Effect of fetal and adult bovine serum on pyocyanin production in Pseudomonas aeruginosa isolated from clinical and soil samples

Document Type: Original Article


Department of Microbiology, Tehran North Branch, Islamic Azad University, Tehran, Iran


Objective(s): Pyocyanin is a blue-greenish redox-active pigment, produced by Pseudomonas aeruginosa, with a wide range of biological and biotechnological applications. Pyocyanin biosynthesis is regulated by the quorum-sensing (QS) system in which the expression of QS genes and QS-controlled virulence genes may be affected by serum as a complex medium. In the current study, effects of adult bovine serum (ABS) and fetal bovine serum (FBS) on the production of pyocyanin were examined in order to develop it.
Materials and Methods: The presence of pyocyanin-producing specific genes and proteins in clinical and soil isolates of P. aeruginosa was confirmed using PCR and SDS-PAGE. Isolates were inoculated to media containing different concentrations of complement-active/-inactivated ABS or FBS and pyocyanin concentration was measured by spectrophotometry. Extracted pigment was characterized by using UV-Visible spectrophotometry. Titration of ABS antibodies against studied isolates was performed by the tube agglutination test.
Results: Adding ABS to P. aeruginosa culture medium decreased pyocyanin production compared to the control, while its production increased in FBS-containing media (113.21±2.581 vs. 55.26±0.827 μg.ml-1 and 126.80±2.036 vs. 30.56±0.382 μg.ml-1 of C11 and E8 pyocyanin concentration in the presence of 10% FBS vs. control, respectively).
Conclusion: In this study, due to the presence of inhibitors such as complement proteins and antibodies in ABS samples, the use of FBS devoid of antibodies was effective to increase pyocyanin production in studied isolates.


1. Jander G, Rahme LG, Ausubel FM. Positive correlation between virulence of Pseudomonas aeruginosa mutants in mice and insects. J Bacteriol 2000; 182:3843-3845.

2. Frank DW. The exoenzyme S regulon of Pseudomonas aeruginosa. Mol Microbiol 1997; 26:621-629.

3. Budzikiewicz H. Secondary metabolites from fluorescent pseudomonads. FEMS Microbiol Rev 1993; 104:209-228.

4. Lau GW, Hassett DJ, Ran H, Kong F. The role of pyocyanin in Pseudomonas aeruginosa infection. Trends Mol Med 2004; 10:599-606.

5. Mavrodi DV, Bonsall RF, Delaney SM, Soule MJ, Phillips G, Thomashow LS. Functional analysis of genes for biosynthesis of pyocyanin and phenazine-1-carboxamide from Pseudomonas aeruginosa PAO1. J Bacteriol 2001; 183:6454-6465.

6. Whiteley M, Lee KM, Greenberg E. Identification of genes controlled by quorum sensing in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 1999; 96:13904-13909.

7. Gross H, Loper JE. Genomics of secondary metabolite production by pseudomonas spp. Nat Prod Rep 2009; 26:1408-1446.

8. Muller M. Pyocyanin induces oxidative stress in human endothelial cells and modulates the glutathione redox cycle. Free Radicals Biol Med 2002; 33:1527-1533.

9. Laursen JB, Nielsen J. Phenazine natural products: biosynthesis, synthetic analogues, and biological activity. Chem Rev 2004; 104:1663-1686.

10. Rahme LG, Ausubel FM, Cao H, Drenkard E, Goumnerov BC, Lau GW, et al. Plants and animals share functionally common bacterial virulence factors. Proc Natl Acad Sci U S A 2000; 97:8815-8821.

11. Anjaiah V, Koedam N, Nowak-Thompson B, Loper JE, Höfte M, Tambong JT, et al. Involvement of phenazines and anthranilate in the antagonism of Pseudomonas aeruginosa PNA1 and Tn5 derivatives toward Fusarium spp. and Pythium spp. Mol Plant-Microbe Interact 1998; 11:847-854.

12. Kerr J, Taylor G, Rutman A, Høiby N, Cole P, Wilson R. Pseudomonas aeruginosa pyocyanin and 1-hydroxyphenazine inhibit fungal growth. J Clin Pathol 1999; 52:385-387.

13. Rabaey K, Verstraete W. Microbial fuel cells: novel biotechnology for energy generation. Trends Biotechnol 2005; 23:291-298.

