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Antimicrobial Agents and Chemotherapy, October 2007, p. 3677-3687, Vol. 51, No. 10
0066-4804/07/$08.00+0     doi:10.1128/AAC.01011-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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{triangledown}

Nadine Hoffmann,1* Baoleri Lee,1 Morten Hentzer,2 Thomas Bovbjerg Rasmussen,2 Zhijun Song,1 Helle Krogh Johansen,1 Michael Givskov,2 and Niels Høiby1

Department of Clinical Microbiology, Rigshospitalet, and Department of Bacteriology, Institute for Medical Microbiology and Immunology, Panum Institute, University of Copenhagen, DK-2100 Copenhagen, Denmark,1 Center for Biomedical Microbiology, BioCentrum-DTU, Building 301, Technical University of Denmark, DK-2800 Lyngby, Denmark2

Received 13 August 2006/ Returned for modification 27 March 2007/ Accepted 30 June 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mucoid Pseudomonas aeruginosa plays an important role in chronic lung infections among patients with CF due to its ability to form alginate-producing biofilms, which enable the bacteria to resist treatments with antibiotics and which affect the actions of the cells of the immune system (23). Macrolides such as azithromycin (AZM) are normally used against gram-positive bacteria, whereas many gram-negative rods, such as Pseudomonas spp., are inherently resistant to macrolides (56). However, several clinical studies from Japan (36, 39, 65) have highlighted the beneficial effects of different macrolides, including AZM, in the treatment of patients with diffuse panbronchiolitis, a disease which is very similar to CF. The first indication that AZM therapy could also benefit the lung function in CF patients was given by Jaffe et al. (27) and was subsequently confirmed by other investigators (9, 16, 58, 74). The mechanisms of action macrolides are multiple and may include an anti-inflammatory effect (36, 40), inhibition of a key enzyme in the alginate synthesis pathway (47), modulation of the production of quorum-sensing (QS) bacterial virulence factors (33, 44, 69), and/or inhibition of protein synthesis after prolonged exposure (67, 68).

In P. aeruginosa, the production of several virulence factors is regulated by cell-to-cell communication, based on the las- and rhl-encoded QS systems (6), which are important for the pathogenesis of P. aeruginosa (61). Sub-MICs of AZM have been shown to inhibit the production of several QS-regulated virulence factors of P. aeruginosa but to have no influence on its proliferation (44), likely by interference with the synthesis of N-acyl homoserine lactone (AHL) signal molecules (69). Recently, microarray analysis demonstrated that AZM exhibited extensive antagonistic activities against the QS system in strain PAO1 (48). Furthermore, AZM was found to retard the biofilm formation of nonmucoid P. aeruginosa strains (11, 14) and to exert bactericidal effects in stationary growth phase (26, 67, 68); thus, development of the AZM resistance phenotype (14) may not be excluded.

A murine study by Nagata et al. (46) that used plastic tubes precoated with P. aeruginosa as a base for the development of a chronic infection has demonstrated that biofilm formation is suppressed by erythromycin. A favorable modulation of the immune response by AZM has been indicated by Moser et al. (45) in a mouse model with seaweed alginate-entrapped P. aeruginosa. Moreover, a mouse study by Nicolau et al. (49) showed that adjunctive AZM had a beneficial effect in the treatment of mucoid P. aeruginosa.

Recently, we established a CF mouse model of chronic P. aeruginosa lung infection based on an alginate-overproducing P. aeruginosa CF isolate without using artificial embedment of the bacteria (21). In the present study, we evaluate the role of AZM in QS, alginate production, and sensitivity to hydrogen peroxide; the sensitivity of certain bacterial growth phases to AZM; and the serum sensitivity of P. aeruginosa in vitro and confirm the therapeutic effect of AZM in our CF mouse model.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
P. aeruginosa isolates. P. aeruginosa NH57388A is a stable mucoid isolate from the sputum of a chronically colonized CF patient attending the Danish CF Center, Rigshospitalet, Copenhagen, Denmark. The isolate shows hyperproduction of alginate due to a deletion in mucA and has functional AHL-based QS systems (21). A lasI mutant of mucoid strain NH57388A was constructed by using previously described knock-out systems (3), and the presence of the mutation was verified by screening for the loss of AHL production by thin-layer chromatography, as described elsewhere (21). Nonmucoid isolates with and without QS are spontaneous isogenic revertants from mucoid strain NH57388A and the lasI mutant, respectively, derived from liquid cultures. The bacteria were cultured in liquid ox bouillon (Statens Serum Institut, Copenhagen, Denmark) for 48 h at 37°C and 180 rpm. The samples were serially diluted and plated on a Blue agar plate (BAP; Statens Serum Institute); Blue agar is a modified Conradi-Drigalski medium selective for gram-negative rods. After incubation at 37°C for 30 to 35 h the spontaneous nonmucoid revertants were selected. QS activity was monitored by using a P. aeruginosa NH57388A strain harboring the QS reporter plasmid pKR-C12 (57). The plasmid can reveal QS-controlled transcriptional activity in bacteria by expression of an unstable green fluorescent protein (GFP) variant under control of the QS-controlled lasB promoter of P. aeruginosa (19). All P. aeruginosa isolates were stored at –80°C in broth supplemented with 10% glycerol.

