Antimicrobial effects of nitric oxide in murine models of Klebsiella pneumonia

Steffen B Wiegand, Lisa Traeger, Huan K Nguyen, Kaitlyn R Rouillard, Anna Fischbach, Francesco Zadek, Fumito Ichinose, Mark H Schoenfisch, Ryan W Carroll, Donald B Bloch, Warren M Zapol, Steffen B Wiegand, Lisa Traeger, Huan K Nguyen, Kaitlyn R Rouillard, Anna Fischbach, Francesco Zadek, Fumito Ichinose, Mark H Schoenfisch, Ryan W Carroll, Donald B Bloch, Warren M Zapol

Abstract

Rationale: Inhalation of nitric oxide (NO) exerts selective pulmonary vasodilation. Nitric oxide also has an antimicrobial effect on a broad spectrum of pathogenic viruses, bacteria and fungi.

Objectives: The aim of this study was to investigate the effect of inhaled NO on bacterial burden and disease outcome in a murine model of Klebsiella pneumonia.

Methods: Mice were infected with Klebsiella pneumoniae and inhaled either air alone, air mixed with constant levels of NO (at 80, 160, or 200 parts per million (ppm)) or air intermittently mixed with high dose NO (300 ppm). Forty-eight hours after airway inoculation, the number of viable bacteria in lung, spleen and blood was determined. The extent of infiltration of the lungs by inflammatory cells and the level of myeloperoxidase activity in the lungs were measured. Atomic force microscopy was used to investigate a possible mechanism by which nitric oxide exerts a bactericidal effect.

Measurements and main results: Compared to control animals infected with K. pneumoniae and breathed air alone, intermittent breathing of NO (300 ppm) reduced viable bacterial counts in lung and spleen tissue. Inhaled NO reduced infection-induced lung inflammation and improved overall survival of mice. NO destroyed the cell wall of K. pneumoniae and killed multiple-drug resistant K. pneumoniae in-vitro.

Conclusions: Intermittent administration of high dose NO may be an effective approach to the treatment of pneumonia caused by K. pneumoniae.

Keywords: Bactericidal; Lung inflammation; Outcome; Treatment.

Conflict of interest statement

WMZ is on the scientific advisory board of Third Pole Inc., which has licensed patents on electric NO generation. All other authors have nothing to declare.

Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.

Figures

Graphical abstract
Graphical abstract
Fig. 1
Fig. 1
Breathing NO reduced Kp CFUs in lung tissue in a dose dependent manner. Mice were infected with 2,000 CFU Kp per animal and treated with NO (160, 200 ppm [black triangle]) or air [white circle], 6 h after inoculation. Mice were sacrificed after 48 h. Inhalation of 160 ppm NO (A) or 200 ppm NO (B) decreased numbers of bacteria in lungs compared to mice treated with air alone (NO 160 ppm: n = 10, air: n = 10 and NO 200 ppm: n = 10, air: n = 10). NO had no effect on the number of CFUs in the spleen or blood of infected mice at any of the NO concentrations that were tested.
Fig. 2
Fig. 2
NO inhalation increased methemoglobin levels, which returned to baseline levels 1.5 h after discontinuation of treatment. Mice were infected with 2,000 CFU Kp per animal and treated with NO or air. Mice were sacrificed after 48 h. (A) Methemoglobin levels were increased in most of the NO treated groups compared to air treated controls (NO 80 ppm: n = 8, NO 160 ppm: n = 5, NO 200 ppm: n = 9, NO 300 ppm: n = 5 and air treated controls: n = 9) (*** = p 

Fig. 3

Breathing high dose NO reduced…

Fig. 3

Breathing high dose NO reduced Kp CFUs in lung tissue, and starting treatment…

Fig. 3
Breathing high dose NO reduced Kp CFUs in lung tissue, and starting treatment with NO early reduced CFUs in both lung and spleen. Mice were infected with 2,000 CFU Kp per animal and treated with intermittent NO (300 ppm) [black triangle] or air [white circle]. Mice were sacrificed after 48 h. (A) Intermittent treatment with 300 ppm NO, starting 6 h after inoculation, reduced bacterial CFUs in lung tissue compared to controls (p < 0.001), but not in spleen (p = 0.795) or blood (p = 0.6) (NO 300 ppm: n = 8, air: n = 8) (*** = p < 0.001) (B) Early treatment, initiated immediately after inoculation, reduced CFUs in lung and splenic tissue, but not in blood (NO 300 ppm: n = 8, air: n = 9) (* = p < 0.05; ** = p < 0.01). There was a trend towards reduction in bacteremia (bacteremia: air: 6/9 vs. NO 300 ppm: 2/8 (chi-square test: † = p = 0.086).

