Skip to main content
SpringerLink
Log in

In Vitro Cytotoxicity of Secondary Metabolites Extracted from Pseudomonas aeruginosa BS25 Strain

  • Research Article - Biological Sciences
  • Published:
Arabian Journal for Science and Engineering Aims and scope Submit manuscript

Abstract

Pseudomonas includes ubiquitous, opportunistic, pathogenic bacteria known to produce a variety of bioactive compounds. Objective of present study was to evaluate bioactive secondary metabolites produced by novel Pseudomonas aeruginosa BS25 strain. The bacterium isolated from patient’s wound was identified via 16S rRNA sequencing. To prepare extracts, nonpolar to polar solvents, i.e., hexane [H], ethyl acetate [E], acetone–ethyl acetate [AE] and water [W], were used. Cytotoxicity screening was performed against three cancer cell lines: HeLa, HepG2 and SHSY5Y. The effect on cell proliferation and mitochondrial activity was measured in HepG2 and Leishmania major. Furthermore, induction of apoptosis, necrosis, singlet oxygen release, lipid peroxidation and DNA binding were evaluated. For chemical composition colorimetric methods, TLC, HPLC and GC–MS analysis were performed. Extract [H] was effective against all cancer cell lines (relative viability: HeLa = 42.97 ± 3.46%, HepG2 = 41.54 ± 4.26%, SHSY5Y = 49.18 ± 2.98%). It strongly inhibited cell proliferation and mitochondrial activity (HepG2 IC50: 168.93 µg/ml), inducing apoptosis (apoptotic cells: 150 µg/ml = 24.89 ± 1.7%, 200 µg/ml = 43.83 ± 2.92%). Furthermore, [H] induced oxidative stress (ΦΔ = 0.5 ± 0.17) and generated TBARs (150 µg/ml = 1.81 ± 0.02 and 200 µg/ml = 2.1 ± 0.2) and DNA degradation. On the other hand, IC50 for L. major was 94.2 µg/ml. Chemical composition analysis indicated the presence of flavonoids, whereas TLC and HPLC revealed two major fractions, and GC–MS showed similarity with l-(+)-ascorbic acid 2,6-dihexadecanoate and 7,9-di-tertbutyl-1-oxaspiro (4,5) deca-6,9-diene-8-one. [H] of BS25 strain exhibits strong biological activity including ROS production, DNA damage and induction of apoptosis. Further investigations on normal cells and in vivo effects are required to fully understand its therapeutic potential.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

References

  1. Baig, N.F.; Dunham, S.J.B.; Morales-Soto, N.; et al.: HHS Public Access. Analyst 140, 6544–6552 (2016)

    Google Scholar 

  2. Drees, S.L.; Fetzner, S.: PqsE of Pseudomonas aeruginosa acts as pathway-specific thioesterase in the biosynthesis of alkylquinolone signaling molecules. Chem. Biol. 22, 611–618 (2015)

    Google Scholar 

  3. Barkal, L.J.; Theberge, A.B.; Guo, C.-J.; et al.: Microbial metabolomics in open microscale platforms. Nat. Commun. 7, 10610 (2016)

    Google Scholar 

  4. Singla, R.K.; Dubey, H.D.; Dubey, A.K.: Therapeutic spectrum of bacterial metabolites. Indo Glob. J. Pharm. 2, 52–64 (2014)

    Google Scholar 

  5. Isnansetyo, A.; Kamei, Y.: Bioactive substances produced by marine isolates of Pseudomonas. J. Ind. Microbiol. Biotechnol. 36, 1239–1248 (2009)

    Google Scholar 

  6. Osman, Y.A.; El-Deep, D.; Younis, S.A.: Azurin: a powerful anticancer from “A” local Pseudomonas aeruginosa isolate. J. Am. Sci. 9, 755–764 (2013)

    Google Scholar 

  7. Leite, G.G.F.; Figueirôa, J.V.; Almeida, T.C.M.; et al.: Production of rhamnolipids and diesel oil degradation by bacteria isolated from soil contaminated by petroleum. Biotechnol. Progr. 32, 262–270 (2016)

    Google Scholar 

  8. Compant, S.; Brader, G.; Muzammil, S.; et al.: Use of beneficial bacteria and their secondary metabolites to control grapevine pathogen diseases. Biocontrol 58, 1–21 (2013)

