Characterization of biofilm-like structures formed by Pseudomonas aeruginosa in a synthetic mucus medium

Cystic fibrosis (CF), an inherited disorder caused by mutations in the gene that encodes the cystic fibrosis transmembrane conductance regulator, affects approximately 30,000 Americans, primarily those of Northern European origin [1, 2]. These mutations cause a deficiency in chloride secretion with ensuing accumulation of thick, stagnant mucus within the lung alveoli of the patients [1–4]. Nutrients in the thick mucus facilitate the colonization of various bacterial pathogens, including Pseudomonas aeruginosa, Staphylococcus aureus, and Haemophilus influenzae[3, 5]. Colonization by these pathogens elicits a strong host inflammatory response which leads to destruction of the lung tissue and, ultimately, death from respiratory failure [1, 6, 7]. P. aeruginosa is one of the significant pathogens in chronic lung infections of CF patients [1, 8].

Among the different factors that contribute to the virulence of P. aeruginosa is its ability to form a biofilm, a community within which bacteria are attached to a substratum or to each other [9]. Within the biofilm, the bacteria are surrounded by extracellular polymeric substance (EPS), which protects them from the effects of the host immune system and from diverse antibiotics [10–12]. Biofilm development occurs in stages that require specific bacterial factors at each stage. For example, during the initial (attachment) stage of biofilm formation, bacteria depend on both the flagellum-mediated swimming motility and the pili-mediated twitching motility [13]. A number of P. aeruginosa infections are associated with biofilm formation, including chronic otitis media, heart valve endocarditis, and chronic lung infections in CF patients [9, 14].

Previously, studies have described synthetic mucin-containing artificial sputum media (ASM) that mimics the thick mucus within the lung of CF patients [15, 16]. When grown in ASM, P. aeruginosa formed in tight microcolonies suspended within the medium rather than attached to the surface or free swimming as in standard broth media [15, 16]. Mucin is the main component of secreted mucus, which also contains a large number of plasma and non-plasma proteins, carbohydrates, amino acids, nucleic acids, lipids, and electrolytes [17, 18]. It has been shown that mucin-P. aeruginosa interactions promote biofilm formation in the continuous culture flow-through system [19].

In this study, we utilized a static microtiter plate culture system to investigate the effect of different conditions on the development of P. aeruginosa biofilms in mucus medium. Within the medium, P. aeruginosa strain PAO1 formed structures that are biofilm-like, but are not attached to the surface. The amount of mucin and extracellular DNA in the medium, as well as the level of environmental oxygen (EO₂), are critical for the development of these biofilm-like structures (BLS). Additionally, one of the P. aeruginosa quorum sensing systems, rhl, affects formation of the BLS. Furthermore, as it develops its BLS, P. aeruginosa eliminates already established S. aureus BLS by a bactericidal mechanism.

CF sputum is a highly viscous secretion in which PAO1 grows readily. PAO1 forms conventional biofilms on abiotic surfaces [13, 19, 35], but it develops macrocolonies, tight aggregates consisting of numerous microcolonies, in ASM and the CF lung [16, 21]. While PAO1 formed a typical flat undifferentiated biofilm that completely covered the substratum with a homogenous distribution of the biovolume in a continuous flow-through system, it grew almost exclusively as discrete microcolonies that eventually formed a mature biofilm on a mucin-covered glass surface [19]. Based on these results, Landry et al. suggested that mucin interacts with specific PAO1 adhesins thereby immobilizing the bacteria onto the glass surface [19]. In our analysis, the observed BLS developed exclusively within the gelatinous mass formed by ASM+ and not on the surface of the well (Figure 1). It is likely that through the initial interaction of these putative adhesins, individual PAO1 bacteria adhere to the mucin glycoprotein forming the nuclei of the microcolonies and leaving no bacteria to adhere to the plastic surface. Individual nuclei would then recruit more PAO1 bacteria, possibly through additional inter-bacterial and glycoprotein-bacterial interactions, growing into well-defined BLS observed over time (Figure 2).

Based on the work of Ghani & Soothill [15] and Sriramulu et al. [16], we utilized 0.5% mucin (1X) in our ASM+. But more recently, Henke et al. [36], showed that the concentrations of MUC5AC and MUC5B, the principal gel-forming mucins, are decreased in airway secretions from CF patients with stable disease and greatly increased during pulmonary exacerbations (by 89% and 908%, respectively). When we reduced the mucin concentration of ASM+ by 50% (0.5X), the gelatinous mass still formed in the well, possibly through the contribution of other ASM+ components (DNA and lecithin) that add to the viscosity. However, the typical multilayered BLS was eliminated and replaced with a structure that appears to consist of small microcolonies amid individual cells and tiny cell clusters distributed throughout most of the gelatinous mass (Figure 4A, B). Surprisingly, the effect of increasing the concentration of mucin to 2X on the development of BLS was similar to that induced by reducing the mucin concentration. Rather than the distinct highly structured BLS architecture, PAO1 produced small microcolonies distributed throughout the ASM+ (Figure 4C). At this time, we do not know if the increase in the availability of mucin glycoprotein interferes with the development of microcolonies that coalesce to form the well-developed BLS.

