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Centre for BioScience and Technology, BioCentrum-DTU, Technical University of Denmark, DK-2800 Lyngby, Denmark
Correspondence
Tim Tolker-Nielsen
ttn{at}biocentrum.dtu.dk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Sauer et al., 2002; Klausen et al., 2003a). The bacteria in biofilms in medical settings often display increased resistance toward host immune responses and enhanced tolerance toward antibiotic treatment (Costerton et al., 1999). Knowledge about the molecular mechanisms that are involved in biofilm development, and the factors that lead to the formation of resistant subpopulations, may be useful for creating strategies to control biofilm formation and eradicate persistent infections.
One of the most distinctive features of biofilms is the extracellular polymeric substances that surround the bacteria and constitute the biofilm matrix. The composition of biofilm matrices varies depending upon the bacterial species and the environmental conditions, but in general they contain components such as polysaccharides, proteins and extracellular DNA (Sutherland, 2001; Whitchurch et al., 2002).
Evidence for a role of extracellular DNA as matrix component in Pseudomonas aeruginosa biofilms has been presented both for the P. aeruginosa PAO1 reference strain and for clinical P. aeruginosa isolates (Whitchurch et al., 2002; Nemoto et al., 2003). P. aeruginosa PAO1 biofilm development in the wells of microtitre plates was attenuated by the presence of DNase I, and biofilm formation by P. aeruginosa PAO1 in flow-chambers was almost absent when the flow-chambers were irrigated with medium containing DNase I (Whitchurch et al., 2002). In addition, young P. aeruginosa PAO1 biofilms that had been grown in flow-chambers irrigated with DNase-free medium dispersed rapidly after addition of DNase I to the flowing medium, whereas mature P. aeruginosa PAO1 biofilms were not dispersed by DNase I treatment, suggesting that components other than extracellular DNA stabilize mature P. aeruginosa PAO1 biofilms (Whitchurch et al., 2002). Matsukawa & Greenberg (2004) investigated the composition of the extracellular matrix of mature P. aeruginosa PAO1 biofilms and found that extracellular DNA was by far the most abundant polymer, although exopolysaccharide appeared to be the most critical structural matrix component. In contrast to the finding that extracellular DNA may not be the primary cell-to-cell interconnecting compound in mature P. aeruginosa PAO1 biofilms, Nemoto et al. (2003) found that mature biofilms formed by four different clinical P. aeruginosa isolates could be dispersed by DNase treatment, suggesting that extracellular DNA is the critical matrix component in mature biofilms formed by these P. aeruginosa strains.
Quorum sensing, a mechanism by which bacteria can monitor their cell population density through the extracellular accumulation of signalling molecules, has been shown to play a role in structural development and stabilization of P. aeruginosa biofilms (Davies et al., 1998). P. aeruginosa employs the three interconnected quorum-sensing systems las, rhl and pqs to control expression of a wide range of virulence factors (Passador et al., 1993; Latifi et al., 1995, 1996; Pesci et al., 1999). The major signal molecules involved are 3-oxo-C12-homoserine lactone (3-oxo-C12-HSL) in the case of the las system, C4-HSL in the case of the rhl system, and 2-heptyl-3-hydroxy-4-quinolone, designated the Pseudomonas quinolone signal (PQS), in the case of the pqs system (Latifi et al., 1995, 1996; Pesci et al., 1999). Synthesis of PQS depends on several putative enzymes encoded by the pqsABCDE, pqsL, phnAB and pqsH genes, as well as on a LysR-type regulator encoded by the pqsR gene (also known as mvfR) (Cao et al., 2001; Gallagher et al., 2002; D'Argenio et al., 2002). Evidence has been presented that the quorum-sensing systems play a role in the formation of extracellular DNA in P. aeruginosa biofilms (Allesen-Holm et al., 2006). Quorum-sensing-regulated DNA release from P. aeruginosa populations presumably involves lysis of a subpopulation of the cells (or lysis of DNA-containing vesicles), as experiments with lacZ-containing P. aeruginosa strains indicated that the formation of extracellular 
-galactosidase correlated with the formation of large amounts of extracellular DNA (Allesen-Holm et al., 2006).
