Potential of oxygen and nitrogen reactive intermediates to disperse Listeria monocytogenes from biofilms

  • Fernanda Barbosa dos Reis-Teixeira
  • Natália Conceição
  • Lilian Pereira da Silva
  • Virgínia Farias Alves
  • Elaine Cristina Pereira De MartinisEmail author
Food Microbiology - Research paper


Studying biofilm dispersal is important to prevent Listeria monocytogenes persistence in food processing plants and to avoid finished product contamination. Reactive oxygen and nitrogen intermediates (ROI and RNI, respectively) may trigger cell detachment from many bacterial species biofilms, but their roles in L. monocytogenes biofilms have not been fully investigated. This study reports on ROI and RNI quantification in Listeria monocytogenes biofilms formed on stainless steel and glass surfaces; bacterial culture and microscopy combined with fluorescent staining were employed. Nitric oxide (NO) donor and inhibitor putative effects on L. monocytogenes dispersal from biofilms were evaluated, and transcription of genes (prfA, lmo 0990, lmo 0807, and lmo1485) involved in ROI and RNI stress responses were quantified by real-time PCR (qPCR). Microscopy detected the reactive intermediates NO, peroxynitrite, H2O2, and superoxide in L. monocytogenes biofilms. Neither NO donor nor inhibitors interfered in L. monocytogenes growth and gene expression, except for lmo0990, which was downregulated. In conclusion, ROI and RNI did not exert dispersive effects on L. monocytogenes biofilms, indicating that this pathogen has a tight control for protection against oxidative and nitrosative stresses.


Dispersal Reactive oxygen intermediate ROI Reactive nitrogen intermediate RNI 


Authors’ contributions

Study concept and design: FBRT, NC, and LPS. Data analysis and interpretation: ECPDM. Manuscript drafting: FBRT, VFA, and ECPDM. Critical revision of the manuscript for important intellectual content: VFA and ECPDM.

