4th International Virtual Congress (IVC-2017).  International E-publication: Publish Projects, Dissertation, Theses, Books, Souvenir, Conference Proceeding with ISBN.  International E-Bulletin: Information/News regarding: Academics and Research

Extraction, purification and characterization of pyocyanin produced by Pseudomonas aeruginosa and evaluation for its antimicrobial activity

Author Affiliations

  • 1Department of Microbiology, Faculty of Biological Sciences, University of Chittagong, Chittagong 4331, Bangladesh
  • 2Department of Microbiology, Faculty of Biological Sciences, University of Chittagong, Chittagong 4331, Bangladesh
  • 3Department of Microbiology, Faculty of Biological Sciences, University of Chittagong, Chittagong 4331, Bangladesh
  • 4Department of Microbiology, Faculty of Biological Sciences, University of Chittagong, Chittagong 4331, Bangladesh
  • 5Department of Microbiology, Faculty of Biological Sciences, University of Chittagong, Chittagong 4331, Bangladesh

Int. Res. J. Biological Sci., Volume 6, Issue (5), Pages 1-9, May,10 (2017)

Abstract

Pseudomonas aeruginosa, a gram negative bacteria, which exerts broad antagonistic activity against other bacterial and fungal pathogens through the production of a secondary metabolite-pyocyanin. In the present study, various clinical samples were taken for P. aeruginosa isolation. From sixteen primary P. aeruginosa isolates, five isolates (PS1, PU5, PU8, PU10 and PP3) were selected, on the basis of pigmentation in cetrimide agar. The screening for antimicrobial activity of P aeruginosain cross streak method showed that Staphylococcus aureus (ATCC 6538), Salmonella enterica (NCTC 6017), and Bacillus cereus (ATCC 14579) were sensitive, Escherichia coli (ATCC 8739) was intermediate, and K. pneumoniae (ATCC 43816) was resistant to the inhibitory action of the selected P. aeruginosa isolates. The antimicrobial pigment pyocyanin was extracted from culture broth following solvent extraction method. Purification of the pigment was done by column and thin layer chromatography. The Rf value was found around 0.81 for all the extracted pigment solution. Confirmation of the pigment as pyocyanin was done through FTIR, and UV-visible spectrophotometric analysis. FTIR analysis revealed different functional groups (-OH, -C=N, -CH3 etc.) which belongs to the aromatic structure of pyocyanin. In UV-Vis spectrophotometric analysis, a maximum absorption was observed at 270-275nm.The modification of media composition enabled to increase pyocyanin production and the highest amount was produced by the isolate PU10 in Medium-B having a concentration of 9.45 &