P. aeruginosa colonizes the lungs of cystic fibrosis (CF) patients and is a major cause of lung deterioration, health decline and death of these patients (Høiby et al., 2001). Several studies indicate that P. aeruginosa forms biofilms in the CF lung (e.g. Lam et al., 1980; Baltimore et al., 1989; Worlitzsch et al., 2002; Høiby et al., 2001), and the biofilm mode of growth is considered the major reason that these bacteria can not be eradicated by host defences or antibiotic treatment (Costerton et al., 1999; Høiby et al., 2001). The conditions that infecting bacteria encounter in the CF lung is of great interest, and artificial CF sputum media have been developed to enable experiments to be carried out under relevant and controllable conditions (Sriramulu et al., 2005; Palmer et al., 2005). P. aeruginosa cells in the CF lung are believed to be in intense competition for iron with the host (Haas et al., 1991; Ratledge & Dover, 2000). Low iron concentrations were shown to promote biofilm formation in artificial CF sputum medium (Sriramulu et al., 2005). In addition to low iron levels, the presence of extracellular DNA was shown to be important for P. aeruginosa biofilm formation in artificial sputum medium (Sriramulu et al., 2005).
Knowledge about the factors that affect DNA release from P. aeruginosa is important for understanding the environmental cues that can lead to biofilm formation and increased pathogenicity of this organism. In this paper we present evidence that the level of iron in the medium affects P. aeruginosa DNA release and biofilm formation. The iron level was shown to have an effect on the amount of biofilm formed in the wells of microtitre trays, as well as on structural biofilm development in flow chambers, and on the development of subpopulations with increased tolerance toward antimicrobial compounds. Evidence is presented that iron affects P. aeruginosa DNA release and biofilm development by affecting the PQS signalling pathway.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. The pqsA, pqsR and pqsL mutants were constructed by D'Argenio et al. (2002) via transposon insertion in PAO1. The gfp-tagged PAO1 strain was constructed by insertion of a mini-Tn7-eGFP-Gmr cassette as described by Klausen et al. (2003b). Escherichia coli strains MT102 and MC1000 were used for standard DNA manipulations. LB medium (Bertani, 1951) was used to cultivate E. coli strains. Batch cultivation of P. aeruginosa was carried out at 30 °C in AB minimal medium (Clark & Maaløe, 1967) supplemented with 30 mg glucose l–1. P. aeruginosa biofilms were cultivated at 30 °C in flow-chambers irrigated with FAB medium (Heydorn et al., 2000) supplemented with 0.3 mM glucose. FeCl3 was added to the media to give the final ferric iron concentrations described in the text. Selective media were supplemented with ampicillin (Ap; 100 mg l–1), gentamicin (Gm; 60 mg l–1), kanamycin (Km; 100 mg l–1), streptomycin (Sm; 100 mg l–1), or tetracycline (Tc; 20 mg l–1). AB medium supplemented with 0.05 mg propidium iodide ml–1 was used in the microtitre trays for quantification of extracellular DNA. DDAO [7-hydroxy-9H-(1,3-dichloro-9,9- dimethylacridin-2-one] was used at a concentration of 1 µM to stain extracellular DNA in flow-chamber-grown biofilms. Propidium iodide was used at a concentration of 0.01 mg ml–1 to stain dead cells in flow-chamber-grown biofilms. Syto62 was used at a concentration of 1 µM to stain bacteria in flow-chamber-grown biofilms.
Measurements of pvdD expression.
A classical -galactosidase assay, described by Miller (1972), was used to measure expression of the pvdD–lacZ fusion in P. aeruginosa PAO1 cells transformed with the low-copy-number plasmid pPM190 : : pvdD (Rombel et al., 1995), which carries the pvdD promoter fused to the E. coli lacZ gene.
