Synthetic furanones inhibit quorum-sensing and enhance bacterial clearance in Pseudomonas aeruginosa lung infection in mice

H. Wu1,*, Z. Song1, M. Hentzer2, J. B. Andersen2, S. Molin2, M. Givskov2 and N. Høiby1

1 Department of Clinical Microbiology, University Hospital of Copenhagen (Rigshospitalet), Juliane Marie Vej 22, Copenhagen Ø, DK-2100; 2 Molecular Microbiology, Building 301, BioCentrum, The Technical University of Denmark, Lyngby, DK-2800, Denmark

Received 20 September 2003; returned 20 November 2003; revised 29 February 2004; accepted 4 March 2004


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Introduction: Antibiotics are used to treat bacterial infections by killing the bacteria or inhibiting their growth, but resistance to antibiotics can develop readily. The discovery that bacterial quorum-sensing regulates bacterial virulence as well as the formation of biofilms opens up new ways to control certain bacterial infections. Furanone compounds capable of inhibiting bacterial quorum-sensing systems have been isolated from the marine macro alga Delisea pulchra.

Objectives: Two synthetic furanones were tested for their ability to attenuate bacterial virulence in the mouse models of chronic lung infection by targeting bacterial quorum-sensing without directly killing bacteria or inhibiting their growth.

Methods: Study I. Mice with Escherichia coli MT102 [luxR-PluxI-gfp(ASV)] lung infection were injected intravenously with N-acyl homoserine lactones with or without furanones to test the interference of furanones with quorum-sensing. Study II. Mice with lung infection by Pseudomonas aeruginosa PAO1 [dsred, lasR-PlasB-gfp(ASV)] were injected intravenously with furanones to evaluate their inhibiting effects on quorum-sensing. Study III. Mice with P. aeruginosa PAO1 lung infection were treated with different doses of furanones to evaluate the therapeutic effects of furanones on the lung infection.

Results: Furanones successfully interfered with N-acyl homoserine lactone and suppressed bacterial quorum-sensing in lungs, which resulted in decreases in expression of green fluorescent protein. Furanones accelerated lung bacterial clearance, and reduced the severity of lung pathology. In a lethal P. aeruginosa lung infection, treatment with furanone significantly prolonged the survival time of the mice.

Conclusion: Synthetic furanone compounds inhibited bacterial quorum-sensing in P. aeruginosa and exhibited favourable therapeutic effects on P. aeruginosa lung infection.

Keywords: antibiotic resistance, acyl-homoserine lactones, green fluorescent protein, GFP, mouse models


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The therapeutic efficacy of antibiotics correlates closely with their bactericidal or bacteriostatic effects. Bacteria can, however, adapt to the selective pressure from antibiotics via genetic alteration leading to development of antibiotic resistance. The efficiency of many traditional antibiotic treatments is currently decreasing, while the occurrence of multiple antibiotic-resistant pathogenic bacteria is increasing. Pseudomonas aeruginosa is one of the most common Gram-negative bacteria found in association with nosocomial infections and in immunocompromised patients. Patients with cystic fibrosis (CF) are especially susceptible to chronic infection with P. aeruginosa, which is responsible for their high morbidity and mortality.1 Although intensive antibiotic treatment allows 90% of CF patients to survive chronic P. aeruginosa lung infections for more than 10 years, the chronic infection cannot be eradicated by antibiotics.2

Many Gram-negative bacterial species utilize N-acyl homoserine lactone (acyl-HSL)3 molecules as signals to coordinate their population behaviour during invasion and colonization of higher organisms (for reviews, see Refs 3–5). The importance of such quorum-sensing systems in bacterial pathogenesis has been documented for several opportunistic human and plant pathogens, where their virulence has been attenuated in quorum-sensing deficient mutants.69 Bacterial quorum-sensing influences bacterial behavioural processes such as the ability of bacteria to form biofilms.1012 Bacteria in biofilms are resistant to disinfectants, antibiotics, and the action of host immune defences. Biofilm formation plays an important role in bacterial pathogenicity during chronic infections.10,13,14

