1 Centre for Biomedical Microbiology, BioCentrum, Technical University of Denmark, DK-2800 Lyngby, Denmark
2 Department of Clinical Microbiology, Rigshospitalet, DK-2100 Copenhagen Ø, Denmark
3 Carlsberg Research Center, Biosector, Gamle Carlsberg Vej 10, DK-2500 Valby, Denmark
4 Institute of Forensic Medicine, University of Copenhagen, DK-2100 Copenhagen Ø, Denmark
5 Bartholin Instituttet, Kommune Hospitalet, Copenhagen, Denmark
6 Department of Microbiology, University of Zürich, CH-8008 Zürich, Switzerland
Correspondence
Michael Givskov
immg{at}pop.dtu.dk
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ABSTRACT |
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INTRODUCTION |
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We have previously demonstrated that a crude extract of garlic specifically inhibits QS-regulated gene expression in P. aeruginosa, as judged from DNA microarray-based transcriptomic analysis (Rasmussen et al., 2005). The expression of 167 genes, 92 of which were regulated by QS, was repressed by the extract (Rasmussen et al., 2005
). The mechanism by which garlic compounds block QS is presently unknown. However, the effect is at a post-transcriptional level, since the amounts of mRNA of neither lasI, lasR, rhlI nor rhlR (the key components of the Las and Rhl QS communication systems in P. aeruginosa) were notably affected by the garlic treatment (Rasmussen et al. 2005
; unpublished results). This suggests that the QSI molecules interact directly with the QS receptors by either a competitive or a non-competitive N-acylhomoserine lactone mechanism. The garlic extract used in this study contained very low amounts of toxic compounds (such as allicin) (Rasmussen et al., 2005
). Treatment of in vitro biofilms with this extract was found to dramatically reduce the tolerance of the bacteria to the antibiotic tobramycin (Rasmussen et al., 2005
). In addition, the extract was capable of attenuating bacterial virulence in a Caenorhabditis elegans nematode infection model (Rasmussen et al., 2005
). We have previously demonstrated that the QS system is in fact a useful drug target, and delivered a proof of concept in which we demonstrated that the blockade of QS with the drug C-30 represents an effective approach for interfering with biofilm tolerance to antibiotic treatments and for attenuating the virulence of P. aeruginosa in a mouse infection model (Wu et al., 2004
; Hentzer et al., 2003
). Recently we have shown that the activation of polymorphonuclear leukocytes (PMNs) is blocked by P. aeruginosa QS signals in vitro (Bjarnsholt et al., 2005
). In addition, QS mutants cause a faster activation of the host defence system in vivo (Bjarnsholt et al., 2005
; Wu et al., 2001
). These factors might account for the rapid clearing of bacteria deficient in QS (either by inactivating mutations or by treatment with QS-blocking drugs) as observed in mouse models of P. aeruginosa pulmonary infections (Wu et al., 2001
, 2004; Hentzer et al., 2003
; Bjarnsholt et al., 2005
).
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METHODS |
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Preparation of garlic extract.
The garlic extract used in all the experiments was prepared, evaluated and found to have no growth inhibitory effect on P. aeruginosa, as described previously (Rasmussen et al., 2005).
Biofilms.
Biofilms were cultivated in continuous-culture once-through flow chambers, perfused with sterile ABtrace minimal medium containing 0·3 mM glucose as described previously (Christensen et al., 1999). The pump was set to 3 ml h1. Garlic extract (2·0 %, v/v) was added to medium as appropriate. The concentration of garlic extract used was the concentration that inhibits QS the most without inhibiting growth. The preparation and concentration calibration of the garlic extract was performed as described previously (Rasmussen et al., 2005
). Biofilm development was examined by scanning confocal laser microscopy (SCLM) using a Zeiss LSM 510 system equipped with an argon laser and a helium-neon laser for excitation of fluorophores. Simulated projections and vertical cross-sections of the biofilms were generated with the Imaris software package (Bitplane). Images were further processed with Photoshop software (Adobe).
Preparation of PMNs.
