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Department of Clinical Microbiology, Rigshospitalet, and Department of Bacteriology, Institute for Medical Microbiology and Immunology, Panum Institute, University of Copenhagen, DK-2100 Copenhagen, Denmark
Center for Biomedical Microbiology, BioCentrum-DTU, Building 301, Technical University of Denmark, DK-2800 Lyngby, Denmark
Department of Microbiological Food Safety, Danish Institute for Food and Veterinary Research, Moerkhoej Bygade 19, 2860 Soeborg, Denmark
ABSTRACT
Biofilms are thought to play a key role in the occurrence of lung infections by Pseudomonas aeruginosa in patients with cystic fibrosis (CF). In this study, 20 nonmucoid P. aeruginosa isolates collected during different periods of chronic infection from eight CF patients were assessed with respect to phenotypic changes and in vitro biofilm formation. The physiological alterations were associated with a loss of motility (35% were nonmotile) and with decreased production of virulence factors (pyocyanin, proteases) and quorum-sensing molecules (45% of the isolates were unable to produce 3-O-C12-homoserine lactone quorum-sensing molecules). Compared with wild-type strain PAO1, most P. aeruginosa isolates demonstrated different degrees of reduction of adherence on polystyrene surfaces. The in vitro biofilm formation of isolates was investigated in a hydrodynamic flow system. Confocal laser scanning microscope analysis showed that the biofilm structures of the P. aeruginosa isolates were highly variable in biomass and morphology. Biofilm development of six genotypically identical sequential isolates recovered from a particular patient at different time points of chronic infection (20 years) and after lung transplantation demonstrated significant changes in biofilm architectures. P. aeruginosa biofilm formation followed a trend of decreased adherence with progression of the chronic lung infection. The results suggest that the adherent characteristic of in vitro biofilm development was not essential for the longitudinal survival of nonmucoid P. aeruginosa during chronic lung colonization.
INTRODUCTION
Biofilm formation and sessile communities are important issues in the pathogenesis of Pseudomonas aeruginosa in chronic lung infections in patients with cystic fibrosis (CF) (9, 21). Chronic infection with P. aeruginosa leads to a decline of lung function, respiratory failure, and ultimately, death in CF patients. P. aeruginosa grows in the CF lung in microcolonies, where bacteria are often embedded in an exopolysaccharide matrix (15, 37). Although maintenance of antibiotic therapy and eventually lung transplantation have significantly improved the survival of CF patients with chronic P. aeruginosa infections, the presence of P. aeruginosa biofilms in the lungs of CF patients is still associated with a poor prognosis (14).
The use of live monitoring systems with confocal laser scanning microscopy (CLSM) has greatly increased our understanding of the complex architectures of bacterial biofilms (6, 49). By using the flow chamber setup, wild-type P. aeruginosa PAO1 biofilms show differentiated mushroom structures with water-filled channels. Biofilm formation in P. aeruginosa is hypothesized to be a process of development that includes initial attachment, the formation of microcolonies, and biofilm maturation (23, 24, 36). Several studies have demonstrated that surface-associated adherence factors such as flagella and type IV pili are essential for the formation of a regular mushroom structure, and maintenance of a fully mature biofilm phenotype involves the las-rhl quorum-sensing system (10, 36).
Intermittent colonization of CF patient airways by P. aeruginosa usually occurs early in the life of the host, and infection starts with a predominant environmental nonmucoid phenotype which presumably is involved in the initial colonization of CF patients (42). During the subsequent infection, certain physiological characteristics of P. aeruginosa associated with its virulence in acute infections undergo alterations. Nonmucoid P. aeruginosa strains give rise to alginate-overproducing mucoid variants, frequently due to mutations in the mucA gene (33, 47). Besides conversion to mucoidy, other phenotypic changes include the loss of flagella or pilus effectory motility, the loss of O-antigen components of the lipopolysaccharide (LPS), the appearance of auxotrophic variants, the loss of pyocyanin production, as well as the emergence of strains multiresistant to antibiotics (2, 27, 28, 32, 38, 44). The phenotypic conversion of P. aeruginosa isolates that occurs during chronic infection probably reflects an adaptive behavior that enables the bacteria to survive in the hostile environment of the CF lung (11, 30, 31).
