Evaluation of Chitosan and Zinc Oxide Nanoparticles Inhibitory Effects on Expression Levels of Virulence Genes at Pseudomonas aeruginosa Clinical Isolates and ATCC27853

Pourjafari, Mozhgan; Kaboosi, Hami; Ghane, Masood; Sadeghi , Babak; Rezaei, Abolhasan

doi:10.30699/ijmm.15.5.571

year 15, Issue 5 (September - October 2021) Iran J Med Microbiol 2021, 15(5): 571-583 | Back to browse issues page

‎ 10.30699/ijmm.15.5.571

Mendeley

Zotero

RefWorks

Pourjafari M, Kaboosi H, Ghane M, Sadeghi B, Rezaei A. Evaluation of Chitosan and Zinc Oxide Nanoparticles Inhibitory Effects on Expression Levels of Virulence Genes at Pseudomonas aeruginosa Clinical Isolates and ATCC27853. Iran J Med Microbiol 2021; 15 (5) :571-583
URL: http://ijmm.ir/article-1-1449-en.html

Evaluation of Chitosan and Zinc Oxide Nanoparticles Inhibitory Effects on Expression Levels of Virulence Genes at Pseudomonas aeruginosa Clinical Isolates and ATCC27853

Mozhgan Pourjafari¹

, Hami Kaboosi

², Masood Ghane³

, Babak Sadeghi⁴

, Abolhasan Rezaei⁵

1- Department of Microbiology, Ayatollah Amoli Branch, Islamic Azad University, Amol, Iran
2- Department of Microbiology, Ayatollah Amoli Branch, Islamic Azad University, Amol, Iran , hkaboosi@gmail.com
3- Department of Microbiology, Tonekabon Branch, Islamic Azad University, Tonekabon, Iran
4- Department of Chemistry, Tonekabon Branch, Islamic Azad University, Tonekabon, Iran
5- Department of Genetics, School of Basic Sciences, Tonekabon Branch, Islamic Azad University, Tonekabon, Iran

Abstract: (2473 Views)

Background and Objective: Quorum sensing (QS) and virulence genes in Pseudomonas aeruginosa causing severe infections in humans. In the present study, the effect of chitosan (CS) and zinc oxide nanoparticles (ZnO-NPs) on the expression level of lasI, exoS and toxA genes during lag, exponential-and stationary growth phases of P. aeruginosa were studied.
Materials and Methods: CS and ZnO-NPs were synthesized by ionic gelation and ultrasonic methods, respectively. The clinical samples were collected from patients suffering from wound infections from 2018-2019, and preliminary identification of P. aeruginosa has done with the standard biochemical tests and 16S rDNA gene. The virulence genes (lasI, exoS and toxA) were detected by polymerase chain reaction (PCR). Finally, real-time reverse transcription-PCR (qRT-PCR) was performed to detect the expression level of QS and virulence-related genes in samples grown at a medium contain minimum inhibitory concentrations (MICs) of CS and ZnO-NPs.
Results: CS and ZnO-NPs were successfully prepared with average particle sizes of 20 and 40 nm. The prevalence rate of virulence-related genes among all clinical isolates originated from burn patients was as follows; toxA (100%), exoS (88%), and lasI (56%). The MIC value of CS was equal to 0.5 mg/mL, which is 8-fold lower than that for ZnO-NPs (4 mg/mL). qRT-PCR analysis revealed CS and ZnO-NPs decrease the expression of lasI, exoS, and toxA in clinical P. aeruginosa isolates during the exponential phase compared to other growth phases. toxA⁺/exoS⁺/lasI⁺ isolates exhibited similar patterns of gene expression changes in response to both NPs at all growth phases. Expression of toxA gene downregulated at exponential-and stationary growth phases of ATCC27853 treated with CS and ZnO-NPs.
Conclusion: The results obtained from this study may provide a better understanding of the expression changes of virulence-related genes throughout different growth phases of P. aeruginosa.