References

  1. Lyczak J.B., Cannon C.L. and Pier G.B. (2000)., Establishment of Pseudomonas aeruginosa infection: lessons from a versatile opportunist., Microbes Infect., 2(9), 1051-1060.
  2. Delden C.V. and Iglewski B.H. (1998)., Cell-to-cell signaling and Pseudomonas aeruginosa infections., Emerg Infect. Dis., 4(4), 551-560.
  3. Mavrodi D.V., Bonsall R.F., Delaney S.M., Soule M.J., Phillips G. and Thomashow L.S. (2001)., Functional analysis of genes for biosynthesis of pyocyanin and phenazine-1-carboxamide from Pseudomonas aeruginosa PAO1., J.Bacteriol., 183(21), 6454-6465.
  4. Wang Y., Wilks J.C., Danhorn T., Ramos I., Croal L. and Newman D.K. (2011)., Phenazine-1-Carboxylic Acid Promotes Bacterial Biofilm Development via Ferrous Iron Acquisition., J.Bacteriol., 193(14), 3606-3617.
  5. Onbasli D. and Aslim B. (2008)., Determination of antimicrobial activity and production of some metabolites by Pseudomonas aeruginosa B1 and B2 in sugar beet molasses., Afr. J. Biotechnol., 7(24), 4614-4619.
  6. Brown V.I. and Lowbury E.J.L. (1965)., Use of an improved cetrimide agar medium and other culture methods for Pseudomonas aeruginosa., J.Clin.Pathol., 18(6), 752-756.
  7. Buchannan R.E. and Gibbson N.E. (1974)., Bergey’s manual of Determinative Bacteriology., Eighth Edition. Williams and Wilkins Co., 217-236.ISBN 13: 9780683011173
  8. Carvajal F. (1947)., Screening Tests for Antibiotics., Mycologia., 39(1), 128-130.
  9. Ingledew W.M. and Campbell J.R. (1969)., A new resuspension medium for pyocyanin production., Can. J.Microbiol., 15(6), 595-598.
  10. Baron S.S. and Rowe J.J. (1981)., Antibiotic action of pyocyanin., Antimicrob. Agents Chemother., 20(6), 814-820. DOI: 10.1128/AAC.20.6.814
  11. El-Shouny W.A., Al-Baidani A.R.H. and Hamza W.T. (2011)., Antimicrobial Activity of Pyocyanin Produced by Pseudomonas aeruginosa Isolated from Surgical Wound-Infections., Intl. J. Pharm. Med. Sci., 1(1), 01-07.
  12. El-Fouly M.Z., Sharaf A.M., Shahin A.A.M., El-Bialy H.A. and Omara A.M.A. (2015)., Biosynthesis of pyocyanin pigment by Pseudomonas aeruginosa., J. Radiat. Res. Appl. Sci., 8(1), 36-48.http://dx.doi.org/10.1016/j.jrras.2014.10.007
  13. Ohfuji K., Sato N., Hamada-Sato N., Kobayashi T., Imada C., Okuma H. and Watanabe E. (2004)., Construction of a glucose sensor based on a screen-printed electrode and a novel mediator pyocyanin from Pseudomonasaeruginosa., Biosens. Bioelectron., 19(10), 1237-1244. DOI: 10.1016/j.bios.2003.11.010
  14. Sudhakar T., Karpagam S., and Shiyama S. (2013)., Analysis of pyocyanin compound and its antagonistic activity against phytopathogens., Int. J. Chem. Tech. Res., 5(3), 1101-1106.
  15. Essar D.W., Eberly L., Hadero A. and Crawford I.P. (1990)., Identification and characterization of genes for a second anthranilate synthase in Pseudomonas aeruginosa: interchangeability of the two anthranilate synthases and evolutionary implications., J.Bacteriol., 172(2), 884-900.
  16. Wu D., Huang W., Duan Q., Li F. and Cheng H. (2014)., Sodium houttuyfonate affects production of N-acyl homoserine lactone and quorum sensing-regulated genes expression in Pseudomonas aeruginosa., Front.Microbiol., 5, 635. DOI: 10.3389/fmicb.2014.00635
  17. Valgas C., Souza S.M.D., Smania E.F.A. and Smania A.J. (2007)., Screening methods to determine antibacterial activity of natural products., Braz. J. Microbiol., 38(2), 369-380. http://dx.doi.org/10.1590/S1517-83822007000200034
  18. Peleg A.Y. and Hooper D.C. (2010)., Hospital-Acquired Infections Due to Gram-Negative Bacteria., N. Engl. J. Med., 362(19), 1804-1813.DOI: 10.1056/NEJMra0904124
  19. Chung G.A., Aktar Z., Jackson S. and Duncan K. (1995)., High-throughput screen for detecting antimycobacterial agents., Antimicrob. Agents Chemother., 39(10), 2235-2238.
  20. Saosoong K., Tongtumma S., Chanthai S., Wongphathanakul W., Bunyatratchata W., and Ruangviriyachai C. (2007)., Isolation and study of chemical properties of pyocyanin produced from Pseudomonas aeruginosa TISTR 782 (ATCC 9027)., KKU Res. J., 12(1)., 24-32.