    Google Scholar 

  9. Buzid, A.; Muimhneacháin, E.Ó.; Reen, F.J.; et al.: Synthesis and electrochemical detection of a thiazolyl-indole natural product isolated from the nosocomial pathogen Pseudomonas aeruginosa. Anal. Bioanal. Chem. 408, 6361–6367 (2016)

    Google Scholar 

  10. Meisel, J.D.; Panda, O.; Mahanti, P.; et al.: Chemosensation of bacterial secondary metabolites modulates neuroendocrine signaling and behavior of C. elegans. Cell. 159, 267–280 (2014)

    Google Scholar 

  11. Spago, F.R.; Mauro, C.I.; Oliveira, A.G.; et al.: Pseudomonas aeruginosa produces secondary metabolites that have biological activity against plant pathogenic Xanthomonas species. Crop Prot. 62, 46–54 (2014)

    Google Scholar 

  12. Hernández-Padilla, L.; Vázquez-Rivera, D.; Sánchez-Briones, L.A.; et al.: The antiproliferative effect of cyclodipeptides from Pseudomonas aeruginosa PAO1 on HeLa cells involves inhibition of phosphorylation of Akt and S6k kinases. Molecules 22, 1024 (2017)

    Google Scholar 

  13. Moayedi, A.; Nowroozi, J.; Akhavan Sepahi, A.: Cytotoxic effect of pyocyanin on human pancreatic cancer cell line (Panc-1). Iran J. Basic Med. Sci. 2018(21), 794–799 (2018)

    Google Scholar 

  14. Fonseca, A.P.; Correia, P.; Sousa, J.C.; et al.: Association patterns of Pseudomonas aeruginosa clinical isolates as revealed by virulence traits, antibiotic resistance, serotype and genotype. FEMS Immunol. Med. Microbiol. 51, 505–516 (2007)

    Google Scholar 

  15. Gill, M.M.; Usman, J.; Kaleem, F.; et al.: Frequency and antibiogram of multi-drug resistant Pseudomonas aeruginosa. J. Coll. Phys. Surg. Pak. 21, 531–534 (2011)

    Google Scholar 

  16. Lister, P.D.; Wolter, D.J.; Hanson, N.D.: Antibacterial-resistant Pseudomonas aeruginosa: clinical impact and complex regulation of chromosomally encoded resistance mechanisms. Clin. Microbiol. Rev. 22, 582–610 (2009)

    Google Scholar 

  17. Vital-Lopez, F.G.; Reifman, J.; Wallqvist, A.: Biofilm formation mechanisms of Pseudomonas aeruginosa predicted via genome-scale kinetic models of bacterial metabolism. PLoS Comput. Biol. 11, 1–24 (2015)

    Google Scholar 

  18. Fujii, A.; Seki, M.; Higashiguchi, M.; et al.: Respiratory medicine case reports pneumonia caused by Pseudomonas aeruginosa. Respir. Med. Case Rep. 12, 30–33 (2014)

    Google Scholar 

  19. Seki, M.; Machida, N.; Yamagishi, Y.; et al.: Nosocomial outbreak of multidrug-resistant Pseudomonas aeruginosa caused by damaged transesophageal echocardiogram probe used in cardiovascular surgical operations. J. Infect. Chemother. 19, 677–681 (2013)

    Google Scholar 

  20. Quick, J.; Cumley, N.; Wearn, C.M.; et al.: Seeking the source of Pseudomonas aeruginosa infections in a recently opened hospital: an observational study using whole-genome sequencing. BMJ Open 4, e006278 (2014)

    Google Scholar 

  21. Shruti, G.; Tanuja, K.; Shweta, Y.: Exposure to ZnO-NPs enhanced gut-associated microbial activity in Eisenia fetida. J. Toxicol. Environ. Health Sci. 7, 9–17 (2015)

    Google Scholar 

  22. Mithun, S.L.V.; Rao, C.S.V.R.: Isolation and molecular characterization of anti-cancerous compound producing marine bacteria by using 16S rRNA sequencing and GC–MS techniques. IJMER 2, 4510–4515 (2012)

    Google Scholar 

  23. Chen, W.; Kuo, T.: A simple and rapid method for the preparation of gram-negative bacterial genomic DNA. Nucl. Acids Res. 21, 2260 (1993)

    Google Scholar 

  24. Null, A.P.; George, L.T.; Muddiman, D.C.: Evaluation of sample preparation techniques for mass measurements of PCR products using ESI-FT-ICR mass spectrometry. J. Am. Soc. Mass. Spectr. 13, 338–344 (2002)