In this study, we attempted to attenuate the pathogenicity of P. aeruginosa by using signal antagonists called furanone compounds to interfere with the quorum-sensing systems of P. aeruginosa. Furanone compounds are isolated from a marine red macro alga Delisea pulchra and have strong anti-fouling effects in nature.15 As natural furanone compounds have limited effect on P. aeruginosa quorum-sensing systems, we used synthetic derivatives with enhanced quorum-sensing inhibitory properties in our study.16


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

The bacterial strains used in this study were wild-type P. aeruginosa PAO1,17 and Escherichia coli MT102, a restriction minus derivative of MC1000 [araD 139, ara-leu)7697 lac, thi, hsdR].18 Bacterial strains were modified and equipped with reporters in order to monitor the expression of bacterial quorum-sensing in mouse lungs, i.e. PAO1 [dsred, lasR-PlasB-gfp(ASV)], and MT102 [luxR-PluxI-gfp(ASV)].18,19

Synthetic furanones and acyl-HSL molecules

Furanone compound 30 (C30) and compound 56 (C56) were chemically synthesized,16 and kindly provided by Professor Kjelleberg, Centre for Marine Biofouling and Bioinnovation, University of New South Wales, Sydney, Australia. N-3-oxo-hexanoyl-homoserine lactone (3-oxo-C6-HSL) and N-3-oxo-dodecanoyl homoserine lactone (3-oxo-C12-HSL) used in the in vivo study were chemically synthesized, and genially supplied by Professor Paul Williams, University of Nottingham, UK. Furanones and acyl-HSL were stored in ethyl acetate and diluted in 0.9% saline before use (Figure 1).



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Figure 1. Chemical structures of the synthetic furanone compounds (C30 and C56) and the signal molecules (C4-HSL and 3-oxo-C12-HSL) produced by P. aeruginosa.

 
Immobilization of bacteria in alginate beads

P. aeruginosa or E. coli were embedded into seaweed alginate beads as described previously,20 Seaweed alginate was dissolved in 0.9% saline to a concentration of 11 mg/mL and the mixture was autoclaved. One millilitre of an overnight bacterial culture was mixed with 9 mL of sterile seaweed alginate. The mixture was forced with air through a channel into a solution containing 0.1 M CaCl2 and 0.1 M Tris–HCl (pH 7.0) to form beads. The solution containing beads was stirred continuously for 1 h. The alginate beads were then collected by centrifugation at low speed (600–800 r.p.m.), and resuspended in 0.9% saline.20 The suspension of bacterial beads was adjusted to yield the bacterial concentrations necessary for each experiment.

Bacterial inoculation and mouse experiments

All mouse experiments were approved by Dyreforsøgstilsynet (The Danish Ministry of Justice for Animal Experiments Inspectorate). Bacterial inoculations were carried out as described previously.21,22 Briefly, all mice were anaesthetized by subcutaneous injection of a mixture of etomidate (Janssen, Birkerød, Denmark) and midazolam (Roche, Hvidovre, Denmark), 1:1, at a dose of 10 mL/kg body weight. Tracheotomy was then carried out, and 0.04 mL of alginate beads (per mouse) was intratracheally instilled using a curved bead-tipped needle. The incision was sutured with silk and healed without any complications. The mice were killed at different time-points by using 20% pentobarbital (DAK, Copenhagen, Denmark) at a dose of 2 mL/kg body weight.

Study protocols

The animal study included three parts: Study I, furanones interfering with acyl-HSL molecules; Study II, furanones inhibiting P. aeruginosa quorum-sensing; Study III, therapeutic effects of furanones on P. aeruginosa lung infection in mice. Table 1 describes the study protocols.


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Table 1. Protocols of mouse experiments for interference of acyl-HSLs, inhibition of quorum-sensing and treatment of lung infection
 
Administration of furanone compounds and acyl-HSL molecules

In Studies I and II (Table 1), furanones and acyl-HSL molecules were given intravenously (iv) to the mice under anaesthetic. In the furanone treatment experiments, as described in Table 1, the furanone compounds were given to the mice either subcutaneously or orally. The amounts of acyl-HSLs and furanones used in each mouse were decided according to the requirements of the study as well as the body weight of the mouse. Pilot experiments using special bacteria tagged with quorum-sensing reporters in lungs were carried out to determine the appropriate and non-toxic dose which could be used for this experiment.

