aeruginosa bacteria appears to be an independent prognostic factor that carries with it an increased risk of death [38, 40]. Strains used here were isolated from sputum samples obtained from multiple patients all of whom had chronic endobronchial infections. Clinical P. aeruginosa isolates were collected between September

2005 and June 2008 from AZD0530 supplier adult patients with confirmed cystic fibrosis who attended one of the seven Ontario adult cystic fibrosis clinics or who attended smaller outreach clinics [24]. These 7 clinics provide secondary and tertiary care to more than 97% of all CF patients in Ontario. Patients were included in the study if they were ≥ 18 years of age, able to spontaneously produce sputum, and if they had a confirmed diagnosis of cystic fibrosis (a sweat chloride value higher than 60 mmol/litre and/or 2 disease-causing mutations). The research ethics board (The Ottawa Hospital Research Ethics Board) of all the participating centers approved

the study, and all participants provided written informed consent. Patients provided sputum samples which were couriered on ice to the central laboratory in Ottawa. To detect P. aeruginosa and other bacterial pathogens, sputum was plated onto the following selective and nonselective media: Columbia blood agar plate (PML), MacConkey agar plate (PML), and Pseudomonas aeruginosa selective agar plate (Oxoid). Plates were incubated at 35°C for 48 hours and P. aeruginosa colonies were identified AZD2281 research buy by oxidase testing,

TSI, arginine and growth at 42°C. If P. aeruginosa was isolated, then two distinct P. aeruginosa colony morphotypes from each sputum were worked up for molecular typing, and five P. aeruginosa isolates derived from each sputum were frozen at -70°C. To prepare for inhibition assays, strains were streaked from frozen on Pseudomonas Isolation Agar (Difco). Single colonies Clomifene were used to inoculate liquid LB (bacto-tryptone 10 g, yeast extract 5 g, NaCl 10 g, dH2O 1000 ml) and incubated under aerobic shaken conditions (150 rpm) at 37°C for 24 to 48 h to yield dense cultures. Estimation of genetic distance Genetic distance was estimated by comparing molecular genotypes of each P. aeruginosa isolate generated through pulsed-field gel electrophoresis (PFGE). PFGE is a well-accepted method [26, 30] that differs from multi-locus sequence typing (MLST)-based approaches in that it includes the entire genome rather than just seven housekeeping genes. MLST profiling of our strains using seven housekeeping genes showed high similarity for those genes; what we would expect since they were all classified as P. aeruginosa. Studies [27] have shown that PFGE is more accurate when typing very closely related strains from the same species. To generate PFGE profiles, genomic DNA was prepared by a modification of a previously described method [48]. P. aeruginosa isolates were grown overnight at 37°C on Tryptone Soya Agar plates containing 5% sheep’s blood.