    Google Scholar 

  25. Thomas, A.T.; Rao, J.V.; Subrahmanyam, V.M.; et al.: In vitro anticancer activity of microbial isolates from diverse habitats. Braz. J. Pharm. Sci. 47, 279–287 (2011)

    Google Scholar 

  26. Jafri, L.; Saleem, S.; Mirza, B.: In vitro assessment of antioxidant potential and determination of polyphenolic compounds of Hedera nepalensis K. Koch. Arab. J. Chem. 10, S3699–S3706 (2014)

    Google Scholar 

  27. Velioglu, Y.S.; Mazza, G.; Gao, L.; et al.: Antioxidant activity and total phenolics in selected fruits, vegetables, and grain products. J. Agric. Food Chem. 46, 4113–4117 (1998)

    Google Scholar 

  28. Wagner, H.; Bladt, S.; Zgainski, E.: Plant Drug Analysis, pp. 225–230. Springer, Berlin (1984)

    Google Scholar 

  29. Anantha, P.S.; Deventhiran, M.; Saravanan, P.; et al.: A comparative GC–MS analysis of bacterial secondary metabolites of Pseudomonas species. J. Pharm. Innov. 5, 84–89 (2016)

    Google Scholar 

  30. Arooj, S.; Nazir, S.; Nadhman, A.; et al.: Novel ZnO: Ag nanocomposites induce significant oxidative stress in human fibroblast malignant melanoma (Ht144) cells. Beilstein J. Nanotechnol. 6, 570–582 (2015)

    Google Scholar 

  31. Mahmoudvand, H.; Sharififar, F.; Rahmat, M.S.; et al.: Evaluation of antileishmanial activity and cytotoxicity of the extracts of Berberis vulgaris and Nigella sativa against Leishmania tropica. J. Vector Borne Dis. 51, 294–299 (2014)

    Google Scholar 

  32. Mascotti, K.; Mccullough, J.; Burger, S.R.: HPC viability measurement: trypan blue versus acridine orange and propidium iodide. Transfusion 40, 693–696 (2000)

    Google Scholar 

  33. Tada, D.B.; Vono, L.L.R.; Duarte, E.L.; et al.: Methylene blue-containing silica-coated magnetic particles: a potential magnetic carrier for photodynamic therapy. Langmuir 23, 8194–8199 (2007)

    Google Scholar 

  34. Bonacin, J.A.; Engelmann, F.M.; Severino, D.; et al.: Singlet oxygen quantum yields (Φd) in water using beetroot extract and an array of LEDs. J. Braz. Chem. Soc. 20, 31–36 (2009)

    Google Scholar 

  35. Ohkawa, H.; Ohishi, N.; Yagi, K.: Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95, 351–358 (1979)

    Google Scholar 

  36. Arslantas, A.; Devrim, A.K.; Necefoglu, H.: The interaction of sheep genomic DNA with a cobalt (ii) complex containing p-nitrobenzoate and n,n′-diethylnicotinamide ligands. Int. J. Mol. Sci. 8, 1225–1233 (2007)

    Google Scholar 

  37. Khan, S.Z.; Amir, M.K.; Abbasi, R.; et al.: New 3D and 2D supramolecular heteroleptic palladium(II) dithiocarbamates as potent anticancer agents. J. Coord. Chem. 69(20), 2999–3009 (2016)

    Google Scholar 

  38. d’Angelo, F.; Santillo, A.; Sevi, A.; Albenzio, M.: A simple salting-out method for DNA extraction from milk somatic cells: investigation into the goat CSN1S1 gene. JDS 90, 3550–3552 (2007)

    Google Scholar 

  39. Zohra, M.; Fawzia, A.: Hemolytic activity of different herbal extracts used in Algeria. IJPSR 5, 495–500 (2014)

    Google Scholar 

  40. Abbasi, R.; Efferth, T.; Kuhmann, C.; et al.: The endoperoxide ascaridol shows strong differential cytotoxicity in nucleotide excision repair-deficient cells. Toxicol. Appl. Pharmacol. 259, 302–310 (2012)

    Google Scholar 

  41. Chauhan, M.; Banerjee, K.; Arjmand, F.: DNA binding studies of novel copper(II) complexes containing l-Tryptophan as chiral auxiliary: in vitro antitumor activity of Cu–Sn2 complex in human neuroblastoma cells. Inorg. Chem. 46, 3072–3082 (2007)

    Google Scholar 

  42. McGaw, L.J.; Bagla, V.P.; Steenkamp, P.A.; et al.: Antifungal and antibacterial activity and chemical composition of polar and non-polar extracts of Athrixia phylicoides determined using bioautography and HPLC. BMC Complement. Altern. Med. 13, 356 (2013)