Funding information

FBR received a Ph.D. fellowship from São Paulo Research Foundation (Process 2010/10051-3). The authors are grateful to São Paulo Research Foundation for a Research Grant (Process 2011/07062-6) to ECPDM and to the Multiuser Laboratory of Confocal Microscopy - FMRP-USP (Process 2004/08868-0). This study was partially funded by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Codes 001.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Silva EP, De Martinis ECP (2013) Current knowledge and perspectives on biofilm formation: the case of Listeria monocytogenes. Appl Microbiol Biotechnol 97:957–968. CrossRefGoogle Scholar
  2. 2.
    Barraud N, Storey MV, Moore ZP, Webb JS, Rice SA, Kjelleberg S (2009) Nitric oxide-mediated dispersal in single- and multi-species biofilms of clinically and industrially relevant microorganisms. Microb Biotechnol 2:370–378. CrossRefGoogle Scholar
  3. 3.
    Watnick P, Kolter R (2000) Biofilm, city of microbes. J Bacteriol 182(10):2675–2679CrossRefGoogle Scholar
  4. 4.
    McDougald D, Rice SA, Barraud N, Steinberg PD, Kjelleberg S (2012) Should we stay or should we go: mechanisms and ecological consequences for biofilm dispersal. Nat Rev Microbiol 10:39–50. CrossRefGoogle Scholar
  5. 5.
    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. CrossRefGoogle Scholar
  6. 6.
    Barraud N, Schleheck D, Klebensberger J, Webb JS, Hassett DJ, Rice SA, Kjelleberg S (2009) Nitric oxide signaling in Pseudomonas aeruginosa biofilms mediates phosphodiesterase activity, decreased cyclic Di-GMP levels, and enhanced dispersal. J Bacteriol 191:7333–7342. CrossRefGoogle Scholar
  7. 7.
    Marvasi M, Chen C, Carrazana M, Durie IA, Teplitski M (2014) Systematic analysis of the ability of nitric oxide donors to dislodge biofilms formed by Salmonella enterica and Escherichia coli O157:H7. AMB Express 4:42. CrossRefGoogle Scholar
  8. 8.
    Webb JS, Givskov M, Kjelleberg S (2003) Bacterial biofilms: prokaryotic adventures in multicellularity. Curr Opin Microbiol 6:578–585CrossRefGoogle Scholar
  9. 9.
    Winkelströter LK, Gomes BC, Thomaz MRS, Souza VM, De Martinis ECP (2011) Lactobacillus sakei 1 and its bacteriocin influence adhesion of Listeria monocytogenes on stainless steel surface. Food Control 22:1404–1407. CrossRefGoogle Scholar
  10. 10.
    Silva S, Teixeira P, Oliveira R, Azeredo J (2008) Adhesion to and viability of Listeria monocytogenes on food contact surfaces. J Food Prot 71:1379–1385CrossRefGoogle Scholar
  11. 11.
    Chae MS, Schraft H (2000) Comparative evaluation of adhesion and biofilm formation of different Listeria monocytogenes strains. Int J Food Microbiol 62:103–111CrossRefGoogle Scholar
  12. 12.
    [CLSI] Clinical and Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved Standard – 6th Edition. 2003. Accessed 28 July 2014
  13. 13.
    Shatalin K, Gusarov I, Avetissova E, Shatalina Y, McQuade LE, Lippard SJ, Nudler E (2008) Bacillus anthracis-derived nitric oxide is essential for pathogen virulence and survival in macrophages. PNAS. 105:1009–1013. CrossRefGoogle Scholar
  14. 14.
    Leriche V, Carpentier B (1995) Viable but nonculturable Salmonella typhimurium in single- and binary-species biofilms in response to chlorine treatment. J Food Prot 58:1186–1191CrossRefGoogle Scholar
  15. 15.
    Sambrook J, Russell DW (2006) Purification of nucleic acids by extraction with phenol:chloroform. CSH Protoc 2006:pdb.prot4455. Google Scholar
  16. 16.
    Sue D, Boor KJ, Wiedmann M (2003) σ B-dependent expression patterns of compatible solute transporter genes opuCA and lmo1421 and the conjugated bile salt hydrolase gene bsh in Listeria monocytogenes. Microbiol. 149:3247–3256CrossRefGoogle Scholar
  17. 17.
    Werbrouck H, Vermeulen A, Coillie EV, Messens W, Herman L, Devlieghere F, Uyttendaele M (2009) Influence of acid stress on survival, expression of virulence genes and invasion capacity into Caco-2 cells of Listeria monocytogenes strains of different origins. Int J Food Microbiol 134:140–146. CrossRefGoogle Scholar
  18. 18.
    Mraheil MA, Billion A, Mohamed W, Rawool D, Hain T, Chakraborty T (2011) Adaptation of Listeria monocytogenes to oxidative and nitrosative stress in IFN-γ-activated macrophages. Int J Med Microbiol 301:547–555. CrossRefGoogle Scholar
  19. 19.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔC T method. Methods. 25:402–408CrossRefGoogle Scholar
  20. 20.
    Herbert KC, Foster SJ (2001) Starvation survival in Listeria monocytogenes: characterization of the response and the role of known and novel components. Microbiol. 147:2275–2284CrossRefGoogle Scholar
  21. 21.
    Reis-Teixeira FBD, Alves VF, De Martinis ECP (2017) Growth, viability and architecture of biofilms of Listeria monocytogenes formed on abiotic surfaces. Braz J Microbiol 48(3):587–591. CrossRefGoogle Scholar
  22. 22.
    Schreiber F, Beutler M, Enning D, Lamprecht-Grandio M, Zafra O, González-Pastor JE, de Beer D (2011) The role of nitric-oxide-synthase-derived nitric oxide in multicellular traits of Bacillus subtilis 3610: biofilm formation, swarming, and dispersal. BMC Microbiol 11:111–121. CrossRefGoogle Scholar
  23. 23.
    Sudhamsu J, Crane BR (2009) Bacterial nitric oxide synthase: what are they good for? Trends Microbiol 17:212–218. CrossRefGoogle Scholar
  24. 24.
    Wei Y, Zhou H, Sun Y, He Y, Luo Y (2007) Insight into the catalytic mechanism of arginine deiminase: functional studies on the crucial sites. Proteins: Struct Func Bioinf 66:740–750CrossRefGoogle Scholar
  25. 25.
    Joseph B, Goebel W (2007) Life of Listeria monocytogenes in the host cells’ cytosol. Microbes Infect 9:1188–1195. CrossRefGoogle Scholar
  26. 26.
    Cole C, Thomas S, Filak H, Henson PM, Lenz LL (2012) Nitric oxide increases susceptibility of toll-like receptor-activated macrophages to spreading Listeria monocytogenes. Immunity. 36:807–820. CrossRefGoogle Scholar
  27. 27.
    Zaitseva J, Granik V, Belik A, Koksharova O, Khmel I (2009) Effect of nitrofurans and NO generators on biofilm formation by Pseudomonas aeruginosa PAO1 and Burkholderia cenocepacia 370. Res Microbiol 160:353–357. CrossRefGoogle Scholar
  28. 28.
    Joannou CL, Cui XY, Rogers N, Vielotte N, Martinez CLT, Vugman NV, Hughes MN, Cammack R (1998) Characterization of the bactericidal effects of sodium nitroprusside and other pentacyanonitrosyl complexes on the food spoilage bacterium Clostridium sporogenes. Appl Environ Microbiol 64:3195–3201Google Scholar

Copyright information

© Sociedade Brasileira de Microbiologia 2019

Authors and Affiliations

  • Fernanda Barbosa dos Reis-Teixeira
    • 1
  • Natália Conceição
    • 1
  • Lilian Pereira da Silva
    • 1
  • Virgínia Farias Alves
    • 2
  • Elaine Cristina Pereira De Martinis
    • 1
    Email author
  1. 1.Faculdade de Ciências Farmacêuticas de Ribeirão PretoUniversidade de São Paulo (FCFRP-USP)Ribeirão PretoBrazil
  2. 2.Faculdade de FarmáciaUniversidade Federal de GoiásGoiâniaBrazil

Personalised recommendations