Synergistic activities of an efflux pump inhibitor and iron chelators against Pseudomonas aeruginosa growth and biofilm formation

Abstract

The efflux pump inhibitor phenyl-arginine-beta-naphthylamide (PAbetaN) was paired with iron chelators 2,2'-dipyridyl, acetohydroxamic acid, and EDTA to assess synergistic activities against Pseudomonas aeruginosa growth and biofilm formation. All of the tested iron chelators synergistically inhibited P. aeruginosa growth and biofilm formation with PAbetaN. PAbetaN-EDTA showed the most promising activity against P. aeruginosa growth and biofilm formation.

FIG. 1.

Growth of P. aeruginosa wild-type PAO1, mexAB-oprM mutant, and pvdA mutant in the presence of different PAβN-Dipy combinations. The optical densities of the cultures at 600 nm (OD 600) were recorded after 0, 2, 4, 6, 8, 10, 12, and 24 h of incubation at 37°C. Each value represents the means and standard deviations from three wells of the microtiter dishes.

FIG. 2.

Growth of P. aeruginosa wild-type PAO1 and pvdA mutant in the presence of different PAβN-acetohydroxamic acid combinations (A) and PAβN-EDTA combinations (B). The optical densities of the cultures were recorded after 0, 2, 4, 6, 8, 10, 12, and 24 h of incubation at 37°C. Each value represents the means and standard deviations from three wells of the microtiter dishes.

FIG. 3.

Biofilm formation at the air-liquid interface of glass slides immersed in culture medium containing medium alone (A), 20 μg/ml Dipy (B), 80 μg/ml acetohydroxamic acid (C), 5 μg/ml EDTA (D), 50 μg/ml PAβN (E), 50 μg/ml PAβN and 20 μg/ml Dipy (F), 50 μg/ml PAβN and 80 μg/ml acetohydroxamic acid (G), and 50 μg/ml PAβN and 5 μg/ml EDTA (H) was observed by confocal laser-scanning microscopy and analyzed by IMARIS (Bitplane AG).

FIG. 4.

Quantification of biofilms by COMSTAT. The results are means of datasets obtained from the analysis of three CLSM images acquired at random positions in each of the biofilms. Standard deviations are shown in parentheses.