Construction of the pqsA : : gfp(ASV) reporter.
The promoter-probe plasmid pAC37, containing a PpqsA : : gfp(ASV) transcriptional fusion, was constructed using standard techniques for DNA manipulations (Sambrook et al., 1989). A P. aeruginosa DNA region containing the PpqsA promoter was PCR amplified by use of the primers PA0996F (5'-GGAATTCTGATCAATGCCGTCGCCCCCTTGGA-3') and PA0996R (5'-GGGTACCCATGACAGAACGTTCCCTCTT-3'). A P. aeruginosa DNA region containing the PrhlR promoter and the rhlR gene was PCR amplified by use of the primers PA3477F (5'-CGTGATCAAACGGCTGGTCCGGCTCG-3') and PA3477R (5'-GGAATTCAAAAAGGCCATCCGTCAGGATGGCCTTCTAGATTATCTACTCAGATGAGACCCAGCGCC-3'). An E. coli T2 transcriptional terminator and a unique XbaI cloning site were introduced in the PCR fragment through use of the PA3477R primer set. pJBA100, consisting of a NotI fragment with the gfp(ASV) gene from pJBA89 (Andersen et al., 2001) cloned in the shuttle vector pUCP22NotI, was used as scaffold. pJBA100 was digested with KpnI and EcoRI; the PCR product containing the PpqsA promoter was digested with BclI and KpnI; and the PCR product containing the rhlR gene was digested with BclI and EcoRI. The expression monitor pAC36 was constructed by ligating the three DNA fragments in one reaction. Plasmid pAC37 was constructed by deleting the rhlR gene on pAC36, via digestion with XbaI and BclI, Klenow treatment, and religation. The pAC37 plasmid was electroporated into P. aeruginosa PAO1 and the P. aeruginosa lasI rhlI mutant.
Measurement of biofilm and extracellular DNA in microtitre trays.
The microtitre tray biofilm formation assay was performed essentially as described by O'Toole & Kolter (1998). Overnight cultures were grown in AB medium and diluted to OD600 0.001 with fresh AB medium. The diluted cultures were transferred to the wells of polystyrene microtitre trays (150 µl per well) and incubated for 24 h at 30 °C. The medium was removed from the wells and they were washed twice with 0.9 % NaCl, stained with 0.1 % crystal violet and washed twice with 0.9 % NaCl; the crystal violet-stained biofilms were then resuspended in 96 % ethanol, and biofilm cell-associated dye was measured as A590. Measurements of extracellular DNA in P. aeruginosa microtitre tray cultures was done as described by Allesen-Holm et al. (2006). Microtitre tray cultures were started by diluting P. aeruginosa overnight cultures to OD600 0.001 in AB-glucose medium supplemented with 0.05 mM propidium iodide and FeCl3 (as indicated) in wells of polystyrene microtitre plates (150 µl per well). The cultures were incubated for 24 h at 37 °C, after which propidium iodide absorbance was measured at 480 nm and cell density was measured at 600 nm by the use of a Wallac microplate reader.
DNase I treatment of biofilms in microtitre trays.
Twenty-four-hour-old microtitre-tray-grown biofilms of PAO1 were washed twice with 0.9 % NaCl and treated with 0.9 % NaCl containing 100 µg DNase I ml–1 for 3 h, after which the amount of biofilm was measured as described above.
Cultivation of biofilms in flow-chambers.
Biofilms were grown in flow-chambers with individual channel dimensions of 1x4x40 mm. The flow system was assembled and prepared as described previously (Sternberg & Tolker-Nielsen, 2005). The flow-chambers were inoculated by injecting 350 µl overnight culture diluted to an OD600 of 0.001 into each flow channel using a small syringe. After inoculation, the flow channels were left without flow for 1 h, after which medium flow was started using a Watson Marlow 205S peristaltic pump. The mean flow velocity in the flow-chambers was 0.2 mm s–1, corresponding to laminar flow with a Reynolds number of 0.02.
Microscopy and image acquisition.
