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Plasma Chemistry and Plasma Processing

, Volume 39, Issue 1, pp 35–49 | Cite as

Regulation of Enterococcus faecalis Biofilm Formation and Quorum Sensing Related Virulence Factors with Ultra-low Dose Reactive Species Produced by Plasma Activated Water

  • Yinglong Li
  • Jie Pan
  • Dan Wu
  • Ying Tian
  • Jue ZhangEmail author
  • Jing Fang
Original Paper
  • 35 Downloads

Abstract

Enterococcus faecalis (E. faecalis) is the species which can cause life-threatening infections in human beings and exhibit highly antibacterial resistance in the nosocomial environment. The purpose of this study was to evaluate the inhibition effect and the mechanisms of ultra-low dose reactive oxygen species produced by plasma activated water (PAW) to the E. faecalis biofilm. The bactericidal effect of E. faecalis planktonic growth was evaluated to find out the maximum processing time. The level of intracellular reactive oxygen species (ROS) and extracellular hydrogen peroxide (H2O2) produced by the PAW without significant killing effect were defined as ultra-low. Then the pre-treated bacteria suspension by the PAW were incubated with brain heart infusion (BHI) broth for 6 h, 12 h, 24 h and 48 h. The biofilm development was evaluated with 96-well polystyrene method (OD570) and confocal laser scanning microscopy (CLSM). Meanwhile, total organic carbon (TOC) was measured with a Shimadzu TOC-L analyzer. The quorum sensing related virulence genes, cylR1, cylA, gelE and sprE, were evaluated with real-time PCR. The PAW did not have significant killing effect within 30 s and the maximum corresponding fluroscence intensity of ROS is under 57.33 ± 0.58 and the concentration of H2O2 is 19.87 ± 2.01 μmol/L. The OD570, CLSM results and TOC results indicated that the biofilm development was effectively inhibited with PAW treatment. Four quorum sensing related virulence genes were all down-regulated. In conclusion, the PAW treatment has potential application to control E. faecalis biofilm development and reduce quorum sensing related virulence genes expression in vitro.

Keywords

Ultra-low dose reactive oxygen species Enterococcus faecalis Biofilm Virulence factors Quorum sensing 

Notes

Acknowledgements

This study was supported by the 985-III program of Peking University. The authors are highly thankful to Peking University for providing financial assistance of this research project.