14. Ohfuji K, Sato N, Hamada-Sato N, Kobayashi T, Imada C, Okuma H, et al. Construction of a glucose sensor based on a screen-printed electrode and a novel mediator pyocyanin from Pseudomonas aeruginosa. Biosens Bioelectron 2004; 19:1237-1244.

15. Hassani HH, Hasan HM, Al-Saadi A, Ali AM, Muhammad MH. A comparative study on cytotoxicity and apoptotic activity of pyocyanin produced by wild type and mutant strains of Pseudomonas aeruginosa. Eur J Exp Biol 2012; 2:1389-1394.

16. Zhao J, Wu Y, Alfred A, Wei P, Yang S. Anticancer effects of pyocyanin on HepG2 human hepatoma cells. Lett Appl Microbiol 2014; 58:541-548.

17. Kruczek C, Qaisar U, Colmer‐Hamood JA, Hamood AN. Serum influences the expression of Pseudomonas aeruginosa quorum‐sensing genes and QS‐controlled virulence genes during early and late stages of growth. MicrobiologyOpen 2014; 3:64-79.

18. Essar D, Eberly L, Hadero A, Crawford I. Identification and characterization of genes for a second anthranilate synthase in Pseudomonas aeruginosa: interchangeability of the two anthranilate synthases and evolutionary implications. J Bacteriol 1990; 172:884-900.

19. Silby MW, Winstanley C, Godfrey SA, Levy SB, Jackson RW. Pseudomonas genomes: diverse and adaptable. FEMS Microbiol Rev 2011; 35:652-680.

20. Chaerun SK, Tazaki K, Asada R, Kogure K. Bioremediation of coastal areas 5 years after the Nakhodka oil spill in the Sea of Japan: isolation and characterization of hydrocarbon-degrading bacteria. Environ Int 2004; 30:911-922.

21. Vives-Flórez M, Garnica D. Comparison of virulence between clinical and environmental Pseudomonas aeruginosa isolates. Int Microbiol 2006; 9:247-252.

22. El-Amine Bendaha M, Mebrek S, Naimi M, Tifrit A, Belaouni H. Isolation and comparison of rhamnolipids production in Pseudomonas aeruginosa P.B: 2 and Pseudomonas fluorescens P.V: 10. Sci Rep 2012; 1:544.

23. Al-Hinai A, Al-Sadi A, Al-Bahry S, Mothershaw A, Al-Said F, Al-Harthi S, et al. Isolation and characterization of Pseudomonas aeruginosa with antagonistic activity against Pythium aphanidermatum. J Plant Pathol 2010; 92:653-660.

24. Karatuna O, Yagci A. Analysis of quorum sensing‐dependent virulence factor production and its relationship with antimicrobial susceptibility in Pseudomonas aeruginosa respiratory isolates. Clin Microbiol Infect 2010; 16:1770-1775.

25. Mohammed HA, Yossef HS, Mohammad FI. The cytotoxicity effect of pyocyanin on human hepatocellular carcinoma cell line (HepG2). Iraqi J Sci 2014; 55:668-674.

26. Vinckx T, Wei Q, Matthijs S, Cornelis P. The Pseudomonas aeruginosa oxidative stress regulator OxyR influences production of pyocyanin and rhamnolipids: protective role of pyocyanin. Microbiology 2010; 156:678-686.

27. Subramaniam L. Rapid diagnosis of Pseudomonas aeruginosa infection by demonstration of pyocyanin & fluorescein. Indian J Med Res 1985; 81:561-566.

28. El-Fouly M, Sharaf A, Shahin A, El-Bialy HA, Omara A. Biosynthesis of pyocyanin pigment by Pseudomonas aeruginosa. J Radiat Res Appl Sci 2015; 8:36-48.

29. Juhas M, Wiehlmann L, Huber B, Jordan D, Lauber J, Salunkhe P, et al. Global regulation of quorum sensing and virulence by VqsR in Pseudomonas aeruginosa. Microbiology 2004; 150:831-841.

30. Wu L, Estrada O, Zaborina O, Bains M, Shen L, Kohler JE, et al. Recognition of host immune activation by Pseudomonas aeruginosa. Science 2005; 309:774-777.