  21. Machan Z.A., Pitt T.L., White W., Watson D., Taylor G.W., Cole P.J. and Wilson R. (1990)., Interaction between Pseudomonas aeruginosa and Staphylococcus aureus: description of an anti-staphylococcal substance., J. Med. Microbiol., 34(4), 213-217.
  22. Saha S., Thavasi R. and Jayalakshmi S. (2008)., Phenazine pigments from Pseudomonas aeruginosa and their application as antibacterial agent and food colourants., Res. J.Microbiol., 3(3), 122-128. DOI:10.3923/jm.2008.122.128
  23. Nansathiti A., Apipattarakul S., Phaosiri C., Pongdontri P., Chanthai S. and Ruangviriyachai C. (2009)., Synthesis, isolation of phenazine derivatives and their antimicrobial activities., Walailak J Sci& Tech., 6(1), 79-91.
  24. Mahajan-Miklos S., Tan M.W., Rahme L.G. and Ausubel F.M. (1999)., Molecular mechanisms of bacterial virulence elucidated using a Pseudomonas aeruginosaCaenorhabditiselegans pathogenesis model., Cell., 96(1), 47-56.
  25. Darby C., Cosma C.L., Thomas J.H. and Manoil C. (1999)., Lethal paralysis of Caenorhabditis elegans by Pseudomonas aeruginosa., Proc. Natl. Acad. Sci., 96(26), 15202-207.DOI: 10.1073/pnas.96.26.15202
  26. Jayaseelan S., Ramaswamy D. and Dharmaraj S. (2014)., Pyocyanin: production, applications, challenges and new insights., World J.Microbiol.Biotechnol.,30(4), 1159-1168. DOI: 10.1007/s11274-013-1552-5
  27. King E.O., Ward M.K. and Raney D.E. (1954)., Two simple media for the demonstration of pyocyanin and fluorescin., J. Lab.Clin. Med., 44(2), 301-307.
  28. Hassett D.J., Charniga L., Bean K., Ohman D.E. and Cohen M.S. (1992)., Response of Pseudomonas aeruginosa to pyocyanin: mechanisms of resistance, antioxidant defenses, and demonstration of a manganese-cofactored superoxide dismutase., Infect. Immun., 60(2), 328-336.
  29. Friedheim E. and Michaelis L. (1931)., Potentiometric study of pyocyanine., J. Biol. Chem., 91(1), 355-368.
  30. Silverstein R.M., Bassler G.C. and Morrill T.C. (1981)., Spectrometric Identification of Organic Compounds., Fourth Edition. John Wiley & Sons Inc. ISBN-13: 978-0471863557
  31. Boulette M.L., Baynham P.J., Jorth P.A., Kukavica-Ibrulj L., Longoria A., Barrera K., Levesque R.C. and Whiteley M. (2009)., Characterization of Alanine Catabolism in Pseudomonas aeruginosa and Its Importance for Proliferation In Vivo., J. Bacteriol., 191(20), 6329-6334.DOI: 10.1128/JB.00817-09
  32. Mac-Faddin J.F. (1985)., Media for Isolation - Cultivation - Identification - Maintenance of Medical Bacteria., Williams and Wilkins. ISBN: 0683053167
  33. Ramalho R., Cunha J., Teixeira P. and Gibbs P.A. (2002)., Modified Pseudomonas agar: new differential medium for the detection/ enumeration of Pseudomonas aeruginosa in mineral water., J.Microbiol.Methods, 49(1), 69-74.
  34. Palumbo S.A. (1972)., Role of iron and sulfur in pigment and slime formation by Pseudomonas aeruginosa., J.Bacteriol., 111(2), 430-436.
  35. Hassan H.M. and Fridorich I. (1980)., Mechanism of the antibiotic action of pyocyanine., J.Bacteriol., 141(1), 156-163.
  36. Kerr J.R., Taylor G.W., Rutman A., Høiby N., Cole P.J. and Wilson R. (1999)., Pseudomonas aeruginosa pyocyanin and 1-hydroxyphenazine inhibit fungal growth., J.Clin.Pathol., 52(5), 385-387.
  37. Dive D. (1973)., Action of exocellular pigments secreted by Pseudomonas aeruginosa on the growth and division of Colpidiumcampylum., Protistologica, 9, 315-318.
  38. Vukomanovic D.V., Zoutman D.E., Stone J.A., Marks G.S., Brien J.F. and Nakatsu K. (1997)., Electrospray mass-spectrometric, spectrophotometric and electrochemical methods do not provide evidence for the binding of nitric oxide by pyocyanine at pH7., Biochem. J., 322(1), 25-29.
  39. Oleiwi S.R. (2015)., Study the Effect of Pyocyanin Extracted from Pseudomonas aeruginosa on DNA Fragmentation of Human Lymphocytes Cells., Iraqi J. Sci., 56(2B), 1366-1371.