Keywords: Chitosan, P. aeruginosa, Quorum sensing, Zinc oxide nanoparticles

Full-Text [PDF 1381 kb] (1253 Downloads) | | Full-Text (HTML) (1321 Views)

Type of Study: Original Research Article | Subject: Microbial Biotechnology
Received: 2021/06/25 | Accepted: 2021/08/10 | ePublished: 2021/09/5

References

1. Diggle SP, Whiteley M. Microbe Profile: Pseudomonas aeruginosa: opportunistic pathogen and lab rat. Microbiology (Reading, England). 2020;166(1):30-3. [DOI:10.1099/mic.0.000860] [PMID] [PMCID]

2. Wu M, Li X. Klebsiella pneumoniae and Pseudomonas aeruginosa. Molecular medical microbiology: Elsevier; 2015. p. 1547-64. [DOI:10.1016/B978-0-12-397169-2.00087-1]

3. LaBauve AE, Wargo MJ. Growth and laboratory maintenance of Pseudomonas aeruginosa. Current protocols in microbiology. 2012;Chapter 6:Unit 6E.1. [DOI:10.1002/9780471729259.mc06e01s25] [PMID] [PMCID]

4. Yousefi-Avarvand A, Khashei R, Sedigh Ebrahim-Saraie H, Emami A, Zomorodian K, Motamedifar M. The Frequency of Exotoxin A and Exoenzymes S and U Genes Among Clinical Isolates of Pseudomonas aeruginosa in Shiraz, Iran. International journal of molecular and cellular medicine. 2015;4(3):167-73.

5. Kroken AR, Chen CK, Evans DJ, Yahr TL, Fleiszig SMJ. The Impact of ExoS on Pseudomonas aeruginosa Internalization by Epithelial Cells Is Independent of fleQ and Correlates with Bistability of Type Three Secretion System Gene Expression. mBio. 2018;9(3). [DOI:10.1128/mBio.00668-18] [PMID] [PMCID]

6. Gellatly SL, Hancock RE. Pseudomonas aeruginosa: new insights into pathogenesis and host defenses. Pathogens and disease. 2013;67(3):159-73. [DOI:10.1111/2049-632X.12033] [PMID]

7. Shaver CM, Hauser AR. Relative contributions of Pseudomonas aeruginosa ExoU, ExoS, and ExoT to virulence in the lung. Infect Immun. 2004;72(12):6969-77. [DOI:10.1128/IAI.72.12.6969-6977.2004] [PMID] [PMCID]

8. Karthikeyan RS, Priya JL, Leal SM, Jr., Toska J, Rietsch A, Prajna V, et al. Host response and bacterial virulence factor expression in Pseudomonas aeruginosa and Streptococcus pneumoniae corneal ulcers. PLoS One. 2013;8(6):e64867. [DOI:10.1371/journal.pone.0064867] [PMID] [PMCID]

9. Subedi D, Vijay AK, Kohli GS, Rice SA, Willcox M. Association between possession of ExoU and antibiotic resistance in Pseudomonas aeruginosa. PLoS One. 2018;13(9):e0204936. [DOI:10.1371/journal.pone.0204936] [PMID] [PMCID]

10. Amirmozafari N, Fallah Mehrabadi J, Habibi A. Association of the Exotoxin A and Exoenzyme S with Antimicrobial Resistance in Pseudomonas Aeruginosa Strains. Arch Iran Med. 2016;19(5):353-8.

11. Rocha AJ, Barsottini MRdO, Rocha RR, Laurindo MV, Moraes FLLd, Rocha SLd. Pseudomonas aeruginosa: virulence factors and antibiotic resistance genes. Brazilian Archives of Biology and Technology. 2019;62. [DOI:10.1590/1678-4324-2019180503]

12. Younes N, Pintus G, Al-Asmakh M, Rasool K, Younes S, Calzolari S, et al. "Safe" Chitosan/Zinc Oxide Nanocomposite Has Minimal Organ-Specific Toxicity in Early Stages of Zebrafish Development. ACS biomaterials science & engineering. 2020;6(1):38-47. [DOI:10.1021/acsbiomaterials.8b01144] [PMID]

