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The concentration-determined and population-specific antimicrobial effects of free nitrous acid on Pseudomonas aeruginosa PAO1

Applied Microbiology and Biotechnology volume 99, pages 2305–2312 (2015)Cite this article

Abstract

There is great potential to use free nitrous acid (FNA/HNO2), the protonated form of nitrite, as an antimicrobial agent due to its bacteriostatic and bactericidal effects on a range of microorganisms. Here, we determine the effects of FNA on the opportunistic pathogen Pseudomonas aeruginosa PAO1, a well-studied denitrifier capable of nitrate/nitrite reduction in its anaerobic respiration. It was seen that lower FNA concentrations in the range of 0.1 to 0.2 mg N/L exerted a temporary inhibitory effect on the growth of P. aeruginosa, while respiratory inhibition was not detected until an FNA concentration of 1.0 mg N/L was applied. The FNA concentration of 5.0 mg N/L caused complete cell killing and likely cell lysis. The results suggest concentration-related and multiple antimicrobial effects of FNA. Differential killing of FNA in the P. aeruginosa subpopulations was detected, suggesting intrastrain heterogeneity, and does not support the idea of specific concentrations of FNA bringing about bacteriostatic and bactericidal effects on this species. A delayed recovery from FNA treatment suggested that FNA caused cell damage which required repair prior to the organism showing cell growth. The results of the study provide insight of the inhibitory and biocidal mechanisms of FNA on this important microorganism.

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References

  • Allaker RP, Silva Mendez LS, Hardie JM, Benjamin N (2001) Antimicrobial effect of acidified nitrite on periodontal bacteria. Oral Microbiol Immunol 16:253–256

    Article  CAS  PubMed  Google Scholar 

  • Almeida JS, Júlio SM, Reis MA, Carrondo MJ (1995) Nitrite inhibition of denitrification by Pseudomonas fluorescens. Biotechnol Bioeng 46:194–201

    Article  CAS  PubMed  Google Scholar 

  • Anthonisen AC, Loehr RC, Prakasam TB, Srinath EG (1976) Inhibition of nitrification by ammonia and nitrous acid. Water Pollut Control Fed 48:835–852

    CAS  Google Scholar 

  • Avery SV (2006) Microbial cell individuality and the underlying sources of heterogeneity. Nat Rev Microbiol 4:577–587

    Article  CAS  PubMed  Google Scholar 

  • Barraud N, Hassett DJ, Hwang SH, Rice SA, Kjelleberg S, Webb JS (2006) Involvement of nitric oxide in biofilm dispersal of Pseudomonas aeruginosa. J Bacteriol 188:7344–7353

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Cammack R, Joannou CL, Cui XY, Torres Martinez C, Maraj SR, Hughes MN (1999) Nitrite and nitrosyl compounds in food preservation. Biochim Biophys Acta 1411:475–488

    Article  CAS  PubMed  Google Scholar 

  • Codling CE, Maillard JY, Russell AD (2003) Aspects of the antimicrobial mechanisms of action of a polyquaternium and an amidoamine. J Antimicrob Chemother 51:1153–1158

    Article  CAS  PubMed  Google Scholar 

  • Dykhuizen RS, Frazer R, Duncan C, Smith CC, Golden M, Benjamin N, Leifert C (1996) Antimicrobial effect of acidified nitrite on gut pathogens: importance of dietary nitrate in host defense. Antimicrob Agents Chemother 40:1422–1425

    PubMed Central  CAS  PubMed  Google Scholar 

  • Fang FC (1997) Perspectives series: host/pathogen interactions. Mechanisms of nitric oxide-related antimicrobial activity. J Clin Invest 99:2818–2825

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Filiatrault MJ, Picardo KF, Ngai H, Passador L, Iglewski BH (2006) Identification of Pseudomonas aeruginosa genes involved in virulence and anaerobic growth. Infect Immun 74:4237–4245

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Firmani MA, Riley LW (2002) Reactive nitrogen intermediates have a bacteriostatic effect on Mycobacterium tuberculosis in vitro. J Clin Microbiol 40:3162–3166