AZM susceptibility. The MIC of AZM was determined with the E-test system (AB Biodisk, Solna, Sweden). Briefly, 1 ml of an overnight ox bouillon culture of P. aeruginosa NH57388A diluted to 10–4 was poured onto a Danish blood agar plate (Statens Serum Institute), and the plate was rotated to ensure even distribution of inoculum. Excess moisture was allowed to absorb before the E-test strip was applied. The plates were incubated overnight at 37°C. The experiment was performed in triplicate.

Killing assay in liquid broth. The bactericidal effect of AZM on P. aeruginosa NH57388A was determined in the exponential phase and the stationary phase (26). After growth in ox bouillon to an optical density (OD) at 600 nm (OD600) of 0.5 (exponential phase) or 2.0 (stationary phase), AZM was added in twofold dilutions (2 to 256 µg/ml) to cells of each phase, and the AZM and cells were coincubated for 8 h at 37°C with orbital shaking (180 rpm). The numbers of CFU were determined by serial dilution of samples plated on BAPs, and the plates were incubated at 37°C for 30 to 35 h. The experiment was performed in triplicate.

Dose-response of lasB expression by AZM. The QS reporter strain P. aeruginosa NH57388A harboring a lasB-gfp fusion was streaked from a frozen stock on an LB plate containing 60 µg/ml gentamicin (Gm). A liquid preculture was prepared by inoculating P. aeruginosa from the plate in 100 ml ox bouillon containing 20 µg/ml Gm, and the culture was incubated overnight at 37°C and 180 rpm. The culture was centrifuged (23,000 x g, 30 min, 4°C), and the pellet was resuspended in 8 ml fresh ox bouillon. The bacteria was diluted to an OD600 of 0.1 in 100-ml Erlenmeyer flasks containing 40 ml ox bouillon supplemented with 2, 4, 8, and 12 µg/ml AZM or not supplemented with AZM (control); and the flasks were incubated at 37°C with orbital shaking (180 rpm). At different time intervals, growth was monitored by determination of the OD600, and the green fluorescence was measured with a fluorometer at an excitation wavelength of 490 nm and an emission wavelength of 515 nm. The experiment was repeated twice and was performed in triplicate. Gm was added to ensure plasmid maintenance. Different concentrations of Gm were used due to the different growth conditions (solid agar surface, 60 µg/ml Gm; liquid culture, 20 µg/ml Gm). Gm was not used together with AZM since the presence of both antibiotics in liquid cultures inhibited the growth of the bacteria. However, the plasmid is rather stable; thus, preculture with Gm was sufficient.

Batch culture conditions. P. aeruginosa NH57388A from a frozen stock was cultured on a BAP. Liquid precultures for growth experiments and virulence factor analysis were prepared by inoculating P. aeruginosa isolates from the plate in 100 ml ox bouillon, and the mixture was incubated overnight at 37°C and 180 rpm. The bacteria were recovered by centrifugation, resuspended, diluted to an OD600 of 0.1 in fresh 80 ml ox bouillon supplemented with (2, 4, 8, and 12 µg/ml) or without (control) AZM, and incubated at 37°C with orbital shaking (180 rpm) for 2 days. Samples were taken at different time intervals and centrifuged (23,000 x g, 30 min, 4°C), and the supernatant was analyzed for virulence factors (see below). The number of bacterial cells was determined by measurement of the OD600 or by determination of the numbers of CFU by using BAPs. The experiment was repeated three times and was performed in triplicate.

Assays of virulence factors. (i) Endochitinase. The endochitinase assay was performed as described previously (50). Briefly, the culture supernatant (200 µl) was mixed with carboxymethyl-chitin-remazol brilliant violet (100 µl; 2 mg ml–1; Loewe Biochemica, Sauerlach, Germany) and sodium acetate buffer (100 µl; 0.05 M; pH 5.0), and the mixture was incubated for 20 min at 50°C in a water bath. The reaction was terminated by addition of HCl (100 µl; 2 M), kept for 10 min on ice, and centrifuged at 10,000 x g at 4°C for 5 min. The absorbance (OD540) of the supernatant was measured. The boiled supernatant with buffer and substrate was used as a blank. Endochitinase activity was expressed in units of the measured A540 x 1,000 x min–1 x ml–1 and was the mean of three independent replicates. The specific activity (enzyme units per unit of cell mass) was calculated as enzyme units of OD540 nm/OD600 nm normalized to 1 ml supernatant.

(ii) Protease. LasB protease (elastase) activity was measured by the method of Ohman et al. (52) by use of the elastin Congo red (Sigma Chemical Co., St. Louis, MO) as the substrate. Elastin Congo red (5 mg ml–1) in 0.1 M Tris-maleate buffer (pH 7.0) and the culture supernatant (500 µl) were incubated for 2 h at 37°C. Sodium phosphate buffer (1 ml; 0.7 M; pH 6.0) was added to the samples, and the mixture was placed on ice for 10 min to stop the reaction. After centrifugation (10,000 x g at 4°C for 15 min), the absorbance (OD495) of the supernatant was measured. The supernatant plus assay buffer was used as a blank. The specific elastase (LasB) activity was measured as the absorbance of the supernatant (OD495) divided by the OD600 of the culture normalized to 1 ml supernatant.

