Environmental Science and Pollution Research

, Volume 25, Issue 34, pp 34765–34776 | Cite as

Mechanistic understanding of cerium oxide nanoparticle-mediated biofilm formation in Pseudomonas aeruginosa

  • Yi Xu
  • Chao Wang
  • Jun HouEmail author
  • Peifang Wang
  • Guoxiang You
  • Lingzhan Miao
Research Article


In this study, the biofilm formation of Pseudomonas aeruginosa in the presence of cerium oxide nanoparticles (CeO2 NPs) was investigated. With the addition of 0.1 mg/L and 1 mg/L CeO2 NPs, the biofilm development was substantially enhanced. During the attachment process, the enhanced surface hydrophobicity and excess production of mannosan and rhamnolipids in CeO2 NP treatments were detected, which were conductive to the colonization of bacterial cells. During the maturation period, the biofilm biomass was accelerated by the improved aggregation percentage as well as the secretion of extracellular DNA and pyocyanin. The reactive oxygen species (ROS) generated by CeO2 NPs were found to activate the N-butyryl homoserine lactone (C4-HSL) and quinolone signals secreted by Pseudomonas aeruginosa. Moreover, the quorum sensing (QS) systems of rhl and pqs were initiated, reflected by the stimulated expression levels of biofilm formation-related genes rhlI-rhlR, rhlAB, and pqsR-pqsA. The addition of a quorum quencher, furanone C-30, significantly declined the activities of QS-controlled catalase and superoxide dismutase. A dose of antioxidant, ascorbic acid, effectively relieved the accelerating effects of NPs on biofilm formation. These results indicated that CeO2 NPs could accelerate biofilm formation through the interference of QS system by generating ROS, which provides possible targets for controlling biofilm growth in the NP exposure environments.


CeO2 nanoparticles Biofilm formation Oxidative stress Quorum sensing Virulence factors Polysaccharide 


Funding information

We are grateful for the grants for project supported by the National Natural Science Funds for Excellent Young Scholar (No. 51722902); the National Science Funds for Creative Research Groups of China (No. 51421006); the Fundamental Research Funds for the Central Universities (2018B671X14); the Key Program of National Natural Science Foundation of China (No. 91647206); the Outstanding Youth Fund of Natural Science Foundation of Jiangsu, China (BK20160038); and the Postgraduate Research and Practice Innovation Program of Jiangsu Province (KYCX18_0636) and PAPD.

Supplementary material

11356_2018_3418_MOESM1_ESM.docx (2.1 mb)
ESM 1 (DOCX 2158 kb)