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Gefen O, Balaban NQ (2009) The importance of being persistent: heterogeneity of bacterial populations under antibiotic stress. FEMS Microbiol Rev 33:704–717

    Article  CAS  PubMed  Google Scholar 

  • Halliwell B, Hu ML, Louie S, Duvall TR, Tarkington BK, Motchnik P, Cross CE (1992) Interaction of nitrogen dioxide with human plasma. Antioxidant depletion and oxidative damage. Febs Lett 313:62–66

    Article  CAS  PubMed  Google Scholar 

  • Harrison JJ, Turner RJ, Ceri H (2005) Persister cells, the biofilm matrix and tolerance to metal cations in biofilm and planktonic Pseudomonas aeruginosa. Environ Microbiol 7:981–994

    Article  CAS  PubMed  Google Scholar 

  • Jiang G, Gutierrez O, Yuan Z (2011) The strong biocidal effect of free nitrous acid on anaerobic sewer biofilms. Water Res 45:3735–3743

    Article  CAS  PubMed  Google Scholar 

  • Juedes MJ, Wogan GN (1996) Peroxynitrite-induced mutation spectra of pSP189 following replication in bacteria and in human cells. Mutat Res 349:51–61

    Article  PubMed  Google Scholar 

  • Keren I, Shah D, Spoering A, Kaldalu N, Lewis K (2004) Specialized persister cells and the mechanism of multidrug tolerance in Escherichia coli. J Bacteriol 186:8172–8180

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Kevil CG, Kolluru GK, Pattillo CB, Giordano T (2011) Inorganic nitrite therapy: historical perspective and future directions. Free Radic Biol Med 51:576–593

    Article  CAS  PubMed  Google Scholar 

  • Maeda S, Okamura M, Kobayashi M, Omata T (1998) Nitrite-specific active transport system of the cyanobacterium Synechococcus sp. strain PCC 7942. J Bacteriol 180:6761–6763

    PubMed Central  CAS  PubMed  Google Scholar 

  • McElroy KE, Hui JG, Woo JK, Luk AW, Webb JS, Kjelleberg S, Rice SA, Thomas T (2014) Strain-specific parallel evolution drives short-term diversification during Pseudomonas aeruginosa biofilm formation. Proc Natl Acad Sci U S A 111:E1419–1427

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Morita Y, Tomida J, Kawamura Y (2014) Responses of Pseudomonas aeruginosa to antimicrobials. Front Microbiol 4:422

    PubMed Central  PubMed  Google Scholar 

  • Nakaki T, Nakayama M, Kato R (1990) Inhibition by nitric oxide and nitric oxide-producing vasodilators of DNA synthesis in vascular smooth muscle cells. Eur J Pharmacol 189:347–353

    Article  CAS  PubMed  Google Scholar 

  • O’Leary V, Solberg M (1976) Effect of sodium nitrite inhibition on intracellular thiol groups and on the activity of certain glycolytic enzymes in Clostridium perfringens. Appl Environ Microbiol 31:208–212

    PubMed Central  PubMed  Google Scholar 

  • Park J-W (1993) S-Nitrosylation of sulfhydryl groups in albumin by nitrosating agents. Arch Pharm Res 16:1–5

    Article  CAS  Google Scholar 

  • Phillips R, Adjei O, Lucas S, Benjamin N, Wansbrough-Jones M (2004) Pilot randomized double-blind trial of treatment of Mycobacterium ulcerans disease (Buruli ulcer) with topical nitrogen oxides. Antimicrob Agents Chemother 48:2866–2870

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Pijuan M, Ye L, Yuan ZG (2010) Free nitrous acid inhibition on the aerobic metabolism of poly-phosphate accumulating organisms. Water Res 44:6063–6072

