Penetration kinetics of four mouthrinses into Streptococcus mutans biofilms analyzed by direct time-lapse visualization



The aim of this study was to determine whether different antiseptic mouthrinses show different penetration kinetics into Streptococcus mutans biofilms.

Materials and methods

The biofilms, grown on glass-based dishes, were exposed to one of four mouthrinses containing chlorhexidine digluconate, essential oil, cetylpyridinium chloride, or isopropylmethylphenol. Then, penetration velocities were determined by monitoring fluorescence loss of calcein AM-stained biofilms with time-lapse confocal laser scanning microscopy. Bactericidal activity was assessed with fluorescent bacterial viable cell (Live/Dead) staining and viable cell counts. Bacterial detachment after the mouthrinse exposure was determined by measuring fluorescence reduction of SYTO9-stained biofilms.


The essential oil-containing mouthrinse showed significantly faster penetration velocity than the other mouthrinses (ANCOVA and Bonferroni test, p < 0.05). However, even 5 min of exposure left the biofilm structure almost intact. After 30 s (consumer rinsing time) of exposure, the essential oil-containing mouthrinse showed the highest log reduction of viable cells (2.7 log CFU) measured by Live/Dead staining, and the mean reduction of total viable cells was 1.41 log CFU measured by viable cell count.


The essential oil-containing mouthrinse showed the best penetration. Within 30 s of exposure, however, no mouthrinses injured all the microorganisms and all mouthrinses left the biofilm structure nearly intact.

Clinical relevance

The mouthrinses tested showed different levels of biofilm penetration. The essential oil rinse was superior to other rinses by all three of the in vitro measurements performed.

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  1. 1.

    Allaker RP, Douglas CWI (2009) Novel anti-microbial therapies for dental plaque-related diseases. Int J Antimicrob Agents 33:8–13

    PubMed  Article  Google Scholar 

  2. 2.

    Marsh PD (2010) Controlling the oral biofilm with antimicrobials. J Dent 38(Suppl 1):S11–S15

    PubMed  Article  Google Scholar 

  3. 3.

    Jeon JG, Rosalen PL, Falsetta ML, Koo H (2011) Natural products in caries research: current (limited) knowledge, challenges and future perspective. Caries Res 45:243–263

    PubMed Central  PubMed  Article  Google Scholar 

  4. 4.

    Socransky SS, Haffajee AD (2000) Dental biofilms: difficult therapeutic targets. Periodontol 2000 28:12–55

    Article  Google Scholar 

  5. 5.

    Kolenbrander PE, Andersen RN, Blehert DS, Egland PG, Foster JS, Palmer RJ Jr (2002) Communication among oral bacteria. Microbiol Mol Biol Rev 66:486–505

    PubMed Central  PubMed  Article  Google Scholar 

  6. 6.

    Marsh PD (2005) Dental plaque: biological significance of a biofilm and community life-style. J Clin Periodontol 32(Suppl 6):7–15

    PubMed  Article  Google Scholar 

  7. 7.

    Stewart PS (2003) Diffusion in biofilms. J Bacteriol 185:1485–1491

    PubMed Central  PubMed  Article  Google Scholar 

  8. 8.

    Thurnheer T, Gmur R, Shapiro S, Guggenheim B (2003) Mass transport of macromolecules within an in vitro model supragingival plaque. Appl Environ Microbiol 69:1702–1709

    PubMed Central  PubMed  Article  Google Scholar 

  9. 9.

    Kim J, Pitts B, Stewart PS, Camper A, Yoon J (2008) Comparison of the antimicrobial effects of chloride, silver ion, and tobramycin on biofilm. Antimicrob Agents Chemother 52:1446–1453

    PubMed Central  PubMed  Article  Google Scholar 

  10. 10.

    Baffone W, Sorgente G, Campana R, Patrone V, Sisti D, Falcioni T (2011) Comparative effect of chlorhexidine and some mouthrinses on bacterial biofilm formation in titanium surface. Curr Microbiol 62:445–451

    PubMed  Article  Google Scholar 

  11. 11.

    Kaneko Y, Thoendel M, Olakanmi O, Britigan BE, Singh PK (2007) The transition metal gallium disrupts Pseudomonas aeruginosa iron metabolism and has antimicrobial and antibiofilm activity. J Clin Invest 117:877–888

    PubMed Central  PubMed  Article  Google Scholar 

  12. 12.

    Davison WM, Pitts B, Stewart PS (2010) Spatial and temporal patterns of biocide action against Staphylococcus epidermidis biofilms. Antimicrob Agents Chemother 54:2920–2927

    PubMed Central  PubMed  Article  Google Scholar 

  13. 13.

    Banin E, Brady KM, Greenberg EP (2006) Chelator-induced dispersal and killing of pseudomonas aeruginosa cells in a biofilm. Appl Environ Microbiol 72:2064–2069

    PubMed Central  PubMed  Article  Google Scholar 

  14. 14.

