THE DETECTION OF PHZS AND PHZM GENES IN PYOCYANIN FORMING AND ANTIBIOTIC RESISTING PSEUDOMONAS AERUGINOSA CLINICAL ISOLATES

Authors

  • F. Q. Falah
  • H. A. A. Alsalim

DOI:

https://doi.org/10.36103/pghpqk75

Keywords:

pyocyanin genes, virulence factors, pathogenic bacteria, antibiotic resistance.

Abstract

This study was aimed to detect the phzS and phzM genes in Pseudomonas aeruginosa clinical isolates that form pyocyanin and resist antibiotics. The study was conducted by collecting 240 samples from different clinical sources (burns, wounds, urinary tract, and ear infections) at Medicine City laboratories Baghdad-Iraq. Only 140 samples were diagnosed as P. aeruginosa, with the major identification depending on morphological characteristics, biochemical tests, the Vitek 2 compact system, and molecular detection of the 16S rRNA gene responsible for this bacterium. Identified isolates were investigated for hemolysin, protease enzymes, and pyocyanin production. The genes phzS and phzM, involved in pyocyanin production, were investigated, and the isolates' sensitivity to 12 antibiotics was also tested. The results showed that all the isolates were able to produce hemolysin, 80% of the isolates were protease enzyme producers, and 72.15% produced pyocyanin. The presence percentage of the phzS gene (90%) was higher than that of the phzM gene (70%), and only the isolates that possessed the two genes producing pyocyanin, while those that contained one of the genes phzS or phzM did not. The highest levels of antibiotic resistance were for colistin (100%) and ceftazidime (97.14%), while the least were for imipenem (26.42%) and piperacillin-tazobactam (22.85%). The isolates producing pyocyanin are more resistant to antibiotics than those unable to produce it.

References

1. Adejumo, S. A., A. N. Oli, E. I. Okoye, C. D. Nwakile, C. M. Ojiako, U. M. Okezie, I. J. Okeke, C. M. Ofomata, A. A. Attama, J. N. Okoyeh, and C. O. Esimone., 2021. Biosurfactant production using mutant strains of Pseudomonas aeruginosa and Bacillus subtilis from agro-industrial wastes. Advanced Pharmaceutical Bulletin, 11(3): 543–556. DOI: 10.34172/apb.2021.063

2. Al-Sajad M. S. and H. A. A. Alsalim., 2024. Detection of genes responsible for heavy metals resistance in locally isolated Pseudomonas spp. Agricultural Sciences. 55(1):361-370.

DOI: https://doi.org/10.36103/wgz9vb91

3. Aqel, H., N. Sannan, R. Foudah, and A. Al-Hunaiti., 2023. Enzyme production and inhibitory potential of Pseudomonas aeruginosa: contrasting clinical and environmental isolates. Antibiotics, 12(9), 1354. DOI.org/10.3390/antibiotics12091354

4. Baron, E. J., 2001. Rapid identification of bacteria and yeast: summary of a national committee for clinical laboratory standards proposed guideline. Clin Infect Dis 33: 220-225. DOI: 10.1086/321816

5. Brisse, S., D. Milatovic, A. C. Fluit, K. Kusters, A. Toelstra, J. Verhoef, and F. J. Schmitz., 2000. Molecular Surveillance of European Quinolone-Resistant Clinical Isolates of Pseudomonas aeruginosa and Acinetobacter spp. using automated ribotyping. Journal of Clinical Microbiology, 38: 3636-3645.

DOI: 10.1128/jcm.38.10.3636-3645.2000

6. Bunyan, I., O. Hadi, and H. Al-Mansoori., 2019. Phenotypic detection and biofilm formation among Pseudomonas aeruginosa isolated from different sites of infection. International Journal of Pharmaceutical Quality Assurance, 10.

DOI: 10.25258/ijpqa.10.2.21

7. Choi, H. J., M. H. Kim, M. S. Cho, B. K. Kim, J. Y, C. Kimkim, and D. S. Park., 2013. Improved PCR for identification of Pseudomonas aeruginosa. Appl Microbiol Biotechnol, 97: 3643-3651. DOI.org/10.1007/s00253-013-4709-0

8. CLSI. 2022. Performance Standards for Antimicrobial Susceptibility Testing. 32nd ed CLSI Supplement M100. Clinical and laboratory Standards Institute. pp: 32-34.

9. Davido B., L. Fellous, C. Lawrence, V. Maxime, M. Rottman, and A. Dinh., 2017. Ceftazidime-avibactam and aztreonam, an interesting strategy to overcome β-Lactam resistance conferred by metallo-β-Lactamases in Enterobacteriaceae and Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy, 61(9):e01008-17.

DOI: 10.1128/AAC.01008-17

10. Elkady, F. M., B. M. Badr, A. H. Hashem, M. S. Abdulrahman, A. M. Abdelaziz, A. A. Al-Askar, G. Abd-Elgayed, and H. R. Hashem., 2024. Unveiling the Launaea nudicaulis (L.) Hook medicinal bioactivities: phytochemical analysis, antibacterial, antibiofilm, and anticancer activities. Frontiers in Microbiology, 15. DOI:10.3389/fmicb.2024.1454623.

