Effects of antibiotics on quorum sensing in Pseudomonas aeruginosa

Abstract

During infection, Pseudomonas aeruginosa employs bacterial communication (quorum sensing [QS]) to coordinate the expression of tissue-damaging factors. QS-controlled gene expression plays a pivotal role in the virulence of P. aeruginosa, and QS-deficient mutants cause less severe infections in animal infection models. Treatment of cystic fibrosis (CF) patients chronically infected with P. aeruginosa with the macrolide antibiotic azithromycin (AZM) has been demonstrated to improve the clinical outcome. Several studies indicate that AZM may accomplish its beneficial action in CF patients by impeding QS, thereby reducing the pathogenicity of P. aeruginosa. This led us to investigate whether QS inhibition is a common feature of antibiotics. We present the results of a screening of 12 antibiotics for their QS-inhibitory activities using a previously described QS inhibitor selector 1 strain. Three of the antibiotics tested, AZM, ceftazidime (CFT), and ciprofloxacin (CPR), were very active in the assay and were further examined for their effects on QS-regulated virulence factor production in P. aeruginosa. The effects of the three antibiotics administered at subinhibitory concentrations were investigated by use of DNA microarrays. Consistent results from the virulence factor assays, reverse transcription-PCR, and the DNA microarrays support the finding that AZM, CFT, and CPR decrease the expression of a range of QS-regulated virulence factors. The data suggest that the underlying mechanism may be mediated by changes in membrane permeability, thereby influencing the flux of N-3-oxo-dodecanoyl-L-homoserine lactone.

FIG. 1.

Screening of antibiotics by the QSIS1 assay. (a) Piperacillin; (b) azithromycin; (c) chloramphenicol; (d) ciprofloxacin; (e) gentamicin; (f) spectinamycin; (g) griseofulvin; (h) kanamycin; (i) tetracycline; (j) tobramycin; (k) ceftazidime; (l) streptomycin.

FIG. 2.

Growth curves for the ΔlasI-ΔrhlI mutant (□) and strain PAO1 grown without antibiotics (×) or PAO1 grown in the presence of 2 μg/ml AZM (Δ), 8 μg/ml AZM (), 16 μg/ml AZM (▴), 0.04 μg/ml CPR (○), 0.08 μg/ml CPR (•), 0.25 μg/ml CFT (⋄), or 0.5 μg/ml CFT (⧫).

FIG. 3.

Hemolysis of autoclaved supernatants from P. aeruginosa PAO1 cultures grown for 20 h. PAO1 was grown without antibiotics (a) and in the presence of 2 μg/ml AZM (b), 8 μg/ml AZM (c), 0.25 μg/ml CFT (d), or 0.04 μg/ml CPR (e). The results for the ΔlasI-ΔrhlI mutant of PAO1 are also shown (f).

FIG. 4.

Rhamnolipid contents, in relative units (RU), in supernatants from P. aeruginosa cultures grown either without antibiotics (black bar) or in the presence of 2 μg/ml AZM (bar with horizontal lines), 8 μg/ml AZM (bar with vertical lines), 0.25 μg/ml CFT (dark gray bar), or 0.04 μg/ml CPR (light gray bar). The ΔlasI-ΔrhlI mutant of PAO1 was also included in the experiment but did not produce a measurable concentration of rhamnolipid (last slot [no column visible]). The concentration of rhamnolipid in the untreated culture was set equal to an index value of 100. The graph is based on the average of the indexes of three independent experiments. *, P < 0.05; **, P < 0.01 (Dunnett's post test).

FIG. 5.

Protease activities of supernatants from P. aeruginosa cultures grown either without antibiotics (black bar) or in the presence of 2 μg/ml AZM (bar with horizontal lines), 8 μg/ml AZM (bar with vertical lines), 0.25 μg/ml CFT (dark gray bar), or 0.04 μg/ml CPR (light gray bar). The ΔlasI-ΔrhlI mutant of PAO1 was also included in the experiment but did not produce a measurable concentration of protease (last slot [no column visible]). The graph is based on the average results of three independent experiments. *, P < 0.05; **, P < 0.01 (Dunnett's test).

FIG. 6.

Elastolytic activities of supernatants from PAO1 cultures grown to an OD₆₀₀ of 2.0 either without antibiotics (black bar) or in the presence of: 2 μg/ml AZM (bar with horizontal lines), 8 μg/ml AZM (bar with vertical lines), 0.25 μg/ml CFT (dark gray bar), or 0.04 μg/ml CPR (light gray bar). The elastolytic activity of the ΔlasI-ΔrhlI mutant of PAO1 is also shown (white bar). The graph is based on the average results of three independent experiments. *, P < 0.05; **, P < 0.01 (Dunnett's post test).

FIG. 7.

Chitinase activities of supernatants from PAO1 cultures grown to an OD₆₀₀ of 2.0 either without antibiotics (black bar) or in the presence of: 2 μg/ml AZM (bar with horizontal lines), 8 μg/ml AZM (bar with vertical lines), 0.25 μg/ml CFT (dark gray bar), or 0.04 μg/ml CPR (light gray bar). The chitinase activity of the ΔlasI-ΔrhlI mutant of PAO1 is also shown (white bar). The graph is based on the average results of three independent experiments. *, P < 0.05; **, P < 0.01 (Dunnett's post test).

FIG. 8.

LasR, RhlR, and LasR-RhlR specificities of AZM (white bars), CFT (black bars), CPR (light gray bars), and C30 (dark gray bars). See the text for the definitions of groups (Gr.) A, B, C, and D.

FIG. 9.

Inhibitors of and signal molecules for P. aeruginosa QS. (a) 4-Nitropyridine-N-oxide (73); (b) C30 (38); (c) 2-heptylthioacetyl homoserine lactone (69a); (d) 3-oxo-C₁₂-2-aminophenol (83a); (e) BHL; (f) patulin (74); (g) OdDHL.

FIG. 10.

Structures of the three antibiotics investigated for potential quorum sensing inhibitory activities. (A) AZM; (B) CFT; (C) CPR.