Human blood samples were obtained from normal healthy volunteers, by venous puncture, and collected in BD vacutainers coated with heparin and lithium (Becton-Dickinson, 388330). The blood was mixed with dextran (T-500) 1 : 5 and the erythrocytes were sedimented for 40 min. The supernatant was applied to Lymphoprep (Axis-Shield) and centrifuged at 2200 r.p.m. for 15 min at 5 °C. The supernatant was discarded and the neutrophils were treated with 2 ml 0·2 % NaCl in order to lyse remaining erythrocytes. Lysis was terminated by adding 2 ml 1·6 % NaCl and 6 ml Eagle-MEM (Bie & Berntsen). The cells were centrifuged at 350 g for 10 min at 5 °C, the supernatant was discarded and the PMNs were resuspended in Eagle-MEM.
PMN treatment of biofilms.
In order to inoculate PMNs into the biofilm chambers, the flow was stopped and the flow cells clamped off. PMNs in the order of 1·5x106 were inoculated in each flow channel. The flow cells were incubated top down in a 37 °C water bath, with shaking, until microscopic inspection.
Monitoring the oxidative burst of PMNs.
The PMNs were incubated for approximately 30 min in Eagle-MEM (10x106 cells ml1) with 10 % normal human AB serum, 5 µM SYTO 62 (Molecular Probes), to stain the nuclei (dsDNA), and 0·1 mg 123-dihydrorhodamine ml1 (Sigma), to stain the H2O2 in the phagosomes (Bassøe et al., 2003). A 100 µl quantity of the PMN mixture was added to each biofilm.
Animals.
Female BALB/c mice were purchased from M & B Laboratory Animals at 10 to 11 weeks of age. The mice were of equal size and were maintained on standard mouse chow and water ad libitum for one week before challenge. All animal experiments were authorised by the National Animal Ethics Committee.
Immobilization of P. aeruginosa in seaweed alginate beads.
Immobilization of P. aeruginosa in seaweed alginate beads was performed as described by Pedersen et al. (1990). The suspension was adjusted to 2·5x108 c.f.u. ml1, confirmed by colony counts, and 0·04 ml of the suspension was instilled in the left lung of each mouse.
Garlic extract administration.
Each mouse was treated with 1·5 % garlic extract (kg body weight)1. The garlic extract (0·3 ml) was mixed with 0·7 ml 0·9 % NaCl and injected subcutaneously. The control groups had 1 ml 0·9 % NaCl injected subcutaneously. The mice were treated two days prior to the bacterial challenge. The treatment was continued until the mice were sacrificed.
Challenge procedure.
The mice were anaesthetized by subcutaneous injection of 0·2 ml Hyp/Mid [2·5 mg Hypnorm ml1 (Janssen) and 1·25 mg midazolam ml1 (Roche) in sterile water]. Sedated mice were fixed and their tracheas were exposed and penetrated with an 18 G needle. The inoculum was installed in the left lung approximately 11 mm from the tracheal penetration site with a curved bead-tipped needle (Moser et al., 1997). The incision was sutured with silk and healed without any complications. Pentobarbital (DAK), 2·0 ml (kg body weight)1, was used to sacrifice the animals. The mice were sacrificed at different time points after infection as mentioned in Results and Discussion.
Bacteriology.
Lungs from mice were prepared for bacteriology examination as described by Moser et al. (1997). Isolated lungs were homogenized on ice. A serial dilution of the lung homogenate was performed and plated on blue agar plates (States Serum Institute), containing selective culture medium for Gram-negative bacilli, for colony counting.
ELISA measurements of cytokines and proteins.
The concentration of the cytokines and proteins was measured in supernatant from lung homogenate by ELISA kits (G-CSF and MIP-2; R&D systems) (GM-CSF, TNF-, IL-6; Becton Dickinson) (Myeloperoxidase, MPO; HyCult Biotechnology). The measurements were performed according to the manufacturer's recommendations.
Histopathology.
For examination of histopathology, the isolated lungs were immediately fixed in PBS with 4 % paraformaldehyde and kept at 5 °C before further preparation.