(iii) Pyocyanin. Pyocyanin was extracted as described by Essar et al. (10). Five milliliters of culture was extracted in 3 ml of chloroform overnight. The blue pigment extracted in the chloroform corresponds to pyocyanin. The chloroform phase was transferred to a clean tube, 1 ml of 0.2 M HCl was added, and the mixture was gently shaken to bring the pyocyanin to a pink aqueous phase. The aqueous solution was centrifuged (20,000 x g at 4°C for 10 min), and the absorbance (OD520) of the supernatant was measured. The pyocyanin concentration was determined by multiplying this measurement by 17.07.

(iv) Alginate. The extraction of exopolysaccharide (alginate) was performed as described by Pedersen et al. (53). Sample cultures (1 ml) were centrifuged at 23,000 x g for 30 min at 4°C. The supernatant was heated for 30 min at 80°C and was centrifuged at 23,000 x g and 4°C. The pellet was discarded, and the supernatant containing the alginate was precipitated with ice-cold 99% ethanol (3x volume). After 1 to 2 h at 4°C, the precipitated alginate was collected. The precipitate was dissolved in 1 ml sterile 0.9% saline. The content of uronic acid polymers (the component of alginate) in the samples was then analyzed by the carbazole-borate assay (35) with D-mannuronlactone (Sigma) as a standard. Briefly, 118 µl of the sample was mixed with 1 ml of borate-sulfuric acid reagent (100 mM H3BO3 in concentrated H2SO4) on ice, and 34 µl of carbazole reagent (0.1% in ethanol) was added. The mixture was heated to 55°C for 30 min, and the absorbance (OD530) was measured.

Dialysis of alginate. Mucoid strain NH57388A was tested for uronic acid secretion. Sample cultures were centrifuged at 23,000 x g for 30 min at 4°C, and the pellet was discarded. For equilibrium dialysis, the method of Jain and Ohman (28) was used. The supernatants (7 ml) were placed in dialysis tubings (molecular weight cutoff, 10,000; 1.8 ml cm–1; Spectra/Por membrane) and dialyzed against an equal volume (7 ml) of 10 mM Tris-HCl, pH 7.6, at 4°C for 16 h. The two fractions corresponding to the dialyzed material (inside the tubing) and the dialysate (outside the tubing) were then tested for their uronic acid contents by the carbazole-borate assay (35).

Susceptibility to hydrogen peroxide. One hundred microliters of a 24-h culture of mucoid P. aeruginosa NH57388A or the isogenic lasI mutant was diluted to 10–4 and plated onto a predried BAP. Filter disks (diameter, 6 mm; Millipore) were soaked with 10 µl of H2O2 (10%; vol/vol) and placed in the center of the plate. Due to the slow growth of the mucoid cells on the BAP, the diameters of the inhibition zones surrounding the disks were measured after 48 h of incubation at 37°C.

Normal human serum (NHS). Blood was obtained by venipuncture from one healthy volunteer and was allowed to clot at 4°C for 2 h. After centrifugation at 3,000 x g at 4°C for 15 min, the serum was harvested, diluted to suitable concentrations in sterile phosphate-buffered saline (PBS), and stored at –80°C.

Serum sensitivity assay. Both mucoid and nonmucoid strains of NH57388A with and without QS were tested. P. aeruginosa were cultured in ox bouillon for 18 h at 37°C and 180 rpm in the presence of 12 µg/ml AZM or without AZM. The cells were harvested, and 20 µl of the bacterial suspensions was mixed with 2 ml of sterile PBS. Samples (20 µl) were removed for CFU determination. Afterwards, 2.5% NHS was added to the bacterial suspensions and the mixtures were rotated at 37°C for 30 min. Activation was terminated by addition of 2 ml of cold PBS, and the numbers of CFU were determined on a BAP. Control samples contained bacteria incubated with 2.5% heat-inactivated NHS (56°C for 30 min).

Experimental animals. Female and male, transgenic homozygote CF CftrtmlUnc-TgN(FABPCFTR) mice (Jackson Laboratories) bred from a mixed genetic background (63) were used. The mice were 16 to 19 weeks old and were housed under specific-pathogen-free conditions at the Royal Veterinary and Agricultural University, Copenhagen, Denmark.

Pharmacokinetics of AZM in mice with CF. The elimination half-life and concentration of AZM in serum in CF mice were determined. The dose of AZM was 500 mg/kg of body weight and was given as one dose orally. For each time point (0.5, 1, 2, and 4 h), blood was collected from three mice for determination of the AZM concentration. The blood was centrifuged (2,000 x g, 4°C, 10 min), and the serum was harvested and stored at –80°C until analysis. A disk diffusion bioassay (22) with Streptococcus sp. strain EB68 as the test strain was used to measure the serum AZM concentration. The controls were sera from animals that did not receive AZM therapy and sera to which defined AZM concentrations were added. All determinations were performed in triplicate. The coefficient of variation of the measurement was 11.8%.

