Co-culture Models of <em>Pseudomonas aeruginosa</em> Biofilms Grown on Live Human Airway Cells

Abstract

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. . CFBE cells should be seeded at a concentration of 10 6 cells/well in a 6-well tissue culture plate or 2 X 10 5 in a 24-well tissue culture plate in minimal essential medium (MEM) supplemented with 10% fetal bovine serum, 2mM L-glutamine, 50 U/mL penicillin, and 50 μg/mL streptomycin. We use 1.5 mL medium per well in 6-well plates and 0.5 mL medium per well in 24-well plates. 2. Cells should be grown at 37°C and 5% CO2-95% air for 7-10 days to form a confluent monolayer before inoculation with bacteria. The medium must be changed every 2-3 days. These conditions have been shown to lead to formation of a confluent monolayer and tight junctions. 3. Grow P. aeruginosa in 5 mL LB for 18 hours at 37°C on an incubator shaker at 200 rpm. Under these conditions, P. aeruginosa cultures will typically reach a density of 5x10 9 CFU/ mL. 4. For bacterial inoculation, remove the medium from CFBE cells and add an equal volume of MEM without phenol red, supplemented with 2 mM L-glutamine (Microscopy medium). Confluent CFBE monolayers are inoculated with P. aeruginosa at a multiplicity of infection of approximately 30:1 relative to the number of CFBE cells originally seeded. This equates to 2 X 10 7 CFU/mL in 1.5 mL MEM/well for 6-well plates and 1.2 X 10 7 CFU/mL in 0.5 mL MEM/well for 24-well plates. 5. Incubate plates for 1 hour at 37°C and 5% CO2-95% air. 6. Following the 1 hour incubation, the supernatant should be removed and replaced with fresh Microscopy medium supplement...

Coculture of Staphylococcus aureus with Pseudomonas aeruginosa Drives S. aureus towards Fermentative Metabolism and Reduced Viability in a Cystic Fibrosis Model

“…For coculture samples, 250 l of 0.1-OD 600 P. aeruginosa and 250 l of 0.1-OD 600 S. aureus were added to triplicate wells of monolayers of CF bronchial epithelial (CFBE) airway cells in a 24-well plate. CFBE monolayers were grown as previously described (54). For monoculture controls, 250 l of 0.1-OD 600 P. aeruginosa or 250 l of 0.1-OD 600 S. aureus and 250 l of MEM ϩ L-Gln were added to triplicate wells.…”

Pseudomonas aeruginosa Biofilm Formation in the CF Lung and Its Implications for Therapy

“…The aggregates appear morphologically similar to biofilms formed on abiotic surfaces (Moreau-Marquis, Bomberger et al 2008;Moreau-Marquis, Redelman et al 2010). Furthermore, formation of these co-culture biofilms requires factors necessary for abiotic biofilm formation, and bacteria within co-culture biofilms display a pattern of gene expression consistent with that found in abiotic biofilms.…”

“…Furthermore, formation of these co-culture biofilms requires factors necessary for abiotic biofilm formation, and bacteria within co-culture biofilms display a pattern of gene expression consistent with that found in abiotic biofilms. In a different study, similar co-culture biofilms were formed by static incubation of P. aeruginosa and CF airway cells in the presence of arginine Moreau-Marquis, Redelman et al 2010). Importantly, in both systems, the antibiotic resistance of the co-culture biofilms was greatly increased compared to both planktonic bacteria and abiotic biofilms Moreau-Marquis, Bomberger et al 2008;Moreau-Marquis, O'Toole et al 2009).…”

“…Dispersion events appear to be influenced by the production of virulent bacteriophage from dormant prophage (Rice, Tan et al 2009). While very few studies investigating P. aeruginosa biofilm formation on living tissue have been performed, it is thought that these steps are conserved during infection (Hoffmann, Rasmussen et al 2005;Anderson, Moreau-Marquis et al 2008;Moreau-Marquis, Bomberger et al 2008;Woodworth, Tamashiro et al 2008;Moreau-Marquis, Redelman et al 2010) (Figure 1). …”

Using Fluorescence Intensity of Enhanced Green Fluorescent Protein to Quantify Pseudomonas aeruginosa

“…Therefore, the ability to differentiate between species, such as bacteria that are genetically modified to express fluorescent proteins of a variety of emission colors, would be beneficial. This assists in tracking the growth of a single species within a complex environment in order to better understand bacterial cell interactions in mixed-species biofilms [42][43][44]. EGFP is a mutant of green fluorescent protein (GFP), and was used in this study due to its higher fluorescence properties [41,45,46].…”