13. García-Lara B, Saucedo-Mora M, Roldán-Sánchez JA, Pérez-Eretza B, Ramasamy M, Lee J, et al. Inhibition of quorum-sensing-dependent virulence factors and biofilm formation of clinical and environmental Pseudomonas aeruginosa strains by ZnO nanoparticles. Letters in applied microbiology. 2015;61(3):299-305. [DOI:10.1111/lam.12456] [PMID]

14. Lee JH, Kim YG, Cho MH, Lee J. ZnO nanoparticles inhibit Pseudomonas aeruginosa biofilm formation and virulence factor production. Microbiological research. 2014;169(12):888-96. [DOI:10.1016/j.micres.2014.05.005] [PMID]

15. Igawa G, Casey M, Sawabe E, Nukui Y, Okugawa S, Moriya K, et al. Comparison of agar dilution and broth microdilution methods for Clostridium difficile antimicrobial susceptibility testing. Journal of global antimicrobial resistance. 2016;7:43-5. [DOI:10.1016/j.jgar.2016.07.007] [PMID]

16. Spilker T, Coenye T, Vandamme P, LiPuma JJ. PCR-based assay for differentiation of Pseudomonas aeruginosa from other Pseudomonas species recovered from cystic fibrosis patients. Journal of clinical microbiology. 2004;42(5):2074-9. [DOI:10.1128/JCM.42.5.2074-2079.2004] [PMID] [PMCID]

17. Wargo MJ, Hogan DA. Examination of Pseudomonas aeruginosa lasI regulation and 3-oxo-C12-homoserine lactone production using a heterologous Escherichia coli system. FEMS microbiology letters. 2007;273(1):38-44. [DOI:10.1111/j.1574-6968.2007.00773.x] [PMID]

18. Macin S, Akyon Y. Phenotypic and genotypic virulence factors in Pseudomonas aeruginosa strains according to pigment presence. ACTA MEDICA MEDITERRANEA. 2017;33(6):1033-8.

19. Amirmozafari N, Fallah Mehrabadi J, Isazadieh K, Habibi A. Molecular analysis of exotoxin A associated with antimicrobial resistance of Pseudomonas aeruginosa strains isolated from patients in Tehran hospitals. Iranian Journal of Medical Microbiology. 2015;8(4):36-43.

20. Calvo P, Remunan‐Lopez C, Vila‐Jato JL, Alonso M. Novel hydrophilic chitosan‐polyethylene oxide nanoparticles as protein carriers. Journal of applied polymer science. 1997;63(1):125-32. https://doi.org/10.1002/(SICI)1097-4628(19970103)63:1<125::AID-APP13>3.0.CO;2-4 [DOI:10.1002/(SICI)1097-4628(19970103)63:13.0.CO;2-4]

21. Haase M, Weller H, Henglein A. Photochemistry and radiation chemistry of colloidal semiconductors. 23. Electron storage on zinc oxide particles and size quantization. The Journal of Physical Chemistry. 1988;92(2):482-7. [DOI:10.1021/j100313a047]

22. Parvekar P, Palaskar J, Metgud S, Maria R, Dutta S. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of silver nanoparticles against Staphylococcus aureus. Biomaterial investigations in dentistry. 2020;7(1):105-9. [DOI:10.1080/26415275.2020.1796674] [PMID] [PMCID]

23. Luo J, Kong JL, Dong BY, Huang H, Wang K, Wu LH, et al. Baicalein attenuates the quorum sensing-controlled virulence factors of Pseudomonas aeruginosa and relieves the inflammatory response in P. aeruginosa-infected macrophages by downregulating the MAPK and NFκB signal-transduction pathways. Drug design, development and therapy. 2016;10:183-203. [DOI:10.2147/DDDT.S97221] [PMID] [PMCID]

24. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402-8. [DOI:10.1006/meth.2001.1262] [PMID]