Preparation of inoculum for lung infection in CF mice. The challenge inoculum was prepared as described previously (21). Briefly, mucoid P. aeruginosa NH57388A was cultured in 80 ml ox bouillon for 28 h at 37°C with shaking (180 rpm). QS reporter strain P. aeruginosa NH57388A harboring a lasB-gfp fusion was streaked from a frozen stock on an LB plate containing 60 µg/ml Gm, and a liquid culture was prepared by inoculating P. aeruginosa from the plate in 80 ml ox bouillon containing 20 µg/ml Gm. The bacteria were harvested and resuspended in 2 ml fresh ox bouillon, and the number of CFU was measured. Afterwards, the cells were adjusted to a challenge inoculum of 108 CFU/ml by dilution (1:10) in pseudomonal alginate solution (purified alginate, 2 mg/ml).

Mouse lung infection model. Before intratracheal challenge with bacteria, mice were anesthesized (subcutaneously) with 0.2 ml of a 1:1 mixture of 25% fentanyl (Hypnorm; Janssen-Cilag), 25% midazolam (Dormicum; Roche), and 50% sterile water. The mice were challenged with 40 µl P. aeruginosa by the use of a curved bead-tipped needle, and the incision was sutured with silk (21).

Drug administration. AZM (Zitromax; Pfizer, Denmark) was dissolved in sterile water immediately before use, according to the instructions of the manufacturer. To record the expression and inhibition of P. aeruginosa QS in vivo, the mouse lungs were challenged with the QS reporter strain NH57388A. The infection was established for 24 h before injection of 100 µl of an AZM solution (250 mg/kg of body weight) or 0.9% saline via the tail vein. Mice with CF were killed 24 h after drug administration. To evaluate the therapeutic effect of AZM, the mice were orally treated with AZM (500 mg/kg of body weight) or 0.9% saline once daily. The treatment was initiated 24 h after challenge and was continued for 5 days. The mice were killed 7 days after challenge by injection of 2 ml 20% pentobarbital kg–1. The usual therapeutic dose of AZM used in CF patients is 250 mg/day (for those with body weights of ≤40 kg) or 500 mg/day (for those with body weights of >40 kg) (9). For the present study, the choice of a high dosage was based on the much faster liver metabolism in mice (elimination half-life of AZM, 2.3 h; see Results) compared to that in humans, in whom the AZM half-life is 68 h (5).

Freeze microtomy. Lungs infected with the mucoid QS reporter strain NH57388A were embedded with Tissue-Tek material and frozen at –20°C to –40°C immediately after removal from the mouse thorax. Frozen sections 20 to 40 µm thick were made at different levels of the lung tissues by freeze microtomy, and subsequently, scanning confocal laser microscopy was used to observe bacterial GFP signals, as described by Wu et al. (75).

Macroscopic description of the lungs. The lungs were described in situ and after removal from the thoracic cavities, as described by Johansen et al. (30). The scores are described as follows: 1, normal; 2, swollen lungs, hypermia, and a low level of atelectasis; 3, pleural adhesion, atelectasis, and multiple small abscesses; and 4, large abscesses, a high level of atelectasis, and hemorrhages. The lungs were then prepared for quantitative bacteriology. The whole lung of each mouse was excised aseptically and homogenized in 5 ml sterile 0.9% saline; and 100 µl of appropriately serial diluted lung homogenate samples was plated on BAPs, and the plates were incubated at 37°C and inspected for P. aeruginosa colonies (CFU) after 35 to 40 h.

Histopathological studies. Randomly selected lungs from half of the mice were used for lung histopathology. The lungs were fixed in formalin buffer (4% formaldehyde, pH 7.0; Bie & Berntsen, Copenhagen, Denmark) for at least 1 week and then embedded in paraffin wax and cut into 5-µm sections. Mounted sections were stained with hematoxylin and eosin (HE) combined with Alcian blue-periodic acid-Schiff stain for the detection of exopolysaccharides. The cellular changes were assigned to acute or chronic inflammation groups by a scoring system (31), based on the proportion of polymorphonuclear leukocytes (PMNs) and mononuclear leukocytes in the inflammatory foci.

Determination of alginate content in mouse lung homogenate. Mouse lung homogenate (500 µl) was extracted with ice-cold 99% ethanol (4x volume) and resuspended in sterile 0.9% saline (500 µl). Lung homogenate from mice challenged with 0.9% saline in purified alginate without bacteria was used as a blank. The uronic acid (alginate) content was quantified by a carbazole-borate assay (35).

Statistical analysis. Paired Student's t test with a two-tailed distribution was used to compare the in vitro data between two groups. The Mann-Whitney U test was used to compare the data for lung bacteriology, lung alginate content, and macroscopic lung pathology between two groups. The categorical data were analyzed by the chi-square test.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
AZM susceptibility. When P. aeruginosa isolate NH57388A was plated onto BAPs and exposed to an E-test strip containing AZM, no inhibition zone was observed (Fig. 1A), showing that the MIC was >256 µg/ml.


Figure 1
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  		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.