11. Fortuna, A., D. Collalto, G. Rampioni, and L. Leoni., 2023. Assays for Studying Pseudomonas aeruginosa Secreted Proteases. In Pseudomonas aeruginosa: Methods and Protocols (pp.137-151). New York, NY: Springer US.

DOI: 10.1007/978-1-0716-3473-8_10

12. Gellatly, S. L., and R. E. Hancock., 2013. Pseudomonas aeruginosa: new insights into pathogenesis and host defenses. Pathogens and Disease, 67: 159-173.

DOI.org/10.1111/2049-632X.12033

13. George, D. and P. Mallery, 2019. IBM SPSS statistics 26 step by step: A simple guide and reference. Routledge.

DOI:10.4324/9780429056765

14. Holt, J., N. Kreig, P. Sneath, J. Staley and S. Williams., 1994. Bergey's manual of determinative bacteriology. 9th (ed.) Williams and Wilkins, U.S.A. pp: 93, 94, 151.

DOI: 10.1007/978-0-387-68572-4

15. Ibrahim, A. H., 2022. Link between some virulnce factors genes and antibacterial resistance of Pseudomonas aeruginosa. Iraqi Journal of Agricultural Sciences, 53(5): 985- 993. DOI.org/10.36103/ijas.v53i5.1612

16. Khudair, A. N., and S. S. Mahmood., 2021. Detection of the antiseptic resistance gene among Pseudomonas aeruginosa isolates. Iraqi Journal of Science, 62(1): 75-82.

DOI: 10.24996/ijs.2021.62.1.7

17. Lau, G. W., H. Ran, F. Kong, D. J. Hassett, and D. Mavrodi., 2004. Pseudomonas aeruginosa pyocyanin is critical for lung infection in mice. Infect Immun, 72, 4275-8.

DOI.org/10.1128/iai.72.7.4275-4278.2004

18. Lister, P. D., D. J. Wolter, and N. D. Hanson., 2009. Antibacterial -Resistant Pseudomonas aeruginosa: clinical impact and complexregulation of chromosomally encoded resistance mechanisms. Clinical Microbiology Reviews. 22(4): 582-610.

DOI:10.1128/cmr.00040-09

19. Liu, H., W. Kong, W. Yang, G. Chen, H. Liang, and Y. Zhang., 2018. Multilocus sequence typing and variations in the oprD gene of Pseudomonas aeruginosa isolated from a hospital in China. Infect Drug Resist, 11: 45-54.

DOI:10.2147/IDR.S152162

20. Mavrodi, D. V., R. F. Bonsall, S. M. Delaney, M. J. Soule, G. Phillips, and L. S. Thomashow., 2001. Functional analysis of genes for biosynthesis of pyocyanin and phenazine-1-carboxamide from Pseudomonas aeruginosa PAO1. J Bacteriol, 183, 6454-65.

DOI: 10.1128/jb.183.21.6454-6465.2001

21. Mitov, I., T. Strateva, and B. Markova., 2010. Prevalence of virulence genes among bulgarian nosocomial and cystic fibrosis isolates of Pseudomonas aeruginosa .Braz. J. Microbiol. 41: 3.

DOI:10.1590/S1517-83822010000300008

22. Mori C.D., E. Déziel, J. Gauthier, R. C. Levesque and G. W. Lau., 2021. An Organ System-Based Synopsis of Pseudomonas aeruginosa Virulence. Virulence, 12 (1): 1469-1507. DOI.org/10.1080/21505594.2021.1926408

23. Najafi, M., M. N. Moghaddam, and E. Yousefi., 2021. The effect of silver nanoparticles on pyocyanin production of Pseudomonas aeruginosa isolated from clinical specimens. Avicenna Journal of Medical Biotechnology, 13(2), 98. DOI: 10.18502/ajmb.v13i2.5529

24. Narvhus J. A., O. N. Bækkelund, E. M. Tidemann, H. M. Østlie, and R. K. Abrahamsen., 2021. Isolates of Pseudomonas spp. from cold-stored raw milk show variation in proteolytic and lipolytic properties. International Dairy Journal,123:105049. DOI:10.1016/j.idairyj.2021.105049

25. Nita, M., and A. Grzybowski., 2016. The role of the reactive oxygen species and oxidative stress in the pathomechanism of the age-related ocular diseases and other pathologies of the anterior and posterior eye segments in adults. Oxid Med Cell Longev, 2016, 3164734. DOI: 10.1155/2016/3164734

26. Nowroozi, J., A. A. Sepahi, and A. Rashnonejad., 2012. Pyocyanine biosynthetic genes in clinical and environmental isolates of Pseudomonas aeruginosa and detection of pyocyanine’s antimicrobial effects with or without colloidal silver nanoparticles. Cell Journal (Yakhteh), 14, 7.

DOI:10.1155/2016/3164734.