25. Janjua HA, Segata N, Bernabò P, Tamburini S, Ellen A, Jousson O. Clinical populations of Pseudomonas aeruginosa isolated from acute infections show a wide virulence range partially correlated with population structure and virulence gene expression. Microbiology (Reading, England). 2012;158(Pt 8):2089-98. [DOI:10.1099/mic.0.056689-0] [PMID]

26. Kumar S, Koh J. Physiochemical, optical and biological activity of chitosan-chromone derivative for biomedical applications. Int J Mol Sci. 2012;13(5):6102-16. [DOI:10.3390/ijms13056102] [PMID] [PMCID]

27. Trung TS, Thein-Han WW, Qui NT, Ng CH, Stevens WF. Functional characteristics of shrimp chitosan and its membranes as affected by the degree of deacetylation. Bioresource technology. 2006;97(4):659-63. [DOI:10.1016/j.biortech.2005.03.023] [PMID]

28. Julkapli NM, Ahmad Z, Akil HM, editors. X‐Ray Diffraction Studies of Cross Linked Chitosan With Different Cross Linking Agents For Waste Water Treatment Application. AIP Conference Proceedings; 2010: American Institute of Physics. [DOI:10.1063/1.3295578]

29. Soleimani H, Baig MK, Yahya N, Khodapanah L, Sabet M, Demiral BM, et al. Synthesis of ZnO nanoparticles for oil-water interfacial tension reduction in enhanced oil recovery. Applied Physics A. 2018;124(2):1-13. [DOI:10.1007/s00339-017-1510-4]

30. Lustriane C, Dwivany FM, Suendo V, Reza M. Effect of chitosan and chitosan-nanoparticles on post harvest quality of banana fruits. Journal of Plant Biotechnology. 2018;45(1):36-44. [DOI:10.5010/JPB.2018.45.1.036]

31. Rosyada A, Sunarharum W, Waziiroh E, editors. Characterization of chitosan nanoparticles as an edible coating material. IOP Conference Series: Earth and Environmental Science; 2019: IOP Publishing. [DOI:10.1088/1755-1315/230/1/012043]

32. Rajendran SP, Sengodan K. Synthesis and characterization of zinc oxide and iron oxide nanoparticles using Sesbania grandiflora leaf extract as reducing agent. Journal of Nanoscience. 2017;2017. [DOI:10.1155/2017/8348507]

33. Mallakpour S, Madani M. Use of silane coupling agent for surface modification of zinc oxide as inorganic filler and preparation of poly (amide-imide)/zinc oxide nanocomposite containing phenylalanine moieties. Bulletin of Materials Science. 2012;35(3):333-9. [DOI:10.1007/s12034-012-0304-8]

34. Handore K, Bhavsar S, Horne A, Chhattise P, Mohite K, Ambekar J, et al. Novel green route of synthesis of ZnO nanoparticles by using natural biodegradable polymer and its application as a catalyst for oxidation of aldehydes. Journal of Macromolecular Science, Part A. 2014;51(12):941-7. [DOI:10.1080/10601325.2014.967078]

35. Nagaraju G, Prashanth S, Shastri M, Yathish K, Anupama C, Rangappa D. Electrochemical heavy metal detection, photocatalytic, photoluminescence, biodiesel production and antibacterial activities of Ag-ZnO nanomaterial. Materials Research Bulletin. 2017;94:54-63. [DOI:10.1016/j.materresbull.2017.05.043]

36. Badawy M, Riad OKM, Taher FA, Zaki SA. Chitosan and chitosan-zinc oxide nanocomposite inhibit expression of LasI and RhlI genes and quorum sensing dependent virulence factors of Pseudomonas aeruginosa. International journal of biological macromolecules. 2020;149:1109-17. [DOI:10.1016/j.ijbiomac.2020.02.019] [PMID]

37. Hosseininassab Nodoushan SA, Yadegari S, Moghim S, Isfahani BN, Fazeli H, Poursina F, et al. Distribution of the Strains of Multidrug-resistant, Extensively Drug-resistant, and Pandrug-resistant Pseudomonas aeruginosa Isolates from Burn Patients. Adv Biomed Res. 2017;6:74. [DOI:10.4103/abr.abr_239_16] [PMID] [PMCID]

38. Nikbin VS, Aslani MM, Sharafi Z, Hashemipour M, Shahcheraghi F, Ebrahimipour GH. Molecular identification and detection of virulence genes among Pseudomonas aeruginosa isolated from different infectious origins. Iranian journal of microbiology. 2012;4(3):118-23.

