Department of Genetics and Microbiology, University of Geneva, CMU, 1, rue Michel-Servet, CH-1211 Geneva 4, Switzerland
Received 6 February 2003; returned 6 April 2003; revised 19 June 2003; accepted 30 June 2003
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Abstract |
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Methods: We used isogenic mutants of wild-type PAO1 strains PAO-BI and PT5 in a static biofilm model. Biofilm formation was quantified using Crystal Violet staining and exopolysaccharide measurements.
Results: Wild-type strain PAO-BI, as a result of its reduced C4-HSL secretion, produced 40% less biofilm compared with the wild-type PAO1 strain PT5. Using isogenic mutants of strain PT5 we have shown that whereas a lasI mutant (deficient in 3-oxo-C12-HSL) produced similar amounts of biofilm to the wild-type, a rhlI mutant (deficient in C4-HSL) produced 70% less biofilm. In the latter strain, biofilm formation could be restored by addition of exogenous C4-HSL. Azithromycin, known to reduce the production of both 3-oxo-C12-HSL and C4-HSL, inhibited biofilm formation of wild-type PT5 by 45%. This inhibition could be reversed by the addition of both cell-to-cell signals.
Conclusions: Our results indicate that C4-HSL also plays a significant role in biofilm formation. Furthermore, we demonstrate the potential of using cell-to-cell signalling blocking agents such as azithromycin to interfere with biofilm formation.
Keywords: quorum sensing, autoinducers, homoserine-lactones, 3-oxo-C12-HSL
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Introduction |
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It has been recently understood that wild-type PAO1 strains from various laboratories might differ in cell-to-cell signal production.14 In particular, strain PAO-BI produces less C4-HSL than other PAO1 strains.14 We have recently described that the macrolide antibiotic azithromycin inhibits virulence factor production of P. aeruginosa by interfering with the cell-to-cell signalling circuit.15 We wondered whether the reduced production of C4-HSL by strain PAO-BI might have affected previous biofilm studies using this strain as a control strain, and whether azithromycin affects biofilm formation by interfering with AHL production.
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Materials and methods |
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Unless otherwise stated, bacteria were freshly streaked from frozen stocks onto LB-agar medium supplemented with antibiotics when appropriate. Liquid LB cultures, containing antibiotics when appropriate, were grown overnight at 37°C with agitation (250 rpm). Minimal AP medium supplemented with 0.3 M NaCl was used for biofilm growth experiments.16 Antimicrobials were supplied at the following concentrations: gentamicin (15 mg/L), tetracycline (50 mg/L), mercury chloride (12.5 mg/L). All chemicals and antimicrobials were purchased from Sigma, except azithromycin which was kindly provided by Pfizer. The bacterial strains and their genetic linkage are presented in Table 1. To construct strain PT918 (PAO-BI mexE::Hg), the mexE::
Hg mutation from strain PT12117 was transferred by transduction using phage E79tv218 into strain PAO-BI.
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For extraction of autoinducers from liquid cultures, bacterial strains were grown in LB medium until they reached stationary phase (6 h). Aliquots of filtered culture supernatants were subjected to two extractions by add-ition of one volume of acidified ethyl acetate (0.01% acetic acid). For extraction of autoinducers from biofilms, polyvinyl chloride (PVC) fragments on which biofilms had been formed were rinsed three times and immersed into 1 mL of 0.9% NaCl. One volume of acidified ethyl acetate was added and the mixture incubated in closed tubes at 4°C overnight. A second extraction was carried out for 1 h, and the two extracts were pooled. The extracted autoinducers were quantified using specific bioassays using E. coli MG4I14 (pPCS1) for 3-oxo-C12-HSL and P. aeruginosa PAO-JP2 (pECP61.5) for C4-HSL as reporter strains.19 ß-Galactosidase (ß-gal) activity was determined as previously described.20
Static biofilm formation assay
Sterile PVC strips of approximately 1 cm2 were cut from original endotracheal devices (Mallinckrodt Inc., St Louis, MO, USA) and biofilm was allowed to develop under static conditions for 3 days as previously described.21 All PVC strips were weighed and experiments were standardized by weight. Bacterial strains were grown in LB medium for 6 h with agitation at 37°C. PVC strips were immersed in the bacterial culture medium for 1 h without agitation to allow bacteria to adhere. PVC strips were then transferred into AP medium and incubated for 3 days without agitation at 37°C. Azithromycin (2 mg/L) and/or autoinducers (10 µM each, once a day) were added as indicated. After the 3 day incubation period, the PVC strips were individually washed twice in 0.9% NaCl and subjected to biofilm measurements (see below). We had previously observed that initial growth of wild-type PT5 was slightly retarded in the presence of 2 mg/L azithromycin when grown under agitation in liquid media.15 This effect was restricted to early and mid-exponential growth phases, when quorum sensing is not yet active. In order to determine whether this small delay could affect final growth in our 3 day static biofilm assay, we measured colony forming units (cfu) of strain PT5 grown without agitation after 3 days in the biofilm medium in the presence and absence of 2 mg/L azithromycin. Azithromycin did not affect the number of viable counts of wild-type strain PT5.