27. O'Donnell J. N., V. Putra, G. M. Belfiore, B. L. Maring, K.Young, and T. P. Lodise., 2022. In vitro activity of imipenem/relebactam plus aztreonam against metallo-β-lactamase-producing, OprD-deficient Pseudomonas aeruginosa with varying levels of Pseudomonas-derived cephalosporinase production. International Journal of Antimicrobial Agents, 99(6),106595. DOI.org/10.1016/j.ijantimicag.2022.106595

28. Ohikhena, F. U., O. A. Wintola, and A. J. Afolayan., 2017. Evaluation of the antibacterial and antifungal properties of Phragmanthera capitata (sprengel) balle (loranthaceae), a mistletoe growing on rubber tree, using the dilution techniques. Scientific World Journal, 2017, 9658598.

DOI:10.1155/2017/9658598

29. Ozdal, M., 2019. A new strategy for the efficient production of pyocyanin, a versatile pigment, in Pseudomonas aeruginosa OG1 via toluene addition. 3 Biotech, 9, 374.

DOI:10.1007/s13205-019-1907-1

30. Qin S, W. Xiao, C. Zhou, Q. Pu, X. Deng, Lefu Lan, H. Liang, X.Song and M. Wu., 2022. Pseudomonas aeruginosa: pathogenesis, virulence factors, antibiotic resistance, interaction with host, technology advances and emerging therapeutics. Signal Transduction and Targeted Therapy. 7:199. DOI.org/10.1038/s41392-022-01056-1

31. Rasamiravaka, T., Q. Labtani, P. Duez, and M. El Jaziri., 2015. The formation of biofilms by pseudomonas aeruginosa: a review of the natural and synthetic compounds interfering with control mechanisms. Biomed res int, 2015, 759348.

DOI.org/10.1155/2015/759348.

32. Rathan, S., and G. Sundararaman., 2020. Computational analysis of pyocyanin (phz gene) from Pseudomonas. Scholars Journal of Applied Medical Sciences, 8: 2120-2126.

DOI: 10.36347/sjams.2020.v08i09.028

33. Snyder, J. W., and R. M. Atlas., 2006. HandBook of Media for Clinical Cicrobiology, CRC press, pp: 1-523.

34. Spilker, T., T. Coenye, P. Vandamme, and J. J. Lipuma., 2004. PCR based assay for differentiation of Pseudomonas aeruginosa from other Pseudomonas species recovered from cystic fibrosis patients. J. Clin. Microbiol. 42(5): 2074-2079.

DOI: 10.1201/9781420005462

35. Talon, D., V. Cailleux, M. Thouverez, and Y. Michel-Briand., 1996. Discriminatory power and usefulness of pulsed field gel electrophoresis in epidemiological studies of Pseudomonas aeruginosaJ Hosp Infect 32: 135-145.

DOI: 10.1016/S0195-6701(96)90055-9

36. Tribelli, P. M., and N. I. López., 2022. Insights into the temperature responses of Pseudomonas species in beneficial and pathogenic host interactions. Applied Microbiology and Biotechnology, 106 (23), 7699-7709.‏

DOI:10.1007/s00253-022-12243-z

37. Verdial, C., I. Serrano, L. Tavares, S. Gil, and M. Oliveira, 2023. Mechanisms of antibiotic and biocide resistance that contribute to Pseudomonas aeruginosa persistence in the hospital environment. Biomedicines, 11(4), 1221.‏

DOI: 10.3390/biomedicines11041221

38. Wang C., Q. Ye, A. Jiang, J. Zhang, Y. Shang, F. Li, B. Zhou, X. Xiang, Q. Gu, R. Pang, Y. Ding, S. Wu, M. Chen, Q. Wu and J. Wang., 2022. Pseudomonas aeruginosa Detection Using Conventional PCR and Quantitative Real-Time PCR Based on Species-Specific Novel Gene Targets Identified by Pangenome Analysi. Front. Microbiol,12:820431. DOI:10.3389/fmicb.2022.820431

39. Xu, J., D. Xiumei, W. Hui, and Z. Qi., 2013. Surveillance and correlation of antimicrobial usage and resistance of Pseudomonas aeruginosa: A Hospital Population-Based Study. PLoS One, 8(11): e78604. DOI: 10.1371/journal.pone.0078604

40. Yan, S. and G. Wu., 2019. Can biofilm be reversed through quorum sensing in Pseudomonas aeruginosa? Frontiers in Microbiology, 10, 1582:1-9. DOI:10.3389/fmicb.2019.01582.

Downloads

Published

2025-10-27

Issue

Section

Articles

How to Cite

Falah , F. Q., & Alsalim, H. A. A. (2025). THE DETECTION OF PHZS AND PHZM GENES IN PYOCYANIN FORMING AND ANTIBIOTIC RESISTING PSEUDOMONAS AERUGINOSA CLINICAL ISOLATES. IRAQI JOURNAL OF AGRICULTURAL SCIENCES, 56(5), 1726-1736. https://doi.org/10.36103/pghpqk75