    Gunther F, Wabnitz GH, Stroh P, Prior B, Obst U, Samstag Y, Wagner C, Hansch GM (2009) Host defence against Staphylococcus aureus biofilms infection: phagocytosis of biofilms by polymorphonuclear neutrophils (PMN). Mol Immunol 46:1805–1813

    PubMed  Article  Google Scholar 

  15. 15.

    Yarwood JM, Bartels DJ, Volper EM, Greenberg EP (2004) Quorum sensing in Staphylococcus aureus biofilms. J Bacteriol 186:1838–1850

    PubMed Central  PubMed  Article  Google Scholar 

  16. 16.

    Rani SA, Pitts B, Beyenal H, Veluchamy RA, Lewandowski Z, Buckingham-Meyer K, Stewart PS (2007) Spatial patterns of DNA replication, protein synthesis and oxygen concentration within bacterial biofilms reveal diverse physiological states. J Bacteriol 189:4223–4233

    PubMed Central  PubMed  Article  Google Scholar 

  17. 17.

    Jefferson KK, Goldmann DA, Pier GB (2005) Use of confocal microscopy to analyze the rate of vancomycin penetration through Staphylococcus aureus biofilms. Antimicrob Agents Chemother 49:2467–2473

    PubMed Central  PubMed  Article  Google Scholar 

  18. 18.

    Takenaka S, Trivedi HM, Corbin A, Pitts B, Stewart PS (2008) Direct visualization of spatial and temporal patterns of antimicrobial action within model oral biofilms. Appl Environ Microbiol 74:1869–1875

    PubMed Central  PubMed  Article  Google Scholar 

  19. 19.

    Corbin A, Pitts B, Parker A, Stewart PS (2011) Antimicrobial penetration and efficacy in an in vitro oral biofilm model. Antimicrob Agents Chemother 55:3338–3344

    PubMed Central  PubMed  Article  Google Scholar 

  20. 20.

    Thurnheer T, van der Ploeg JR, Giertsen E, Guggenheim B (2006) Effects of Streptococcus mutans gtfC deficiency on mixed oral biofilms in vitro. Caries Res 40:163–171

    PubMed  Article  Google Scholar 

  21. 21.

    Bowen WH, Koo H (2011) Biology of Streptococcus mutans-derived glucosyltransferases: role in extracellular matrix formation of cariogenic biofilms. Caries Res 45:69–86

    PubMed Central  PubMed  Article  Google Scholar 

  22. 22.

    Sedlacek MJ, Walker C (2007) Antibiotic resistance in an in vitro subgingival biofilm model. Oral Microbiol Immunol 22:333–339

    PubMed Central  PubMed  Article  Google Scholar 

  23. 23.

    Pan P, Barnett ML, Coelho J, Brogdon C, Finnegan MB (2000) Determination of the in situ bactericidal activity of an essential oil mouthrinse using a vital stain method. J Clin Periodontol 27:256–261

    PubMed  Article  Google Scholar 

  24. 24.

    Tadokoro K, Yamaguchi T, Kawamura K, Shimizu H, Egashira T, Minabe M, Yoshino T, Oguchi H (2010) Rapid quantification of periodontitis-related bacteria using a novel modification of Invader PLUS technologies. Microbiol Res 165:43–49

    PubMed  Article  Google Scholar 

  25. 25.

    Zhang Z, Nadezhina E, Wilkinson KJ (2011) Quantifying diffusion in a biofilm of Streptococcus mutans. Antimicrob Agents Chemother 55:1075–1081

    PubMed Central  PubMed  Article  Google Scholar 

  26. 26.

    Marcotte L, Therien-Aubin H, Sandt C, Barbeau J, Lafleur M (2004) Solute size effects on the diffusion in biofilms of Streptococcus mutans. Biofouling 20:189–201

    PubMed  Article  Google Scholar 

  27. 27.

    Peulen TO, Wilkinson KJ (2011) Diffusion of nanoparticles in a biofilm. Environ Sci Technol 45:3367–3373

    PubMed  Article  Google Scholar 

  28. 28.

    Nichols WW, Dorrington SM, Slack MPE, Walmskey HL (1988) Inhibition of tobramycin diffusion by binding to alginate. Antimicrob Agents Chemother 32:518–523

    PubMed Central  PubMed  Article  Google Scholar 

  29. 29.

    Robinson C (2011) Mass transfer of therapeutics through natural human plaque biofilms: a model for therapeutic delivery to pathological bacterial biofilms. Arch Oral Biology 56:829–836

    Article  Google Scholar 

  30. 30.

    Guggenheim B, Guggenheim M, Gmur R, Giertsen E, Thurnheer (2004) Application of the Zurich biofilm model to problems of cariology. Caries Res 38:212–222

    PubMed  Article  Google Scholar 

  31. 31.