One of the hallmarks of the CF syndrome is the overproduction of mucin within the lung alveoli [1, 3, 7]. Yet during P. aeruginosa infection of the CF lung alveoli, the level of mucin may vary [36]. P. aeruginosa LPS induces the production of reactive oxygen intermediates, which cause release of transforming growth factor α; TGF-α then up-regulates the expression of MUC-5 AC thereby causing excessive mucin production[37–39]. However, P. aeruginosa produces other factors that may reduce the amount of mucus within its immediate vicinity; alveolar mucin is degraded by P. aeruginosa extracellular serine proteases such as LasB [40]. Ultimately, the interaction of all these factors would produce a net mucin concentration suitable for the full development of the BLS, while any imbalance in the production of these factors that reduces or increases mucin concentration would prevent the establishment of the BLS. Alternatively, BLS may form in the initial stages of P. aeruginosa infection in the CF lung. Treatment that reduces the amount of mucin present would disperse the bacteria making them more susceptible to antibacterial treatment (stable disease). Alternatively, mucin may reduce the chances of forming new BLS.

Extracellular DNA is another contributor to the viscosity of CF sputum [15, 16]. Within the CF lung, there are several sources for this extracellular DNA – dead host immune cells, lysed bacteria, QS-regulated release of P. aeruginosa DNA, and outer membrane vesicles that contain DNA [41, 42]. Like mucin, DNA contributes to biofilm formation as well; a similar biofilm was detected on a glass surface coated with DNA [41]. Previous studies using other biofilm development media, such as LB or minimal medium, indicated that extracellular DNA is critical for the initial establishment of P. aeruginosa biofilms [42]. The levels of extracellular DNA also vary within CF sputum, ranging from 0.3 to 9.5 mg/ml in one study of 167 CF sputum samples [43]. Variations in the level of extracellular DNA in ASM+ affected the development of BLS much more dramatically than variations in the level of mucin. In ASM+ with 0.5X DNA (2 mg/ml), a well developed BLS was visible (Figure 5B), but the biovolume and total surface area occupied were considerably less (Table 1 and 2). When the amount of DNA was increased to 1.5X (6 mg/ml), PAO1 did not produce detectable structures; rather, the gelatinous mass formed by the ASM+ contained scattered individual cells (Figure 4C). However, at this time it is not clear how an increase in the external DNA reduces the number of BLS within the gelatinous mass of ASM+.

Within the lung of CF patients and during other chronic lung infections, P. aeruginosa survives under microaerobic (10% EO₂) to anaerobic (0% EO₂) conditions. A steep oxygen gradient exists within the P. aeruginosa infected alveolar mucus [5, 21]. Within the mucus, P. aeruginosa secretes compounds that lower the oxygen transfer rate generating optimum conditions for microaerobic growth [22, 44]. We showed previously that lower oxygen tension also influences the expression of P. aeruginosa virulence genes [45]. Compared with aerobic conditions, the expression of pyoverdine genes was reduced under microaerobic conditions; in contrast, the expression of the exotoxin A gene, toxA was increased [45]. Compared with 20% EO₂ and 0% EO₂, microaerobic (10% EO₂) conditions are optimal for the development of P. aeruginosa BLS in ASM+. BLS developed under 10% EO₂ had a greater mean thickness and a larger biovolume than those developed under either 20% or 0% EO₂ (Figure 6, Table 1 and 2). In the absence of EO₂, PAO1 required 6 days to develop rudimentary BLS (Figure 6C) indicating that a low level of oxygen is essential for the full development of these structures.