While significant attention has been given to the emergence of the alginate-overproducing mucoid phenotype, longitudinal studies shown that the initially acquired nonmucoid P. aeruginosa strain resides in the CF lung for many years (30, 40). The persistence mechanisms and phenotypic heterogeneity of nonmucoid P. aeruginosa strains associated with the chronic infection process have not been fully understood. In the study described in this report, we examined 20 nonmucoid CF isolates recovered during different phases of chronic lung infection from eight CF patients. The isolates were investigated for phenotypic variations in regard to motility and the production of virulence factors and quorum-sensing molecules. Furthermore, the entire collection of strains was screened for their adherence and different capacities of biofilm formation. The biofilm structural morphology and dynamic behavior were monitored by CLSM. Our data show that the capacity of P. aeruginosa isolates to form biofilms is progressively reduced during the chronic infection. The results indicate that the longitudinal survival of P. aeruginosa in CF lungs may involve strategies not related to biofilm development, as it is described from in vitro investigations.
MATERIALS AND METHODS
CF patients and bacterial isolates. The clinical strains of P. aeruginosa included in the present study were isolated from sputum samples of chronically colonized CF patients attending the Danish CF Center, Rigshospitalet, Copenhagen. All patients are monitored on a monthly basis by evaluation of the clinical status, pulmonary function, height, weight, and microbiology of lower airway secretions. Chronic P. aeruginosa infection is defined as the persistent presence of P. aeruginosa for at least 6 consecutive months or less when the persistence is combined with the presence of two or more precipitating anti-P. aeruginosa antibodies in the serum (20). The sputum samples were obtained by expectoration or endolaryngeal suction, followed by Gram staining and examination under a microscope to confirm their origin from the lower airway. P. aeruginosa was identified by conventional biochemical tests, and all P. aeruginosa isolates collected were stored at –80°C in broth supplemented with 10% glycerol. All eight patients included in the study had nonmucoid P. aeruginosa isolates at the initial acquisition of P. aeruginosa in the lung and in the early phase of the colonization, and both nonmucoid and mucoid P. aeruginosa colony phenotypes were detected from patient sputum samples during the chronic infection. However, in this study, we focused on the phenotypic heterogeneity of nonmucoid isolates among different CF patients and within one individual over different periods of chronic lung infection. In total, 20 nonmucoid isolates from eight CF patients were examined. Two isolates (CF patients 2 to 8) to six isolates (CF patient 1; five sequential isolates collected during chronic infection and one isolate collected in the post-lung transplantation phase) per patient were included. The isolates data are summarized in Table 1.
PFGE analysis. The relatedness of the isolated strains was assessed by pulsed-field gel electrophoresis (PFGE), as described previously (41). P. aeruginosa cells were embedded in agarose blocks and treated with proteinase K and EDTA. Before electrophoresis, the DNA was digested with the restriction enzyme SpeI (BioLabs, Inc.). Bacteriophage lambda ladders were applied as molecular size markers. PFGE was carried out by contour-clamped homogeneous electric field electrophoresis (CHEF-DR III apparatus; Bio-Rad, Munich, Germany). After PFGE, the banding patterns were visualized by ethidium bromide staining and then photographed (GelDoc imaging system; Bio-Rad). Evaluation of similarity was done as described by Tenover et al. (51).
Colony morphology. The colony phenotype of each isolate grown on Luria-Bertani (LB) agar plates (24 h incubation at 37°C) was examined by using a Leica microscope (Ernst Leitz Wetzlar GmbH, Germany) with a 2.5/0.06x achromatic plan objective.
