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...

Staphylococcus aureus forms pathogenic biofilms. Previous studies have indicated that ethanol supplementation during S. aureus biofilm formation results in increased biofilm formation and changes in gene expression. However, the impact of alcohols on preformed S. aureus biofilms has not been studied. In this study, we formed S. aureus biofilms on PVC plastic plates and then treated these preformed biofilms with five different alcohols. We observed that alcohol treatment of preformed S. aureus biofilms led to significant increases in biofilm levels after 24 h of treatment. Many bacteria within these biofilms were found to be alive and metabolically active. Alcohol treatment also resulted in increased transcription of the biofilm-promoting genes icaA and icaD, as well as several antibiotic resistance genes. These results demonstrate that treatment of S. aureus preformed biofilms with alcohols enhances biofilm levels if maintained for extended periods. Thus, alcohols might be of limited usefulness for the eradication of preformed S. aureus biofilms.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen with the capacity to cause serious disease, including chronic biofilm infections in the lungs of cystic fibrosis (CF) patients. These infections are treated with high concentrations of antibiotics. Virulence modulation is an important tool utilized by P. aeruginosa to propagate infection and biofilm formation in the CF airway. Many different virulence modulatory pathways and proteins have been identified, including the magnesium transporter protein MgtE. We have recently found that isogenic deletion of mgtE leads to increased cytotoxicity through effects on the type III secretion system. To explore the role of the CF lung environment in MgtE activity, we investigated mgtE transcriptional regulation following antibiotic treatment. Utilizing quantitative real-time-PCR, we have demonstrated an increase in mgtE transcript levels following antibiotic treatment with most of the 12 antibiotics tested. To begin to determine the regulatory network governing mgtE expression, we screened a transposon-mutant library of P. aeruginosa to look for mutants with potentially altered mgtE activity, using cytotoxicity as a readout. In this screen, we observed that AlgR, which regulates production of the biofilm polysaccharide alginate, alters MgtE-mediated cytotoxicity. This cross-talk between MgtE and AlgR suggests that AlgR is involved in linking external inducing signals (e.g. antibiotics) to mgtE transcription and downstream virulence and biofilm activities. Analysing such interactions may lead to a better understanding of how the CF lung environment shapes P. aeruginosa biofilm infections.

In nature, bacteria exist in and adapt to different environments by forming microbial communities called “biofilms.” We propose simple, inquiry-based laboratory exercises utilizing a biofilm formation assay, which allows controlled biofilm growth. Students will be able to qualitatively assess biofilm growth via staining. Recently, we developed a biofilm assay exercise for a high school biology class and found success in increasing students’ interest in biology and explaining ecological concepts. Because of this success, we feel that biofilm assay laboratory exercises would be an excellent addition to any science teacher’s curriculum.