Antibiofilm Activity of α-Amylase from Bacillus subtilis S8-18 Against Biofilm Forming Human Bacterial Pathogens

Abstract

The extracellular α-amylase enzyme from Bacillus subtilis S8-18 of marine origin was proved as an antibiofilm agent against methicillin-resistant Staphylococcus aureus (MRSA), a clinical strain isolated from pharyngitis patient, Vibrio cholerae also a clinical isolate from cholera patient and Pseudomonas aeruginosa ATCC10145. The spectrophotometric and microscopic investigations revealed the potential of α-amylase to inhibit biofilm formation in these pathogens. At its BIC level, the crude enzyme caused 51.81–73.07% of biofilm inhibition. Beyond the inhibition, the enzyme was also effective in degradation of preformed mature biofilm by disrupting the exopolysaccharide (EPS), an essential component in biofilm architecture. Furthermore, the enzyme purified to its homogeneity by chromatographic techniques was also effective in biofilm inhibition (43.83–61.68%) as well as in degradation of EPS. A commercial α-amylase enzyme from B. subtilis was also used for comparative purpose. Besides, the effect of various enzymes and temperature on the antibiofilm activity of amylase enzymes was also investigated. This study, for the first time, proved that α-amylase enzyme alone can be used to inhibit/disrupt the biofilms of V. cholerae and MRSA strains and beholds much promise in clinical applications.

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References

  1. 1.

    Hall-Stoodley, L., Costerton, J. W., & Stoodley, P. (2004). Bacterial biofilms: From the nature environment to infectious diseases. Nature Reviews Microbiology, 2, 95–108.

    Article  CAS  Google Scholar 

  2. 2.

    Bakkiyaraj, D., & Pandian, S. K. (2010). In vitro and in vivo antibiofilm activity of a coral associated actinomycete against drug resistant Staphylococcus aureus biofilms. Biofouling, 26, 711–717.

    Article  CAS  Google Scholar 

  3. 3.

    Faruque, S. M., Biswas, K., Udden, S. M., Ahmad, Q. S., Sack, D. A., Nair, G. B., et al. (2006). Transmissibility of cholera: In vivo-formed biofilms and their relationship to infectivity and persistence in the environment. Proceedings of the National Academy of Sciences (USA), 103, 6350–6355.

    Article  CAS  Google Scholar 

  4. 4.

    Ikeno, T., Fukudo, K., Ogawa, M., Honda, M., Tanebe, T., & Taniguchu, H. (2007). Small and rough colony Pseudomonas aeruginosa with elevated biofilm formation ability isolated in hospitalized patients. Microbiology and Immunology, 51, 929–938.

    CAS  Google Scholar 

  5. 5.

    de Carvalho, C. C. C. C. R. (2007). Biofilms: Recent developments on an old battle. Recent Patents on Biotechnology, 1, 49–57.

    Article  Google Scholar 

  6. 6.

    Zhang, T., Ke, S. Z., Liu, Y., & Fang, H. P. (2005). Microbial characteristics of a methanogenic phenol-degrading sludge. Water Science Technology, 52, 73–78.

    CAS  Google Scholar 

  7. 7.

    Allison, D. G., McBain, A., & Gilbert, P. (2000). Biofilms: Problems of their control—Community and co-operation in biofilms (pp. 309–327). Cambridge: Cambridge University Press. Society for General Microbiology.

    Google Scholar 

  8. 8.

    Xavier, J. B., Picioreanu, C., Rani, S. A., Von Loosdrecht, M. C. M., & Stewart, P. S. (2005). Biofilm control strategies based on enzymatic disruption of the extracellular polymeric substance matrix—A modeling study. Journal of Microbiology, 151, 3817–3832.

    Article  CAS  Google Scholar 

  9. 9.

    Thenmozhi, R., Nithyanand, P., Rathna, J., & Pandian, S. K. (2009). Antibiofilm activity of coral-associated bacteria against different clinical M serotypes of Streptococcus pyogenes. FEMS Immunology and Medical Microbiology, 57, 284–294.

    Article  CAS  Google Scholar 

  10. 10.

    Nithya, C., & Pandian, S. K. (2009). Isolation of heterotrophic bacteria from Palk Bay sediments showing heavy metal tolerance and antibiotic production. Microbiological Research, 165, 578–593.

    Article  Google Scholar 

  11. 11.

    Nithya, C., Begum, M. F., & Pandian, S. K. (2010). Marine bacterial isolates inhibit biofilm formation and disrupt mature biofilms of Pseudomonas aeruginosa PAO1. Applied Microbiology and Biotechnology, 88, 341–358.

    Article  CAS  Google Scholar 

  12. 12.