Determination of hypermutable phenotypes. The hypermutability of the isolates was determined as described by Oliver et al. (35) by plating aliquots of serial dilutions of bacterial cells harvested from an overnight culture on LB plates with and without rifampin (300 μg/ml) and streptomycin (500 μg/ml). Counting of the bacterial CFU was performed after 1 or 2 days of incubation. The mutation frequencies were calculated, and isolates were considered mutators when the corresponding mutation frequencies with both rifampin and streptomycin were 20-fold higher than those observed for PAO1.
Motility assays. (i) Swimming. Cells were inoculated by use of a sterile toothpick into 5 mm ABT plates (AB medium [8] containing 2.5 mg/liter thiamine) containing 0.3% Bacto agar, 0.2% Casamino Acids, and 30 mM glucose. The swimming zone was measured after 48 h incubation at room temperature.
(ii) Twitching motility. Cells were stab inoculated with a toothpick through a thin 2-mm ABT medium supplemented with 0.2% Casamino Acids, 30 mM glucose, and 1.5% Bacto agar to the bottom of the petri dish. After incubation for 24 and 48 h at 30°C, the diameter of the hazy zone of growth was measured.
(iii) Swarming. Swarm plates were composed of 0.4% Bacto agar and ABT supplement with 0.5% Casamino Acids and 0.5% glucose. The plates were dried for 2 h at room temperature. A total of 5 μl of an overnight culture was inoculated, the plates were incubated at 37°C for 36 h, and the surface locomotion of the bacteria was observed. All the motility assays were performed in triplicate.
Detection of quorum-sensing signals. The production and secretion of quorum-sensing signal molecules (N-acylhomoserine lactone) were investigated by cross-streaking P. aeruginosa isolates against two different monitor strains. Escherichia coli MH297 contains P. aeruginosa lasB fused to the luxCDABE reporter cassette of Vibrio fischeri (5). The construct responds to 3-OC12-HSL. E. coli MH298 contains the Aeromonas hydrophila ahyR and the ahyI promoter fused to luxCDABE (50). The construct responds to C4-HSL. After incubation of the plates at 30°C for 24 h, AHL production was detected by inspecting the bioluminescence of MH297 and MH298 with a highly sensitive photo-counting C2400-40 charge-coupled device camera (Hamamatsu Photonics).
Production of virulence factors. (i) Pyocyanin production. Bacterial isolates were grown in LB medium at 37°C for 16 h. The pyocyanin was extracted from 5 ml culture supernatant with 3 ml of chloroform. The blue pigment extracted in the chloroform corresponds to pyocyanin. The pigment was further extracted with 0.2 M HCl as a pink to deep red solution, and the optical density at 520 nm (OD520) was measured (56).
(ii) Skim milk protease assay. The production of protease was performed as described by the manufacturer (Loewe Biochemica). One milliliter of culture supernatant (24 h) was applied to skim milk plates, followed by overnight incubation at 37°C. The clearing zone surround the inoculation spot indicates the ability of isolates to produce proteases.
LPS analysis. LPSs from P. aeruginosa isolates were prepared as described previously (13), separated on sodium dodecyl sulfate-polyacrylamide gels, and detected by silver staining.
Static culture biofilm assay. Biofilm formation in a polystyrene microtiter plate was assayed by the methods of O'Toole and Kolter (36), with modifications. The diluted overnight culture from LB medium was inoculated in 150 μl fresh medium with a multiprong device and incubated at 37°C for 48 h; and after removal of the medium and two washes with 0.9% NaCl, the biofilm cells were stained with 0.1% crystal violet solution and solubilized in 96% ethanol. The biofilm cell-associated dye was measured by determination of the OD590.
Plasmid construction. Initially, a 2-kb NotI fragment encoding the RBSII-gfpmut3-T0-T1 cassette of pJBA25 (49) preceded by the PlacUV5 promoter was isolated from pJBA34 (J. B. Andersen, unpublished data) and treated with Klenow polymerase. The resulting blunt-end fragments were subsequently ligated to the 8.3-kb Klenow-treated HindIII fragment of pME6031 (16) to give pJBA128. Finally, pJBA142 was constructed by ligating the 0.85-kb SmaI fragment of pUCGm encoding the aacC1 gene (which confers resistance to gentamicin) to the 8.4-kb EcoRV fragment of pJBA128.
