Increased Susceptibility of Pseudomonas aeruginosa to Macrolides and Ketolides in Eukaryotic Cell Culture Media and Biological Fluids Due to Decreased Expression of oprM and Increased Outer-Membrane Permeability

Abstract

Macrolides show high minimum inhibitory concentrations (MICs) against Pseudomonas aeruginosa when tested in recommended media (cation-adjusted Muller-Hinton broth [CA-MHB]). Nevertheless, azithromycin is successfully used in cystic fibrosis patients, supposedly because of "nonantibiotic effects." CA-MHB and Roswell Park Memorial Institute (RPMI) 1640 medium (used for growing eukaryotic cells) were compared for measuring azithromycin MICs (with or without Phe-Arg-β-naphthylamide [PAβN], an efflux inhibitor), [(14)C]-clarithromycin accumulation, azithromycin-induced protein synthesis inhibition, oprM (encoding the outer-membrane protein coupled with MexAB and MexXY efflux systems) expression, outer-membrane permeability (tested with 1-N-phenylnaphthylamine and nitrocefin), and synergy (determined by checkerboard assay) between azithromycin and outer-membrane disrupting agents. Key experiments were repeated with CA-MHB supplemented with serum, mouse bronchoalveolar lavage fluid, other macrolides, and other gram-negative bacteria. Azithromycin MICs were ≥128 mg/L in CA-MHB, compared with 1-16 mg/L in RPMI 1640 medium, CA-MHB supplemented with serum, or bronchoalveolar lavage fluid (repeated for RPMI 1640 medium with clarithromycin, other macrolides, and other gram-negative bacteria). [(14)C]-clarithromycin accumulation was 2.2-fold higher in RPMI 1640 medium, compared with CA-MHB. Inhibition of >95% of protein synthesis was obtained with azithromycin at 16 mg/L in RPMI 1640 medium, compared with >512 mg/L in CA-MHB. Strains not expressing oprM showed an MIC of 4 mg/L in CA-MHB. PAβN decreased MICs in CA-MHB but not in RPMI 1640 medium. Real-time polymerase chain reaction showed downregulation of oprM by azithromycin in RPMI 1640 medium. Outer-membrane permeability was 3-4.5 times higher in RPMI 1640 medium or bronchoalveolar lavage fluid, compared with CA-MHB. Azithromycin combined with outer-membrane disrupting agents were synergistic in CA-MHB but indifferent in RPMI 1640 medium. Macrolides show antimicrobial activity against P. aeruginosa in eukaryotic media through increased uptake and reduced efflux. These data may help explain the clinical efficacy of macrolides against pseudomonal infections.