Freeze microtomy

The lung tissues with pathological changes were embedded with Tissue-Tek and frozen at –20 to –40°C immediately after removal from the mouse thorax. Frozen sections of 40–50 µm thickness were made at different levels of the lung tissues using freeze microtomy.23

Epifluorescence microscopy

An axioplan epifluorescence microscope (Leitz ARISTOPLAN E Camera System, type-307–148.002) was used to observe the bacterial green or red fluorescent protein (GFP or RFP). The microscope was equipped with a 100 W mercury lamp, and filter sets no. 10 (Carl Zeiss) to visualize GFP. A slow-scan charge-coupled device (CCD) camera CH250 (Photometrics, Tucson, AZ, USA) equipped with a KAF 1400 chip (pixel size, 608 x 608 µm), was used for capturing digital images (PMIS). The camera was operated at –40°C, and the chip was read out in 12 bits (4096 intensity levels) at a rate of 200 kHz.

Evaluation of lung pathology

The qualitative analysis of macroscopic lung pathology included abscess, consolidation, atelectasis and haemorrhage. The quantitative analysis of the macroscopic lung pathology was expressed as the lung index of macroscopic pathology (LIMP = the lung area with pathologic changes divided by the area of the whole lung).9,24 The lung tissues with pathologic changes were cut into 5–10 µm sections, and stained with haematoxylin–eosin (HE) to evaluate the severity of microscopic pathology by light microscopy. The evaluation was done blindly to avoid bias.

Detection of lung bacteriology

Lung samples were prepared for quantitative bacteriological examination as previously described.25 The lungs were removed and immediately put into sterile containers, which were then stored at 4°C for less than 2 h before homogenization. The lung samples were homogenized in 5 mL of cold sterile phosphate-buffered saline at 4°C. Appropriately diluted samples were plated on agar plates to determine the cfu per lung after 20–24 h of incubation at 37°C.

Statistical analyses

The categorical data were analysed by the {chi}2 test. Both Mann–Whitney U-test and analysis of variance (ANOVA) were used to compare the data obtained from groups in this study. Correlation between two parameters was analysed by simple regression. Statistical analyses were carried out using Statview, a computer program for statistics, from Abacus Concepts, Inc. (www.abacus.com).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The tested dosages of furanone C30 and C56 used in this study did not inhibit bacterial growth in vitro (data not shown), in concordance with other reports.19,2629

Study I. Synthetic furanones interfered with acyl-HSL molecules in mouse lungs

E. coli MT102 carrying the luxR-PluxI-gfp based quorum-sensing cassette has been shown to express GFP in response to acyl-HSL,18,23 and was introduced intratracheally to mouse lungs with an inoculum of 4 x 107 cfu/mouse (Figure 2a). 3-oxo-C6-HSL (14 µg/g), injected iv into mice, successfully activated luxR-PluxI-gfp expression (Figure 2b). In contrast, 3-oxo-C12-HSL, injected iv into mice, failed to induce any expression of GFP, even at a concentration of 160 µg/g, which is more than 10 times higher than the functional dosage of 3-oxo-C6-HSL (data not shown). Actually, only 4 µg/L of 3-oxo-C12-HSL is sufficient to light up the luxR-PluxI-gfp sensor in liquid medium in vitro.23 This indicates that the long-chain 3-oxo-C12-HSL did not diffuse well through the wall of the blood vessels and lung tissues, or that special active influx and efflux channels are needed for its transportation.30



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Figure 2. Photomicrographs showing the interaction of C56 with 3-oxo-C6-HSL using E.coli MT102 [luxR-PluxI-gfp(ASV)] after a 2 h exposure in a mouse model of lung infection. Upper panel: reflection images; Lower panel: epifluorescence images. (a) Control (saline only); (b) 14 µg/g of 3-oxo-C6-HSL only; (c) 3.5 µg/g of furanone C56 plus 14 µg/g of 3-oxo-C6-HSL; (d) 3.5 µg/g of furanone C56 plus 56 µg/g of 3-oxo-C6-HSL.