All microscopy observations and image acquisitions were done with a Zeiss LSM510 confocal laser scanning microscope (CLSM) equipped with detectors and filter sets for monitoring of Gfp, propidium iodide Syto62 and DDAO fluorescence. Images were obtained using a 63x/1.4 objective or a 40x/1.3 objective. Simulated three-dimensional images and sections were generated using the IMARIS software package (Bitplane AG).
Assessment of extracellular DNA in flow-chamber-grown biofilms.
DDAO was added to the medium to a final concentration of 1 µM to fluorescently stain extracellular DNA in flow-chamber-grown biofilms. DDAO fluorescence in different biofilms was recorded with fixed settings of the CLSM.
Antimicrobial treatment.
Biofilm tolerance to EDTA or ciprofloxacin was assessed by irrigating 4-day-old flow-chamber-grown P. aeruginosa biofilms with medium containing 50 mM EDTA or 50 µg ciprofloxacin ml–1 for 24 h, followed by staining of the dead cells with propidium iodide, and CLSM image acquisition. To quantify survival rates, the bacteria from flow-chamber biofilms were recovered after EDTA treatment, ciprofloxacin treatment, or no antimicrobial treatment, by pumping bead-containing (Sigma glass-beads 212–300 µm) medium rapidly in and out of the flow-chambers using syringes. The bead-containing bacterial suspensions were vortex mixed, and the bacteria were plated on LB plates; c.f.u. were determined and survival rates calculated.
COMSTAT image analysis.
CLSM images were analysed by use of the computer program COMSTAT (Heydorn et al., 2000). A fixed threshold value and connected volume filtration were used for all image stacks.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). We measured -galactosidase activity in microtitre tray cultures of P. aeruginosa harbouring the low-copy-number plasmid pPM190 : : pvdD, which carries the pvdD promoter fused to the E. coli lacZ gene (Rombel et al., 1995). The -galactosidase activity (Miller units) was 7757, 4944, 2773 and 2206 (three replicates, SD 581, 458, 347 and 58) in cultures supplemented with 5, 10, 50 and 100 µM FeCl3. Accordingly, the selected range of iron concentrations in our medium evidently affected the iron status of the P. aeruginosa cells.
When P. aeruginosa PAO1 was cultivated in microtitre trays, a high cell density was reached in wells containing AB-glucose medium supplemented with 100 or 50 µM iron, whereas a slightly lower cell density was reached in wells containing AB-glucose medium supplemented with 10 µM iron, and a significantly lower cell density was reached in wells containing AB-glucose medium supplemented with 5 µM iron (Fig. 1A). However, the lowest iron concentration used did not limit growth dramatically, and the highest iron concentration used did not inhibit growth.
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). Because extracellular DNA is a major matrix component of P. aeruginosa biofilms (Whitchurch et al., 2002; Nemoto et al., 2003; Matsukawa & Greenberg, 2004; Allesen-Holm et al., 2006), we found it of interest to investigate whether the presence of high levels of iron might inhibit the generation of extracellular DNA from P. aeruginosa. In accordance with the work of Musk et al. (2005), we found that biofilm formation by P. aeruginosa PAO1 in microtitre trays decreased with increasing iron concentrations (Fig. 1B
). In order to quantify the extracellular DNA generated in the microtitre tray cultures (from planktonic as well as from biofilm cells), we supplemented the medium with propidium iodide (which fluoresces when it is bound to DNA, and does not penetrate live bacteria) and measured the level of fluorescence in the cultures after 1 day of growth. The amount of extracellular DNA in P. aeruginosa PAO1 microtitre tray cultures was high in medium supplemented with 5 µM iron, and decreased with increasing iron concentrations (Fig. 1C). Treatment of 1-day-old P. aeruginosa PAO1 biofilms with DNase I led to dispersal of large amounts of the biofilms (Fig. 1D), showing that extracellular DNA plays a critical role in biofilm formation in the experimental setup used.