    Google Scholar 

  43. Brandhagen, B.N.; Tieszen, C.R.; Ulmer, T.M.; et al.: Cytostasis and morphological changes induced by mifepristone in human metastatic cancer cells involve cytoskeletal filamentous actin reorganization and impairment of cell adhesion dynamics. BMC Cancer 13, 35 (2013)

    Google Scholar 

  44. Du, K.; Ramachandran, A.; Jaeschke, H.: Oxidative stress during acetaminophen hepatotoxicity: sources, pathophysiological role and therapeutic potential. Redox Biol. 1, 148–156 (2016)

    Google Scholar 

  45. Kryston, T.B.; Georgiev, A.B.; Pissis, P.; et al.: Mutation research/fundamental and molecular mechanisms of mutagenesis role of oxidative stress and dna damage in human carcinogenesis. Mutat. Res. Fund. Mol. Mech. 711, 193–201 (2011)

    Google Scholar 

  46. Cherrak, S.A.; Mokhtari-Soulimane, N.; Berroukeche, F.; et al.: In vitro antioxidant versus metal ion chelating properties of flavonoids: a structure-activity investigation. PLoS ONE 11, e0165575 (2016)

    Google Scholar 

  47. Semwal, D.K.; Semwal, R.B.; Combrinck, S.; et al.: Myricetin: a dietary molecule with diverse biological activities. Nutrients 8, 1–31 (2016)

    Google Scholar 

  48. Ta, C.A.K.; Arnason, J.T.: Mini review of phytochemicals and plant taxa with activity as microbial biofilm and quorum sensing inhibitors. Molecules (Basel, Switzerland) 21, E29 (2015)

    Google Scholar 

  49. Gibellini, L.; Pinti, M.; Nasi, M.; et al.: Interfering with ROS metabolism in cancer cells: the potential role of quercetin. Cancers 2, 1288–1311 (2010)

    Google Scholar 

  50. Zhang, L.; Li, J.; Zong, L.; et al.: Reactive oxygen species and targeted therapy for pancreatic cancer. Oxid. Med. Cell. Longev. 2016, 1616781 (2016)

    Google Scholar 

Download references

Acknowledgements

The study was supported by the Higher Education Commission (HEC), Pakistan (Project ID: PMIPFP/HRD/HEC/2010/1815), and the Institute of Biomedical and Genetic Engineering, Islamabad, Pakistan. The authors wish to thank Prof. Dr. Z. K. Shinwari (Molecular Systematics and Applied Ethnobotany lab, Department of Biotechnology, Quaid-i-Azam University) for his advice and support, and N. Khan (Institute of Biomedical and Genetic Engineering, Islamabad), H. Shakeel (International Islamic University, Islamabad) and A. Anwar (PMAS-University of Arid Agriculture, Rawalpindi) for their excellent technical assistance.

Author information

Authors and Affiliations

  1. Institute of Biomedical and Genetic Engineering, G-9/1, Islamabad, Pakistan

    Sadaf Mushtaq, Abdul Hameed, Asma Umar Khayam, Samra Irum, Mohammad Ismail, Nafees Ahmad & Rashda Abbasi

  2. Department of Biotechnology, International Islamic University, Islamabad, Pakistan

    Sadaf Mushtaq, Bushra Uzair & Samra Irum

  3. Department of Biochemistry, Quaid-i-Azam University, Islamabad, Pakistan

    Asma Umar Khayam

  4. Department of Physics, Air University, Sector E-9, Islamabad, Pakistan

    Khuram Shahzad

  5. Faculty of Pharmacy, Gomal University, Dera Ismail Khan, Pakistan

    Barkat Ali Khan

Authors
  1. Sadaf Mushtaq
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bushra Uzair
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Abdul Hameed
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Asma Umar Khayam
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Samra Irum
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Khuram Shahzad
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Barkat Ali Khan
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Mohammad Ismail
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Nafees Ahmad
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Rashda Abbasi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sadaf Mushtaq.

Ethics declarations

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Mushtaq, S., Uzair, B., Hameed, A. et al. In Vitro Cytotoxicity of Secondary Metabolites Extracted from Pseudomonas aeruginosa BS25 Strain. Arab J Sci Eng 45, 81–94 (2020). https://doi.org/10.1007/s13369-019-04092-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13369-019-04092-2

Keywords

Search

Navigation