 
Effect of AZM on P. aeruginosa in different growth phases. A study of nonmucoid strain PAO1 has shown that AZM renders the bacteria sensitive in the stationary phase and not in the exponential phase (26). We elucidated how AZM affected the sensitivity of our mucoid strain NH57388A in these two growth phases. As illustrated in Fig. 1B, the reduction in viable counts was 88%, even with 2 µg/ml AZM, and was 99% with 16 µg/ml AZM in the stationary phase. In contrast, similar reductions required much higher AZM concentrations in the exponential phase (87% at 64 µg/ml AZM and 99% at 256 µg/ml AZM, respectively).

Dose-response of lasB expression by AZM. To investigate the effect of AZM on QS by using molecular technology, a lasB-gfp (avian sarcoma virus) fusion was expressed in mucoid P. aeruginosa NH57388A. Being a type IV QS gene, lasB is induced in the late exponential-early stationary phase (72). Figure 2a shows that the addition of AZM repressed GFP expression in a concentration-dependent manner, and at all concentrations tested (2 to 12 µg/ml AZM) the fluorescence was significantly (P < 0.01) reduced compared to that in the control without affecting the growth rate (Fig. 2b). This indicates that AZM specifically blocks P. aeruginosa QS.


Figure 2
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  		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); {square}, 2 µg/ml AZM; {Delta}, 4 µg/ml AZM; x, 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.

 
Inhibition of QS-regulated virulence factor and alginate production in vitro. The study from which the data presented above were obtained used the fusion of a reporter to the lasB promoter to study the effects of N-(3-oxodode canoyl)homoserine lactone and AZM. A limitation to this approach is the restriction of analysis to the level of transcription. Therefore, we investigated the efficacy of AZM directly on the production of virulence factors, including elastase, pyocyanin, and chitinase, which was assessed after 24 h. As shown in Table 1, concentrations of 2 to 12 µg/ml AZM suppressed the virulence factors of mucoid P. aeruginosa isolates in a concentration-dependent manner.


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  		TABLE 1. Effect of AZM on virulence factor production by P. aeruginosa over 24 h of culture

 
Inhibition and dialysis of alginate. It has been shown that the loss of the AlgK and AlgG proteins encoded by the alginate operon results in the secretion of dialyzable uronic acids instead of polymerized alginate (28, 29). As shown in Table 2, the supernatant of mucoid strain NH57388A contained large amounts of uronic acids and had it almost exclusively in the dialyzed fraction due to a polymeric (alginate) form (Fig. 3), which is too large to pass through the dialysis membrane. In contrast, the supernatant of the mucoid strain treated with 12 µg/ml AZM had significantly smaller amounts of uronic acids and contained more uronic acids that had diffused out of the dialysis sac both with equilibrium dialysis (P < 0.01) and exhaustive dialysis (P < 0.05) (Table 2). These results explain the results of our initial tests, which showed that the culture supernatants of NH57388A grown with 12 µg/ml AZM had a lower pH than those without AZM (pH 7.99 and 8.73, respectively). When the bacteria were plated on BAPs, both treated and nontreated P. aeruginosa bacteria from liquid cultures (24 h) displayed mucoid colonies, but the treated bacteria appeared as small-colony mucoid variants that grew more slowly than the untreated bacteria. Overall, the results show that AZM inhibits alginate synthesis and may cause a defect in alginate polymer formation.


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  		TABLE 2. Equilibrium dialysis of uronic acids secreted by P. aeruginosa NH57388A when it was cultivated in the presence and absence of 12 µg/ml AZM

 

Figure 3
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  		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.

 
Effect of AZM on sensitivity of P. aeruginosa to hydrogen peroxide. To determine whether AZM affected the sensitivity of mucoid P. aeruginosa to reactive oxygen intermediates, the bacteria were exposed to 10% H2O2. As illustrated in Fig. 4, the isogenic mucoid strains with and without QS showed similar H2O2 sensitivities when they were not treated with AZM. AZM, however, increased the sensitivity to H2O2 of the mucoid strain with QS and even more that of the strain without QS (zones of inhibition, 31% [P < 0.01] and 43% [P < 0.01] larger, respectively) compared to the sensitivity of the untreated bacteria.


Figure 4
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  		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).

 
Effect of AZM on serum sensitivity of P. aeruginosa. Alginate and QS-regulated virulence factors, e.g., elastase, are known to confer resistance to host phagocytes and complement (34, 55). We evaluated the effect of AZM on the serum sensitivity of mucoid (with or without QS) and nonmucoid (with or without QS) P. aeruginosa. Human serum at 2.5% was chosen as a source of complement since higher concentrations showed significant killing of P. aeruginosa at ≥5% (data not shown). As shown in Table 3, the mucoid strain with QS treated with 12 µg/ml AZM became significantly more sensitive to 2.5% serum than bacteria not treated with AZM, with a 58,000-fold reduction in the numbers of CFU/ml (2.5 x 102 and 1.45 x107 CFU/ml, respectively). Similar results were obtained with the nonmucoid strain with QS, where the total clearance of AZM-treated bacteria was observed after incubation with serum. Notably, the rate of survival was higher for mucoid strains than for nonmucoid strains when incubation was with 2.5% serum, regardless of either QS capability or AZM treatment, supporting the fact that alginate is a key factor in enhancing the survival of P. aeruginosa. Interestingly, there were no significant changes in sensitivity to serum between AZM-treated and untreated bacteria for both mucoid and nonmucoid strains without QS (8.6 x 105 versus 7.3 x105 CFU/ml AZM-treated mucoid and nonmucoid strains, respectively, and 2.0 x 102 versus 2.1 x104 CFU/ml for untreated mucoid and nonmucoid strains, respectively), indicating that QS mutants (lasI) are more resistant.