Tagging of P. aeruginosa isolates with gfp. Gfpmut3 tagging of P. aeruginosa isolates was carried out by triparental matings with the mobilizable plasmid pJBA 142 and helper E. coli plasmid pRK600 (22). The fluorescent exoconjugants with gentamicin resistance-conferring cassettes were selected on Pseudomonas isolation agar (Difco) supplemented with an appropriate concentration of gentamicin. The fluorescently tagged strains showed no phenotypic changes compared with the phenotypes of the parental strains.
P. aeruginosa biofilms. Biofilms were grown at 30°C in three-channel flow cells with individual channel dimensions of 0.3 by 4 by 40 mm and supplied with FB minimal medium [1 mM MgCl2, 0.1 mM CaCl2, nonchelated trace elements (53a), 2 g of (NH4)2SO4 per liter, 6 g of Na2HPO4 · 2H2O per liter, 3 g of KH2PO4 per liter, 3 g of NaCl per liter] supplemented with 0.02% Casamino Acids. The flow system was assembled and prepared as described by Christensen et al. (6). The substratum consisted of a microscope glass coverslip (24 by 50 mm; st1; Knittel Glser, Braunschweig, Germany). Gfp-tagged P. aeruginosa isolates were streaked onto LB plates with gentamicin and incubated for 24 h at 37°C. A single colony was used for inoculation of 10 ml 10% LB medium. The cultures were incubated at 30°C overnight; cultures diluted in sterile 0.9% NaCl to an OD600 of 0.001 were used for inoculation of the flow channels. A Watson-Marlow 205S peristaltic pump was used to keep the medium flow at a constant rate of 3 ml h–1.
Image acquisition and analysis. All microscopic observations were performed on a Zeiss LSM510 scanning confocal laser microscope (Carl Zeiss, Jena, Germany) equipped with an argon laser and detector and filter sets for monitoring of gfp expression (excitation, 488 nm; emission, 517 nm). Images were obtained by using a 40x/1.3 Plan-Neofluar oil objective. Multichannel simulated fluorescence projection (a shadow projection) image and vertical cross section through the biofilm were generated by using the IMARIS software package (Bitplane AG, Zurich, Switzerland) running on a personal computer. The images were further processed for display by using PhotoShop software (Adobe, Mountain View, Calif.).
RESULTS
Macrorestriction analysis of CF isolates. Twenty nonmucoid P. aeruginosa isolates from eight patients included in this study were characterized by PFGE analysis. The closely related isolates from individual patients displayed identical or highly similar macrorestriction fragment patterns. These PFGE profiles indicate that each patient was persistently colonized by these respective P. aeruginosa clones. The PFGE profile of SpeI-digested chromosomal DNA of the P. aeruginosa isolates from patient 1 indicates that the patient carried the same clone throughout the chronic infection phase and subsequent to lung transplantation (data not shown).
Phenotypic characterization of P. aeruginosa isolates. The different phenotypic features of the clinical P. aeruginosa isolates were characterized (Table 1). Colony morphology was categorized as smooth or rough, and different isolates (those from CF patients 1, 2, 3, and 8) showed variations in their colony morphologies. The isolates changed from the nonhypermutable to the hypermutable phenotype during the long-term colonization. Three types of motilities were assessed for the isolates collected. Nonmotile isolates were predominant during the course of chronic infection. Overall, 14 isolates (70%) lacked swimming motility, and 10 isolates (50%) lacked both twitching and swarming motilities. Seven (35%) isolates (six isolates recovered from the late chronic infection phase and one isolate, isolate 1395b/2003, recovered during the posttransplantation phase) had lost all three types of motility. Differences in the production of AHL (3-O-C12-HSL and C4-HSL) were found. Nine isolates (45%) were unable to produce 3-O-C12-HSL, while the majority (80%) of isolates were able to produce C4-HSL. The pathogenesis of P. aeruginosa is related to the production of a number of extracellular virulence determinants. We investigated the production of pyocyanin and proteases. The loss of pigmentation and proteases was found in the later samples of isolates from five of eight patients. Note that isolate 1395b/2003 from the posttransplantation phase had phenotypic properties similar to those of the isolate (67903b/1999) derived just before lung transplantation.
