Inhibitory effect of zinc oxide nanoparticles on pseudomonas aeruginosa biofilm formation

Document Type: Research Paper


1 Department of Biology, Damghan Branch, Islamic Azad University, Damghan, Iran

2 Department of Biology, Mashhad Branch, Islamic Azad University, Mashhad, Iran



Bacterial biofilm formation causes many persistent and chronic infections. The matrix protects biofilm bacteria from exposure to innate immune defenses and antibiotic treatments. The purpose of this study was to evaluate the biofilm formation of clinical isolates of Pseudomonas aeruginosa and the activity of zinc oxide nanoparticles (ZnO NPs) on biofilm.
Materials and Methods:
After collecting bacteria from clinical samples of hospitalized patients, the ability of organisms were evaluated to create biofilm by tissue culture plate (TCP) assay. ZnO NPs were synthesized by sol gel method and the efficacy of different concentrations (50- 350 µg/ml) of ZnO NPs was assessed on biofilm formation and also elimination of pre-formed biofilm by using TCP method.
The average diameter of synthesized ZnO NPs was 20 nm. The minimum inhibitory concentration of nanoparticles was 150- 158 μg/ml and the minimum bactericidal concentration was higher (325 µg/ml). All 15 clinical isolates of P. aeruginosa were able to produce biofilm. Treating the organisms with nanoparticles at concentrations of 350 μg/ml resulted in more than 94% inhibition in OD reduction%. Molecular analysis showed that the presence of mRNA of pslA gene after treating bacteria with ZnO NPs for 30 minutes.
The results showed that ZnO NPs can inhibit the establishment of P. aeruginosa biofilms and have less effective in removing pre-formed biofilm. However the tested nanoparticles exhibited anti-biofilm effect, but mRNA of pslA gene could be still detected in the medium by RT-PCR technique after 30 minutes treatment with ZnO.


1. Pileni MP. Self-assemblies of nanocrystals: fabrication and collective properties. In: Feldheim DL, Foss CA Jr (eds). Metal nanoparticles: synthesis, characterization, and applications. New York. Marcel Dekker. 2002.   

2. Shuxia Liu, Junhui He, Jianfeng Xue. Wenjun Ding. Efficient fabrication of transparent antimicrobial poly (vinyl alcohol) thin films. J Nanopart Res. 2009; 11: 553–560.

3. Toshima N, Yonezawa T. Bimetallic nanoparticlesnovel materials for chemical and physical applications. New J Chem. 1998; 22: 1179–1201.

4. Pati R, Mehta RK, Mohanty S, Padhi A, Sengupta M, Vaseeharan B, et al. Topical application of zinc oxide nanoparticles reduces bacterial skin infection in mice and exhibits antibacterial activity by inducing oxidative stress response and cell membrane disintegration in macrophages. Nanomedicine. 2014; 10(6): 1195-208.

5. Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: A common cause of persistent infections. Science. 1999; 284: 1318–1322.

6. Izano E, Amarante M, Kher W, Kaplan J. Differential roles of poly-N-acetylglucosamine surface polysaccharide and extracellular DNA in Staphylococcus aureus and Staphylococcus epidermidis biofilms. Appl Environ Microbiol. 2008; 74: 470–476.

7. Otto M. Staphylococcus epidermidis—the accidental pathogen. Nat Rev Microbiol. 2009; 7: 555–567.

8. Cerca N, Jefferson KK, Oliveira R, Pier GB, Azeredo J. Comparative antibody- mediated phagocytosis of Staphylococcus epidermidis cells grown in a biofilm or in the planktonic  state. Infect Immun. 2006; 74: 4849–4855.

9. Vuong C, Voyich JM, Fischer ER, Braughton KR, Whitney AR, DeLeo FR, et al. Polysaccharide intercellular adhesin (PIA) protects Staphylococcus epidermidis against major components of the human innate immune system. Cell Microbiol. 2004; 6: 269–275.

10. Govan JR, Deretic V. Microbial pathogenesis in cystic fibrosis: Mucoid P. aeruginosa and Burkholderia cepacia. Microbiol Rev. 1996; 60: 539-574.

11. Foxman B. The epidemiology of urinary tract infection. Nat Rev Urol. 2010; 7: 653–660.

12. Kuramitsu HK,Wang BY. The whole is greater than the sum of its parts: Dental plaque bacterial interactions can affect the virulence properties of cariogenic Streptococcus mutans. Am J Dent. 2011; 24: 153–154.

13. Fey PD. Modality of bacterial growth presents unique targets: How do we treat biofilm-mediated infections? Curr Opin Microbiol. 2010; 13: 610–615.

14. Kostakioti M, Hadjifrangiskou M, Hultgren SJ. Bacterial biofilms: development, dispersal, and therapeutic strategies in the dawn of the post-antibiotic era. Cold Spring Harb Perspect Med. 2013; 3(4): a010306.

15. Gellatly SL, Hancock RE. Pseudomonas aeruginosa: new insights into pathogenesis and host defenses. Pathog Dis. 2013; 67(3): 159-73.

16. Krieg, Noel. Bergey's Manual of Systematic Bacteriology. Volume 1. Baltimore: Williams & Wilkins. 1984.

17. Presterl E, Suchomel M, Eder M, Reichmann S, Lassnigg A, Graninger W, et al. Effects of alcohols, povidone-iodine and hydrogen peroxide on biofilms of Staphylococcus epidermidis. J Antimicrob Chemother. 2007; 60: 417–420.

18. Christensen GD, Simpson WA, Younger JJ, Baddour LM, Barrett FF, Melton DM, et al. Adherence of coagulase-negative taphylococci to plastic tissue culture plates: a quantitative model for the adherence of staphylococci to medical devices. Microbiol. 1985; 22: 996–1006.

19. Yanping X, Yiping H, Peter LI, Tony J, Xianming S. Antibacterial activityand mechanism of action of zinc oxide nanoparticles against Campylobacter jejuni. Appl Environ Microbiol. 2011; 77: 2325- 2331.

20. Wei Q, Ma LZ. Biofilm matrix and its regulation in Pseudomonas aeruginosa. Int J Mol Sci. 2013; 14: 20983-21005.

21. Saadat M, Roudbar Mohammadi S, Yadegari M, Eskandari M, Khavari-nejad R. An assessment of antibacterial activity of ZnO nanoparticles, Catechin, and EDTA on standard strain of pseudomonas aeruginosa. JJUMS. 2012; 10: 13-19.

22. Hoseinzadeh E, Samargandi M, Alikhani M, Roshanaei G, Asgari G. Antimicrobial efficacy of zinc oxide nanoparticles suspension against Gram negative and Gram positive bacteria. IJHE. 2012; 5: 331-342.

23. Lee JH, Kim YG, Cho MH, Lee J. ZnO nanoparticles inhibit Pseudomonas aeruginosa biofilm formation and virulence factor production. Microbiol Res. 2014; [in press].

24. Applerot G, Lellouche J, Perkas N, Nitzan Y, Gedanken A, Banin E. ZnO nanoparticle-coated surfaces inhibit bacterial biofilm formation and increase antibiotic susceptibility. RSC Adv. 2012; 2: 2314-2321.

25. Khameneh B, Zarei H, Fazly Bazzaz BS. The effect of silver nanoparticles on Staphylococcus epidermidis biofilm biomass and cell viability. Nanomed J. 2014; 1: 302-307