Fig. 4

Treatment with NO decreased lung…

Fig. 4

Treatment with NO decreased lung inflammation and MPO levels in lung extracts. Mice…
Fig. 4
Treatment with NO decreased lung inflammation and MPO levels in lung extracts. Mice were inoculated with 2000 CFU of Kp and received NO 200 ppm (continuously) or 300 ppm (intermittently) or air alone for 48 h. (A–D) Lung sections were fixed and stained with H&E and examined under light microscopy. Representative sections showing (A) no infiltration of neutrophils and (B) mild infiltration (Score:1), (C) intermediate infiltration (Score:2) and (D) widespread infiltration (Score:4). No section in this collection of tissues had an inflammation score of 3. (NO 200 ppm: n = 10, NO 300 ppm: n = 10, air: n = 15) (E) Myeloperoxidase activity in mice receiving 200 or 300 ppm nitric oxide 6 h after inoculation or immediately after inoculation (#) were significantly reduced compared to control mice ((NO 200 ppm+6 h: n = 9, NO 300 ppm+6 h: n = 8, NO 200 ppm#: n = 8, NO300 ppm#: n = 8, air: n = 37 (* = p < 0.05; ** = p < 0.01; (*** = p < 0.001). There was no difference among the different NO groups.

Fig. 5

High dose intermittent NO treatment…

Fig. 5

High dose intermittent NO treatment improved overall survival compared to air breathing controls.…
Fig. 5
High dose intermittent NO treatment improved overall survival compared to air breathing controls. Mice were inoculated with (A) 2000 CFU or (B) 3500 CFU and received either 12min intermittent treatment with 300 ppm NO every 3 h for 48 h [full line] or air [dashed line]. Overall survival was increased in mice receiving treatment with NO compared to mice in control group ((A) Air breathing mice: n = 10, intermittent NO-treated-mice: n = 9 (* = p < 0.05); (B) Air breathing mice: n = 4, intermittent NO-treated-mice: n = 4) (** = p < 0.01).

Fig. 6

NO from an NO donor…

Fig. 6

NO from an NO donor compound (spermine NONOate), led to degradation of Kp…
Fig. 6
NO from an NO donor compound (spermine NONOate), led to degradation of Kp cell wall in-vitro. (A) Kp exposed to (A) PBS or (B) spermine, which does not release NO, showed a smooth intact cell surface. (C) NO 0.1 μmol and (D) 1 μmol had no effect on the appearance of Kp cell wall. (E) NO 10 μmol led to degradation of the cell wall of Kp and spillage of organelles.