 
Furanone C56 (3.5 µg/g), co-administered iv with 3-oxo-C6-HSL (14 µg/g), inhibited the expression of GFP from the luxR-PluxI-gfp cassette (Figure 2c). The inhibition exerted by C56 was removed by increasing the dosage of 3-oxo-C6-HSL from 14 to 56 µg/g (Figure 2d). In the C30 experiment, co-administration of C30 in 0.35 µg/g with 3-oxo-C6-HSL (14 µg/g) showed significant inhibition of GFP, which was 10-fold lower than the dose of C56 used. Similarly, the inhibition of GFP was released by increasing the doses of 3-oxo-C6-HSL (data not shown). The results showed that furanones interfered with quorum-sensing but did not kill the bacteria since they expressed GFP again by increasing the concentration of 3-oxo-C6-HSL molecules. It also suggested that the synthetic furanones were transported by blood circulation to the lungs, penetrated the lung tissues, entered the bacteria and inhibited 3-oxo-C6-HSL-dependent gene expression, which is dependent on the amount of quorum-sensing signals produced.

Study II. Synthetic furanones inhibited P. aeruginosa quorum-sensing in mouse lungs

The experiments were carried out by infecting mice with P. aeruginosa PAO1 [dsred, lasR-PlasB-gfp(ASV), 2 x 107 cfu/mouse]. The infection was established for 24 h, followed by iv injection of furanone C56 (17 µg/g) through the tail vein. Over a time-span of 4–6 h after the administration of C56, the expression of lasR-PlasB-gfp in PAO1 was significantly reduced (Figure 3), whereas 8 h after the injection of C56, GFP expression reappeared. These results indicate that C56 inhibited the expression of lasR-PlasB-gfp and later, when C56 was gradually cleared, the lasR-PlasB-gfp expression recommenced (Figure 3). A lower C56 concentration (5 µg/g) did not inhibit the expression of lasR-PlasB-gfp (data not shown).



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Figure 3. Photomicrographs showing the inhibition of furanone C56 (17 µg/g) in P. aeruginosa quorum-sensing in a mouse model of lung infection. Upper panel: a dsred expression cassette provides a red fluorescent tag on the bacteria for easy identification in tissue samples. Lower panel: the PAO1 contains a lasR-PlasB-gfp(ASV) reporter fusion for detection of PAO1 quorum-sensing.

 
In another experiment, C30 at either 1 or 2 µg/g, injected iv into mice, the dosage of C30 being 17-fold lower than that of C56, significantly inhibited the expression of lasR-PlasB-gfp in PAO1 (data not shown). It is known that C30 is a more efficient quorum-sensing inhibitor than C56 in conditions in vitro.28 Our data indicate that this conclusion can be extended to the conditions in vivo as well.

Study III. Therapeutic effects of synthetic furanones on P. aeruginosa lung infection

Based on the above results, we examined the therapeutic effects of furanones on P. aeruginosa lung infection in mice by feeding with C56 or by subcutaneous injection with C30.

Survival time

In general, the mortality of the infected mice depends on the concentration of inoculum. With an inoculum of 1.0 x 107 cfu/mouse, PAO1 could cause lung infection of mice without killing them. However, when the inoculum was increased to 2.0 x 107 cfu/mouse, 88% of the mice (15 out of 17) in the placebo group died from the lethal lung infection 48 h post-inoculation. For the furanone C56 (5 µg/g) treated mice, only 55% (10 out of 18) died, which is significantly lower than the placebo control (P < 0.03). Although none of the mice survived 72 h post-inoculation, the results indicate that C56 treatment significantly prolonged the survival time of the mice in the lethal infection.

Clearance of bacteria from the lungs

In the following experiments, all mice were infected with a sub-lethal dose of P. aeruginosa PAO1 (1 x 107 cfu/mouse). In the C56 treatment experiments, the mice received C56 (5 µg/g) or 0.9% saline immediately post-inoculation by gastric intubation, thrice a day for 3 days. The mice were killed 1 day after the treatment had stopped, i.e. on day 4 post-inoculation. The median of lung bacterial cfu in the C56-treated group was only one-fifth of that in the placebo group, and the difference is significant (P < 0.05) (Figure 4), indicating that C56 could influence the colonization and persistence of P. aeruginosa in the lungs. In C30 treatment experiments, mice were injected subcutaneously with different concentrations of C30, i.e. 0.25 (low), 0.5 (medium) and 0.75 (high) µg/g, or 0.9% saline immediately post-inoculation, thrice a day for 3 days, and killed on day 7 post-inoculation. The value of the lung bacterial cfu in the high dosage treatment group is approximately 1000 times lower than the values of the lung bacterial cfu in the low dosage treatment (P = 0.01) and the placebo (P = 0.0035) groups (Figure 5). The lung bacterial cfu in the medium dosage treatment group was also significantly lower than that in the placebo group (P < 0.04). In contrast, the value of lung bacterial cfu in the low dosage treatment group did not differ significantly from the value of lung bacterial cfu in the placebo and the medium dosage treatment groups, and the difference between the values of lung bacterial cfu in the medium and high dosage treatment groups is also not significant (Figure 5). Regression analysis shows a clear negative correlation between the bacterial lung cfu and the concentrations of C30 (r = –0.456, P < 0.0001).