Screening of biofilm formation of P. aeruginosa isolates in polystyrene microtiter plates. Initially, we examined and compared the biofilm formation of 20 P. aeruginosa isolates in 96-well polystyrene microtiter plates. As shown in Fig. 1, the P. aeruginosa isolates from different individuals as well as from the same patient expressed different levels of biofilm formation. Compared with reference strain PAO1, the majority of isolates had a reduced capacity to adhere to the polystyrene plate surface; only one isolate, isolate 1738b/1985 from patient 3, showed adherence equivalent to that of PAO1. Compared with the P. aeruginosa isolates recovered from the earlier chronic infection phase, different degrees of reduction of biofilm-forming capacities were observed from the isolates collected from the late chronic infection stage of all eight patients.
Biofilm development by P. aeruginosa sequential isolates. To characterize the biofilm architecture under more defined conditions, we compared the biofilm formation of five sequential isolates and one isolate from the post-lung transplantation phase from patient 1. Parallel flow chambers were also inoculated with reference strain PAO1. Top views of the CLSM images of the flow chamber cultivations are shown in Fig. 2. Images were acquired on days 1 (24 h), 3 (72 h), 5 (120 h), and 7 (148 h) at random positions in the flow channels. Visual inspection revealed that the P. aeruginosa isolates formed biofilms that were significantly different from those of wild-type strain PAO1. The formation of the PAO1 biofilm was characterized by the multiplication of cells, which formed a continuous layer that covered the substratum during the first 3 days of growth, followed by the formation of microcolonies by days 5 to 7. An obvious difference in biofilm development was observed for the sequential isolates recovered at different periods of the chronic infection. Isolates 14889a/1980, 19193a/1984, and 476a/1988 attached and began to form monolayers of cells within the first 24 h. After 5 to 7 days of growth, isolates 14889a/1980, 19193a/1984, and 476a/1988 developed a rather flat biofilm structure associated with a high level of coverage of the substratum. In contrast, the later isolates, isolates 15278a/1994, 67903b/1999, and 1395b/2003, showed very little attachment to the coverslip; and the biofilms were composed of inconsistent cell aggregates that failed to cover the entire surface during the course of the experiment.
Biofilm architectures of P. aeruginosa isolates are dependent on the motility properties. The flow chamber biofilm architectures of the remaining isolates were also examined, and the biofilms were established in the same way as described above. The biofilms differed in their architectures possibly due to the motility properties of each isolate (Table 1). Twitching motility plays an important role in shaping the biofilm architectures (23). Isolates that possessed twitching motility (isolates 14889a/1980, 19193a/1984, 467a/1988, 374d/1985, 54514a/1997, 21168a/1984, 16020/1999, and 15761/1978) were able to develop flat uniform biofilm structures consisting of a homogeneous layer of cells. A moderate heterogeneous biofilm structure with irregular microcolonies was observed for isolates that lacked twitching motility (isolates 15278a/1994, 64691c/1999, 19696/1984, 68000d/1999, 20688a/1984, 5284a/1995, and 15164/1997).
Abnormal biofilm architectures of P. aeruginosa isolates. The development of abnormal biofilm architectures from three P. aeruginosa isolates was observed, as shown in Fig. 3. Isolate 1738a/1985 showed up as microcolonies at day 1, and further growth of the microcolonies developed a unique biofilm structure in which the biofilm cells mingled together, thus resulting in a number of uncovered round or irregular void spaces. At day 5, the biofilm became thicker and the void areas developed into larger exposed spaces due to cell detachment. Isolate 65608a/1999 exhibited enhanced microcolony formation. Following a period of initial microcolony formation at day 3, cell proliferation occurred at a fixed position, and by day 6 the bacterial biofilm developed into a structure with massive elevated perpendicular microcolonies. An alternative biofim architecture consisting of "rivulet" structures stretched in the direction of medium flow was observed for isolate 15357a/1994.