References

  1. 1.
    Paphitou NI (2013) Antimicrobial resistance: action to combat the rising microbial challenges. Int J Antimicrob Agents 42:S25–S28CrossRefGoogle Scholar
  2. 2.
    Hall-Stoodley L, Costerton JW, Stoodley P (2004) Bacterial biofilms: from the natural environment to infectious diseases. Nat Rev Microbiol 2:95–108CrossRefGoogle Scholar
  3. 3.
    Hoiby N, Bjarnsholt T, Givskov M, Molin S, Ciofu O (2010) Antibiotic resistance of bacterial biofilms. Int J Antimicrob Ag 35:322–332CrossRefGoogle Scholar
  4. 4.
    Davies D (2003) Understanding biofilm resistance to antibacterial agents. Nat Rev Drug Discov 2:114–122CrossRefGoogle Scholar
  5. 5.
    Kalia VC, Purohit HJ (2011) Quenching the quorum sensing system: potential antibacterial drug targets. Crit Rev Microbiol 37:121–140CrossRefGoogle Scholar
  6. 6.
    Antunes LCM, Ferreira RBR, Buckner MMC, Finlay BB (2010) Quorum sensing in bacterial virulence. Microbiology 156:2271–2282CrossRefGoogle Scholar
  7. 7.
    Gilmore MS, Lebreton F, van Schaik W (2013) Genomic transition of enterococci from gut commensals to leading causes of multidrug-resistant hospital infection in the antibiotic era. Curr Opin Microbiol 16:10–16CrossRefGoogle Scholar
  8. 8.
    Arias CA, Murray BE (2012) The rise of the Enterococcus: beyond vancomycin resistance. Nat Rev Microbiol 10:266–278CrossRefGoogle Scholar
  9. 9.
    Wang L, Dong M, Zheng JB, Song QY, Yin W, Li Y, Niu WD (2011) Relationship of biofilm formation and gelE Gene expression in Enterococcus faecalis recovered from root canals in patients requiring endodontic retreatment. J Endod 37:631–636CrossRefGoogle Scholar
  10. 10.
    Zhang CJ, Du JR, Peng ZX (2015) Correlation between Enterococcus faecalis and persistent intraradicular infection compared with primary intraradicular infection: a systematic review. J Endod 41:1207–1213CrossRefGoogle Scholar
  11. 11.
    Rocas IN, Siqueira JF, Santos KRN (2004) Association of Enterococcus faecalis with different forms of periradicular diseases. J Endod 30:315–320CrossRefGoogle Scholar
  12. 12.
    Lu ZS, Meng L, Liu ZH, Ren GH, Sun AJMLX (2013) Expression of quorum-sensing related genes during Enterococcus faecalis biofilm formation. Chin J Stomatol 48:485–489Google Scholar
  13. 13.
    Haas W, Shepard BD, Gilmore MS (2002) Two-component regulator of Enterococcus faecalis cytolysin responds to quorum-sensing autoinduction. Nature 415:84–87CrossRefGoogle Scholar
  14. 14.
    Coburn PS, Gilmore MS (2003) The Enterococcus faecalis cytolysin: a novel toxin active against eukaryotic and prokaryotic cells. Cell Microbiol 5:661–669CrossRefGoogle Scholar
  15. 15.
    Thurlow LR, Thomas VC, Narayanan S, Olson S, Fleming SD, Hancock LE (2010) Gelatinase contributes to the pathogenesis of endocarditis caused by Enterococcus faecalis. Infect Immun 78:4936–4943CrossRefGoogle Scholar
  16. 16.
    Park SY, Kim KM, Lee JH, Seo SJ, LEE IH (2007) Extracellular gelatinase of Enterococcus faecalis destroys a defense system in insect hemolymph and human serum. Infect Immun 75:1861–1869CrossRefGoogle Scholar
  17. 17.
    Hancock LE, Perego M (2004) The Enterococcus faecalis fsr two-component system controls biofilm development through production of gelatinase. J Bacteriol 186:5629–5639CrossRefGoogle Scholar
  18. 18.
    Kristich CJ, Li YH, Cvitkovitch DG, Dunny GM (2004) Esp-independent biofilm formation by Enterococcus faecalis. J Bacteriol 186:154–163CrossRefGoogle Scholar
  19. 19.
    Lalucque H, Silar P (2003) NADPH oxidase: an enzyme for multicellularity? Trends Microbiol 11:9–12CrossRefGoogle Scholar
  20. 20.
    Cap M, Vachova L, Palkova Z (2012) Reactive oxygen species in the signaling and adaptation of multicellular microbial communities. Oxid Med Cell Longev 11:976753Google Scholar
  21. 21.
    Isbary G, Shimizu T, Li YF, Stolz W, Thomas HM, Morfill GE, Zimmermann JL (2013) Cold atmospheric plasma devices for medical issues. Expert Rev Med Dev 10:367–377CrossRefGoogle Scholar
  22. 22.