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Figure 4. Effect of C56 (5 µg/g) on bacterial clearance in a mouse model of P. aeruginosa lung infection. The box-plots show the median and range. Lung bacterial cfu is lower in the treated group (P < 0.05).

 


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Figure 5. Effect of different concentrations of C30 on lung bacterial clearance in a mouse model of P. aeruginosa lung infection. The box-plots show the median and range.

 
Macroscopic lung pathology

In concordance with the results of the lung bacteriology study, milder lung pathology was also observed for the furanone-treated groups. Much milder lung pathology was found in the C56-treated group than the placebo group (P = 0.002) (Figure 6). The evaluation carried out on day 4 post-infection indicated that C56 significantly restricted the lung pathologic changes to a smaller area during the acute inflammation.



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Figure 6. Effect of furanone C56 (5 µg/g) on macroscopic lung pathology in a mouse model of P. aeruginosa lung infection. Macroscopic lung pathology is expressed as lung index of macroscopic pathology (LIMP, median and range).

 
In C30 treatment experiments, a longer observation period was chosen to allow lung abscess formation in the animals, and the evaluation was carried out on day 7 (3 days later than in the experiments with C56). A significantly higher frequency of lung abscesses was noted in both the placebo and the low dosage treatment groups (P < 0.04) than in the medium and high dosage treatment groups, in which 80% of the mice showed lung atelectasis and only 20% of mice showed lung abscesses. In contrast, the incidence of lung abscesses was 64% in both the placebo and low dosage treatment groups, and this value is significantly higher than the value obtained for the medium and high dosage treatment groups (P < 0.04) (Figure 7).



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Figure 7. Effect of C30 on frequencies of lung abscesses in a mouse model of P. aeruginosa lung infection.

 
Microscopic lung pathology

In the C30 treatment experiments, lung abscesses with significant tissue damage were found mainly in the placebo and the low dosage treatment groups (Figure 8a). In the medium or high dosage treatment group, the polymorphonuclear leucocyte (PMN) infiltration was limited mostly to the bronchia with a low density of inflammatory cells, and chronic inflammation was a predominant change in the lung tissues (Figure 8b and c). In the placebo and the low dosage treatment groups, a significantly higher density of PMNs with haemorrhage was found in both bronchia and lung tissues indicating a severe acute inflammation (Figure 8a and c).



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Figure 8. Effect of C30 on microscopic lung pathology in a mouse model of P. aeruginosa lung infection. (a) Photomicrographs of lung section from a placebo control mouse indicating a clear lung abscess (shown in circle, x25) and the polymorphonuclear leucocytes (PMNs) or pus cells along the abscess wall (x400, HE staining). (b) Lung section from a furanone C30-treated mouse indicating that the PMN infiltration in the lung focus is not significant and the lung tissues exhibit a chronic inflammation (centre: x100, right: x400, HE staining). (c) The lung sections show the infiltration of PMNs within the bronchia: Left picture is from a mouse lung without furanone treatment; high density of PMNs in the bronchia and slight lung haemorrhage are seen. Right picture is from a mouse lung with furanone C30 treatment; much lower density of PMN infiltration is found in the bronchia (x100, HE staining).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study, two different quorum-sensing inhibitors (C30 and C56) were used, which showed rather similar effects, although the purpose of this study was not to compare these inhibitors. Our results showed that the synthetic furanones C30 and C56 interfered with the actions of acyl-HSL molecules and inhibited gene expression regulated by quorum-sensing systems in P. aeruginosa in vivo. Furthermore, C30 and C56 also exerted favourable therapeutic effects on P. aeruginosa lung infections in mice. Although our results did not demonstrate directly how the synthetic furanones inhibit quorum-sensing in vivo, they showed that C30 and C56 acted in a dosage-dependent manner. Synthetic furanones significantly increased the survival time of mice with lethal P. aeruginosa infections, assisted bacterial clearance by the host, and reduced the lung pathology (Figures 48), thus indicating that they could function as acyl-HSL antagonists and as quorum-sensing inhibitors in vivo (Figures 2 and 3), in concordance with the findings reported from studies in vitro.19,27 All these observations demonstrate that bacterial virulence can be partially attenuated by inhibition of quorum-sensing.