DISCUSSION
In this report we have demonstrated that clinical P. aeruginosa isolates display a high degree of phenotypic variability (Table 1). For example, the isogenic serial isolates recovered from patient 1 displayed significant phenotypic alterations during the course of chronic infection, although the PFGE fingerprint remained stable, suggesting that different P. aeruginosa phenotypes evolve within the CF respiratory tract. P. aeruginosa isolates recovered from CF patients early in the course of infection expressed flagellar and pilus motilities in a conventional manner, whereas the loss of motility was observed after the establishment of chronic infection. The phenotypic changes also correlated with the decreased production of virulence factors, such as pyocyanin and proteases, controlled by the las quorum-sensing system. The loss of motility and virulence factor production have been suggested to confer a survival advantage in CF patients (27, 30, 31), and with minimal invasion of lung tissue, the bacteria are able to persist for prolonged periods of time (31, 47). It is interesting, however, that posttransplantation isolate1395b/2003 from patient 1 maintained the genotype and phenotypes described from the pretransplantation-phase isolates. The reoccurrence of this strain from the chronic colonization is most likely due to the presence of persistent clones in the upper part of the patient's own respiratory tract, notably, the sinuses (52). This recolonization indicates that the loss of virulence does not impair new lung colonization. Moreover, the LPS profiles of six sequential isolates from patient 1 were analyzed, and loss of the O antigen (the B band) was observed throughout the chronic infection (data not shown). LPS is an important component of the bacterial outer membrane, and it has been implicated in initial surface attachment and biofilm formation in P. aeruginosa (43). Changes in LPS may affect the initial attachment, and the loss of the B-band LPS reduced the cell's ability to interact with hydrodynamic surfaces (32).
The detection of a reduction of in vitro biofilm formation of P. aeruginosa isolates compared to that of wild-type isolate PAO1 during our initial screening on a polystyrene microtiter plate was unexpected. However, the results of flow cell biofilm experiments of the serial isolates from patient 1 support the adherence data. The reduction of the biofilm formation capacity of sequential isolates is an important indicator for the phenotypic conversion of P. aeruginosa during chronic infection (Fig. 2). Isolates from the early periods of chronic infection (isolates 14889a/1980, 19193a/1984, and 476a/1988) were able to form uniform flat biofilms where cells appear densely packed. In contrast, isolates recovered from the late phase of chronic infection and from the post-lung transplantation phase (isolates 15278a/1994, 67903b/1999, and 1395b/2003) exhibited less adherence and essentially no formation of mature biofilm. This finding is particularly obvious for biofilms that consist mainly of cell aggregates with large uncolonized areas at the glass substratum. Isolates 15278a/1994, 67903b/1999, and1395b/2003 were deficient in factors suggested to play a role in the formation of mature biofilms, such as motility and expression of las quorum sensing (Table 1). It has been shown that flagella and type IV pili are important for biofilm development (36). The early isolates that lacked swimming motility (isolates 14889a/1980, 19193a/1984, and 476a/1988) were still able to adhere and develop mature biofilm (Fig. 2), indicating that flagella do not play a role in the attachment of P. aeruginosa to a glass surface, and this is in agreement with previous work from Klausen et al. (23). Heydorn et al. demonstrated that biofilm development of a lasI mutant was indistinguishable from that of wild-type PAO1 cells (18), whereas other studies suggest that although lasI cell signaling may not be required for biofilm formation, it probably plays a role in the structural heterogeneity of the biofilm (12, 58). Thus, a lack of motility and lasI quorum sensing does not necessarily result in decreased or unstructured biofilm formation.