  1. Arocho A, Chen B, Ladanyi M (2006) Validation of the 2-ΔΔCt calculation as an alternate method of data analysis for quantitative PCR of BCR-ABL P210 transcripts. Diagn Mol Pathol 1:56–61CrossRefGoogle Scholar
  2. Barken KB, Pamp SJ, Yang L, Gjermansen M, Bertrand JJ, Klausen M, Givskov M, Whitchurch CB, Engel JN, Tolker NT (2008) Roles of type IV pili, flagellum-mediated motility and extracellular DNA in the formation of mature multicellular structures in Pseudomonas aeruginosa biofilms. Environ Microbiol 10:2331–2343CrossRefGoogle Scholar
  3. Barton LE, Auffan M, Bertrand M, Barakat M, Santaella C, Masion A, Borschneck D, Olivi L, Roche N, Wiesner MR, Bottero JY (2014) Transformation of pristine and citrate-functionalized CeO2 nanoparticles in a laboratory-scale activated sludge reactor. Environ Sci Technol 48(13):7289–7296CrossRefGoogle Scholar
  4. Bridier A, Briandet R, Thomas V, Dubois-Brissonnet F (2011) Resistance of bacterial biofilms to disinfectants: a review. Biofouling 27(9):1017–1032CrossRefGoogle Scholar
  5. Camara M, Daykin M, Chhabra SR (1998) Detection, purification, and synthesis of N-acylhomoserine lactone quorum sensing signal molecules. Methods Microbiol 27:319–330CrossRefGoogle Scholar
  6. Cáp M, Váchová L, Palková Z (2012) Reactive oxygen species in the signaling and adaptation of multicellular microbial communities. Oxidative Med Cell Longev 11:976753Google Scholar
  7. Cappitelli F, Principi P, Sorlini C (2006) Biodeterioration of modern materials in contemporary collections: can biotechnology help? Trends Biotechnol 24(8):350–354CrossRefGoogle Scholar
  8. Cassee FR, van Balen EC, Singh C, Green D, Muijser H, Weinstein J, Dreher K (2011) Exposure, health and ecological effects review of engineered nanoscale cerium and cerium oxide associated with its use as a fuel additive. Crit Rev Toxicol 41:213–229CrossRefGoogle Scholar
  9. Castillo-Juarez I, Garcia-Contreras R, VelazquezGuadarrarma N, Soto-Hernandez M, MartinezVazquez M (2013) Amphypterygium adstringens anacardic acid mixture inhibits quorum sensing-controlled virulence factors of Chromobacteriurn violaceum and Pseudomonas aeruginosa. Arch Med Res 44:488–494CrossRefGoogle Scholar
  10. Cerrillo C, Barandika G, Igartua A, Areitioaurtena O, Mendoza G (2016) Towards the standardization of nanoecotoxicity testing: natural organic matter ‘camouflages’ the adverse effects of TiO2 and CeO2 nanoparticles on green microalgae. Sci Total Environ 543:95–104CrossRefGoogle Scholar
  11. Chen X, Schauder S, Potier N, Van Dorsselaer A, Pelczer I, Bassler BL, Hughson FM (2002) Structural identification of a bacterial quorum-sensing signal containing boron. Nature 415(6871):545–549CrossRefGoogle Scholar
  12. Davies DG, Parsek MR, Pearson JP, Iglewsji BH, Costerton JW, Greenberg EP (1998) The involvement of cell-to-cell signals in the development of a bacterial biofilm. Science 280:295–298CrossRefGoogle Scholar
  13. Drenkard E, Ausubel FM (2002) Pseudomonas biofilm formation and antibiotic resistance are linked to phenotypic variation. Nature 416(6882):740–743CrossRefGoogle Scholar
  14. Drumm B, Neumann AW, Policova Z, Sherman PM (1989) Bacterial cell surface hydrophobicity properties in the mediation of in vitro adhesion by the rabbit enteric pathogen Escherichia coli strain RDEC-1. J Clin Invest 84:1588–1594CrossRefGoogle Scholar
  15. Essar DW, Eberly L, Hadero A, Crawford IP (1990) Identification and characterization of genes for a second anthranilate synthase in Pseudomonas aeruginosa: interchangeability of the two anthranilate synthases and evolutionary implications. J Bacteriol 172:884–900CrossRefGoogle Scholar
  16. Fang XH, Yu R, Li BQ, Somasundaran P, Chandran K (2010) Stresses exerted by ZnO, CeO2 and anatase TiO2 nanoparticles on the Nitrosomonas europaea. J Colloid Interface Sci 348:329–334CrossRefGoogle Scholar
  17. Frey R, He L, Cui Y, Decho A, Kawaguchi T, Ferguson P, Ferry J (2010) Reaction of N-acylhomoserine lactones with hydroxyl radicals: rates, products, and effects on signaling activity. Environ Sci Technol 44:7465–7469CrossRefGoogle Scholar
  18. Friedman L, Kolter R (2004) Two genetic loci produce distinct carbohydrate-rich structural components of the Pseudomonas aeruginosa biofilm matrix. J Bacteriol 186:4457–4465CrossRefGoogle Scholar