    Traylor MJ, Pavlovich MJ, Karim S, Hait P, Sakiyama Y, Clark DS, Graves DB (2011) Long-term antibacterial efficacy of air plasma-activated water. J Phys D Appl, Phys, p 44Google Scholar
  23. 23.
    Stoffels E, Sakiyama Y, Graves DB (2008) Cold atmospheric plasma: charged species and their interactions with cells and tissues. IEEE Trans Plasma Sci 36:1441–1457CrossRefGoogle Scholar
  24. 24.
    Mai-Prochnow A, Bradbury M, Ostrikov K, Murphy AB (2015) Pseudomonas aeruginosa biofilm response and resistance to cold atmospheric pressure plasma is linked to the redox-active molecule phenazine. PLoS ONE 10:e0130373CrossRefGoogle Scholar
  25. 25.
    Turan NB, Chormey DS, Buyukpinar C, Engin GO, Bakirdere S (2017) Quorum sensing: little talks for an effective bacterial coordination. Trac-Trend Anal Chem 91:1–11CrossRefGoogle Scholar
  26. 26.
    Ziuzina D, Boehm D, Patil S, Cullen PJ, Bourke P (2015) Cold plasma inactivation of bacterial biofilms and reduction of quorum sensing regulated virulence factors. PLoS ONE 10:0138209CrossRefGoogle Scholar
  27. 27.
    Vandervoort KG, Brelles-Marino G (2014) Plasma-mediated inactivation of Pseudomonas aeruginosa biofilms grown on borosilicate surfaces under continuous culture system. PLoS ONE 9:e108512CrossRefGoogle Scholar
  28. 28.
    Wang ZJ, Shen Y, Haapasalo M (2017) Antibiofilm peptides against oral biofilms. J Oral Microbiol 9:1327308CrossRefGoogle Scholar
  29. 29.
    Jiao NZ, Yang YJ, Luo TW (2004) Membrane potential based characterization by flow cytometry of physiological states in an aerobic anoxygenic phototrophic bacterium. Aquat Microb Ecol 37:149–158CrossRefGoogle Scholar
  30. 30.
    Yu S, Chen QZ, Liu JH, Wang KL, Jiang Z, Sun ZL, Zhang J, Fang J (2015) Dielectric barrier structure with hollow electrodes and its recoil effect. Appl Phys Lett 106:87Google Scholar
  31. 31.
    Li YL, Sun K, Ye GP, Liang YD, Pan H, Wang GM, Zhao YJ, Pan J, Zhang J, Fang J (2015) Evaluation of cold plasma treatment and safety in disinfecting 3-week root canal Enterococcus faecalis biofilm in vitro. J Endodont 41:1325–1330CrossRefGoogle Scholar
  32. 32.
    Fletcher M (1988) Attachment of pseudomonas-fluorescens to glass and influence of electrolytes on bacterium-substratum separation distance. J Bacteriol 170:2027–2030CrossRefGoogle Scholar
  33. 33.
    Barraud N, Hassett DJ, Hwang SH, Rice SA, Kjelleberg S, Webb JS (2006) Involvement of nitric oxide in biofilm dispersal of Pseudomonas aeruginosa. J Bacteriol 188:7344–7353CrossRefGoogle Scholar
  34. 34.
    Stepanovic S, Vukovic D, Hola V, Di Bonaventura G, Djukic S, Cirkovic I, Ruzicka F (2007) Quantification of biofilm in microtiter plates: overview of testing conditions and practical recommendations for assessment of biofilm production by staphylococci. Apmis 115:891–899CrossRefGoogle Scholar
  35. 35.
    Kroukamp O, Wolfaardt GM (2009) CO2 Production as an Indicator of Biofilm Metabolism. Appl Environ Microbol 75:4391–4397CrossRefGoogle Scholar
  36. 36.
    Schafer AI, Nghiem LD, Oschmann N (2006) Bisphenol A retention in the direct ultrafiltration of greywater. J Membr Sci 283:233–243CrossRefGoogle Scholar
  37. 37.
    Alekshun MN, Levy SB (1999) The mar regulon: multiple resistance to antibiotics and other toxic chemicals. Trends Microbiol 7:410–413CrossRefGoogle Scholar
  38. 38.
    Kohanski MA, DePristo MA, Collins JJ (2010) Sublethal antibiotic treatment leads to multidrug resistance via radical-induced mutagenesis. Mol Cell 37:311–320CrossRefGoogle Scholar
  39. 39.
    Banerjee G, Ray K, Kumar R (2016) Effect of temperature on lateral gene transfer efficiency of multi-antibiotics resistant bacterium, Alcaligenes faecalis. Sains Malays 45:909–914Google Scholar
  40. 40.
    Dryden MS, Cooke J, Salib RJ, Holding RE, Biggs T, Salamat AA, Allan RN, Newby RS, Halstead F, Oppenheim B (2017) Reactive oxygen: a novel antimicrobial mechanism for targeting biofilm-associated infection. J Glob Antimicrob Resist 8:186–191CrossRefGoogle Scholar
  41. 41.