Crystal Violet staining and exopolysaccharide determinations
PVC strips, on which biofilm had developed, were incubated for 15 min in a Crystal Violet staining solution (0.1% in distilled water),22 and washed three times in distilled water. The stain was then dissolved in ethanol and absorbance was measured at 570 nm as previously described.22
Exopolysaccharide determinations were carried out by total carbohydrate assays.23 PVC strips were washed in 0.9% NaCl, and incubated in an equal volume of 5% phenol to which 5 volumes of H2SO4 containing hydrazine sulphate was added. The mixture was incubated for 1 h in the dark and absorbance was measured at 490 nm.
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Results and discussion |
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Most in vitro studies have investigated the potential role of cell-to-cell signals in the formation of P. aeruginosa biofilm using a continuous flow system.9,10,24,25 In such bioreactors a constant medium flux brings new nutrients and eliminates metabolic products. These experimental conditions are therefore very different from in vivo situations such as biofilm formation on endotracheal intubation devices. Indeed the bacteria growing inside the secretions that accumulate above the cuff of intubation devices are in static growth conditions without regular change of their growth medium. We therefore decided to adopt a static in vitro biofilm model21 using sterile PVC strips derived from original intubation devices. Previous experiments, using continuous flow systems, have shown that the total amount of biofilm formed by the lasI mutant PAO-JP1 is not reduced compared with its parent strain.9,25 As experimental conditions greatly influence biofilm formation,25 we first compared biofilm formation by mutant PAO-JP1 and its parent strain the PAO1 wild-type strain PAO-BI in our static biofilm assay. Biofilm measurements using Crystal Violet staining gave similar results for both strains (Figure 1a), confirming that the lasI mutation does not affect total biofilm for-mation in our biofilm model.
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When biofilm production was assessed, the lasI mutant PT466 produced slightly more biofilm than strain PT5 (Figure 1b). In contrast, the biofilm formed by the rhlI mutant PT454 was reduced by 70% compared with its parent strain (Figure 1b), correlating with the reduction in biofilm formed by the rhlI mutant PDO100 (Figure 1a). Furthermore, the double lasI/rhlI mutant PT502 formed a similar amount of biofilm as the rhlI mutant PT454 (Figure 1b). These results indicate that C4-HSL is required for full biofilm formation. To confirm this hypothesis, we complemented mutant PT454 by adding exogenous C4-HSL during the biofilm formation assay. As shown in Figure 1(b), addition of exogenous C4-HSL partially restored the biofilm formation of rhlI mutant PT454 from 30% to 65% of the wild-type PT5 level. Similarly, in the presence of both exogenous AHLs, the biofilm formed by mutant PT502 increased from 30% to 75% of the PT5 level (Figure 1b). The lack of complete complementation of both mutants through addition of a constant concentration of exogenous autoinducers to the biofilm assay is likely to result from suboptimal AHL concentrations as these vary both in time and space during biofilm formation.10 We do not believe that secondary mutations in vfr and algC in our cell-to-cell signalling mutants could be responsible for the lack of complete complementation. Mutations in these genes affect twitching motility, which is cell-to-cell signalling in-dependent.27,28 As twitching motility did not differ between parent strain PT5 and its derived cell-to-cell signalling mutants PT466, PT454 and PT502 (data not shown), such secondary mutations are unlikely to have occurred in our strains.