  19. Fuqua WC, Winans SC, Greenberg EP (1994) Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators. J Bacteriol 176:269–275CrossRefGoogle Scholar
  20. Gagliano MC, Ismail SB, Stams AJM, Plugge CM, Temmink H, Van Lier JB (2017) Biofilm formation and granule properties in anaerobic digestion at high salinity. Water Res 121:61–71CrossRefGoogle Scholar
  21. García-Lara B, Saucedo-Mora MÁ, Roldán-Sánchez JA, Pérez-Eretza B, Ramasamy M, Lee J, Coria-Jimenze R, Tapia M, Varela-Guerrero V, García-Contreras R (2015) Inhibition of quorum-sensing-dependent virulence factors and biofilm formation of clinical and environmental Pseudomonas aeruginosa strains by ZnO nanoparticles. Lett Appl Microbiol 61(3):299–305CrossRefGoogle Scholar
  22. Golowczyc MA, Mobili P, Garrote GL, de los Angeles SM, Abraham AG, De Antoni GL (2009) Interaction between Lactobacillus kefir and Saccharomyces lipolytica isolated from kefir grains: evidence for lectin-like activity of bacterial surface proteins. J Dairy Res 76:111–116CrossRefGoogle Scholar
  23. Hassett DJ, Ma JF, Elkins JG, McDermott TR, Ochsner UA, West SE, Huang CT, Fredericks J, Burnett S, Stewart PS, Mcfeters G, Passador L, Iglewski BH (1999) Quorum sensing in Pseudomonas aeruginosa controls expression of catalase and superoxide dismutase genes and mediates biofilm susceptibility to hydrogen peroxide. Mol Microbiol 34(5):1082–1093CrossRefGoogle Scholar
  24. Henkel M, Schmidberger A, Kühnert C, Beuker J, Bernard T, Schwartz T, Syldatk C, Hausmann R (2013) Kinetic modeling of the time course of N-butyryl-homoserine lactone concentration during batch cultivations of Pseudomonas aeruginosa PAO1. Appl Microbiol Biotechnol 97(17):7607–7616CrossRefGoogle Scholar
  25. Ibáñez de Aldecoa AL, Zafra O, González-Pastor JE (2017) Mechanisms and regulation of extracellular DNA release and its biological roles in microbial communities. Front Microbiol 8:1390CrossRefGoogle Scholar
  26. Izano EA, Amarante MA, Kher WB, Kaplan JB (2008) Differential roles of poly-N-acetylglucosamine surface polysaccharide and extracellular DNA in Staphylococcus aureus and Staphylococcus epidermidis biofilms. Appl Environ Microbiol 74(2):470–476CrossRefGoogle Scholar
  27. Jayaraman A, Wood TK (2008) Bacterial quorum sensing: signals, circuits, and implications for biofilms and disease. Annu Rev Biomed Eng 10:145–167CrossRefGoogle Scholar
  28. Johnson LR (2008) Microcolony and biofilm formation as a survival strategy for bacteria. J Theor Biol 251:24–34CrossRefGoogle Scholar
  29. Keller L, Surette MG (2006) Communication in bacteria: an ecological and evolutionary perspective. Nat Rev Microbiol 4(4):249–258CrossRefGoogle Scholar
  30. Keller AA, McFerran S, Lazareva A, Suh S (2013) Global life cycle releases of engineered nanomaterials. J Nanopart Res 15:1–17CrossRefGoogle Scholar
  31. Klausen M, Aaes-Jorgensen A, Molin S, Tolker-Nielsen T (2003) Involvement of bacterial migration in the development of complex multicellular structures in Pseudomonas aeruginosa biofilms. Mol Microbiol 50:61–68CrossRefGoogle Scholar
  32. Kohler T, Curty LK, Barja F, van Delden C, Pechere JC (2000) Swarming of Pseudomonas aeruginosa is dependent on cell-to-cell signaling and requires flagella and pili. J Bacteriol 182:5990–5996CrossRefGoogle Scholar
  33. Lazareva A, Keller AA (2014) Estimating potential life cycle releases of engineered nanomaterials from wastewater treatment plants. ACS Sustain Chem Eng 2(7):1656–1665CrossRefGoogle Scholar
  34. Lipke PN, Klotz SA, Dufrene YF, Jackson DN, Garcia-Sherman MC (2018) Amyloid-like β-aggregates as force-sensitive switches in fungal biofilms and infections. Microbiol Mol Biol Rev 82(1):e00035–e00017Google Scholar
  35. Maeda T, Garcıa-Contreras R, Pu M, Sheng L, Garcia LR, Tomas M, Wood TK (2012) Quorum quenching quandary: resistance to antivirulence compounds. ISME J 6:493–501CrossRefGoogle Scholar
  36. McCann KS (2000) The diversity-stability debate. Nature 405:228–233CrossRefGoogle Scholar
  37. McLean RJ, Whiteley M, Stickler DJ, Fuqua WC (1997) Evidence of autoinducer activity in naturally occurring biofilms. FEMS Microbiol Lett 154(2):259–263CrossRefGoogle Scholar
