Azithromycin blocks quorum sensing and alginate polymer formation and increases the sensitivity to serum and stationary-growth-phase killing of Pseudomonas aeruginosa and attenuates chronic P. aeruginosa lung infection in Cftr(-/-) mice

Abstract

The consequences of O-acetylated alginate-producing Pseudomonas aeruginosa biofilms in the lungs of chronically infected cystic fibrosis (CF) patients are tolerance to both antibiotic treatments and effects on the innate and the adaptive defense mechanisms. In clinical trials, azithromycin (AZM) has been shown to improve the lung function of CF patients. The present study was conducted in accordance with previous in vitro studies suggesting that the effect of AZM may be the inhibition of alginate production, blockage of quorum sensing (QS), and increased sensitivity to hydrogen peroxide and the complement system. Moreover, we show that AZM may affect the polymerization of P. aeruginosa alginate by the incomplete precipitation of polymerized alginate and high levels of readily dialyzable uronic acids. In addition, we find that mucoid bacteria in the stationary growth phase became sensitive to AZM, whereas cells in the exponential phase did not. Interestingly, AZM-treated P. aeruginosa lasI mutants appeared to be particularly resistant to serum, whereas bacteria with a functional QS system did not. We show in a CF mouse model of chronic P. aeruginosa lung infection that AZM treatment results in the suppression of QS-regulated virulence factors, significantly improves the clearance of P. aeruginosa alginate biofilms, and reduces the severity of the lung pathology compared to that in control mice. We conclude that AZM attenuates the virulence of P. aeruginosa, impairs its ability to form fully polymerized alginate biofilms, and increases its sensitivity to complement and stationary-phase killing, which may explain the clinical efficacy of AZM.

FIG. 1.

E-test determining the MIC of AZM (gradient, 0.016 to 256 μg/ml) for P. aeruginosa. (A) No inhibition zone observed. (B) Numbers of viable P. aeruginosa NH57338A isolates cocultured with AZM. The bacteria were grown to exponential phase (open circles) or stationary growth phase (closed circles) and cocultured with 2 to 256 μg/ml AZM, and after coincubation for 8 h, the numbers of CFU were determined. Values are means ± standard errors of three replicates.

FIG. 2.

Dose-response curves for AZM. Different concentrations of AZM were incubated with the QS monitor of P. aeruginosa NH57388A (lasB-GFP). Fluorescence (a) and growth (b) were monitored over 14 h. Symbols: *, no AZM (control); □, 2 μg/ml AZM; Δ, 4 μg/ml AZM; ×, 8 μg/ml AZM; +, 12 μg/ml AZM. Values are means ± standard errors of three replicates. P was <0.01 for the treated cultures compared to the results for the control. RFU, relative fluorescent units.

FIG. 3.

Alcohol precipitation of alginate from liquid ox bouillon cultures of P. aeruginosa NH57388A after 24 h of incubation at 37°C. The precipitation of high-weight polymers of alginate in untreated cultures (a) versus that in cultures treated with 12 μg AZM/ml (b) was negative.

FIG. 4.

Effect of AZM treatment on sensitivity of mucoid P. aeruginosa NH57388A with QS and the isogenic mucoid mutant without QS (lasI) to H₂O₂. Sensitivity was recorded as the mean ± standard error (n = 3) of the diameter of the growth inhibition. Bar 1, NH57388A with QS (without AZM); bar 2, NH57388A with QS (with 12 μg AZM/ml); bar 3, NH57388A without QS (without AZM); bar 4, NH57388A without QS (with 12 μg AZM/ml) (P < 0.01).

FIG. 5.

Reflection and epifluorescence images of mouse lung tissues infected with the QS reporter strain of P. aeruginosa NH57388A expressing GFP in response to ongoing AHL production. The infection was established for 24 h before injection of 100 μl of 0.9% saline (a and b) or an AZM solution (250 mg/kg) (c and d) via the tail vein. Green fluorescence indicates active transcription of the lasB gene. The border of probably alginate biofilms (black arrows) is clearly seen (b and d) by the reflection images.

FIG. 6.

Bacterial CFU in the lungs of mice at 7 days postinfection after treatment with AZM and saline (P < 0.01) (A); alginate content in lung homogenates from mice treated with AZM and saline (P < 0.0001) (B). Horizontal lines indicate medians.

FIG. 7.

Effect of AZM on lung pathology in mice with CF after P. aeruginosa infection. (a to d) AZM-treated mouse indicating mild pathology with no abscess (a) and probably remnants of a biofilm (black arrow) with a pronounced PMN infiltration (white arrow) (magnification, ×40) (b), and residues of alginate (blue color) without bacteria could be found (magnification, ×100) (c). HE and Alcian blue staining were used. (d) Lung homogenate without alginate precipitation. (e to h) Saline-treated mouse showing a big lung abscess (black arrows) (e) and large P. aeruginosa biofilms (black arrows) in the alveoli (f and g) encapsulated in alginate (blue color) surrounded by PMNs (white arrow). Magnifications, ×40 (f) and ×100 (g). HE and Alcian blue staining was used. (h) Alcohol precipitation of alginate from mouse lung homogenate (white arrow).