Degrading of the Pseudomonas Aeruginosa Biofilm by Extracellular Levanase SacC from Bacillus subtilis

  • Elena TriznaEmail author
  • Mikhail I Bogachev
  • Airat Kayumov


Pseudomonas aeruginosa is an opportunistic pathogenic bacterium causing variety of biofilm-related infections in patients with burns, lung cancer, chronic obstructive pulmonary disease, and cystic fibrosis. Here, we show that extracellular levanase SacC from Bacillus subtilis disrupts the matrix biofilm of P. aeruginosa and this way increasing the efficacy of antibiotics against biofilm-embedded bacteria. In particular, the biofilm thickness decreased twofold after 2 h of treatment with levanase at 1 mg/ml, while 5 mg/ml of cellulase was required for the same effect. Next, in the presence of SacC, the efficacy of ciprofloxacin against biofilm-embedded P. aeruginosa increased fourfold. While the efficacy of amikacin in the presence of SacC increased fourfold against detached cell clumps, it remained unchanged against biofilm-embedded cells. These data suggest that extracellular levanase from B. subtilis could appear an alternative to other glycoside hydrolases reported to be active against biofilms of P. aeruginosa agent for external wound treatment to suppress biofilm formation and reduce reinfection risks.


Pseudomonas aeruginosa Biofilms Antimicrobial activity Biofilm destruction 



This work was supported by the Ministry of Science and Higher Education of the Russian Federation (assignment 2.5475.2017/6.7 to Mikhail I Bogachev) and the Russian Government Program to support the competitive development of Kazan Federal University.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.


