Azithromycin inhibits quorum sensing in Pseudomonas aeruginosa

Abstract

We report that 2 microg of azithromycin/ml inhibits the quorum-sensing circuitry of Pseudomonas aeruginosa strain PAO1. Addition of synthetic autoinducers partially restored the expression of the trancriptional activator-encoding genes lasR and rhlR but not that of the autoinducer synthase-encoding gene lasI. We propose that azithromycin interferes with the synthesis of autoinducers, by an unknown mechanism, leading to a reduction of virulence factor production.

FIG. 1

Effect of azithromycin on growth and elastase and rhamnolipid production. (A) Bacterial strains were grown in Luria-Bertani (LB) medium in the absence (squares) or in the presence (circles, 2 μg/ml; upside triangles, 3 μg/ml; downside triangles, 4 μg/ml; diamonds, 5 μg/ml) of azithromycin. Growth curve determinations were repeated five times and the graph shows results from one typical experiment. (B) Supernatants of cells, grown either in the absence (squares) or the presence (circles) of 2 μg of azithromycin/ml, were collected at regular intervals and elastase activity was determined by the elastin Congo red assay. (C) PAO1(pECP60) (rhlA′-lacZ) was grown in LB medium either in the absence (squares) or the presence (circles) of 2 μg of azithromycin/ml, and β-Gal activities were assayed at regular time intervals. Inserted graphs in panels B and C show the corresponding growth curves. Results are the mean ± SD of three independent experiments performed in duplicate.

FIG. 1

Effect of azithromycin on growth and elastase and rhamnolipid production. (A) Bacterial strains were grown in Luria-Bertani (LB) medium in the absence (squares) or in the presence (circles, 2 μg/ml; upside triangles, 3 μg/ml; downside triangles, 4 μg/ml; diamonds, 5 μg/ml) of azithromycin. Growth curve determinations were repeated five times and the graph shows results from one typical experiment. (B) Supernatants of cells, grown either in the absence (squares) or the presence (circles) of 2 μg of azithromycin/ml, were collected at regular intervals and elastase activity was determined by the elastin Congo red assay. (C) PAO1(pECP60) (rhlA′-lacZ) was grown in LB medium either in the absence (squares) or the presence (circles) of 2 μg of azithromycin/ml, and β-Gal activities were assayed at regular time intervals. Inserted graphs in panels B and C show the corresponding growth curves. Results are the mean ± SD of three independent experiments performed in duplicate.

FIG. 2

Azithromycin affects transcription of quorum-sensing genes. The expression of the transcriptional activator and the autoinducer synthase genes was measured by β-Gal determinations in strain PAO1 cultures grown for 10 h in the absence (azithromycin −) or presence (azithromycin +) of 2 μg of azithromycin/ml, using plasmids pPCS1001(lasR′-lacZ) and pPCS1002(rhR′-lacZ) (A) and plasmids pPCS223(lasI′-lacZ) and pLPRI(rhlI′-lacZ) (B). Exogenous autoinducers were added to the cell culture as indicated (autoinducers +). Results are the mean ± SD of three independent experiments performed in duplicate.

FIG. 3

Autoinducers are reduced in the presence of azithromycin and partially restore rhlAB expression and elastase production. (A) Strain PAO1 cultures were grown for 12 h in Luria-Bertanimedium, either in the absence (azithromycin −) or presence (azithromycin +) of 2 μg of azithromycin/ml. The supernatants were extracted with ethyl acetate, and the 3-oxo-C₁₂-HSL and C₄-HSL concentrations were measured using specific bioassays. Results are the mean ± SD of three independent experiments. (B) Cells were grown for 10 h in the absence (azithromycin −) or presence (azithromycin +) of 2 μg of azithromycin/ml and in the absence (autoinducers −) or presence (autoinducers +) of exogenous autoinducers. The expression of the rhlAB gene was determined by measuring the β-Gal activity of the rhlA′-lacZ reporter fusion pECP60. The production of elastase was determined by the elastin Congo red assay performed on culture supernatants. Results are expressed as the percentage of the maximum observed in each individual experiment and represent the mean ± SD of three independent experiments performed in duplicate.