  38. Nakao R, Ramstedt M, Wai SN, Uhlin BE (2012) Enhanced biofilm formation by Escherichia coli LPS mutants defective in Hep biosynthesis. PLoS One 7:e51241CrossRefGoogle Scholar
  39. O’Toole G, Kaplan HB, Kolter R (2000) Biofilm formation as microbial development. Annu Rev Microbiol 54(1):49–79CrossRefGoogle Scholar
  40. Ohman DE, Cryz SJ, Iglewski BH (1980) Isolation and characterization of Pseudomonas aeruginosa PAO mutant that produces altered elastase. J Bacteriol 142:836–842Google Scholar
  41. Ramalingam B, Sekar R, Boxal JB, Biggs C (2013) Aggregation and biofilm formation of bacteria isolated from domestic drinking water. Water Sci Technol 13:1016Google Scholar
  42. Rogers NJ, Franklin NM, Apte SC, Batley GE, Angel BM, Lead JR, Baalousha M (2010) Physico-chemical behaviour and algal toxicity of nanoparticulate CeO2 in freshwater. Environ Chem 7:50–60CrossRefGoogle Scholar
  43. Schuster M, Greenberg EP (2006) A network of networks: quorum-sensing gene regulation in Pseudomonas aeruginosa. Int J Med Microbiol 296(2):73–81CrossRefGoogle Scholar
  44. Sendra M, Yeste PM, Moreno-Garrido I, Gatica JM, Blasco J (2017) CeO2 NPs, toxic or protective to phytoplankton? Charge of nanoparticles and cell wall as factors which cause changes in cell complexity. Sci Total Environ 590:304–315CrossRefGoogle Scholar
  45. Sheng GP, Yu HQ (2006) Chemical-equilibrium-based model for describing the strength of sludge: taking hydrogen-producing sludge as an example. Environ Sci Technol 40:1280–1285CrossRefGoogle Scholar
  46. Singh PK, Schaefer AL, Parsek MR, Moninger TO, Welsh MJ, Greenberg EP (2000) Quorum-sensing signals indicate that cystic fibrosis lungs are infected with bacterial biofilms. Nature 407(6805):762–764CrossRefGoogle Scholar
  47. Thill A, Zeyons O, Spalla O, Chauvat F, Rose J, Auffan M, Flank AM (2006) Cytotoxicity of CeO2 nanoparticles for Escherichia coli. Physico-chemical insight of the cytotoxicity mechanism. Environ Sci Technol 40:6151–6156CrossRefGoogle Scholar
  48. Thomann A, de Mello Martins AG, Brengel C, Empting M, Hartmann RW (2016) Application of dual inhibition concept within looped autoregulatory systems toward antivirulence agents against Pseudomonas aeruginosa infections. ACS Chem Biol 11(5):1279–1286CrossRefGoogle Scholar
  49. Wang D, Jin Q, Xiang H (2011) Transcriptional and functional analysis of the effects of magnolol: inhibition of autolysis and biofilms in Staphylococcus aureus. PLoS One 6:e26833CrossRefGoogle Scholar
  50. Whitchurch CB, Tolker-Nielsen T, Ragas PC, Mattick JS (2002) Extracellular DNA required for bacterial biofilm formation. Science 295(5559):1487–1487CrossRefGoogle Scholar
  51. Wilhelm S, Gdynia A, Tielen P, Rosenau F, Jaeger KE (2007) The autotransporter esterase EstA of Pseudomonas aeruginosa is required for rhamnolipid production, cell motility, and biofilm formation. J Bacteriol 189(18):6695–6703CrossRefGoogle Scholar
  52. Xu Y, Wang C, Hou J, Wang P, You G, Miao L, Lv B, Yang Y (2017) Effects of cerium oxide nanoparticles on the species and distribution of phosphorus in enhanced phosphorus removal sequencing batch biofilm reactor. Bioresour Technol 227:393–397CrossRefGoogle Scholar
  53. Yang X, Pan H, Wang P, Zhao FJ (2017) Particle-specific toxicity and bioavailability of cerium oxide (CeO2) nanoparticles to Arabidopsis thaliana. J Hazard Mater 322:292–300CrossRefGoogle Scholar
  54. You G, Hou J, Wang P, Xu Y, Wang C, Miao L, Lv B, Yang Y, Luo H (2016) Effects of CeO2 nanoparticles on sludge aggregation and the role of extracellular polymeric substances—explanation based on extended DLVO. Environ Res 151:698–705CrossRefGoogle Scholar
  55. Zeyons O, Thill A, Chauvat F, Menguy N, Cassier-Chauvat C, Oréar C, Daraspe J, Auffan M, Rose J, Spalla O (2009) Direct and indirect CeO2 nanoparticles toxicity for Escherichia coli and Synechocystis. Nanotoxicology 3:284–295CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Key Laboratory of Integrated Regulation and Resources Development on Shallow Lakes of Ministry of Education, College of EnvironmentHohai UniversityNanjingPeople’s Republic of China

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