These results clearly demonstrate that, at least under our experimental conditions, C4-HSL is required for optimal biofilm formation by P. aeruginosa. Indeed it appears that a previously undiscovered reduced expression of rhlI, leading to lower C4-HSL production, in the wild-type strain PAO-BI, is responsible for an underestimation of the role of this autoinducer in biofilm formation. The importance of C4-HSL is indeed not visible when PAO-BI is used as the control strain,9 or when carrying an rhlI reporter fusion.10 In contrast, the importance of this autoinducer becomes apparent when both the parent and the rhlI mutant do not overexpress the MexEF-OprN efflux system. These results correlate well with the recent observation that the rhl system is activated during the maturation stage of biofilm development.29 It appears that while the las system (LasR/ 3-oxo-C12-HSL) is essential for biofilm differentiation,9 and plays a role during the irreversible attachment stage,29 the rhl system (RhlR/C4-HSL) also plays an important role.
The finding that P. aeruginosa strains overexpressing the MexEF-OprN multidrug efflux system are impaired in biofilm formation is interesting. Such strains can be selected during quinolone treatment in an animal model,30 and are less virulent in a rat model of acute pneumonia.31 Whether such multidrug-resistant isolates could be selected during antimicrobial treatment of CF patients, and affect the evolution of the disease deserves further investigation.
Azithromycin inhibits biofilm formation by reducing cell-to-cell signalling
The potential clinical value of antibacterials that would control and/or prevent P. aeruginosa infections by interfering with cell- to-cell signalling has recently been underlined.2,32 The macrolide antibiotic azithromycin is neither bactericidal nor bacteriostatic against P. aeruginosa (MIC 64128 mg/L) at achievable tissue concentrations (14 mg/L).33,34 However, even at low concentrations (12 mg/L) azithromycin reduced the adherence of P. aeruginosa to polystyrene,35 as well as the production of exopolysaccharides by P. aeruginosa, and has therefore been suggested to inhibit biofilm formation.36 We have recently shown that 2 mg/L azithromycin inhibits the production of both 3-oxo-C12-HSL and C4-HSL by P. aeruginosa.15 In view of the above presented results we wondered whether azithromycin inhibits biofilm formation by interfering with autoinducer synthesis. In the presence of 2 mg/L azithromycin, the biofilm formed by strain PT5 was reduced by 45% as quantified by Crystal Violet staining (Figure 2a). This decrease was associated with a 50% reduction in exopolysaccharide production as measured by the total carbohydrate assay. In contrast 2 mg/L azithromycin did not affect biofilm production by mutant PT502 (27% of wild-type in the absence versus 25% in the presence of 2 mg/L azithromycin), suggesting that azithromycin exerts its effect on biofilm formation via inhibition of cell-to-cell signalling.
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Acknowledgements |
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Footnotes |
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Corresponding author. Tel: +41-22-702-56-39; Fax: +41-22-702-57-02; E-mail: Christian.VanDelden{at}medecine.unige.ch
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References |
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![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Van Delden, C. & Iglewski, B. H. (1998). Cell-to-cell signaling and Pseudomonas aeruginosa infections. Emerging Infectious Diseases 4, 55160.[ISI][Medline]
3 . Geisenberger, O., Givskov, M., Riedel, K. et al. (2000). Production of N-acyl-l-homoserine lactones by P. aeruginosa isolates from chronic lung infections associated with cystic fibrosis. FEMS Microbiology Letters 184, 2738.[CrossRef][ISI][Medline]
4 . Singh, P. K., Schaefer, A. L., Parsek, M. R. et al. (2000). Quorum-sensing signals indicate that cystic fibrosis lungs are infected with bacterial biofilms. Nature 407, 7624.[CrossRef][ISI][Medline]
5
.
Wu, H., Song, Z., Hentzer, M. et al. (2000). Detection of N-acylhomoserine lactones in lung tissues of mice infected with Pseudomonas aeruginosa. Microbiology 146, 248193.
6
.
Stickler, D. J., Morris, N. S., McLean, R. J. et al. (1998). Biofilms on indwelling urethral catheters produce quorum-sensing signal molecules in situ and in vitro. Applied and Environmental Microbiology 64, 348690.
7 . Middleton, B., Rodgers, H. C., Camara, M. et al. (2002). Direct detection of N-acylhomoserine lactones in cystic fibrosis sputum. FEMS Microbiology Letters 207, 17.[CrossRef][ISI][Medline]
8
.