Fig. 7

Exposure to NO released from…

Fig. 7

Exposure to NO released from an NO donor (DETA NONOate) reduced growth of…
Fig. 7
Exposure to NO released from an NO donor (DETA NONOate) reduced growth of multiple drug resistant Kp in a dose-dependent manner in-vitro. (A) After a 6 h incubation period, bacterial CFUs of multiple-drug resistant Kp increased and addition of amoxicillin/clavulanic acid 32/16 μg/mL or meropenem 2 μg/mL had no effect on the number of CFUs compared to bacteria grown in control media. Cefepime 8 μg/mL has a small effect on Kp CFUs (7.4 log10 CFU/mL (7.2; 7.6), vs. 9.1 log10 CFU/mL (9.0; 9.5), * = p < 0.05) compared to media alone. Exposure with NO 1 μmol/L (6.9 log10 CFU/mL (6.8; 7.1)); 4 μmol/L (5.3 log10 CFU/mL (5.1; 5.6)); 7.5 μmol/L (4.7 log10 CFU/mL (4.6; 4.8)); and 15 μmol/L (4.2 log10 CFU/mL (4.1; 4.3)) reduced Kp CFUs compared to media alone (9.1 log10 CFU/mL (9.0; 9.5)) (each group: n = 10, except controls: n = 20). NO 7.5 μmol/L and 15 μmol/L were bactericidal.; (*** = p < 0.001). (B) Exposure with sulpho NONOate at 2.5 mg/mL, which releases nitrous oxide instead of nitric oxide, had no effect on CFUs compared to bacteria grown in media (8.7 log10 CFU/mL (8.7: 8.8) vs. 8.7 log10 CFU/mL (8.7; 8.8); p = 0.96), but 2.5 mg/mL of DETA NONOate, which corresponds to 15 μmol/L NO, was bactericidal (*** = p < 0.001) (each group: n = 5).
All figures (8)
Fig. 3
Fig. 3
Breathing high dose NO reduced Kp CFUs in lung tissue, and starting treatment with NO early reduced CFUs in both lung and spleen. Mice were infected with 2,000 CFU Kp per animal and treated with intermittent NO (300 ppm) [black triangle] or air [white circle]. Mice were sacrificed after 48 h. (A) Intermittent treatment with 300 ppm NO, starting 6 h after inoculation, reduced bacterial CFUs in lung tissue compared to controls (p < 0.001), but not in spleen (p = 0.795) or blood (p = 0.6) (NO 300 ppm: n = 8, air: n = 8) (*** = p < 0.001) (B) Early treatment, initiated immediately after inoculation, reduced CFUs in lung and splenic tissue, but not in blood (NO 300 ppm: n = 8, air: n = 9) (* = p < 0.05; ** = p < 0.01). There was a trend towards reduction in bacteremia (bacteremia: air: 6/9 vs. NO 300 ppm: 2/8 (chi-square test: † = p = 0.086).
Fig. 4
Fig. 4
Treatment with NO decreased lung inflammation and MPO levels in lung extracts. Mice were inoculated with 2000 CFU of Kp and received NO 200 ppm (continuously) or 300 ppm (intermittently) or air alone for 48 h. (A–D) Lung sections were fixed and stained with H&E and examined under light microscopy. Representative sections showing (A) no infiltration of neutrophils and (B) mild infiltration (Score:1), (C) intermediate infiltration (Score:2) and (D) widespread infiltration (Score:4). No section in this collection of tissues had an inflammation score of 3. (NO 200 ppm: n = 10, NO 300 ppm: n = 10, air: n = 15) (E) Myeloperoxidase activity in mice receiving 200 or 300 ppm nitric oxide 6 h after inoculation or immediately after inoculation (#) were significantly reduced compared to control mice ((NO 200 ppm+6 h: n = 9, NO 300 ppm+6 h: n = 8, NO 200 ppm#: n = 8, NO300 ppm#: n = 8, air: n = 37 (* = p < 0.05; ** = p < 0.01; (*** = p < 0.001). There was no difference among the different NO groups.
Fig. 5
Fig. 5
High dose intermittent NO treatment improved overall survival compared to air breathing controls. Mice were inoculated with (A) 2000 CFU or (B) 3500 CFU and received either 12min intermittent treatment with 300 ppm NO every 3 h for 48 h [full line] or air [dashed line]. Overall survival was increased in mice receiving treatment with NO compared to mice in control group ((A) Air breathing mice: n = 10, intermittent NO-treated-mice: n = 9 (* = p < 0.05); (B) Air breathing mice: n = 4, intermittent NO-treated-mice: n = 4) (** = p < 0.01).
Fig. 6
Fig. 6
NO from an NO donor compound (spermine NONOate), led to degradation of Kp cell wall in-vitro. (A) Kp exposed to (A) PBS or (B) spermine, which does not release NO, showed a smooth intact cell surface. (C) NO 0.1 μmol and (D) 1 μmol had no effect on the appearance of Kp cell wall. (E) NO 10 μmol led to degradation of the cell wall of Kp and spillage of organelles.
Fig. 7
Fig. 7
Exposure to NO released from an NO donor (DETA NONOate) reduced growth of multiple drug resistant Kp in a dose-dependent manner in-vitro. (A) After a 6 h incubation period, bacterial CFUs of multiple-drug resistant Kp increased and addition of amoxicillin/clavulanic acid 32/16 μg/mL or meropenem 2 μg/mL had no effect on the number of CFUs compared to bacteria grown in control media. Cefepime 8 μg/mL has a small effect on Kp CFUs (7.4 log10 CFU/mL (7.2; 7.6), vs. 9.1 log10 CFU/mL (9.0; 9.5), * = p < 0.05) compared to media alone. Exposure with NO 1 μmol/L (6.9 log10 CFU/mL (6.8; 7.1)); 4 μmol/L (5.3 log10 CFU/mL (5.1; 5.6)); 7.5 μmol/L (4.7 log10 CFU/mL (4.6; 4.8)); and 15 μmol/L (4.2 log10 CFU/mL (4.1; 4.3)) reduced Kp CFUs compared to media alone (9.1 log10 CFU/mL (9.0; 9.5)) (each group: n = 10, except controls: n = 20). NO 7.5 μmol/L and 15 μmol/L were bactericidal.; (*** = p < 0.001). (B) Exposure with sulpho NONOate at 2.5 mg/mL, which releases nitrous oxide instead of nitric oxide, had no effect on CFUs compared to bacteria grown in media (8.7 log10 CFU/mL (8.7: 8.8) vs. 8.7 log10 CFU/mL (8.7; 8.8); p = 0.96), but 2.5 mg/mL of DETA NONOate, which corresponds to 15 μmol/L NO, was bactericidal (*** = p < 0.001) (each group: n = 5).