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  		TABLE 3. Effect of 12 µg/ml AZM on sensitivity of P. aeruginosa to serum

 
Pharmacokinetics in CF mice. In healthy mice the elimination half-life of AZM is 43 min (7). The elimination half-life of AZM determined in CF mice was 2.3 h. The concentrations of AZM in serum were 2.9 ± 3.0, 2.9 ± 0.8, 3.1 ± 0.6, and 1.7 ± 0.9 µg/ml at 0.5, 1, 2, and 4 h, respectively.

Expression of QS in lungs of CF mice. To record the expression of P. aeruginosa QS in vivo, mouse lungs were challenged with the QS reporter strain of NH57388A. GFP expression by P. aeruginosa was detected in the lungs (Fig. 5a and b) and increased over time due to bacterial proliferation. Control experiments showed that the GFP signal could not be found in lung tissue when the mice were killed at 0.5 h postchallenge, probably due to the initial low bacterial concentration (approximately 4 x 106 CFU/ml) (data not shown). These results demonstrate that the QS of mucoid P. aeruginosa bacteria is functional in the lungs of CF mice.


Figure 5
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  		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.

 
Inhibition of QS in lungs of CF mice with AZM. The ability of AZM to suppress P. aeruginosa QS in vivo was tested by challenging mouse lungs with the QS reporter strain. The infection was established for 24 h before injection of 100 µl of an AZM solution (250 mg/kg) through the tail vein. Saline (0.9%) was used as a control. The GFP signal was markedly reduced in AZM-treated mice (Fig. 5c and d) compared to that in saline-treated mice (Fig. 5a and b) 24 h after drug administration.

Therapeutic effect of AZM in mice with CF and chronic P. aeruginosa lung infection. On the basis of the results of the in vitro and in vivo studies presented above, the therapeutic effect of AZM was evaluated in our new model of CF in mice. The mice were challenged with P. aeruginosa, and after 24 h treatment was initiated. The bacteriology and pathology of the lungs were assessed at 7 days postinfection, 1 day after the treatment was finished.

(i) Mortality. The mice in both groups were monitored for mortality every day from day 1 to day 7 postinfection. In the group of AZM-treated mice, the cumulative mortality was 24% (7/29), whereas it was 33% (9/27) in the saline-treated group (P was not significant).

(ii) Bacteriology and alginate content. P. aeruginosa was cleared from the lungs of 5 of 12 AZM-treated mice (42%), and the bacterial density was significantly lower (P < 0.01) in this group than in the saline-treated group (medians, 50 CFU/lung and 7.5 x 105 CFU/lung, respectively) (Fig. 6A). This finding correlates with a significantly lower (P < 0.0001) alginate content in the lung homogenates of mice treated with AZM than in the saline-treated group (medians, 10 µg/ml and 366 µg/ml, respectively) (Fig. 6B).


Figure 6
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  		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.

 
(iii) Macroscopic lung pathology. The macroscopic lung pathology was significantly milder (P < 0.01), as judged by lung scoring (Table 4), and the frequency of lung abscesses was especially lower (P < 0.05) in the group treated with AZM than in the group treated with saline (32% and 67%, respectively).


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  		TABLE 4. Macroscopic lung pathology at 7 days postinfection after intratracheal P. aeruginosa challenge of mice with CF

 
(iv) Histopathology. Pathological examinations of the lungs of mice treated with AZM (Fig. 7a to d) demonstrated crippling of the P. aeruginosa alginate biofilm, in which debris-like granular and fibrous spongy-like material surrounded by numerous PMNs was observed (Fig. 7b). In some AZM-treated mice, scattered residues of alginate without bacteria could be found (Fig. 7c). In contrast, saline-treated mice (Fig. 7e to h) showed abscess formation (Fig. 7e) and extensive focal endobronchial and alveolar P. aeruginosa biofilms with PMN infiltration. P. aeruginosa was present in the alveoli as microcolonies encapsulated in a matrix of alginate (Fig. 7f and g) which could be precipitated with alcohol (Fig. 7 h).