The biofilm structures obtained from P. aeruginosa isolates were quite heterogeneous and most often different from the biofilms formed by wild-type strain PAO1. The fundamental difference in biofilm structures may be attributed to the role of flagella and pili, factors that are important during surface colonization. It has been suggested by Klausen et al. (23) that the combination of growth and efficient type IV pilus-driven migration on the substratum results in flat wild-type biofilms. P. aeruginosa isolates from CF patients with twitching motility developed flat biofilms, whereas isolates that lacked twitching motility developed distinct microcolonies (rough biofilms). The development of very special biofilm structures was also observed for some of the P. aeruginosa isolates (Fig. 3).
It has been shown that alginate is a significant exopolysaccharide involved in establishing P. aeruginosa biofilm structures in the lungs of CF patients (56). In the present investigation we have chosen to examine nonmucoid P. aeruginosa biofilms, although alginate-overproducing mucoid P. aeruginosa variants were also isolated from patient 1 during the chronic colonization and post-lung transplantation phases. No significant difference in the fragment patterns compared to that of the nonmucoid clone was seen in the PFGE analysis (data not shown). The major reason for this choice is that, by far, most of the available in vitro investigations of P. aeruginosa biofilms available for comparison were performed with such nonmucoid strains. Furthermore, in vitro investigations also indicate that alginate is not required for biofilm formation (48, 55), although studies of P. aeruginosa laboratory and clinical strains showed that the biofilms produced by mucoid strains were much thicker than those produced by isogenic nonmucoid strains (17, 34).
Infections of P. aeruginosa in the lungs of CF patients have for a long time been associated with the biofilm lifestyle of these bacteria based on, e.g., direct microscopic observations of samples from patients and autopsy materials from CF lungs studied by light and electron microscopy (1, 19, 25). Additionally, the homoserine-lactone quorum-sensing molecules detected in sputum from CF patients with chronic P. aeruginosa infection were characteristic for the biofilm mode of growth (45). It has been shown that biofilm development caused by mucoid, alginate-producing P. aeruginosa isolates is an important virulence factor for the organism (7, 15, 37). Compared with nonmucoid P. aeruginosa isolates, isolates with the alginate-overproducing mucoid P. aeruginosa phenotype are often associated with a poor prognosis and tissue damage in CF patients (25, 29, 37, 39). The improved persistence of mucoid P. aeruginosa strains relative to that of nonmucoid strains in mouse lungs has been reported from in vivo studies (3, 19, 46, 57). Previous work from Hoffmann et al. (19) and Worlitzsch et al. (54) has demonstrated that nonmucoid P. aeruginosa is a niche specialist which often splits off from the mucoid P. aeruginosa, and phenotypic conversion occurs, especially under anaerobic conditions in the conductive airways. It has also been shown that in the CF airway, the lumen and sputum are largely composed of dead polymorphonuclear leukocytes, where cellular components released from necrotic neutrophils can serve as a biological matrix to facilitate P. aeruginosa biofilm formation (4, 26, 53).
Microbial biofilm formation has been studied with increasing intensity during the last 5 to 10 years, and it is now generally recognized that bacterial life on surfaces is often the dominant lifestyle. P. aeruginosa has attracted particular interest as a model system for biofilm development, partly because it is a frequent pathogen in humans and partly because it is a well-characterized organism with a very versatile capacity to persist and proliferate in many different environments. The understanding about the biofilm development route and its control may constitute a platform for the design of strategies that can be used to combat and eradicate the infection (9, 21). Our present results suggest that nonmucoid P. aeruginosa cells loose their biofilm formation capacity over time in the CF lung, and this leads to the conclusion that the biofilm lifestyle as we know it from in vitro analysis is not important for the nonmucoid phenotype during the chronic infection state.
ACKNOWLEDGMENTS
This work was supported by the Danish Cystic Fibrosis Foundation and Danish Research Agency.
We thank Ulla Johansen (Rigshospitalet), Tina Wasserman (Panum Institute), and Tove Johansen (BioCetrum-DTU) for excellent technical assistance.
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