1. Clin Infect Dis. 2012 Aug;55(4):534-42. Epub 2012 May 9.

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.

Buyck JM, Plésiat P, Traore H, Vanderbist F, Tulkens PM, Van Bambeke F.

Pharmacologie cellulaire et moléculaire, Louvain Drug Research Institute,
Université catholique de Louvain.

Background. 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."
Methods. 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. Results. 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. Conclusions. 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.

PMID: 22573850  [PubMed - in process]