Sodium nitrite-mediated killing of the major cystic fibrosis pathogens Pseudomonas aeruginosa, Staphylococcus aureus, and Burkholderia cepacia under anaerobic planktonic and biofilm conditions

Abstract

A hallmark of airways in patients with cystic fibrosis (CF) is highly refractory, chronic infections by several opportunistic bacterial pathogens. A recent study demonstrated that acidified sodium nitrite (A-NO(2)(-)) killed the highly refractory mucoid form of Pseudomonas aeruginosa, a pathogen that significantly compromises lung function in CF patients (S. S. Yoon et al., J. Clin. Invest. 116:436-446, 2006). Therefore, the microbicidal activity of A-NO(2)(-) (pH 6.5) against the following three major CF pathogens was assessed: P. aeruginosa (a mucoid, mucA22 mutant and a sequenced nonmucoid strain, PAO1), Staphylococcus aureus USA300 (methicillin resistant), and Burkholderia cepacia, a notoriously antibiotic-resistant organism. Under planktonic, anaerobic conditions, growth of all strains except for P. aeruginosa PAO1 was inhibited by 7.24 mM (512 μg ml(-1) NO(2)(-)). B. cepacia was particularly sensitive to low concentrations of A-NO(2)(-) (1.81 mM) under planktonic conditions. In antibiotic-resistant communities known as biofilms, which are reminiscent of end-stage CF airway disease, A-NO(2)(-) killed mucoid P. aeruginosa, S. aureus, and B. cepacia; 1 to 2 logs of cells were killed after a 2-day incubation with a single dose of ∼15 mM A-NO(2)(-). Animal toxicology and phase I human trials indicate that these bactericidal levels of A-NO(2)(-) can be easily attained by aerosolization. Thus, in summary, we demonstrate that A-NO(2)(-) is very effective at killing these important CF pathogens and could be effective in other infectious settings, particularly under anaerobic conditions where bacterial defenses against the reduction product of A-NO(2)(-), nitric oxide (NO), are dramatically reduced.

Figures

FIG. 1.

A-NO2−-mediated killing of P. aeruginosa FRD1 under anaerobic conditions. NaNO2 concentrations tested were 0 μg ml−1 (closed circles), 64 μg ml−1 (open circles), 128 μg ml−1 (squares), 256 μg ml−1 (triangles), 512 μg ml−1 (diamonds), and 1,024 μg ml−1 (stars).The graphs display the averages and standard deviations of three independent cultures analyzed concurrently.

FIG. 2.

A-NO2−-mediated killing of P. aeruginosa PAO1 under anaerobic conditions. NaNO2 concentrations tested were 0 μg ml−1 (closed circles), 256 μg ml−1 (open circles), 512 μg ml−1 (squares), 1,024 μg ml−1 (triangles), 2,048 μg ml−1 (diamonds), and 4,096 μg ml−1 (stars). The graphs display the averages and standard deviations of three independent cultures analyzed concurrently.

FIG. 3.

A-NO2−-mediated killing of S aureus USA300 under anaerobic conditions. NaNO2 concentrations tested were 0 μg ml−1 (closed circles), 512 μg ml−1 (open circles), 1,024 μg ml−1 (squares), and 2,048 μg ml−1 (triangles). The graphs represent the averages of three independent cultures analyzed concurrently.

FIG. 4.

A-NO2−-mediated killing of B. cepacia under anaerobic conditions. NaNO2 concentrations tested were 0 μg ml−1 (closed circles), 128 μg ml−1 (open circles), 256 μg ml−1 (squares), 512 μg ml−1 (triangles), and 1,024 μg ml−1 (diamonds). The graphs display the averages and standard deviations of three independent cultures analyzed concurrently.

FIG. 5.

A-NO2−-mediated killing of several B. cepacia isolates. (A) Effect of 0 μg ml−1 NaNO2 on six B. cepacia isolates anaerobically maintained at 37°C for 0 and 24 h. (B) Effect of 512 μg ml−1 NaNO2 on six B. cepacia isolates anaerobically maintained at 37°C for 0 and 24 h.

FIG. 6.

A-NO2−-mediated killing of biofilm bacteria. Biofilms were grown for 1 day under aerobic conditions in MH medium. Following A-NO2− addition, biofilms were maintained under anaerobic conditions for an additional 2 days. The results represent CFU per ml of biofilms at different NaNO2 concentrations (means ± standard errors of means are shown; n = 3). (A) P. aeruginosa FRD1; (B) P. aeruginosa PAO1; (C) S. aureus USA300; (D) B. cepacia.

FIG. 7.

A-NO2−-mediated killing of P. aeruginosa PAO1 biofilms. Biofilms were grown as described for Fig. 6, except for the NaNO2 concentrations in each sample. The − sign represents control biofilms, and those with a + sign were treated with 4,096 μg ml−1 NaNO2.