Physiological levels of nitrate support anoxic growth by denitrification of Pseudomonas aeruginosa at growth rates reported in cystic fibrosis lungs and sputum

© 2014 Line, Alhede, Kolpen, Kuhl, Ciofu, Bjarnsholt, Moser, Toyofuku, Nomura, H0i'by and Jensen. Chronic Pseudomonas aeruginosa lung infection is the most severe complication in patients with cystic fibrosis (CF). The infection is characterised by the formation of biofilm surrounded by numerous polymorphonuclear leukocytes (PMNs) and strong O2 depletion in the endobronchial mucus. We have reported that O2 is mainly consumed by the activated PMNs, while O2 consumption by aerobic respiration is diminutive and nitrous oxide (N2O) is produced in infected CF sputum. This suggests that the reported growth rates ofP. aeruginosa in lungs and sputum may result from anaerobic respiration using denitrification. The growth rate of P. aeruginosa achieved by denitrification at physiological levels (~400 μM) of nitrate (NO3-) is however, not known. Therefore, we have measured growth rates of anoxic cultures of PAO1 and clinical isolates (n = 12) in LB media supplemented with NO3- and found a significant increase of growth when supplementing PAO1 and clinical isolates with > 150 μM NO3- and 100 μM NO3-, respectively. An essential contribution to growth by denitrification was demonstrated by the inability to establish a significantly increased growth rate by a denitrification deficient ΔnirS-N mutant at <1 mM of NO3-. Activation of denitrification could be achieved by supplementation with as little as 62.5 μM of NO3- according to the significant production of N2O by the nitrous oxide reductase deficient ΔnosZ mutant. Studies of the promoter activity, gene transcripts and enzyme activity of the four N-oxide reductases in PAO1 (Nar, Nir, Nor, Nos) further verified the engagement of denitrification, showing a transient increase in activation and expression and rapid consumption of NO3- followed by a transient increase of NO2-. Growth rates obtained by denitrification in this study were comparable to our reported growth rates in the majority of P. aeruginosa cells in CF lungs and sputum. Thus, we have demonstrated that denitrification is required for P. aeruginosa growth in infected endobronchial CF mucus

Nitrous oxide production in sputum from cystic fibrosis patients with chronic Pseudomonas aeruginosa lung infection

Chronic lung infection by Pseudomonas aeruginosa is the major severe complication in cystic fibrosis (CF) patients, where P. aeruginosa persists and grows in biofilms in the endobronchial mucus under hypoxic conditions. Numerous polymorphonuclear leukocytes (PMNs) surround the biofilms and create local anoxia by consuming the majority of O2 for production of reactive oxygen species (ROS). We hypothesized that P. aeruginosa acquires energy for growth in anaerobic endobronchial mucus by denitrification, which can be demonstrated by production of nitrous oxide (N2O), an intermediate in the denitrification pathway. We measured N2O and O2 with electrochemical microsensors in 8 freshly expectorated sputum samples from 7 CF patients with chronic P. aeruginosa infection. The concentrations of NO 3- and NO2- in sputum were estimated by the Griess reagent. We found a maximum median concentration of 41.8 μM N2O (range 1.4-157.9 μM N2O). The concentration of N2O in the sputum was higher below the oxygenated layers. In 4 samples the N2O concentration increased during the initial 6 h of measurements before decreasing for approximately 6 h. Concomitantly, the concentration of NO3- decreased in sputum during 24 hours of incubation. We demonstrate for the first time production of N2O in clinical material from infected human airways indicating pathogenic metabolism based on denitrification. Therefore, P. aeruginosa may acquire energy for growth by denitrification in anoxic endobronchial mucus in CF patients. Such ability for anaerobic growth may be a hitherto ignored key aspect of chronic P. aeruginosa infections that can inform new strategies for treatment and prevention. © 2014 Kolpen et al