    Article  CAS  PubMed  Google Scholar 

  • Platt MD, Schurr MJ, Sauer K, Vazquez G, Kukavica-Ibrulj I, Potvin E, Levesque RC, Fedynak A, Brinkman FSL, Schurr J, Hwang S-H, Lau GW, Limbach PA, Rowe JJ, Lieberman MA, Barraud N, Webb J, Kjelleberg S, Hunt DF, Hassett DJ (2008) Proteomic, microarray, and signature-tagged mutagenesis analyses of anaerobic Pseudomonas aeruginosa at pH 6.5, likely representing chronic, late-stage cystic fibrosis airway conditions. J Bacteriol 190:2739–2758

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Poole RK (2005) Nitric oxide and nitrosative stress tolerance in bacteria. Biochem Soc Trans 33:176–180

    Article  CAS  PubMed  Google Scholar 

  • Rao A, Jump RL, Pultz NJ, Pultz MJ, Donskey CJ (2006) In vitro killing of nosocomial pathogens by acid and acidified nitrite. Antimicrob Agents Chemother 50:3901–3904

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Rhoades ER, Orme IM (1997) Susceptibility of a panel of virulent strains of Mycobacterium tuberculosis to reactive nitrogen intermediates. Infect Immun 65:1189–1195

    PubMed Central  CAS  PubMed  Google Scholar 

  • Rogstam A, Larsson JT, Kjelgaard P, von Wachenfeldt C (2007) Mechanisms of adaptation to nitrosative stress in Bacillus subtilis. J Bacteriol 189:3063–3071

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Rowe JJ, Yarbrough JM, Rake JB, Eagon RG (1979) Nitrite inhibition of aerobic bacteria. Curr Microbiol 2:51–54

    Article  CAS  Google Scholar 

  • Rubbo H, Radi R, Trujillo M, Telleri R, Kalyanaraman B, Barnes S, Kirk M, Freeman BA (1994) Nitric oxide regulation of superoxide and peroxynitrite-dependent lipid peroxidation. Formation of novel nitrogen-containing oxidized lipid derivatives. J Biol Chem 269:26066–26075

    CAS  PubMed  Google Scholar 

  • Schurek KN, Breidenstein EB, Hancock RE (2012) Antibiotic discovery and development. Springer, New York

    Google Scholar 

  • Stover CK, Pham XQ, Erwin AL, Mizoguchi SD, Warrener P, Hickey MJ, Brinkman FS, Hufnagle WO, Kowalik DJ, Lagrou M, Garber RL, Goltry L, Tolentino E, Westbrock-Wadman S, Yuan Y, Brody LL, Coulter SN, Folger KR, Kas A, Larbig K, Lim R, Smith K, Spencer D, Wong GK, Wu Z, Paulsen IT, Reizer J, Saier MH, Hancock RE, Lory S, Olson MV (2000) Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406:959–964

    Article  CAS  PubMed  Google Scholar 

  • Vadivelu VM, Keller J, Yuan ZG (2006a) Effect of free ammonia and free nitrous acid concentration on the anabolic and catabolic processes of an enriched Nitrosomonas culture. Biotechnol Bioeng 95:830–839

    Article  CAS  PubMed  Google Scholar 

  • Vadivelu VM, Yuan Z, Fux C, Keller J (2006b) The inhibitory effects of free nitrous acid on the energy generation and growth processes of an enriched Nitrobacter culture. Environ Sci Technol 40:4442–4448

    Article  CAS  PubMed  Google Scholar 

  • Wang Q, Ye L, Jiang G, Jensen P, Batstone D, Yuan Z (2013a) Free nitrous acid (FNA)-based pre-treatment enhances methane production from waste activated sludge. Environ Sci Technol 47:11897–11904

    Article  CAS  PubMed  Google Scholar 

  • Wang Q, Ye L, Jiang G, Yuan Z (2013b) A free nitrous acid (FNA)-based technology for reducing sludge production. Water Res 47:3663–3672

    Article  CAS  PubMed  Google Scholar 

  • Webb JS, Thompson LS, James S, Charlton T, Tolker-Nielsen T, Koch B, Givskov M, Kjelleberg S (2003) Cell death in Pseudomonas aeruginosa biofilm development. J Bacteriol 185:4585–4592