  1. 1.
    O’Toole, G. A., & Kolter, R. (1998). Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Molecular Microbiology, 30(2), 295–304.CrossRefGoogle Scholar
  2. 2.
    Valderrey, A. D., Pozuelo, M. J., Jimenez, P. A., Macia, M. D., Oliver, A., & Rotger, R. (2010). Chronic colonization by Pseudomonas aeruginosa of patients with obstructive lung diseases: cystic fibrosis, bronchiectasis, and chronic obstructive pulmonary disease. Diagn Microbiol Infect Dis, 68(1), 20–27.CrossRefGoogle Scholar
  3. 3.
    Page, M. J., & Di Cera, E. (2008). Serine peptidases: classification, structure and function. Cell Mol Life Sci, 65(7–8), 1220–1236.CrossRefGoogle Scholar
  4. 4.
    Ryder, C., Byrd, M., & Wozniak, D. J. (2007). Role of polysaccharides in Pseudomonas aeruginosa biofilm development. Current Opinion in Microbiology, 10(6), 644–648.CrossRefGoogle Scholar
  5. 5.
    Moreau-Marquis, S., Stanton, B. A., & O’Toole, G. A. (2008). Pseudomonas aeruginosa biofilm formation in the cystic fibrosis airway. Pulmonary Pharmacology & Therapeutics, 21(4), 595–599.CrossRefGoogle Scholar
  6. 6.
    Matsukawa, M., & Greenberg, E. P. (2004). Putative exopolysaccharide synthesis genes influence Pseudomonas aeruginosa biofilm development. Journal of Bacteriology, 186(14), 4449–4456.CrossRefGoogle Scholar
  7. 7.
    Hentzer, M., Teitzel, G. M., Balzer, G. J., Heydorn, A., Molin, S., Givskov, M., & Parsek, M. R. (2001). Alginate overproduction affects Pseudomonas aeruginosa biofilm structure and function. Journal of Bacteriology, 183(18), 5395–5401.CrossRefGoogle Scholar
  8. 8.
    Ma, L. Y., Lu, H. P., Sprinkle, A., Parsek, M. R., & Wozniak, D. J. (2007). Pseudomonas aeruginosa PSl is a galactose- and mannose-rich exopolysaccharide. Journal of Bacteriology, 189(22), 8353–8356.CrossRefGoogle Scholar
  9. 9.
    Kayumov, A. R., Nureeva, A. A., Trizna, E. Y., Gazizova, G. R., Bogachev, M. I., Shtyrlin, N. V., Pugachev, M. V., Sapozhnikov, S. V., & Shtyrlin, Y. G. (2015). New derivatives of pyridoxine exhibit high antibacterial activity against biofilm-embedded Staphylococcus cells. BioMed Research International.Google Scholar
  10. 10.
    Baidamshina, D. R., Trizna, E. Y., Holyavka, M. G., Bogachev, M. I., Artyukhov, V. G., Akhatova, F. S., Rozhina, E. V., Fakhrullin, R. F., & Kayumov, A. R. (2017). Targeting microbial biofilms using Ficin, a nonspecific plant protease. Sci Rep, 7.Google Scholar
  11. 11.
    Trizna E, Baydamshina D, Kholyavka M, Sharafutdinov I, Hairutdinova A, Khafizova F, Zakirova E, Hafizov R, Kayumov A: Soluble and immobilized papain and trypsin as destroyers of bacterial biofilms. Genes Cells. vol. 10; 2016: 106–112.Google Scholar
  12. 12.
    Whitchurch, C. B., Tolker-Nielsen, T., Ragas, P. C., & Mattick, J. S. (2002). Extracellular DNA required for bacterial biofilm formation. Science, 295(5559), 1487–1487.CrossRefGoogle Scholar
  13. 13.
    Flemming, H. C., & Wingender, J. (2010). The biofilm matrix. Nature Reviews Microbiology, 8(9), 623–633.CrossRefGoogle Scholar
  14. 14.
    Hall-Stoodley, L., Costerton, J. W., & Stoodley, P. (2004). Bacterial biofilms: from the natural environment to infectious diseases. Nature Reviews Microbiology, 2(2), 95–108.CrossRefGoogle Scholar
  15. 15.
    Rumbaugh, K. P., Diggle, S. P., Watters, C. M., Ross-Gillespie, A., Griffin, A. S., & West, S. A. (2009). Quorum sensing and the social evolution of bacterial virulence. Current Biology, 19(4), 341–345.CrossRefGoogle Scholar
  16. 16.
    Kayumov, A. R., Khakimullina, E. N., Sharafutdinov, I. S., Trizna, E. Y., Latypova, L. Z., Lien, H. T., Margulis, A. B., Bogachev, M. I., & Kurbangalieva, A. R. (2015). Inhibition of biofilm formation in Bacillus subtilis by new halogenated furanones. Journal of Antibiotics, 68(5), 297–301.CrossRefGoogle Scholar
  17. 17.
    Trizna, E. Y., Khakimullina, E. N., Latypova, L. Z., Kurbangalieva, A. R., Sharafutdinov, I. S., Evtyugin, V. G., Babynin, E. V., Bogachev, M. I., & Kayumov, A. R. (2015). Thio derivatives of 2(5H)-furanone as inhibitors against Bacillus subtilis biofilms. Acta Naturae, 7(2), 102–107.Google Scholar
  18. 18.
    Trizna, E., Latypova, L., Kurbangalieva, A., Bogachev, M. I., & Kayumov, A. (2016). 2(5H)-Furanone derivatives as inhibitors of staphylococcal biofilms. Bionanoscience, 6(4), 423–426.CrossRefGoogle Scholar
  19. 19.
    Gibson, D. G., Young, L., Chuang, R. Y., Venter, J. C., Hutchison, C. A., & Smith, H. O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods, 6(5), 343–U341.CrossRefGoogle Scholar
  20. 20.
    Fedorova, K., Kayumov, A., Ilinskaja, O., & Forchhammer, K. (2013). Transcription factor TnrA inhibits the biosynthetic activity of glutamine synthetase in Bacillus subtilis. FEBS Journal, 280, 228–228.Google Scholar
  21. 21.
    Leclercq, R., Canton, R., Brown, D. F. J., Giske, C. G., Heisig, P., MacGowan, A. P., Mouton, J. W., Nordmann, P., Rodloff, A. C., Rossolini, G. M., et al. (2013). EUCAST expert rules in antimicrobial susceptibility testing. Clinical Microbiology and Infection, 19(2), 141–160.CrossRefGoogle Scholar
  22. 22.
    ECfASTotESoCMaID: EUCAST Definitive Document E.Def 1.2. (2000). Terminology relating to methods for the determination of susceptibility of bacteria to antimicrobial agents. Clin Microbiol Infect, 6(9), 503–508.CrossRefGoogle Scholar
  23. 23.
    Herigstad, B., Hamilton, M., & Heersink, J. (2001). How to optimize the drop plate method for enumerating bacteria. Journal of Microbiological Methods, 44(2), 121–129.CrossRefGoogle Scholar
  24. 24.
    Sharafutdinov, I. S., Trizna, E. Y., Baidamshina, D. R., Ryzhikova, M. N., Sibgatullina, R. R., Khabibrakhmanova, A. M., Latypova, L. Z., Kurbangalieva, A. R., Rozhina, E. V., Klinger-Strobel, M., et al. (2017). Antimicrobial effects of sulfonyl derivative of 2(5&ITH&IT)-furanone against planktonic and biofilm associated methicillin-resistant and -susceptible&IT Staphylococcus aureus&IT. Frontiers in Microbiology, 8.Google Scholar
  25. 25.
    O’Toole, G. A. (2011). Microtiter dish biofilm formation assay. Journal of Visualized Experiments, 47.
  26. 26.
    Fleming, D., & Rumbaugh, K. P. (2017). Approaches to dispersing medical biofilms. Microorganisms, 5(2).Google Scholar
  27. 27.
    Wilton, M., Charron-Mazenod, L., Moore, R., & Lewenza, S. (2016). Extracellular DNA acidifies biofilms and induces aminoglycoside resistance in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy, 60(1), 544–553.CrossRefGoogle Scholar
  28. 28.
    Abdi-Ali, A., Mohammadi-Mehr, M., & Alaei, Y. A. (2006). Bactericidal activity of various antibiotics against biofilm-producing Pseudomonas aeruginosa. International Journal of Antimicrobial Agents, 27(3), 196–200.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Elena Trizna
    • 1
    Email author
  • Mikhail I Bogachev
    • 2
  • Airat Kayumov
    • 1
  1. 1.Institute of Fundamental Medicine and BiologyKazan Federal UniversityKazanRussia
  2. 2.Biomedical Engineering Research CentreSaint Petersburg Electrotechnical UniversitySt. PetersburgRussia

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