By microscopy, slides with two tissue sections mounted on each were used for evaluation. Based on an overview at low magnification (x62·5), and detailed studies of five different areas of each tissue section under high magnification (x500), the degree of inflammation was scored as 0 (no inflammation), 1 (mild inflammation), 2 (moderate to severe inflammation) and 3 (severe inflammation with necrosis or with severe inflammation throughout the lung). The histopathological evaluation was done blindly.
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RESULTS AND DISCUSSION |
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Clearance of the infecting bacteria was assessed in a similar experimental setup, but with a reduced bacterial challenge (1x107 c.f.u. per lung). Garlic and placebo treatments were given as described for the previous experiment. Mice were sacrificed on days 1, 3 and 5 post-challenge, and the bacterial content in the lungs of the animals was determined. On average, the garlic-treated group displayed three orders of magnitude fewer c.f.u. per lung. On day 5, the lungs of 9/10 mice in the garlic-treated group were sterile, whereas the placebo group still contained a mean of 1x105 c.f.u. per lung (Fig. 3).
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We also detected a significantly lower production of G-CSF in the garlic-extract-treated group of mice on day 1 (P<0·04) and day 3 (P<0·05). G-CSF, being a major mobilizer of PMNs, contributes to impaired lung function during the chronic P. aeruginosa lung infection in CF patients (Jensen, 2003). A reduction in G-CSF was shown to correlate with improved lung function. We link the reduced G-CSF production in the garlic-extract-treated group of mice to an adequate immune response brought about by the blocked QS system.
The higher levels of both MIP-2 and G-CSF seen in the untreated group of mice compared to the treated group of mice correlate to the state of chronic infection observed in CF patients. MPO was measured to determine the amount of PMNs present in the lungs (data not shown). No difference in MPO level was determined between the two groups, indicating that the same amount of PMNs is present in the lung homogenate despite the treatment. Since bacteria are cleared in the treated group, and we detect more PMNs inside the lungs, we hypothesize that QS signals might function to block the influx of PMNs into the lung. The extravasation of PMNs is possibly reduced in the untreated group and the PMNs are stuck to endothelial cells where they cause collateral damage to the lung tissue.
The above cytokines were also measured in uninfected, garlic-treated mice, as well as uninfected, saline-treated mice. Neither saline nor garlic treatment alone leads to changes in cytokine production (data not shown). Similarly, neither inflammatory nor pathological changes were observed (data not shown).
Conclusion
The present report demonstrates that a QS-blocking garlic treatment is a possible approach to the attenuation of virulence and control of P. aeruginosa infections. Studies carried out at the Danish CF Center in Copenhagen (Ciofu et al., 1994) have shown the development of bacterial resistance to antibiotics to be a serious side-effect of the current anti-pseudomonal treatment, and it highlights the importance of the development of therapies that limit the formation of persistent biofilms in the lungs. A balance between colonization and clearance causes the slow development of chronic P. aeruginosa infection. When the balance is pushed in the direction of colonization, the biofilm mode of growth protects the colonizing bacteria from the host defence system and increases the tolerance to antibiotics. It is therefore interesting that QS-blocking drugs that efficiently eradicate pulmonary infections, in our animal model, also enable a more appropriate PMN response in the presence of P. aeruginosa biofilms. The administration of such drugs is expected not only to lead to the development of less persistent biofilms but also to inhibit the expression of bacterial virulence determinants that actively degrade components of the defence system (Kharazmi et al., 1986
). Taken together with the synergistic effect of QS blockage and PMN activation, this might suffice to reverse the delicate balance in favour of the host clearance mechanism, and thereby reverse the severity of infection and improve the lung function. For younger CF individuals without chronic P. aeruginosa infections, an early prophylactic treatment based on these drugs might prevent the formation of persistent and damaging biofilms in the lung. However, in order to achieve the garlic-extract dose used for the treatment of the infectious mouse model, an 80 kg person would have to ingest 50 whole bulbs of garlic every day! Work is currently in progress to isolate and characterize the pure compounds responsible for blocking the P. aeruginosa QS systems.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 9 February 2005;
revised 14 July 2005;
accepted 18 August 2005.
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