Erickson, D. L., Endersby, R., Kirkham, A. et al. (2002). Pseudomonas aeruginosa quorum-sensing systems may control virulence factor expression in the lungs of patients with cystic fibrosis. Infection and Immunity 70, 178390.
9
.
Davies, D. G., Parsek, M. R., Pearson, J. P. et al. (1998). The involvement of cell-to-cell signals in the development of a bacterial biofilm. Science 280, 2958.
10
.
De Kievit, T. R., Gillis, R., Marx, S. et al. (2001). Quorum-sensing genes in Pseudomonas aeruginosa biofilms: their role and expression patterns. Applied and Environmental Microbiology 67, 186573.
11 . Favre-Bonte, S., Pache, J. C., Robert, J. et al. (2002). Detection of Pseudomonas aeruginosa cell-to-cell signals in lung tissue of cystic fibrosis patients. Microbial Pathogenesis 32, 1437.[CrossRef][ISI][Medline]
12 . Yoon, S. S., Hennigan, R. F., Hilliard, G. M. et al. (2002). Pseudomonas aeruginosa anaerobic respiration in biofilms: relationships to cystic fibrosis pathogenesis. Developmental Cell 3, 593603.[ISI][Medline]
13
.
Davey, M. E., Caiazza, N. C. & OToole, G. A. (2003). Rhamnolipid surfactant production affects biofilm architecture in Pseudomonas aeruginosa PAO1. Journal of Bacteriology 185, 102736.
14
.
Kohler, T., Van Delden, C., Curty, L. K. et al. (2001). Overexpression of the MexEF-OprN multidrug efflux system affects cell-to-cell signaling in Pseudomonas aeruginosa. Journal of Bacteriology 183, 521322.
15
.
Tateda, K., Comte, R., Pechere, J. C. et al. (2001). Azithromycin inhibits quorum sensing in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 45, 19303.
16 . Terry, J. M., Pina, S. E. & Mattingly, S. J. (1992). Role of energy metabolism in conversion of nonmucoid Pseudomonas aeruginosa to the mucoid phenotype. Infection and Immunity 60, 132935.[Abstract]
17 . Kohler, T., Michea-Hamzehpour, M., Henze, U. et al. (1997). Characterization of MexE-MexF-OprN, a positively regulated multidrug efflux system of Pseudomonas aeruginosa. Molecular Microbiology 23, 34554.[ISI][Medline]
18
.
Kohler, T., Curty, L. K., Barja, F. et al. (2000). Swarming of Pseudomonas aeruginosa is dependent on cell-to-cell signaling and requires flagella and pili. Journal of Bacteriology 182, 59906.
19
.
Van Delden, C., Comte, R. & Bally, A. M. (2001). Stringent response activates quorum sensing and modulates cell density-dependent gene expression in Pseudomonas aeruginosa. Journal of Bacteriology 183, 537684.
20 . Miller, J. H. (1972). Experiments in Molecular Genetics, pp. 352355. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA.
21 . Takeoka, K., Ichimiya, T., Yamasaki, T. et al. (1998). The in vitro effect of macrolides on the interaction of human polymorphonuclear leukocytes with Pseudomonas aeruginosa in biofilm. Chemotherapy 44, 1907.[CrossRef][ISI][Medline]
22 . OToole, G. A. & Kolter, R. (1998). Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Molecular Microbiology 30, 295304.[CrossRef][ISI][Medline]
23 . Kintner, P. K. & Van Buren, J. P. (1982). Carbohydrate interference and its correction in pectin analysis using the m-hydroxydiphenyl method. Journal of Food Science 47, 75664.[ISI]
24
.
Shih, P. C. & Huang, C. T. (2002). Effects of quorum-sensing de-ficiency on Pseudomonas aeruginosa biofilm formation and antibiotic resistance. Journal of Antimicrobial Chemotherapy 49, 30914.
25
.
Heydorn, A., Ersboll, B., Kato, J. et al. (2002). Statistical analysis of Pseudomonas aeruginosa biofilm development: impact of mutations in genes involved in twitching motility, cell-to-cell signaling, and stationary-phase sigma factor expression. Applied and Environmental Microbiology 68, 200817.
