Sub-lethal concentrations of antibiotics increase mutation frequency in the cystic fibrosis pathogen Pseudomonas aeruginosa.

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, ß-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 ß-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.

Metabolic adaptations of Pseudomonas aeruginosa during cystic fibrosis chronic lung infections.

Pseudomonas aeruginosa forms chronic infections in the lungs of cystic fibrosis (CF) patients, and is the leading cause of morbidity and mortality in patients with CF. Understanding how this opportunistic pathogen adapts to the CF lung during chronic infections is important to increase the efficacy of treatment and is likely to increase insight into other long-term infections. Previous studies of P. aeruginosa adaptation and divergence in CF infections have focused on the genetic level, both identifying characteristic mutations and patterns of gene expression. However, these approaches are not sufficient to fully understand the metabolic changes that occur during long-term infection, as metabolic regulation is complex and takes place on different biological levels. We used untargeted metabolic profiling (metabolomics) of cell supernatants (exometabolome analysis, or metabolic footprinting) to compare 179 strains, collected over time periods ranging from 4 to 24 years for the individual patients, representing a series of mostly clonal lineages from 18 individual patients. There was clear evidence of metabolic adaptation to the CF lung environment: acetate production was highly significantly negatively associated with length of infection. For amino acids, which are available to the bacterium in the lung environment, the tendency of isolates to evolve more efficient uptake was related to the biosynthetic cost of producing each metabolite; conversely, for the non-mammalian metabolite trehalose, isolates had significantly reduced tendency to utilize this compound with length of infection. However, as well as adaptation across patients, there was also a striking degree of metabolic variation between the different clonal lineages: in fact, the patient the strains were isolated from was a greater source of variance than length of infection for all metabolites observed. Our data highlight the potential for metabolomic investigation of complex phenotypic adaptations during infection.

Cyanogenesis by the entomopathogenic bacterium Pseudomonas entomophila.

AIMS: To investigate whether the entomopathogenic bacterium Pseudomonas entomophila can synthesize hydrogen cyanide (HCN). METHODS AND RESULTS: Cyanide production was assayed for during the growth of P. entomophila in liquid culture and during colonial growth. Pseudomonas entomophila produced HCN at a concentration of up to 40 micromol l(-1) during growth in liquid cultures and its production was found to be affected by oxygen availability, with levels increasing as the oxygen-transfer coefficient decreased. Pseudomonas entomophila made HCN during colonial growth at levels greater (approximately threefold) than those made by the well studied cyanogenic bacterium Pseudomonas aeruginosa. CONCLUSIONS: This study demonstrated unequivocally that P. entomophila can synthesize HCN, placing it among the small number of cyanogenic bacteria. Our data indicate that HCN production in P. entomophila is regulated by oxygen availability. SIGNIFICANCE AND IMPACT OF THE STUDY: Pseudomonas entomophila was recently identified to be the only pseudomonad that naturally infects and induces lethality of Drosophila melanogaster. The virulence factors which contribute to entomopathogenicity exerted by this species are largely unknown. In this study, we demonstrate that P. entomophila produces HCN, a secondary metabolite implicated in biocontrol properties and pathogenicity exerted by other bacteria.

Pseudomonas aeruginosa, cyanide accumulation and lung function in CF and non-CF bronchiectasis patients.

In patients with cystic fibrosis (CF) and non-CF bronchiectasis, Pseudomonas aeruginosa is the most important respiratory pathogen. It is able to synthesise hydrogen cyanide, a potent inhibitor of cellular respiration. The present study investigated whether cyanide is present in the sputum of CF and non-CF bronchiectasis patients infected with P. aeruginosa, and whether the detection of cyanide affected lung function. Cyanide was measured in sputum using a cyanide ion selective electrode. Cyanide was detected in sputum from 15 out of 25 CF and non-CF bronchiectasis patients with current P. aeruginosa infection; however, it was not detected in any of the 10 patients without this organism. Maximum levels were 130 microM (mean+/-SE 72+/-6.6 microM). Concurrent lung function data were available on all 21 P. aeruginosa-infected CF patients; the group with measurable sputum cyanide (n = 11) was not different from those without (n = 10) on the basis of age or sex. However, those with detectable cyanide had significantly poorer lung function than those without (forced expiratory volume in one second (% predicted) 26.8+/-3.8 versus 46.0+/-6.7%; forced vital capacity (% pred) 44.4+/-4.9 versus 60.1+/-7.7%). Cyanide is detectable in sputum from cystic fibrosis and non-cystic fibrosis bronchiectasis patients infected with Pseudomonas aeruginosa, and is also associated with impaired lung function.