References

    1. Moncada S., Palmer R.M., Higgs E.A. Biosynthesis of nitric oxide from L-arginine. A pathway for the regulation of cell function and communication. Biochem. Pharmacol. 1989;38:1709–1715. doi: 10.1016/0006-2952(89)90403-6.
    1. Fang F.C. Antimicrobial reactive oxygen and nitrogen species: concepts and controversies. Nat. Rev. Microbiol. 2004;2:820. doi: 10.1038/nrmicro1004.
    1. Rossaint R., Falke K.J., López F., Slama K., Pison U., Zapol W.M. Inhaled nitric oxide for the adult respiratory distress syndrome. N. Engl. J. Med. 1993;328:399–405. doi: 10.1056/NEJM199302113280605.
    1. Wysocki M., Delclaux C., Roupie E., Langeron O., Liu N., Herman B., Lemaire F., Brochard L. Additive effect on gas exchange of inhaled nitric oxide and intravenous almitrine bismesylate in the adult respiratory distress syndrome. Intensive Care Med. 1994;20:254–259. doi: 10.1007/bf01708960.
    1. Roberts J.D., Polaner D.M., Lang P., Zapol W.M. Inhaled nitric oxide in persistent pulmonary hypertension of the newborn. Lancet. 1992;340:818–819.
    1. Zapol W.M. Nitric oxide story. Anesthesiology. 2019;130:435–440. doi: 10.1097/ALN.0000000000002579.
    1. Akerström S., Gunalan V., Keng C.T., Tan Y.-J., Mirazimi A. Dual effect of nitric oxide on SARS-CoV replication: viral RNA production and palmitoylation of the S protein are affected. Virology. 2009;395:1–9. doi: 10.1016/j.virol.2009.09.007.
    1. Akaberi D., Krambrich J., Ling J., Luni C., Hedenstierna G., Järhult J.D., Lennerstrand J., Lundkvist Å. Mitigation of the replication of SARS-CoV-2 by nitric oxide in vitro. Redox Biology. 2020:101734. doi: 10.1016/j.redox.2020.101734.
    1. Thomas D.D., Ridnour L.A., Isenberg J.S., Flores-Santana W., Switzer C.H., Donzelli S., Hussain P., Vecoli C., Paolocci N., Ambs S., Colton C.A., Harris C.C., Roberts D.D., Wink D.A. The chemical biology of nitric oxide: implications in cellular signaling. Free Radic. Biol. Med. 2008;45:18–31. doi: 10.1016/j.freeradbiomed.2008.03.020.
    1. Nathan C., Shiloh M.U. Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens. Proc. Natl. Acad. Sci. U.S.A. 2000;97:8841.
    1. Joannou C.L., Cui X.-Y., Rogers N., Vielotte N., Torres Martinez C.L., Vugman N.V., Hughes M.N., Cammack R. Characterization of the bactericidal effects of sodium nitroprusside and other pentacyanonitrosyl complexes on the food spoilage bacterium Clostridium sporogenes. Appl. Environ. Microbiol. 1998;64:3195–3201.
    1. Jones-Carson J., Laughlin J., Hamad M.A., Stewart A.L., Voskuil M.I., Vázquez-Torres A. Inactivation of [Fe-S] metalloproteins mediates nitric oxide-dependent killing of burkholderia mallei. PloS One. 2008;3 doi: 10.1371/journal.pone.0001976.
    1. Lepoivre M., Fieschi F., Coves J., Thelander L., Fontecave M. Inactivation of ribonucleotide reductase by nitric oxide. Biochem. Biophys. Res. Commun. 1991;179:442–448. doi: 10.1016/0006-291X(91)91390-X.
    1. Nairz M., Schleicher U., Schroll A., Sonnweber T., Theurl I., Ludwiczek S., Talasz H., Brandacher G., Moser P.L., Muckenthaler M.U., Fang F.C., Bogdan C., Weiss G. Nitric oxide–mediated regulation of ferroportin-1 controls macrophage iron homeostasis and immune function in Salmonella infection. J. Exp. Med. 2013;210:855–873. doi: 10.1084/jem.20121946.