Iron affects the Pseudomonas quinolone signal (PQS) pathway
In a previous report, we presented evidence that release of extracellular DNA from P. aeruginosa populations was regulated through the pqs quorum-sensing system (Allesen-Holm et al., 2006). To investigate whether the observed effect of iron on DNA release from P. aeruginosa populations occurs through the PQS system, we initially tested DNA release from different PQS mutants in microtitre tray cultures supplemented with different concentrations of iron. The mutants pqsA and pqsR were found to release less extracellular DNA than the wild-type in an iron-independent manner, whereas a pqsL mutant was found to release more extracellular DNA than the wild-type via a pathway that was suppressed by high iron concentrations (Fig. 2). These results are in agreement with evidence reported by D'Argenio et al. (2002) that the pqsA and pqsR mutants were deficient in PQS production and autolysis, whereas the pqsL mutant overproduced PQS and was hyper-autolysing.
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) and the pqsA : : gfp(ASV) reporter-construct is thus useful for monitoring pqsABCDE expression. The plasmid with the reporter-construct was transferred to the P. aeruginosa wild-type as well as to an isogenic P. aeruginosa lasI rhlI quorum-sensing mutant, and pqsA : : gfp(ASV) expression was measured in microtitre tray cultures. The experiments confirmed that the pqsABCDE operon is under N-acylhomoserine lactone (AHL) quorum-sensing control, as expression of the pqsA : : gfp(ASV) reporter consistently was lower in the lasI rhlI quorum-sensing mutant than in the wild-type (Fig. 3A). Transcription of the pqsABCDE operon in the P. aeruginosa wild-type was found to be high in medium supplemented with 5 µM iron, and decreased with increasing iron concentrations (Fig. 3A). In the lasI rhlI quorum-sensing mutant there was also an inverse relation between pqsA : : gfp(ASV) expression and the level of iron in the medium (Fig. 3A), suggesting that the effect of iron on pqsABCDE expression to some extent is independent of the AHL quorum-sensing system. DNA release in the microtitre tray cultures was found to correlate with the expression of the pqsABCDE operon (Fig. 3B). Based on these results, we suggest that iron affects P. aeruginosa DNA release and biofilm formation by affecting the PQS signalling pathway.
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Iron affects P. aeruginosa DNA release and structural biofilm development in flow-chambers
In order to examine the effect of iron concentration on DNA release in P. aeruginosa biofilms, we grew the PAO1 wild-type in flow-chambers irrigated with normal FAB medium (containing 1 µM iron) or FAB100 medium (containing 100 µM iron). Because of the continuous supply of medium, the amount of iron available to the bacteria will be higher in the flow-chamber setup than in the microtitre tray setup, and we therefore chose to use FAB, containing 1 µM iron, as the medium with the lowest iron concentration in the investigations. After 4 days of growth, the extracellular DNA in the biofilms was visualized by staining with the compound DDAO followed by CLSM. In agreement with our previous study (Allesen-Holm et al., 2006), the biofilms grown in FAB medium contained large amounts of extracellular DNA in the part of the biofilm close to the substratum and in the outer region of the stalk portions of the mushroom-shaped multicellular structures (Fig. 4A). In agreement with the quantifications of extracellular DNA and biofilm formation in the microtitre tray assays (described above), the biofilms grown in FAB100 medium contained less extracellular DNA and less biomass than the biofilms grown in normal FAB medium (Fig. 4B). In addition, biofilms grown in FAB100 medium appeared less spatially organized and did not contain the mushroom-shaped multicellular structures typical of the biofilms grown in normal FAB medium (Fig. 4). The difference in biomass and spatial structure between biofilms grown in normal FAB medium and FAB100 medium was confirmed by COMSTAT analysis (Table 1).
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) and reached the highest expression level at day 2 where it was localized mainly in the outer part of the microcolonies (Fig. 5C). After day 2, expression of the pqsA : : gfp(ASV) fusion decreased, and at day 4 only a weak green signal could be detected in the stalk portion of the mushroom-shaped multicellular structures (Fig. 5E). In FAB100 medium, there was significantly less expression of the pqsA : : gfp(ASV) fusion, and this expression appeared less coordinated in time and space (Fig. 5B, D and F).