26 . Latifi, A., Foglino, M., Tanaka, K. et al. (1996). A hierarchical quorum-sensing cascade in Pseudomonas aeruginosa links the transcriptional activators LasR and RhIR (VsmR) to expression of the stationary-phase sigma factor RpoS. Molecular Microbiology 21, 113746.[ISI][Medline]
27
.
Beatson, S. A., Whitchurch, C. B., Semmler, A. B. T. et al. (2002). Quorum sensing is not required for twitching motility in Pseudomonas aeruginosa. Journal of Bacteriology 184, 3598604.
28
.
Beatson, S. A., Whitchurch, C. B., Sargent, J. L. et al. (2002). Differential regulation of twitching motility and elastase production by Vfr in Pseudomonas aeruginosa. Journal of Bacteriology 184, 360513.
29
.
Sauer, K., Camper, A. K., Ehrlich, G. D. et al. (2002). Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm. Journal of Bacteriology 184, 114054.
30
.
Join-Lambert, O. F., Michea-Hamzehpour, M., Kohler, T. et al. (2001). Differential selection of multidrug efflux mutants by trovafloxacin and ciprofloxacin in an experimental model of Pseudomonas aeruginosa acute pneumonia in rats. Antimicrobial Agents and Chemotherapy 45, 5716.
31
.
Cosson, P., Zulianello, L., Join-Lambert, O. et al. (2002). Pseudomonas aeruginosa virulence analyzed in a Dictyostelium discoideum host system. Journal of Bacteriology 184, 302733.
32 . Hassett, D. J., Cuppoletti, J., Trapnell, B. et al. (2002). Anaerobic metabolism and quorum sensing by Pseudomonas aeruginosa biofilms in chronically infected cystic fibrosis airways: rethinking antibiotic treatment strategies and drug targets. Advanced Drug Delivery Reviews 54, 142543.[CrossRef][ISI][Medline]
33 . Retsema, J., Girard, A., Schelkly, W. et al. (1987). Spectrum and mode of action of azithromycin (CP-62,993), a new 15-membered-ring macrolide with improved potency against gram-negative organisms. Antimicrobial Agents and Chemotherapy 31, 193947.[ISI][Medline]
34 . Kucers, A., Crowe, S. M., Grayson, M. L. et al. (1997). Azithromycin. In The Use of Antibiotics, pp. 653663. Butterworth-Heinemann, Oxford, UK.
35 . Vranes, J. (2000). Effect of subminimal inhibitory concentrations of azithromycin on adherence of Pseudomonas aeruginosa to polystyrene. Journal of Chemotherapy 12, 2805.[ISI][Medline]
36 . Ichimiya, T., Takeoka, K., Hiramatsu, K. et al. (1996). The influence of azithromycin on the biofilm formation of Pseudomonas aeruginosa in vitro. Chemotherapy 42, 18691.[ISI][Medline]
37 . Jaffe, A., Francis, J., Rosenthal, M. et al. (1998). Long-term azithromycin may improve lung function in children with cystic fibrosis. Lancet 351, 420.
38
.
Wolter, J., Seeney, S., Bell, S. et al. (2002). Effect of long term treatment with azithromycin on disease parameters in cystic fibrosis: a randomised trial. Thorax 57, 2126.
39 . Equi, A., Balfour-Lynn, I. M., Bush, A. et al. (2002). Long term azithromycin in children with cystic fibrosis: a randomised, placebo-controlled crossover trial. Lancet 360, 97884.[CrossRef][ISI][Medline]
40 . Seed, P. C., Passador, L. & Iglewski, B. H. (1995). Activation of the Pseudomonas aeruginosa lasI gene by LasR and the Pseudomonas autoinducer PAI: an autoinduction regulatory hierarchy. Journal of Bac-teriology 177, 6549.[Abstract]
41 . Pearson, J. P., Pesci, E. C. & Iglewski, B. H. (1997). Role of Pseudomonas aeruginosa las and rhl quorum-sensing systems in the control of elastase and rhamnolipid biosynthesis genes. Journal of Bacteriology 179, 575667.[Abstract]
42 . Brint, J. M. & Ohman, D. E. (1995). Synthesis of multiple exoproducts in Pseudomonas aeruginosa is under the control of RhlR-RhlI, another set of regulators in strain PAO1 with homology to the autoinducer-responsive LuxR-LuxI family. Journal of Bacteriology 177, 715563.[Abstract]