    1. Bogdan C. Nitric oxide synthase in innate and adaptive immunity: an update. Trends Immunol. 2015;36:161–178. doi: 10.1016/j.it.2015.01.003.
    1. Ito C., Saito Y., Nozawa T., Fujii S., Sawa T., Inoue H., Matsunaga T., Khan S., Akashi S., Hashimoto R., Aikawa C., Takahashi E., Sagara H., Komatsu M., Tanaka K., Akaike T., Nakagawa I., Arimoto H. Endogenous nitrated nucleotide is a key mediator of autophagy and innate defense against bacteria. Mol. Cell. 2013;52:794–804. doi: 10.1016/j.molcel.2013.10.024.
    1. Kacmarek R.M., Ripple R., Cockrill B.A., Bloch K.J., Zapol W.M., Johnson D.C. Inhaled nitric oxide. A bronchodilator in mild asthmatics with methacholine-induced bronchospasm. Am. J. Respir. Crit. Care Med. 1996;153:128–135. doi: 10.1164/ajrccm.153.1.8542105.
    1. Salathe M. Regulation of mammalian ciliary beating. Annu. Rev. Physiol. 2007;69:401–422. doi: 10.1146/annurev.physiol.69.040705.141253.
    1. Howlin R.P., Cathie K., Hall-Stoodley L., Cornelius V., Duignan C., Allan R.N., Fernandez B.O., Barraud N., Bruce K.D., Jefferies J., Kelso M., Kjelleberg S., Rice S.A., Rogers G.B., Pink S., Smith C., Sukhtankar P.S., Salib R., Legg J., Carroll M., Daniels T., Feelisch M., Stoodley P., Clarke S.C., Connett G., Faust S.N., Webb J.S. Low-dose nitric oxide as targeted anti-biofilm adjunctive therapy to treat chronic Pseudomonas aeruginosa infection in cystic fibrosis. Mol. Ther. 2017;25:2104. doi: 10.1016/j.ymthe.2017.06.021.
    1. Peleg A.Y., Hooper D.C. Hospital-acquired infections due to gram-negative bacteria. N. Engl. J. Med. 2010;362:1804–1813. doi: 10.1056/NEJMra0904124.
    1. Cassini A., Högberg L.D., Plachouras D., Quattrocchi A., Hoxha A., Simonsen G.S., Colomb-Cotinat M., Kretzschmar M.E., Devleesschauwer B., Cecchini M., Ouakrim D.A., Oliveira T.C., Struelens M.J., Suetens C., Monnet D.L., Strauss R., Mertens K., Struyf T., Catry B., Latour K., Ivanov I.N., Dobreva E.G., Andraševic A.T., Soprek S., Budimir A., Paphitou N., Žemlicková H., Olsen S.S., Sönksen U.W., Märtin P., Ivanova M., Lyytikäinen O., Jalava J., Coignard B., Eckmanns T., Sin M.A., Haller S., Daikos G.L., Gikas A., Tsiodras S., Kontopidou F., Tóth Á., Hajdu Á., Guólaugsson Ó., Kristinsson K.G., Murchan S., Burns K., Pezzotti P., Gagliotti C., Dumpis U., Liuimiene A., Perrin M., Borg M.A., de Greeff S.C., Monen J.C., Koek M.B., Elstrøm P., Zabicka D., Deptula A., Hryniewicz W., Caniça M., Nogueira P.J., Fernandes P.A., Manageiro V., Popescu G.A., Serban R.I., Schréterová E., Litvová S., Štefkovicová M., Kolman J., Klavs I., Korošec A., Aracil B., Asensio A., Pérez-Vázquez M., Billström H., Larsson S., Reilly J.S., Johnson A., Hopkins S. Attributable deaths and disability-adjusted life-years caused by infections with antibiotic-resistant bacteria in the EU and the European Economic Area in 2015: a population-level modelling analysis. Lancet Infect. Dis. 2019;19:56–66. doi: 10.1016/S1473-3099(18)30605-4.
