Approximately 80% of adult patients with cystic fibrosis (CF) become chronically infected with Pseudomonas aeruginosa and consequently require antibiotic therapy at intervals throughout their lives. Achieving lethal concentrations of antibiotics in the lung remains a challenge. Recent evidence from Escherichia coli and Staphylococcus aureus suggests that the generation of hydroxyl radicals by sublethal concentrations of antibiotics may induce mutagenesis and confer bacteria with resistance to a wide range of antimicrobials. As Ps. aeruginosa can persist for many years following colonization of the airways and during this time it is repeatedly exposed to bactericidal antibiotics, we tested whether its exposure to sublethal levels increases mutation frequency. We demonstrate that sublethal levels of three classes of bactericidal antibiotics commonly used against Ps. aeruginosa infections, β-lactams, aminoglycosides and quinolones lead to an increase in mutation frequency, varying between c. threefold increase with aminoglycosides and a c. 14-fold increase in mutation frequency with β-lactam antibiotics. These findings could be clinically significant because exposure to sublethal concentrations of antibiotics during chronic infection leading to increased mutation frequency may facilitate adaptive radiation of pathogenic bacteria in the heterogeneous environment of the CF lung.

Background: Phenotypic profiling of knockout libraries is a possible functional genomics strategy.Results: Gluconate excretion is a novel phenotype of the Pseudomonas aeruginosa rpoN mutant, which is also weakly associated with antibiotic susceptibility in a clinical strain panel.Conclusion: The rpoN phenotype results from catabolite repression deregulation of 6-phosphogluconate dehydratase.Significance: NMR profiling can uncover novel gene functions with potential clinical relevance.

We have previously reported cyanide at concentrations of up to 150 mM in the sputum of cystic fibrosis patients infected with Pseudomonas aeruginosa and a negative correlation with lung function. Our aim was to investigate possible mechanisms for this association, focusing on the effect of pathophysiologically relevant cyanide levels on human respiratory cell function.Ciliary beat frequency measurements were performed on nasal brushings and nasal air-liquid interface (ALI) cultures obtained from healthy volunteers and cystic fibrosis patients.Potassium cyanide decreased ciliary beat frequency in healthy nasal brushings (n56) after 60 min (150 mM: 47% fall, p,0.0012; 75 mM: 32% fall, p,0.0001). Samples from cystic fibrosis patients (n53) showed similar results (150 mM: 55% fall, p50.001). Ciliary beat frequency inhibition was not due to loss of cell viability and was reversible. The inhibitory mechanism was independent of ATP levels. KCN also significantly inhibited ciliary beat frequency in ALI cultures, albeit to a lesser extent. Ciliary beat frequency measurements on ALI cultures treated with culture supernatants from P. aeruginosa mutants defective in virulence factor production implicated cyanide as a key component inhibiting the ciliary beat frequency.If cyanide production similarly impairs mucocilliary clearance in vivo, it could explain the link with increased disease severity observed in cystic fibrosis patients with detectable cyanide in their airway. @ERSpublications Cyanide levels detectable in sputum inhibit ciliary function

Cyanide is produced by a few bacterial species, including Pseudomonas aeruginosa, and it has a role in the opportunistic infections of this bacterium including in cystic fibrosis lung infections. We describe two methods for determining cyanide in culture and patient sputum samples. One uses an ion-selective electrode to provide a convenient, rapid method of cyanide quantitation in culture or sputum, and the second is a semiquantitative method using Feigl-Anger paper that is useful for screening large numbers of bacterial strains for cyanide production.

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