Depending on conditions under which the biofilms were developed (medium, the biofilm development system, and the biofilm substrate), previous studies indicated the involvement of the QS systems in the development of P. aeruginosa biofilm [29, 30, 35, 46]. In those studies, the deficiency in biofilm development was associated with either a lasI or rhlI mutation. We tested mutants defective in all three known P. aeruginosa QS systems in ASM+. PAO-R1 (ΔlasR), PAO-JP1 (ΔlasI), and PW2798::pqsA-lacZ (ΔpqsA) produced BLS that were visually and architecturally similar to each (Figure 8). In contrast, PDO111 (ΔrhlR) BLS were visually, architecturally, and structurally dissimilar to PAO1 BLS, in that they had a smaller biovolume and mean thickness (Figure 8, Tables 3 and 4). The loss of rhlI (PDO100) had the most profound effect on the BLS produced by PAO1 in ASM+. Both PDO100 (ΔrhlI) and PDO111 (ΔrhlR) produced BLS that were significantly smaller (biovolume, mean thickness) than PAO1 BLS (Figure 8, Tables 3 and 4). However, BLS produced by these two strains were more heterogeneous than PAO1 BLS (a significant increase in roughness coefficient) (Figure 8, Tables 3 and 4). Additionally, more regions of the PDO100 and PDO111 BLS were exposed to nutrients than PAO1 BLS (a significantly higher surface to biovolume values) (Figure 8, Tables 3 and 4). Our results suggest that the production and maturation of the fully-developed complex BLS requires a potential P. aeruginosa factor that is stringently controlled by the rhl and not the las or the pqs systems. Among the P. aeruginosa factors that are stringently controlled by the rhl system are the rhamnolipid biosurfactants [47, 48]. The rhamnolipids encoded by the rhlAB operon contribute to biofilm development in P. aeruginosa through multiple mechanisms including maintaining open channels by affecting cell-to-cell interaction [28], promoting microcolony formation in the initial stages of biofilm development [49], and dispersing cells from the mature biofilms [50]. Analysis of PAOΔrhlA and/or PAOΔrhlB mutants in ASM+ should allow us to determine if rhamnolipid plays a role in the development of the BLS. Interestingly, PA103, which is known to have a deletion in lasR[51], produced BLS with reduced biovolume and mean thickness (compared with those produced by PAO1 or PAO-R1) (Figure 7, Tables 3 and 4). This suggests that the observed differences between the BLS produced by PAO1 and PA103 are not due to the loss of the lasR gene in PA103. CI-4, a clinical isolate obtained from a patient who had been continuously infected with P. aeruginosa for 30 days, has deletions in both lasR and rhlR[27]. This strain produced BLS that had less biovolume, mean thickness and covered less total surface area that PAO1; visually, the BLS were also unique in appearance among all the QS mutants, numerous small microcolonies distributed throughout the medium (Figure 7, Tables 3 and 4). This suggests that there is a complex interaction among the QS systems in controlling BLS production within ASM+.

Using ASM+, which has the same components as our ASM+, Sriramula et al. [16] examined the growth of PAO1, PAOΔlasR, and PAOΔrhlR. Both PAO1 and PAOΔrhlR formed macroscopically visible clumps or aggregates, which they termed tight microcolonies, that could not be disturbed even with vigorous pipetting [16]. In contrast, PAOΔlasR failed to develop these tight microcolonies [16]. In our study, neither PAO1, nor any other tested strain produced macroscopically visible structures. In part, this is due to the turbidity of ASM+. Similar to the tight microcolonies described by Sriramula et al. [16], the BLS we observed in our ASM+ did not attach to a surface. The BLS are adherent when fully-developed, but cells within the BLS can be dispersed by vortexing. Differences between the two studies, despite the similarity in the growth medium, are most likely due to differences in other growth conditions. While Sriramula et al. [16], grew their cultures under 20% EO₂ with shaking, we grew our cultures under static conditions regardless of the EO₂ concentration. Given these differences, it is not practical to directly compare the bacterial structures observed in the two studies with respect to the role of the QS systems in their formation.

Biofilms at different infection sites often consist of multiple species of bacterial pathogens [52, 53]. These bacterial species may either compete with each other or support each other’s growth. Qin et al. [54] previously showed that P. aeruginosa inhibited the planktonic growth of Staphylococcus epidermidis through a QS-related mechanism. Additionally, using the static chamber cultivation system (microtiter plate assay), they demonstrated that P. aeruginosa extracellular polysaccharide disrupted an already established S. epidermidis biofilm [54]. Disruption of these biofilms, however, does not occur through the bactericidal effect observed with the planktonic cells; instead the bacteria within the biofilm were dispersed alive [54]. When we co-cultured P. aeruginosa and S. aureus statically under 20% EO₂ in TSBDC or ASM+, P. aeruginosa eliminated S. aureus by day 2 (Figure 10). Furthermore, and similar to the findings by Qin et al. with S. epidermidis[54], the addition of P. aeruginosa to S. aureus BLS established in ASM+ disrupted the S. aureus BLS (11a, b). However, P. aeruginosa disrupted the S. aureus BLS through an bactericidal effect rather than dispersion. By 56-h post addition of PAO1, no CFU of AH133 were recovered (Figure 11C), although it is remotely possible that our failure to detect S. aureus is due to their existence in a viable but nonculturable state. This effect is similar to the clinical observations of CF lung infections where S. aureus, an early colonizer, is gradually replaced by P. aeruginosa. The nature of the PAO1 bactericidal factor that eliminates the S. aureus BLS is under investigation.

In this study, we have demonstrated that thick, viscous ASM+ containing mucin and extracellular DNA and incubated under static conditions with lowered oxygen tension (10% EO₂) – constituents and conditions similar to those within the lung alveoli of CF patients – induces the formation of biofilm-like structures by P. aeruginosa and S. aureus, two of the pathogens most commonly seen in the infected lungs of these patients. The BLS are not attached to the surface, but form within the medium as has been reported for the development of macrocolonies within the mucus in CF lungs. Thus, ASM+ represents an in vitro medium in which the effect of changing levels of substances produced by the host and the bacteria can be analyzed to determine the effect on such structures and on the susceptibility of the bacteria within the BLS to various treatments.