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1Department of Physiology, Dartmouth College, 2Department of Biology, Indiana University Purdue University Indianapolis
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This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.
Moreau-Marquis, S., Redelman, C. V., Stanton, B. A., Anderson, G. G. Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells. J. Vis. Exp. (44), e2186, doi:10.3791/2186 (2010).
Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.
1. Static Co-culture Biofilm Model
2. Flow Cell Co-culture Biofilm Model
3. Representative Results
We use a strain of P. aeruginosa containing the pSMC21 plasmid which allows for constitutive expression of GFP6. For this reason, GFP-labeled biofilms growing on a CFBE monolayer can be visualized by epifluorescence microscopy. Alternatively, visualization of the biofilm can be achieved by staining unlabeled P. aeruginosa with a 1% calcofluor white solution for 1 hour at 37°C1.
For imaging biofilm formation on CFBE cells in the flow assay, we use a custom-made stage adapter, but several companies can now provide specially fitted stage adapters. We work with an Olympus IX70 inverted microscope equipped with an ORCA-AG deep cooling CCD camera and an x60 oil-immersion objective (numerical aperture 1.40). The filter wheel is equipped with a 480/40 nm band pass excitation filter and a 535/30 nm band pass emission filter. Digital images were acquired with the OpenLab 4.0.3 software package (Improvision) and volumes were deconvolved by iterative restoration using the Volocity 3.5.1 software (Improvision). Quantitative analysis of 3D biofilm structures was achieved with the COMSTAT image analysis software package7,8.
For any co-culture model considered, one must pay particular attention to the cytotoxicity developing between the components of the model. In both the static and the flow cell assays, we found that the CFBE monolayer could withstand the presence of P. aeruginosa for up to 8 hours after inoculation without any sign of alteration. Epithelial monolayer integrity can be assessed by phase-contrast microscopy using an inverted microscope1 (Figure 1A) or by differential interference contrast (DIC) microscopy throughout the experiment5. Over time, P. aeruginosa will produce toxins and virulence factors which accumulate and can damage the epithelial cell monolayer fully or in sections (Figure 1B-1C). Cytotoxicity can be quantitatively assessed using various kits to measure the release of lactate dehydrogenase (LDH) from the epithelial cells. LDH is a stable cytosolic enzyme released in the extracellular milieu upon cell lysis or cell death.
When the integrity of the airway monolayer is not compromised (typically for ~8 h following inoculation), P. aeruginosa biofilms can successfully form and develop at the apical surface of airway cells in both co-culture models described (Figure 2A-2B). Following 3D reconstruction (Figure 2C) and quantification, biomass accumulation can be accurately determined. We have also used these co-culture biofilms in several phenotypic assays. For instance, as mentioned above, biofilm CFU can be easily determined by lysing the epithelial cells, serially diluting the lysate, and plating on agar plates. We have found this technique advantageous in determining the resistance of different strains to antibiotic treatment. Furthermore, biofilm toxicity toward the epithelial cells can be measured using commercially available LDH detection kits. In this manner, the role of virulence factors in toxicity of biofilms can be assessed. These model systems also support a number of gene expression applications, including promoter fusion studies, RT-PCR, and microarray analysis1,5,9.
Figure 1. Monolayer of CFBE cells and representative images of compromised and damaged airway cell monolayers due to P. aeruginosa biofilm growth. (A) Representative image of a confluent monolayer of CFBE cells grown in tissue culture plates, assessed by phase-contrast microscopy. Scale bar, 120 μm. (B) Example of a compromised CFBE monolayer. Even though the monolayer does not display obvious visible signs of damage yet, P. aeruginosa bacteria are seen spreading between the tight junctions of the epithelial cells and gaining access to the basolateral membranes. Biofilm formation is typically not achieved under these conditions due to the monolayer deteriorating. Scale bar, 20 μm. (C) Example of an overgrown P. aeruginosa biofilm observed 24 h post-inoculation. After successfully supporting biofilm formation, the CFBE monolayer was damaged beyond repair and is now virtually absent. Residual biofilm, growing as a flat layer of bacteria, is shown attaching to the glass coverslip. Scale bar, 20 μm.
