Abstract 943 Pulmonary: Cystic Fibrosis Poster Symposium, Tuesday, 5/4

Pseudomonas aeruginosa infections in cystic fibrosis (CF) are characterised by acute exacerbations of lower respiratory symptoms. Established infection is marked by transition from a non-mucoid to mucoid phenotype, sustained airway inflammation and deteriorating lung function. The in-vivo regulation of P. aeruginosa virulence factors is poorly understood and difficulty collecting lower respiratory secretions from children means little is known about these early infecting strains. Methods used for testing virulence, such as laboratory batch cultures or isogenic mutant pairs in animals, may not accurately model the CF lung. This is critical where bacteria like, P. aeruginosa, have complex quorum sensing systems. Also the limited number of isolates able to be tested may not represent the total bacterial population. The recently described technique of population transcript accumulation (PTA) addresses some of these problems by extracting mRNA from the total bacterial population within the specimen and by using homologous DNA probes to measure virulence factor transcription. To further examine the role of P. aeruginosa in early CF lung disease, since 1992 we have conducted a prospective study using bronchoalveolar lavage (BAL) to collect lower respiratory secretions annually from CF infants identified by a statewide neonatal screening programme. P. aeruginosa infection (≥ 105 CFU/ml) was diagnosed in 26/124 (21%) cohort members. A subset of 26 BAL samples from 21 (16 female) infected subjects, aged 8-57 months, was stratified for clinical severity according to whether the BAL was performed as an outpatient (n=16) or during hospitalisation for severe respiratory illness (n=10). The 2 groups were comparable for age, gender, CF genotype, antibiotics, P. aeruginosa colony counts and colonial morphology. PFGE typing showed that while 18 subjects had a unique P. aeruginosa strain, 3 others shared the same isolate. Specific DNA probes for P. aeruginosa exotoxin A (toxA), elastase (lasB) and alginate (algD) examined the PTA of these products within BAL fluid. While 10/10 BAL specimens from hospitalised subjects yielded both toxA and algD transcriptional activity, only 3/16 and 2/16 BAL specimens from outpatient subjects had detectable toxA and algD mRNA respectively (p < 0.001). Semi-quantitative analysis by densitometry and standardising bacterial counts to 107 confirmed these findings. Relative intensities of toxA and algD transcription were highly correlated (rs = 0.844, p < 0.001). LasB transcription was detected in sputa of severely ill children but not in BAL specimens. Finally, hospitalised subjects had greater neutrophil and IL-8 concentrations (p < 0.02) in their BAL fluid. We have shown that within the lungs of young CF children, up-regulation of certain P. aeruginosa virulence factors is associated with increased airway inflammation and clinical severity.