Real-time PCR detection and PFGE typing of Pseudomonas aeruginosa from cystic fibrosis patients

Pseudomonas aeruginosa (PA) infection is the main cause of mortality in Cystic Fibrosis (CF) patients. Rapid and sensitive PA detection is pivotal to improve the prognosis. This thesis includes two parts. The first focuses on the development of a Real-time PCR protocol to detect dormant- non-culturable PA in sputum samples from CF patients (CFPA). 12 culture-positive (CP) and 29 culture-negative (CN) samples were analyzed by a qPCR targeting ecfX, 24% resulted positive (P), with bacterial counts (102 - 106 cells/ml) also higher than those obtained for CP samples. A significant association of culture-negative/PCR-positive samples from chronic patients with symptoms relapse (P=0.018) and following culture-positive samples (P=0.034) was found. These findings stress the value of qPCR approaches in monitoring P. aeruginosa colonization and foreseeing symptom recurrence in CF patients. In second part we investigated the epidemiological relatedness of CFPA. 77 CFPA and 19 PA from non-CF patients (NCFPA) were analysed by pulsed field gel electrophoresis (PFGE) profile, antibiotic resistance (AR) and mucous production. Population structure analysis showed an overall polyclonality, more evident among CF strains. 100% similarity was only found within each of 10 CFPA pairs from the same patient, and 95% between 2 CFPA isolates from different patients who never met each other in hospital, suggesting a common source of infection rather than cross-contamination. Among NCFPA four isolates from as many inpatients and two from as many outpatients showed the same pulsotype (H and M, respectively). AR was more common among CFPA and nonmucoid phenotype. In particular, a significant (P=0.016) association was found between gentamycin-resistance CFPA and nonmucoid phenotype. AR patterns were however highly variable, also among PA belonging to the same pulsotype; suggesting the lack of involvement of multiple antibiotic resistance (MAR) in cross-colonization.