    Zhang Q, Ma RN, Tian Y, Su B, Wang KL, Yu S, Zhang J, Fang J (2016) Sterilization efficiency of a novel electrochemical disinfectant against Staphylococcus aureus. Environ Sci Technol 50:3184–3192CrossRefGoogle Scholar
  42. 42.
    Khlyustova A, Khomyakova N, Sirotkin N, Marfin Y (2016) The effect of pH on OH radical generation in aqueous solutions by atmospheric pressure glow discharge. Plasma Chem Plasma Process 36:1229–1238CrossRefGoogle Scholar
  43. 43.
    Shih KY, Locke BR (2010) Chemical and physical characteristics of pulsed electrical discharge within gas bubbles in aqueous solutions. Plasma Chem Plasma Process 30:1–20CrossRefGoogle Scholar
  44. 44.
    Pan J, Li YL, Liu CM, Tian Y, Yu S, Wang KL, Zhang J, Fang J (2017) Investigation of cold atmospheric plasma-activated water for the dental unit waterline system contamination and safety evaluation in vitro. Plasma Chem Plasma Process 37:1091–1103CrossRefGoogle Scholar
  45. 45.
    Sies H (2017) Hydrogen peroxide as a central redox signaling molecule in physiological oxidative stress: oxidative eustress. Redox Biol 11:613–619CrossRefGoogle Scholar
  46. 46.
    Treberg JR, Munro D, Banh S, Zacharias P, Sotiri E (2015) Differentiating between apparent and actual rates of H2O2 metabolism by isolated rat muscle mitochondria to test a simple model of mitochondria as regulators of H2O2 concentration. Redox Biol 5:216–224CrossRefGoogle Scholar
  47. 47.
    Marinho HS, Real C, Cyrne L, Soares H, Antunes F (2014) Hydrogen peroxide sensing, signaling and regulation of transcription factors. Redox Biol 2:535–562CrossRefGoogle Scholar
  48. 48.
    Imlay JA (2015) Transcription factors that defend bacteria against reactive oxygen species. Annu Rev Microbiol 69:93–108CrossRefGoogle Scholar
  49. 49.
    Nakajo K, Komori R, Ishikawa S, Ueno T, Suzuki Y, Iwami Y, Takahashi N (2006) Resistance to acidic and alkaline environments in the endodontic pathogen Enterococcus faecalis. Oral Microbiol Immun 21:283–288CrossRefGoogle Scholar
  50. 50.
    Costerton JW, Lewandowski Z, Caldwell DE, Korber DR, Lappinscott HM (1995) Microbial biofilms. Annu Rev Microbiol 49:711–745CrossRefGoogle Scholar
  51. 51.
    Flemming HC, Neu TR, Wozniak DJ (2007) The EPS matrix: the “house of biofilm cells”. J Bacteriol 189:7945–7947CrossRefGoogle Scholar
  52. 52.
    Stoodley P, Sauer K, Davies DG, Costerton JW (2002) Biofilms as complex differentiated communities. Annu Rev Microbiol 56:187–209CrossRefGoogle Scholar
  53. 53.
    Bassler BL, Losick R (2006) Bacterially speaking. Cell 125:237–246CrossRefGoogle Scholar
  54. 54.
    Fuqua C, Greenberg EP (2002) Listening in on bacteria: acyl-homoserine lactone signalling. Nat Rev Mol Cell Biol 3:685–695CrossRefGoogle Scholar
  55. 55.
    Sakuragi Y, Kolter R (2007) Quorum-sensing regulation of the biofilm matrix genes (pel) of Pseudomonas aeruginosa. J Bacteriol 189:5383–5386CrossRefGoogle Scholar
  56. 56.
    Otto M (2013) Staphylococcal infections: mechanisms of biofilm maturation and detachment as critical determinants of pathogenicity. Annu Rev Med 64:175CrossRefGoogle Scholar
  57. 57.
    Li ZL, Lu PL, Zhang DJ, Chen GC, Zeng SW, He Q (2016) Population balance modeling of activated sludge flocculation: investigating the influence of Extracellular Polymeric Substances (EPS) content and zeta potential on flocculation dynamics. Sep Purif Technol 162:91–100CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Yinglong Li
    • 1
    • 2
  • Jie Pan
    • 2
  • Dan Wu
    • 3
  • Ying Tian
    • 1
  • Jue Zhang
    • 1
    • 4
    Email author
  • Jing Fang
    • 1
    • 4
  1. 1.Academy for Advanced Interdisciplinary StudiesPeking UniversityBeijingPeople’s Republic of China
  2. 2.Department of General DentistryPeking University School and Hospital of StomatologyBeijingPeople’s Republic of China
  3. 3.College of Environmental Sciences and EngineeringPeking UniversityBeijingPeople’s Republic of China
  4. 4.College of EngineeringPeking UniversityBeijingPeople’s Republic of China

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