Figure 2. P. aeruginosa biofilms grown at the apical surface of confluent CFBE cells using the Static Co-culture Biofilm Model and the Flow Cell Co-culture Biofilm Model. (A) Representative image of a GFP-expressing P. aeruginosa biofilm grown on a confluent monolayer of CFBE cells using the Static Co-culture Biofilm Model, assessed by epifluorescence microscopy. Image is an overlay of the phase contrast channel and the fluorescence channel. Scale bar, 35 μm. (B) Representative image of a GFP-labeled P. aeruginosa biofilm grown for 6 h on a confluent monolayer of CFBE cells using the Flow Cell Co-culture Biofilm Model. To facilitate the visualization of the airway monolayer, nuclei were stained with 10 μg/mL Hoechst 33342 (Molecular Probes) for 30 min prior to inoculation with P. aeruginosa. Merged and pseudocolored images were viewed by differential interference contrast (DIC), and the corresponding fluorescent images are shown. Biofilms, presenting as green clumps attached to the apical surface of the CFBE cells, are dispersed across the airway cells. Scale bar, 20 μm. (C) Three-dimensional reconstruction of z-series image stacks showing the typical mushroom-like structures of 6 h-old P. aeruginosa biofilms forming on a CFBE cell monolayer. Scale bar, 10 μm.
Biofilms are communities of bacteria that form in response to environmental stimuli. These environmental signals lead to global regulatory changes within each bacterium, resulting in binding to a surface, aggregation, production of exopolysaccharides, and other phenotypes such as increased antibiotic resistance10. Over the last couple of decades, much evidence has supported the hypothesis that biofilms play a large role in the pathogenesis of chronic infections. For instance, it is well accepted that P. aeruginosa is able to establish chronic infections in the lungs of Cystic Fibrosis (CF) patients by forming biofilms11. CF is a genetic disorder, where mutations in the CFTR gene lead to improper chloride secretion12. CF patients typically experience altered airway physiology leading to thick mucus plugs in the airways and, concurrently, to microbial infection13. By late adolescence, the dominating infectious agent of CF airways is P. aeruginosa, leading to a chronic biofilm infection that is resistant to antibiotics14.
The protocols described in this paper have been developed to act as model systems for studying biofilm formation on living lung cells. Bacterial biofilms are implicated in many disease states and, yet, have mostly been studied on non-living solid surfaces, such as glass and plastic15,16. By studying biofilm formation and growth on abiotic surfaces only, one is missing important interactions between pathogen and host that can only occur with a model system such as described in the protocols above. Specifically, the Static Co-culture Biofilm Model and the Flow Cell Co-culture Biofilm Model take advantage of the ability of P. aeruginosa to interact with and bind to the epithelial surface of the lung. Using these models, we have shown that the response of the co-culture biofilms to antibiotic treatment is unique and that these models are more likely to accurately reflect the infectious state1,5. In this regard, we have reported that the resistance to tobramycin increases by >25-fold when P. aeruginosa biofilms are grown on airway cells compared to biofilms grown on abiotic surfaces such as glass5. It is likely that these techniques reflect the events that occur during early colonization of the lung17. Therefore, the co-culture model systems represent innovative tools for understanding early infection of the CF airway epithelium by P. aeruginosa1.
Tissue culture systems have commonly been employed to understand interactions between host and pathogen. We have taken these interactions a step further by forming biofilms on live human epithelial cells. In the future, modified versions of the models described here could potentially be used to investigate biofilm formation of different bacteria in other infectious states.
No conflicts of interest declared.
We would like to thank G. O Toole for guidance and suggestions in developing these models. This work was supported by the Cystic Fibrosis Foundation (ANDERS06F0 to G.G.A., STANTO07RO and STANTO08GA to B.A.S.), the National Institutes of Health (T32A107363 to G.G.A. and R01-HL074175 to B.A.S.), and the National Center for Research Resources Centers for Biomedical Research Excellence (COBRE P20-RR018787 to B.A.S.).
|FCS2 (Focht Live-Cell) chamber||Bioptechs||060319131616|
|FCS2 chamber controller||Bioptechs||060319-2-0303|
|40 mm glass coverslips||Bioptechs||PH 40-1313-0319|
|MEM without phenol red||Mediatech, Inc.||Mediatech, Manassass, VA|
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