    1. Rice L.B. Federal funding for the study of antimicrobial resistance in nosocomial pathogens: No ESKAPE. J. Infect. Dis. 2008;197:1079–1081.
    1. Maechler F., Peña Diaz L., Schröder C., Geffers C., Behnke M., Gastmeier P. Prevalence of carbapenem-resistant organisms and other Gram-negative MDRO in German ICUs: first results from the national nosocomial infection surveillance system (KISS) Infection. 2015;43:163–168. doi: 10.1007/s15010-014-0701-6.
    1. Nathan C. Neutrophils and immunity: challenges and opportunities. Nat. Rev. Immunol. 2006;6:173–182. doi: 10.1038/nri1785.
    1. Webert K., Vanderzwan J., Duggan M., Scott J., McCormack D., Lewis J., Mehta S. Effects of inhaled nitric oxide in a rat model of Pseudomonas aeruginosa pneumonia. Crit. Care Med. 2000;28:2397–2405.
    1. Jean D., Maître B., Tankovic J., Meignan M., Adnot S., Brun-Buisson C., Harf A., Delclaux C. Beneficial effects of nitric oxide inhalation on pulmonary bacterial clearance. Crit. Care Med. 2002;30:442–447.
    1. Workman A.D., Carey R.M., Kohanski M.A., Kennedy D.W., Palmer J.N., Adappa N.D., Cohen N.A. Relative susceptibility of airway organisms to antimicrobial effects of nitric oxide. International Forum of Allergy & Rhinology. 2017;7:770. doi: 10.1002/alr.21966.
    1. El-Solh A.A., Pietrantoni C., Bhat A., Aquilina A.T., Okada M., Grover V., Gifford N. Microbiology of severe aspiration pneumonia in institutionalized elderly. Am. J. Respir. Crit. Care Med. 2003;167:1650–1654. doi: 10.1164/rccm.200212-1543OC.
    1. Kang J.L., Park W., Pack I.S., Lee H.S., Kim M.J., Lim C.-M., Koh Y. Inhaled nitric oxide attenuates acute lung injury via inhibition of nuclear factor-κB and inflammation. J. Appl. Physiol. 2002;92:795–801. doi: 10.1152/japplphysiol.00202.2001.
    1. Wu C.-H., Chen T.-L., Chen T.-G., Ho W.-P., Chiu W.-T., Chen R.-M. Nitric oxide modulates pro- and anti-inflammatory cytokines in lipopolysaccharide-activated macrophages. Journal of Trauma and Acute Care Surgery. 2003;55:540–545. doi: 10.1097/01.TA.0000033496.62796.3B.
    1. Da J., Chen L., Hedenstierna G. Nitric oxide up-regulates the glucocorticoid receptor and blunts the inflammatory reaction in porcine endotoxin sepsis*. Crit. Care Med. 2007;35:26–32. doi: 10.1097/.
    1. Deupree S.M., Schoenfisch M.H. Morphological analysis of the antimicrobial action of nitric oxide on Gram-negative pathogens using atomic force microscopy. Acta Biomater. 2009;5:1405–1415. doi: 10.1016/j.actbio.2009.01.025.
    1. Deppisch C., Herrmann G., Graepler-Mainka U., Wirtz H., Heyder S., Engel C., Marschal M., Miller C.C., Riethmüller J. Gaseous nitric oxide to treat antibiotic resistant bacterial and fungal lung infections in patients with cystic fibrosis: a phase I clinical study. Infection. 2016;44:513–520. doi: 10.1007/s15010-016-0879-x.
    1. Elsayed N.M. Toxicity of nitrogen dioxide: an introduction. Toxicology. 1994;89:161–174. doi: 10.1016/0300-483x(94)90096-5.
    1. 1988 OSHA PEL Project - Nitrogen Dioxide. NIOSH | CDC; 2020. accessed September 9, 2020.

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