grade 3 (iv) S. aureus corneal infection. (B) Disease severity scores after 72-h treatments with BSS (negative control), moxifloxacin, PT (alone), rifampin (alone), and PT plus rifampin. (C) CFU recovered from inoculated animals after 3-day treatment with either BSS, moxifloxacin, polymyxin B-trimethoprim (PT), rifampin, or polymyxin B-trimethoprim plus rifampin. Each symbol represents an individual animal. The lower limit of detection is indicated with the horizontal dashed line, and each group average is indicated by a horizontal line. ***, P < 0.05; **, P < 0.1; *, P < 0.15 by Student's t test.