Synthetic furanones could efficiently inhibit the expression of luxR-PluxI-gfp(ASV) in bacterial reporter, in which the production of GFP is inducible by acyl-HSL molecules. The tested concentrations of furanones interfering with 3-oxo-C6-HSL molecules did not kill the bacterial reporters because the bacteria expressed GFP again when the concentration of 3-oxo-C6-HSL was increased (Figure 2). The concentration-dependent manner of furanone action supported the view that furanones could displace 3-oxo-C6-HSL signals from the LuxR protein.26 Attenuation of bacterial virulence rather than killing the pathogen might become a new concept for control of bacterial infections. This mode of action might not impose a selective pressure for the development of bacterial resistance to the antagonist.

The synthetic furanone C56 has been shown to specifically repress the expression of lasR-PlasB-gfp, controlled by PAO1 quorum-sensing system, in PAO1 without affecting the bacterial growth rate and the general protein synthesis, and to reduce the production of several virulence factors in vitro.19 In this study, the synthetic furanones C30 and C56 successfully suppressed the transcription of lasR-PlasB-gfp in mouse lungs. Higher furanone concentrations showed more efficient inhibition of the transcription of lasR-PlasB-gfp. C56, at 5 µg/g, did not show the inhibition of GFP synthesis in mouse lungs (data not shown); however, at 17 µg/g, C56 inhibited it (Figure 3). In contrast, C30 at 1 and 2 µg/g, inhibited lasR-PlasB-gfp transcription in mouse lungs. The mice injected with the higher dosage of C30 (2 µg/g) exhibited much weaker GFP signals, indicating stronger inhibition.

Our results showed that mice treated with furanones carried much lower bacterial number in lungs (Figures 4 and 5), indicating that the ability of the bacteria to colonize the host may be reduced owing to the inhibition of quorum-sensing systems or because the bacterial clearance in the hosts is improved by furanones. The effect of furanones on bacterial clearance correlated negatively with the furanone concentrations, which further confirmed that the action of furanones was dosage-dependent. Furanones could interfere with acyl-HSL signals that may influence the type of host immune responses and reduce the inflammation in vivo since 3-oxo-C12-HSL, an important quorum-sensing signal molecule produced by P. aeruginosa, has been shown to have immunomodulatory and inflammatory effects.31,32

Our results showed that furanones inhibited bacterial quorum-sensing by interfering with acyl-HSL signals, and simultaneously reduced the severity of the lung infection. Consequently, the inhibition of quorum-sensing attenuates the virulence of the pathogen and impairs its colonization ability. In conclusion, synthetic furanone compounds appear to be promising novel antimicrobial agents that possess solely quorum-sensing inhibition and sub-MIC properties and lead to increased clearance of the bacteria from the lungs and decreased lung pathology. This agrees with the recent proposal that quorum-sensing is a target for treatment of Gram-negative bacterial infections.33,34


    Acknowledgements
 
We thank Ms Jette Pedersen for her expert assistance in histopathological examination. We are grateful to the University Hospital of Copenhagen (Rigshospitalet) for the financial support to H.W. This work was also supported by grants from the Danish Technical Research Council, the Danish Medical Research Council, the Plasmid Foundation, and the Villum Kann-Rasmussen Foundation to M.G. We are also very grateful to Professor Koh Chong-Lek for kindly checking the English of the manuscript.


    Footnotes
 
* Corresponding author. Tel: +45-35456428; Fax: +45-35456412; E-mail: wuhong{at}get2net.dk Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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