Anaerobic killing of mucoid Pseudomonas aeruginosa by acidified nitrite derivatives under cystic fibrosis airway conditions
Sang Sun Yoon, … , Richard C. Boucher, Daniel J. Hassett
Sang Sun Yoon, … , Richard C. Boucher, Daniel J. Hassett
Published February 1, 2006
Citation Information: J Clin Invest. 2006;116(2):436-446. https://doi.org/10.1172/JCI24684.
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Research Article Pulmonology

Anaerobic killing of mucoid Pseudomonas aeruginosa by acidified nitrite derivatives under cystic fibrosis airway conditions

Abstract

Mucoid, mucA mutant Pseudomonas aeruginosa cause chronic lung infections in cystic fibrosis (CF) patients and are refractory to phagocytosis and antibiotics. Here we show that mucoid bacteria perish during anaerobic exposure to 15 mM nitrite (NO2–) at pH 6.5, which mimics CF airway mucus. Killing required a pH lower than 7, implicating formation of nitrous acid (HNO2) and NO, that adds NO equivalents to cellular molecules. Eighty-seven percent of CF isolates possessed mucA mutations and were killed by HNO2 (3-log reduction in 4 days). Furthermore, antibiotic-resistant strains determined were also equally sensitive to HNO2. More importantly, HNO2 killed mucoid bacteria (a) in anaerobic biofilms; (b) in vitro in ultrasupernatants of airway secretions derived from explanted CF patient lungs; and (c) in mouse lungs in vivo in a pH-dependent fashion, with no organisms remaining after daily exposure to HNO2 for 16 days. HNO2 at these levels of acidity and NO2– also had no adverse effects on cultured human airway epithelia in vitro. In summary, selective killing by HNO2 may provide novel insights into the important clinical goal of eradicating mucoid P. aeruginosa from the CF airways.

Authors

Sang Sun Yoon, Ray Coakley, Gee W. Lau, Sergei V. Lymar, Benjamin Gaston, Ahmet C. Karabulut, Robert F. Hennigan, Sung-Hei Hwang, Garry Buettner, Michael J. Schurr, Joel E. Mortensen, Jane L. Burns, David Speert, Richard C. Boucher, Daniel J. Hassett

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Figure 7

Applications of HNO2-mediated killing of mucoid P. aeruginosa to clinical specimens.

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Applications of HNO2-mediated killing of mucoid P. aeruginosa to clinica...
(A) Killing of FRD1 by NO2– in sterile ultrasupernatants of CF airway secretions derived from explanted CF lungs. Bacteria were incubated anaerobically for 24 hours, and 15 mM NO2– was added. CFUs were determined (n = 3) and plotted as the X ± SEM versus time. *P < 0.01 versus CFU decrease before adding NO2–. (B) NO generation in CF ASL by 15 mM NO2–. Except for the media and pH, experimental conditions were identical to those used in Figure 4B. (C) Effects of NO2– on killing of FRD1 and FRD1/pmucA in mouse lungs. CD1 mice were infected with FRD1 or FRD1/pmucA as described in Methods. Infected mice were treated with buffer (black bars) and buffered NO2– (white bars) daily, and viable bacteria from the lung homogenates were enumerated. n = 8 per group. *P < 0.01 versus buffer alone. (D) Effects of long-term NO2– treatment on killing of FRD1 in mouse lungs. Another group of FRD1-infected mice were treated daily with buffer (–, 50 mM sodium phosphate, pH 6.5) or buffer with 15 mM NO2– (+) for 16 days (n = 8 per group). Organisms surviving treatment with buffer and NO2– were shown in logarithmic scale. *P < 0.01 versus buffer alone. (E) CI experiments with 106 FRD1 and FRD1/pmucA intratracheally instilled into CD1 mouse airways and incubated for 6 days prior to harvesting of mouse lungs and enumeration of CFUs after homogenization.