Murepavadin Enhances the Killing Efficacy of Ciprofloxacin against Pseudomonas aeruginosa by Inhibiting Drug Efflux

Abstract

Pseudomonas aeruginosa is a multidrug-resistant Gram-negative pathogen and one of the leading causes of ventilator-associated pneumonia and infections in patients with chronic obstructive pulmonary disease and cystic fibrosis. Murepavadin is a peptidomimetic that specifically targets outer-membrane lipopolysaccharide transport protein LptD of P. aeruginosa. In this study, we find that murepavadin enhances the bactericidal efficacy of ciprofloxacin. We further demonstrate that murepavadin increases intracellular accumulation of ciprofloxacin by suppressing drug efflux. In addition, the murepavadin-ciprofloxacin combination exhibits a synergistic bactericidal effect in an acute murine pneumonia model. In conclusion, our results identify an effective drug combination for the treatment of P. aeruginosa infections.

Keywords: Pseudomonas aeruginosa; ciprofloxacin; murepavadin.

Figure 1

The time–killing curves of murepavadin (0.5 μg/mL) and ciprofloxacin (0.25 μg/mL) alone and in combination against PA14. *, p < 0.05; **, p < 0.01 compared with the bacteria treated with the single antibiotic at the corresponding time points by Student’s t-test. The data are the means ± SD of three independent samples. Mur, murepavadin; Cip, ciprofloxacin.

Figure 2

Murepavadin increases the intracellular accumulation of ciprofloxacin. (a) Wild type PA14 was treated with 10 µg/mL ciprofloxacin with or without 0.25 µg/mL murepavadin in Mueller–Hinton Broth (MHB) for 15 min, followed by measurement of fluorescence of intracellular ciprofloxacin. The relative fluorescence was normalized by the OD₆₀₀. (b) PA14 cells were treated with or without murepavadin (0.03125 μg/mL) and ciprofloxacin (0.025 μg/mL), individually or in combination and grown to OD₆₀₀ = 1 at 37 °C. The mRNA levels of PA0614, PA0629, and recA were determined by qRT-PCR. The data are the means ± SD of three independent samples. *, p < 0.05; **, p < 0.01 by Student’s t-test. Mur, murepavadin; Cip, ciprofloxacin.

Figure 3

Effects of CCCP on intracellular accumulation of ciprofloxacin and ethidium bromide. (a) Wild type PA14 was treated with 10 µg/mL ciprofloxacin with or without 0.25 µg/mL murepavadin and 40 µg/mL CCCP in MHB for 15 min, followed by measurement of the fluorescence of intracellular ciprofloxacin. (b) Wild type PA14 was incubated with or without 0.25 µg/mL murepavadin, 40 µg/mL CCCP and 2 μg/mL ethidium bromide in PBS for 30 min at 37 °C. The relative fluorescence was normalized by the OD₆₀₀. ns, not significant; *, p < 0.05; ***, p < 0.001 by Student’s t-test. Mur, murepavadin.

Figure 4

Bactericidal effects of murepavadin in combination with ciprofloxacin against clinical strains. (a–l) The bacteria were treated with murepavadin (0.5 μg/mL) and ciprofloxacin (0.25 μg/mL) individually or in combination at 37 °C with shaking. At the indicated time points, the live bacteria numbers were determined by plating. The strain numbers were shown in each of the figures. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with the bacteria treated with the single antibiotic at the corresponding time points by Student’s t-test. The data are the means ± SD of three independent samples. Mur, murepavadin; Cip, ciprofloxacin.

Figure 5

The murepavadin–ciprofloxacin combination displayed synergistic effect in vivo. Mice were inoculated intranasally with 4 × 10⁶ CFU of wild type PA14. Three hours later, NaCl, murepavadin, ciprofloxacin, or the murepavadin–ciprofloxacin combination were administered intranasally. Sixteen hours post-infection, the bacterial loads in the lungs were determined. ***, p < 0.001 by Student’s t-test. Mur, murepavadin; Cip, ciprofloxacin.