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), whereas EDTA treatment caused killing of the cells located within the mushroom-shaped structures (Fig. 6E). In 4-day-old P. aeruginosa PAO1 biofilms grown in FAB100 medium, treatment with ciprofloxacin or EDTA appeared to kill a large proportion of the bacteria, and there was no special area for the ciprofloxacin- or EDTA-mediated killing (Fig. 6D, F). Propidium iodide staining of biofilms that had not been treated with antibiotic showed that virtually no dead cells were present in the biofilms before antibiotic treatment, and that the propidium iodide staining did not visualize extracellular DNA with the setting of the CLSM that was used (Fig. 6A, B). (Propidium iodide does not give as high a signal as DDAO, and with normal settings of the CLSM it will therefore mainly visualize dead cells where the concentration of DNA is high compared to in the biofilm matrix.)
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Banin et al., 2005). On the other hand, high concentrations of iron were shown to suppress P. aeruginosa biofilm formation in both microtitre tray and flow-chamber systems (Musk et al., 2005), and low iron concentrations were shown to be required for P. aeruginosa biofilm (microcolony) formation in artificial sputum medium (Sriramulu et al., 2005). In agreement with these previous studies, we found in the present study that in our microtitre tray and flow-chamber setups low iron levels promoted biofilm formation while high iron levels suppressed biofilm formation. Experiments involving DNase I treatment of biofilms in microtitre trays (this study) or flow-chambers (Allesen-Holm et al., 2006) suggested that extracellular DNA is an important constituent of the biofilm matrix. Furthermore, evidence was presented that the formation of extracellular DNA in P. aeruginosa biofilms is regulated via the PQS quorum-sensing system (Allesen-Holm et al., 2006). In this paper, we present evidence that low iron concentrations promote P. aeruginosa biofilm development via up-regulation of the pqs genes and the formation of extracellular DNA, and that high levels of iron suppress P. aeruginosa biofilm development via down-regulation of the pqs genes and the formation of extracellular DNA. In agreement with our present and previous work, a study comparing different microarray results identified the PQS regulator gene pqsR as being among genes that are induced at low iron levels and also positively controlled by quorum sensing (Cornelis & Aendekerk, 2004).
Under specific conditions in flow-chamber systems (e.g. the glucose FAB medium used in the present study), P. aeruginosa forms heterogeneous biofilms with mushroom-shaped multicellular structures via a process that involves formation of microcolonies in the initial phase and subsequent formation of cap-shaped structures on top of the microcolonies in the later phase (e.g. Klausen et al., 2003a). In agreement with the present study, the extracellular DNA in such P. aeruginosa biofilms was shown to be present in high concentrations close to the substratum and in the outer part of the microcolonies in the initial phase of biofilm development, and in the outer region of the stalk portion of the mushroom-shaped multicellular structures in the later phase of biofilm development (Allesen-Holm et al., 2006). In the present study we observed that when P. aeruginosa formed biofilms in flow-chambers irrigated with FAB medium, expression of a pqsA : : gfp(ASV) fusion was detectable in 1-day-old biofilms and reached the highest expression level at day 2, where it was localized mainly in the outer part of the microcolonies. After day 2, expression of the pqsA : : gfp(ASV) fusion in the biofilms decreased, and at day 4 only a weak green signal could be detected in the stalk portion of the mushroom-shaped multicellular structures. This spatio-temporal expression pattern of pqsABCDE is in accordance with the location of extracellular DNA in P. aeruginosa biofilms reported here and by Allesen-Holm et al. (2006). In the medium containing a high iron concentration, there was significantly less expression of the pqsA : : gfp(ASV) fusion, and this expression appeared less coordinated in time and space. In agreement with the quantifications of extracellular DNA and biofilm formation in the microtitre tray assays, and in agreement with the role of the pqs genes in DNA release (Allesen-Holm et al., 2006), the biofilms grown at high iron concentration contained less extracellular DNA and less biomass than the biofilms grown in normal FAB medium. In addition, biofilms grown at high iron concentration appeared less spatially organized and did not develop mushroom-shaped multicellular structures.
