THE STUDY OF THE GENE(S) INVOLVED IN THE PROTECTION OF THE PSEUDOMONAS AERUGINOSA MUCOID S

Pulmonary infections involving Pseudomonas aeruginosa (PA) are a complication faced by cystic fibrosis (CF) patients. The bacteria mutate to a mucoid state and form a biofilm that creates an anaerobic environment, decreasing the effectiveness of antibiotics. Acidified nitrite (NO2-) has been shown to be an effective antibacterial agent against the PA mucoid mutant, mucA22. This experiment used six double mutants, in an anaerobic environment mimicking the conditions in the lungs of CF patients, to investigate the gene(s) involved in protecting the bacteria from NO2- toxicity. It was hypothesized that at least one double mutant would show increased susceptibility to NO2-. An overnight culture of bacteria was diluted 100 fold into lysogeny broth (LB) (pH 6.5, 50 mM phosphate buffer) containing either 15 mM KNO3 (control) or 15 mM KNO3 plus 15 mM of NaNO2 (experiment). Cells were incubated in an anaerobic chamber at 37 °C. Tenfold serial dilution was performed at 0, 24 and 48 hours. The diluted samples were spotted on LB agar plates and incubated an additional 24 hours before counting and reporting the colony forming units (CFUs). The experiment was repeated four times. Results showed that the double mutants mucA22gal, mucA22gip, mucA22oprH and mucA22prlC were one to two orders of magnitude more susceptible to NO2- compared to mucA22 while mucA22fdnG and mucA22uspK were not. It was expected that gene(s) would be identified that played a role in protecting mucA22 from NO2-, and instead 4 gene alterations were found that increased susceptibility to NO2- toxicity.