Figure 7
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  		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, x40) (b), and residues of alginate (blue color) without bacteria could be found (magnification, x100) (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, x40 (f) and x100 (g). HE and Alcian blue staining was used. (h) Alcohol precipitation of alginate from mouse lung homogenate (white arrow).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of this study show that sub-MICs of AZM suppress the QS-controlled lasB expression (Fig. 2) of mucoid P. aeruginosa NH57388A in parallel with the inhibition of QS-regulated virulence factors and alginate (Table 1), which is in accordance with the findings of other studies (25, 44, 48, 69). Furthermore, AZM makes the mucoid bacteria susceptible to killing in stationary growth phase (Fig. 1B) and sensitive to hydrogen peroxide (Fig. 4) and complement (Table 3). Therefore, AZM exerts favorable therapeutic effects on mucoid P. aeruginosa lung infection in mice with CF by resulting in the improved clearance of the alginate-producing bacteria. The present data are in accordance with the findings of clinical studies that AZM treatment improves lung function in CF patients (58) and may inhibit in vivo biofilm formation, as shown by a decrease in mucoid P. aeruginosa from sputum samples from CF patients (16). The mechanisms underlying the effect of AZM are multiple. AZM is anti-inflammatory (36, 40) and interferes with QS via the reduction of AHL molecules (11, 69), which may reduce the inflammation in vivo (70). A bactericidal effect on P. aeruginosa through interaction with the outer membrane (26) and inhibition of protein synthesis (67) after prolonged AZM exposure have also been shown.

The matrix of the bacterial biofilm is composed of exopolysaccharides (e.g., alginate), proteins (64), and DNA (71). Biofilms formed by nonmucoid P. aeruginosa are not composed of alginate and normally demonstrate a uniform flat and densely packed structure, whereas alginate biofilms have a thick and highly structured architecture (18, 51). The O-acetylation of alginate plays an important role in the ability of mucoid P. aeruginosa to form stable biofilms (51) and to resist complement-mediated phagocytosis (55). Therefore, eradication of mucoid bacteria may be improved by crippling the polymerized O-acetylated alginate matrix, making the bacteria sensitive to phagocytosis. The suggestion that AZM may affect polymerization was demonstrated in our study by the incomplete precipitation of polymerized alginate (Fig. 3) and the high levels of readily dialyzable uronic acids in supernatants compared to the results for the untreated supernatants (Table 2). Small amounts of uronic acids, however, were retained by exhaustive dialysis in the AZM-treated supernatant, assuming that the uronic acids produced included some high-molecular-weight polymers and, thus, that at least some polymerization took place. These results reflect previous findings that deletions of algK and algG in mucoid P. aeruginosa isolates block alginate polymer formation, leading to uronic acid secretion and the nonmucoid phenotype (28, 29). A change to the nonmucoid phenotype after AZM treatment was not observed in the present study; however, the treated bacteria displayed slow growth and small-colony mucoid variants on BAPs. This finding may be explained by the high concentration of unpolymerized uronic acids, which is detrimental to bacterial growth, as considered by Jain and Ohman (28).

The inflammatory response to chronic P. aeruginosa lung infection is mainly characterized by the constant influx of PMNs (2), resulting in the exposure of the bacteria to toxic free oxygen radicals, but the alginate matrix of mucoid P. aeruginosa protects the bacteria from free radicals (59). Additionally, it has been demonstrated that QS participates in the resistance of P. aeruginosa to H2O2 due to the production of the antioxidants catalase and superoxide dismutase (8, 17). We show that AZM makes mucoid isogenic strains with and without QS more susceptible to H2O2, in agreement with the results for nonmucoid strain PAO1 (48). This effect of AZM may be explained in part by the inhibition of alginate (Table 1) and, thus, abrogation of the scavenging effect, in combination with the down regulation of las-controlled virulence factors (Fig. 2), e.g., catalase, which make the bacteria susceptible to H2O2. Notably, Bjarnsholt et al. (4) found significant killing of a nonmucoid PAO1QS mutant compared to that of the wild type after H2O2 treatment. However, no difference in the sensitivity to killing of mucoid strains with and without QS by H2O2 was found (Fig. 4) in the present study. This emphasizes that alginate may be a crucial key factor and that the loss of functional QS is not of critical importance regarding resistance to oxygen radicals in mucoid strains.

The LasB elastase is required during the synthesis of alginate (32), and microarray analysis demonstrated a link between lasB elastase and alginate (13). O-acetylation of P. aeruginosa alginate is crucial to resistance to complement-mediated phagocytosis (55). In addition, bacterial elastase impairs the host defense by cleaving complement components and important surface cell receptors of the complement systems (34). Thus, mucoid P. aeruginosa strains with QS may be able to escape phagocytosis by dampening the complement. In this study, 2.5% human serum was chosen as a source of complement to test whether AZM treatment rendered P. aeruginosa more sensitive to serum. We show that AZM treatment led to the pronounced serum-mediated killing of both mucoid and nonmucoid bacterial strains with QS. For untreated bacteria, mucoid strains exhibited higher levels of resistance to serum than nonmucoid strains (Table 3), supporting the possibility that alginate may act as a barrier blocking access to the immune system (41, 54, 59). Remarkably, strains without QS remained significantly more resistant to serum after AZM treatment than strains with QS. It has been demonstrated, however, that QS-negative (lasR) mutants of P. aeruginosa can avoid cell lysis and death, whereas wild-type strains cannot, questioning whether under some environmental conditions the loss of QS might give P. aeruginosa a selective advantage (20, 43). In fact, naturally occurring QS mutants have been isolated from the clinical environment (15, 21, 62). Interestingly, recent work by Smith et al. (60) showed that QS (lasR) mutants are found in the majority of infections in CF patients, suggesting that inactivation of QS may play an important role in the adaptive processes of P. aeruginosa in the airways of patients with CF. Overall, AZM treatment may increase the effects of complement by inhibiting and incompletely polymerizing alginate (Table 2; Fig. 3) and by reducing the production of QS-regulated virulence factors, e.g., elastase (Table 1; Fig. 2), leading to enhanced phagocytosis and clearance of the bacteria. Other mechanisms, such as changes in lipopolysaccharides and outer membrane proteins through which AZM is known to enhance the serum sensitivity of P. aeruginosa (66), were, however, not investigated in the present study.