The cap-forming and the stalk-forming subpopulation of the mushroom-shaped multicellular structures in P. aeruginosa biofilms in many cases display differential tolerance to antimicrobial compounds. For example, the antibiotic tobramycin was shown to kill preferentially bacteria located in the outer part of the cap portion of the mushroom-shaped structures, whereas the antibiotic colistin, the detergent SDS and the chelator EDTA were shown to kill preferentially bacteria in the stalk portion of the mushroom-shaped structures (Bjarnsholt et al., 2005; Banin et al., 2006; Haagensen et al., 2007). Since we had found that the level of iron in the medium may control whether a structured or an unstructured P. aeruginosa biofilm is formed, we found it of interest to investigate tolerance to antimicrobial compounds in P. aeruginosa biofilms grown in FAB medium or in FAB100 high-iron medium. Ciprofloxacin treatment and EDTA treatment caused killing of cells located in the cap portion and stalk portion, respectively, of the mushroom-shaped structures of 4-day-old P. aeruginosa biofilms grown in FAB medium, whereas there was no special area for ciprofloxacin- or EDTA-mediated killing in 4-day-old biofilms grown in high-iron medium. In addition, the structured P. aeruginosa biofilms, grown in FAB medium, displayed higher tolerance to the antimicrobial treatment than the unstructured P. aeruginosa biofilms, grown in high-iron medium.
The formation of P. aeruginosa extracellular DNA is evidently regulated by both quorum sensing and the level of available iron. Other processes that appear to be subject to regulation by the same cues include elastase production and Mn-superoxide dismutase (SOD) production, which were both shown to be quorum-sensing controlled (Hassett et al., 1999; Winson et al., 1995), and also regulated by iron so that they were induced under low-iron conditions but repressed under high-iron conditions (Bjorn et al., 1979; Hassett et al., 1997; Sokol et al., 1982; Bollinger et al., 2001). The fact that iron regulation of these processes to some extent is independent of quorum-sensing regulation makes nutritional override of quorum sensing possible. Other examples of possible nutritional override of quorum sensing include pyocyanin production, which is positively controlled by quorum sensing (Brint & Ohman, 1995), but can also be induced by phosphate starvation (Hassett et al., 1992). The possibility of nutritional override of quorum sensing should be taken into account when possible strategies for manipulating quorum sensing for controlling bacterial infections are considered.
Evidence was recently presented that biofilm formation by Staphylococcus aureus is induced at low iron concentrations and repressed at high iron concentrations via a mechanism that does not affect expression of polymeric N-acetylglucosamine, which is the major exopolysaccharide implicated in S. aureus biofilm formation (Johnson et al., 2005). Our unpublished results suggest that extracellular DNA plays a role in S. aureus biofilm formation, and that formation of S. aureus extracellular DNA occurs at high levels in low-iron medium and at low levels in high-iron medium. It is possible, therefore, that there is a connection between iron concentration, extracellular DNA and biofilm formation also for the Gram-positive organism S. aureus.
The finding that high amounts of iron may inhibit P. aeruginosa biofilm formation raises the question whether iron might be used to treat biofilm infections in medical settings. However, because the lack of free iron in the human body in most cases is limiting bacterial growth (Weinberg, 1999), treatment of biofilm-based infections with iron might not be feasible. Nevertheless, the possibility exists that a combination treatment involving antibiotics and iron might be useful. The finding that iron can suppress biofilm formation may motivate a search for similar compounds that can suppress biofilm formation but do not stimulate bacterial growth.
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ACKNOWLEDGEMENTS |
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Edited by: P. E. Kolenbrander
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Received 4 December 2006;
revised 11 January 2007;
accepted 16 January 2007.
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