39. Lee NY, Ko WC, Hsueh PR. Nanoparticles in the Treatment of Infections Caused by Multidrug-Resistant Organisms. Front Pharmacol. 2019;10:1153. [DOI:10.3389/fphar.2019.01153] [PMID] [PMCID]

40. Sirelkhatim A, Mahmud S, Seeni A, Kaus NHM, Ann LC, Bakhori SKM, et al. Review on Zinc Oxide Nanoparticles: Antibacterial Activity and Toxicity Mechanism. Nano-micro letters. 2015;7(3):219-42. [DOI:10.1007/s40820-015-0040-x] [PMID] [PMCID]

41. Qi L, Xu Z, Jiang X, Hu C, Zou X. Preparation and antibacterial activity of chitosan nanoparticles. Carbohydrate research. 2004;339(16):2693-700. [DOI:10.1016/j.carres.2004.09.007] [PMID]

42. Ali SG, Ansari MA, Alzohairy MA, Alomary MN, Jalal M, AlYahya S, et al. Effect of Biosynthesized ZnO Nanoparticles on Multi-Drug Resistant Pseudomonas Aeruginosa. Antibiotics (Basel, Switzerland). 2020;9(5). [DOI:10.3390/antibiotics9050260] [PMID] [PMCID]

43. Bayroodi E, Jalal R. Modulation of antibiotic resistance in Pseudomonas aeruginosa by ZnO nanoparticles. Iranian journal of microbiology. 2016;8(2):85-92.

44. Al-Shabib NA, Husain FM, Ahmed F, Khan RA, Ahmad I, Alsharaeh E, et al. Biogenic synthesis of Zinc oxide nanostructures from Nigella sativa seed: Prospective role as food packaging material inhibiting broad-spectrum quorum sensing and biofilm. Sci Rep. 2016;6:36761. [DOI:10.1038/srep36761] [PMID] [PMCID]

45. Cremonini E, Zonaro E, Donini M, Lampis S, Boaretti M, Dusi S, et al. Biogenic selenium nanoparticles: characterization, antimicrobial activity and effects on human dendritic cells and fibroblasts. Microbial biotechnology. 2016;9(6):758-71. [DOI:10.1111/1751-7915.12374] [PMID] [PMCID]

46. Souza RCd, Haberbeck LU, Riella HG, Ribeiro DH, Carciofi BA. Antibacterial activity of zinc oxide nanoparticles synthesized by solochemical process. Brazilian Journal of Chemical Engineering. 2019;36:885-93. [DOI:10.1590/0104-6632.20190362s20180027]

47. Saleh MM, Sadeq RA, Latif HKA, Abbas HA, Askoura M. Zinc oxide nanoparticles inhibits quorum sensing and virulence in Pseudomonas aeruginosa. Afr Health Sci. 2019;19(2):2043-55. [DOI:10.4314/ahs.v19i2.28] [PMID] [PMCID]

48. Prateeksha, Rao CV, Das AK, Barik SK, Singh BN. ZnO/Curcumin Nanocomposites for Enhanced Inhibition of Pseudomonas aeruginosa Virulence via LasR-RhlR Quorum Sensing Systems. Molecular pharmaceutics. 2019;16(8):3399-413. [DOI:10.1021/acs.molpharmaceut.9b00179] [PMID]

49. Singh BR, Singh BN, Singh A, Khan W, Naqvi AH, Singh HB. Mycofabricated biosilver nanoparticles interrupt Pseudomonas aeruginosa quorum sensing systems. Sci Rep. 2015;5:13719. [DOI:10.1038/srep13719] [PMID] [PMCID]