Quantitation of Pseudomonas aeruginosa Swimming Motility

Pseudomonas aeruginosa rarely causes lung infections in healthy people but is the leading cause of death in patients with cystic fibrosis (CF). Therefore it is important to understand how P. aeruginosa is controlled in normal airways so we can treat and perhaps prevent infection in CF airways. Altered CF epithelial cell secretions are a potential contributor to the increased infection rate. The purpose of this study was to develop a quantitative method to investigate the effects of normal secretions from Calu-3 cells, a human airway epithelial cell line, on P. aeruginosa swimming motility.  P. aeruginosa motility is associated with increased virulence. The motility of two P. aeruginosa strains, PAO1 and PA14, were visually examined using a simple wet mount under oil immersion with 1000x total magnification. An analog camera connected to the microscope and a computer with VirtualDub software were used to capture 10-second videos of swimming P. aeruginosa in the presence or absence of Calu-3 secretions. Analysis of the swimming patterns of individual P. aeruginosa was performed with Manual Tracking (an ImageJ plug-in), and quantitative data was analyzed using Microsoft Excel.  Visual examination of the tracings qualitatively showed inhibition in overall motility and speed when bacteria were treated with Calu-3 airway secretions. Calu-3 secretions decreased the percent of motile PAO1 (44% ± 23% without vs. 18% ± 11% with secretions) and PA14 (50% ± 27% without vs. 27% ± 27% with secretions) strains. Additionally, of the bacteria that remained motile, the average swimming speed was decreased in the presence of Calu-3 supernatants. In conclusion, we were successfully able to quantitate P. aeruginosa swimming motility using relatively simple equipment and readily available software. Using this process, we demonstrate that normal airway secretions inhibit P. aeruginosa motility and thus may play a role in prevention of infection.