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Weller R, Price RJ, Ormerod AD, Benjamin N, Leifert C (2001) Antimicrobial effect of acidified nitrite on dermatophyte fungi, Candida and bacterial skin pathogens. J Appl Microbiol 90:648–652

    Article  CAS  PubMed  Google Scholar 

  • Woods LF, Wood JM (1982) A note on the effect of nitrite inhibition on the metabolism of Clostridium botulinum. J Appl Bacteriol 52:109–110

    Article  CAS  PubMed  Google Scholar 

  • Woods LF, Wood JM, Gibbs PA (1981) The involvement of nitric oxide in the inhibition of the phosphoroclastic system in Clostridium sporogenes by sodium nitrite. J Gen Microbiol 125:399–406

    CAS  PubMed  Google Scholar 

  • Wu Q, Stewart V (1998) NasFED proteins mediate assimilatory nitrate and nitrite transport in Klebsiella oxytoca (pneumoniae) M5al. J Bacteriol 180:1311–1322

    PubMed Central  CAS  PubMed  Google Scholar 

  • Ye L, Pijuan M, Yuan Z (2013) The effect of free nitrous acid on key anaerobic processes in enhanced biological phosphorus removal systems. Bioresour Technol 130:382–9

    Article  CAS  PubMed  Google Scholar 

  • Ye L, Pijuan M, Yuan ZG (2010) The effect of free nitrous acid on the anabolic and catabolic processes of glycogen accumulating organisms. Water Res 44:2901–2909

    Article  CAS  PubMed  Google Scholar 

  • Zhou Y, Ganda L, Lim M, Yuan ZG, Kjelleberg S, Ng WJ (2010) Free nitrous acid (FNA) inhibition on denitrifying poly-phosphate accumulating organisms (DPAOs). Appl Microbiol Biotechnol 88:359–369

    Article  CAS  PubMed  Google Scholar 

  • Zhou Y, Oehmen A, Lim M, Vadivelu V, Ng WJ (2011) The role of nitrite and free nitrous acid (FNA) in wastewater treatment plants. Water Res 45:4672–4682

    Article  CAS  PubMed  Google Scholar 

  • Zhou Y, Pijuan M, Yuan Z (2007) Free nitrous acid inhibition on anoxic phosphorus uptake and denitrification by poly-phosphate accumulating organisms. Biotechnol Bioeng 98:903–912

    Article  CAS  PubMed  Google Scholar 

  • Zhou Y, Pijuan M, Zeng RJ, Yuan Z (2008) Free nitrous acid inhibition on nitrous oxide reduction by a denitrifying-enhanced biological phosphorus removal sludge. Environ Sci Technol 42:8260–8265

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

We acknowledge the Australian Research Council for funding support of project DP120102832 (Biofilm Control in Wastewater Systems using Free Nitrous Acid—a Renewable Material from Wastewater) and scholarship support for Shu-Hong Gao from the China Scholarship Council. We thank Dr. Beatrice Keller and Jianguang Li, University of Queensland for FIA analysis and Dr. Michael Nefedov, University of Queensland for assistance with the BD FACSAria™ II flow cytometer and data analysis.

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The authors declare that they have no conflict of interest.

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  1. Advanced Water Management Centre, The University of Queensland, Level 4 Gehrmann Building, St. Lucia, Brisbane, QLD, 4072, Australia

    Shu-Hong Gao, Lu Fan, Zhiguo Yuan & Philip L Bond

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  1. Shu-Hong Gao
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  2. Lu Fan
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  3. Zhiguo Yuan
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  4. Philip L Bond
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Correspondence to Lu Fan or Philip L Bond.

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Gao, SH., Fan, L., Yuan, Z. et al. The concentration-determined and population-specific antimicrobial effects of free nitrous acid on Pseudomonas aeruginosa PAO1. Appl Microbiol Biotechnol 99, 2305–2312 (2015). https://doi.org/10.1007/s00253-014-6211-8

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  • DOI: https://doi.org/10.1007/s00253-014-6211-8

Keywords

  • Free nitrous acid
  • Bacteriostatic
  • Bactericidal
  • Pseudomonas aeruginosa PAO1