Occurrence of hypermutable Pseudomonas aeruginosa in cystic fibrosis patients is associated with the oxidative stress caused by chronic lung inflammation

Abstract

Oxidative stress caused by chronic lung inflammation in patients with cystic fibrosis (CF) and chronic lung infection with Pseudomonas aeruginosa is characterized by the reactive oxygen species (ROS) liberated by polymorphonuclear leukocytes (PMNs). We formulated the hypothesis that oxidation of the bacterial DNA by ROS presents an increased risk for the occurrence of hypermutable P. aeruginosa. The occurrence of hypermutable P. aeruginosa isolates was investigated directly in the sputum of 79 CF patients and among 141 isolates collected from 11 CF patients (10 to 15 isolates/patient) collected from the 1st and up to the 25th year of their chronic lung infection. The level of oxidized guanine moiety 8-oxo-2'-deoxyguanosine (8-oxodG), which is a frequently investigated DNA oxidative lesion, was measured. Hypermutable P. aeruginosa isolates were found in the sputum bacterial population of 54.4% of the CF patients. The earliest mutator P. aeruginosa isolates were found after 5 years from the onset of the chronic lung infection, and once they were present in the CF lung, the prevalence increased with time. The hypermutable isolates were significantly more resistant to antipseudomonal antibiotics than nonhypermutable isolates (P<or=0.001). The level of 8-oxodG/10(6) deoxyguanosine (dG) was significantly higher in hypermutable P. aeruginosa isolates (87+/-38) than in nonhypermutable P. aeruginosa isolates (59.4+/-17) (P=0.02), and an increase to 86.84 from 21.65 8-oxodG/10(6) dG was found after exposure of the reference strain PAO1 to activated PMNs. Our results suggest that the chronic PMN inflammation in the CF lung promotes oxidative stress and is associated with the occurrence of hypermutable bacteria in the lung. The hypermutable phenotype can associate with mutations that confer adaptation of the bacteria in the lung and persistence of the infection.

FIG. 1.

Mutation frequency after exposure to rifampin of 141 P. aeruginosa isolates collected in five periods of chronic lung infection (0 to 5, 5 to 10, 10 to 15, 15 to 20, and 20 to 25 years) from 11 patients with CF. The results for hypermutable (hp; ▴) and nonhypermutable (nhp; ○) P. aeruginosa isolates are presented. The mutation frequency after exposure to rifampin of reference strain PAO1 is shown.