Our results with mice with CF demonstrated that AZM significantly improved the pulmonary bacterial clearance (Fig. 6A), reduced the extent of lung abscesses, and decreased the severity of lung pathology (Table 4), probably by interference with QS (Fig. 5) and crippling of the alginate biofilms (Fig. 7). Apart from the suppression of virulence factors, the effect of AZM in rendering the bacteria susceptible to killing in stationary growth phase (Fig. 1A) (26) might be of major relevance and could be another possibility that explains the reduced bacterial load in vivo. Moreover, Tateda et al. (67) suggest that bacteria continuously exposed to AZM for prolonged periods may be sensitized to the serum bactericidal effect. On the other hand, the induction of virulence by AZM has been found since AZM-treated strain PAO1 cultures showed enhanced expression of the type III secretion system (TTSS), leading to increased cytotoxicity (48). However, a functional TTSS is repressed in mucoid P. aeruginosa strains (76) and is active only in nonmucoid cells during initial colonization and not during the chronic stage of infection (42). Thus, over time P. aeruginosa strains in the CF lung accumulate a number of mutations that reduce bacterial toxicity to the host (42, 60). It has previously been shown (37) that inoculation of AZM-treated P. aeruginosa PAO1 significantly enhanced mortality in mice, and this was explained by an acute toxic effect rather the proliferation of the bacteria. In contrast, administration of AZM after the inoculation of P. aeruginosa did not increase the rate of mortality in mice (37), in accordance with the findings of our study.

In the respiratory zone and alveolar spaces, P. aeruginosa establishes alginate biofilms surrounded by numerous PMNs (Fig. 7f and g) (21). When P. aeruginosa multiplies in the lungs, QS is activated (Fig. 5a) (12, 75) and triggers the activation of virulence factors that may injure host cells, initiating local inflammation and tissue damage during frustrated phagocytosis (38). The serum AZM concentration does not reflect the AZM concentration at the site of infection since AZM concentrates and persists in alveolar cells (5), epithelial lining fluid (1), PMNs, and sputum (73). PMNs, for example, appear to target and concentrate at sites of infection. Drug-loaded PMNs can transport large amounts of active AZM to tissue foci of infection (Fig. 7b and c), leading to the effective attenuation of QS, crippling of the mucoid biofilm, and thus, a milder lung pathology (Table 4).

In summary, we demonstrated that AZM repressed the QS-controlled lasB expression and the alginate production of P. aeruginosa and affected the polymerization of alginate. Furthermore, AZM rendered the bacteria sensitive to killing during stationary-phase growth and by H2O2 and serum. In addition, AZM-treated QS mutants appeared to be resistant to serum. AZM treatment of mice with CF significantly improved the clearance of mucoid P. aeruginosa biofilms and reduced the severity of the lung pathology compared with that in mice that received placebo. Taken together, the attenuation of bacterial virulence, killing during stationary growth phase, and enhancement of complement sensitivity by AZM may be responsible for the beneficial effects observed in our mice with CF and in patients with CF.

In a recent microarray study (48), nonmucoid P. aeruginosa PAO1 was used to analyze the impact of AZM. PAO1 was originally a wound isolate, and the genomic profile was not similar to that of the majority of clinical isolates from patients with CF (24). It is unclear how representative this genome is compared to those of CF isolates, and therefore, microarray analyses of QS and alginate genes in CF isolates for determination of the specificity of AZM are in progress in our laboratory. In addition, future studies comparing the effect of AZM on P. aeruginosa and isogenic QS mutants in our mouse model may improve the evidence regarding QS inhibition. Finally, the data that indicated that AZM-treated QS mutants exhibited serum resistance have interesting implications, as this finding may be related to the apparent selection for QS mutants in the airways of patients with CF and requires further studies.


   ACKNOWLEDGMENTS
 
We thank Jette Pedersen and Ulla Johansen for excellent technical assistance with the histological samples and the disk diffusion assay, respectively.

This work was supported by a grant from the Villum Kann Rasmussen Foundation to M.G.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Bacteriology, Institute for Medical Microbiology and Immunology, Panum Institute 24.1, University of Copenhagen, DK-2100 Copenhagen, Denmark. Phone: (45) 3532 7890. Fax: (45) 3532 7693. E-mail: nadinehof{at}hotmail.com Back

{triangledown} Published ahead of print on 9 July 2007. Back


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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