    Swain, M. R., Kar, S., Padmaja, G., & Ray, R. C. (2006). Partial characterization and optimization of extra-cellular α-amylase from Bacillus subtilis isolated from cow dung microflora. Polish Journal of Microbiology, 55, 289–296.

    CAS  Google Scholar 

  13. 13.

    Baldassarri, L., Creti, R., Recchia, S., Imperi, M., Facinelli, B., Giovanetti, E., et al. (2006). Therapeutic failures of antibiotics used to treat macrolide-susceptible Streptococcus pyogenes infections may be due to biofilm formation. Journal of Clinical Microbiology, 44, 2721–2727.

    Article  CAS  Google Scholar 

  14. 14.

    Clinical and Laboratory Standards Institute (2006). Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; Approved standard, 7th edn. Clinical and Laboratory Standards Institute document M7-A7. Clinical and Laboratory Standards Institute, Wayne, PA.

  15. 15.

    You, J., Xue, X., Cao, L., Lu, X., Wang, J., Zhang, L., et al. (2007). Inhibition of Vibrio biofilm formation by a marine actinomycete strain A66. Applied Microbiology and Biotechnology, 76, 1137–1144.

    Article  CAS  Google Scholar 

  16. 16.

    Augustine, S. K., Bhavsar, S. P., & Kapadnis, B. P. (2005). A non-polyene antifungal antibiotic from Streptomyces albidoflavus PU 23. Journal of Biosciences, 30, 201–211.

    Article  CAS  Google Scholar 

  17. 17.

    Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Analytical Chemistry, 28, 350–356.

    Article  CAS  Google Scholar 

  18. 18.

    Lowry, O. H., Rosenbrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193, 265–275.

    CAS  Google Scholar 

  19. 19.

    Johansen, C., Falholt, P., & Gram, L. (1997). Enzymatic removal and disinfection of bacterial biofilms. Applied and Environmental Microbiology, 63, 3724–3728.

    CAS  Google Scholar 

  20. 20.

    Lequette, Y., Boelsb, G., Clarissea, M., & Faille, C. (2010). Using enzymes to remove biofilms of bacterial isolates sampled in the food-industry. Biofouling, 26, 421–431.

    Article  CAS  Google Scholar 

  21. 21.

    Leroy, C., Delbarrea, C., Ghillebaertb, F., Comperec, C., & Combes, D. (2008). Effects of commercial enzymes on the adhesion of a marine biofilm forming bacterium. Biofouling, 24, 11–22.

    Article  CAS  Google Scholar 

  22. 22.

    Orgaz, B., Kives, J., Pedregosa, A. M., Monistrol, I. F., Laborda, F., & SanJose, C. (2006). Bacterial biofilm removal using fungal enzymes. Enzyme and Microbial Technology, 40, 51–56.

    Article  CAS  Google Scholar 

  23. 23.

    Wiatr, C.L. (1991). Application of multiple enzymes blend to control industrial slime on equipments surfaces. United States Patent, Patent No.5071765.

  24. 24.

    Wai, S. N., Mizunoe, Y., Takade, A., Kawabata, S. I., & Yoshida, S. I. (1998). Vibrio cholerae O1 strain TSI-4 produces the exopolysaccharide materials that determine colony morphology, stress resistance, and biofilm formation. Applied and Environmental Microbiology, 64, 3648–3655.

    CAS  Google Scholar 

  25. 25.

    Yildiz, F. H., & Schoolnik, G. K. (1999). Vibrio cholerae O1 El Tor: identification of a gene cluster required for the rugose colony type, exopolysaccharide production, chlorine resistance, and biofilm formation. Proceedings of the National Academy of Sciences (USA), 96, 4028–4033.

    Article  CAS  Google Scholar 

Download references

Acknowledgement

The authors gratefully acknowledge the computational and bioinformatics facility provided by the Alagappa University Bioinformatics Infrastructure Facility (funded by DBT, GOI; grant no. BT/BI/25/001/2006). Financial support provided to Balu Jancy Kalpana in the form of Innovation in Scientific Pursuit for Inspired Research (INSPIRE) Fellowship by Department of Science and Technology, Government of India (DST/INSPIRE Fellowship/2010 [IF10448]) is thankfully acknowledged. Financial assistance rendered by CSIR for carrying out this work is gratefully acknowledged.

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Correspondence to Shunmugiah Karutha Pandian.

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Kalpana, B.J., Aarthy, S. & Pandian, S.K. Antibiofilm Activity of α-Amylase from Bacillus subtilis S8-18 Against Biofilm Forming Human Bacterial Pathogens. Appl Biochem Biotechnol 167, 1778–1794 (2012). https://doi.org/10.1007/s12010-011-9526-2

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Keywords

  • α-Amylase
  • Bacillus subtilis S8-18
  • Biofilms
  • Confocal Laser Scanning Microscopy (CLSM)
  • Exopolysaccharide (EPS)