    Auschill TM, Hellwig E, Sculean A, Hein N, Arweiler NB (2004) Impact of the intraoral location on the rate of biofilm growth. Clin Oral Invest 8:97–101

    Article  Google Scholar 

  32. 32.

    Shapiro S, Giertsen E, Guggenheim B (2002) An in vitro oral biofilm model for comparing the efficacy of antimicrobial mouthrinses. Caries Res 36:93–100

    PubMed  Article  Google Scholar 

  33. 33.

    Guggenheim B, Giertsen E, Schüpbach P, Shapiro S (2001) Validation of an in vitro biofilm model of supragingival plaque. J Dent Res 80:363–370

    PubMed  Article  Google Scholar 

  34. 34.

    Foster JS, Pan PC, Kolenbrander PE (2004) Effects of antimicrobial agents on oral biofilms in a saliva-conditioned flowcell. Biofilms 1:5–12

    Article  Google Scholar 

  35. 35.

    Ouhayoun JP (2003) Penetrating the plaque biofilm: impact of essential oil mouthwash. J Clin Periodontol 30(Suppl 5):10–12

    PubMed  Article  Google Scholar 

  36. 36.

    Hope CK, Wilson M (2004) Analysis of the effects of chlorhexidine on oral biofilm vitality and structure based on viability profiling and an indicator of membrane integrity. Antimicrob Agents Chemother 48:1461–1468

    PubMed Central  PubMed  Article  Google Scholar 

  37. 37.

    Pratten J, Wilson M (1999) Antimicrobial susceptibility and composition of microcosm dental plaques supplemented with sucrose. Antimicrob Agents Chemother 43:1595–1599

    PubMed Central  PubMed  Google Scholar 

  38. 38.

    von Ohle C, Gieseke A, Nistico L, Decker EM, DeBeer D, Stoodley P (2010) Real-time microsensor measurement of local metabolic activities in ex vivo dental biofilms exposed to sucrose and treated with chlorhexidine. Appl Environ Microbiol 76:2326–2334

    Article  Google Scholar 

  39. 39.

    Sandt C, Barbeau J, Gagnon MA, Lafleur M (2007) Role of the ammonium group in the diffusion of quaternary ammonium compounds in Streptococcus mutans biofilms. J Antimicrob Chemother 60:1281–1287

    PubMed  Article  Google Scholar 

  40. 40.

    Vitkov L, Hermann A, Krautgartner WD, Herrmann M, Fuchs K, Klappacher M, Hannig M (2005) Chlorhexidine-induced ultrastructural alterations in oral biofilm. Microsc Res Tech 68:85–89

    PubMed  Article  Google Scholar 

  41. 41.

    Cheung HY, Wong MM, Cheung SH, Liang LY, Lam YW, Chiu SK (2012) Differential actions of chlorhexidine on the cell wall of Bacillus subtilis and Escherichia coli. PLoS One 7:e36659

    PubMed Central  PubMed  Article  Google Scholar 

  42. 42.

    Stewart PS, Franklin MJ (2008) Physiological heterogeneity in biofilms. Nat Rev Microbiol 6:199–210

    PubMed  Article  Google Scholar 

  43. 43.

    Joux F, Lebaron P (2000) Use of fluorescent probes to assess physiological functions of bacteria atsingle-cell level. Microbes Infect 2(12):1523–1535

    PubMed  Article  Google Scholar 

  44. 44.

    Decker EM (2001) The ability of direct fluorescence-based, two-colour assays to detect different phythiological states or oral streptococci. Lett Appl Microbiol 33(3):188–192

    PubMed  Article  Google Scholar 

  45. 45.

    Sträuber H, Müller S (2010) Viability states of bacteria- specific mechanisms of selected probes. Cytometry A 77(7):623–634

    PubMed  Article  Google Scholar 

  46. 46.

    Davey HM (2011) Life, death, and in-between: meanings and methods in microbiology. Appl Environ Microbiol 77(16):5571–5576

    PubMed Central  PubMed  Article  Google Scholar 

  47. 47.

    Tawakoli PN, Al-Ahmad A, Hoth-Hannig W, Hannig M, Hannig C (2012) Comparison of different live/dead stainings for detection and quantification of adherent microorganisms in the initial oral biofilm. Clin Oral Investig 17(3):841–850. doi:10.1007/s00784-012-0792-3

    PubMed  Article  Google Scholar 

  48. 48.

    Berney M, Hammes F, Bosshard F, Weilenmann HU, Egli T (2007) Assessment and interpretation of bacterial viability by using the LIVE/DEAD BacLight in combination with flow cytometry. Appl Environ Microbiol 73:3283–3290

    PubMed Central  PubMed  Article  Google Scholar 

  49. 49.

    Shi L, Günther S, Hübschmann T, Wick LY, Harms H, Müller S (2007) Limits of propidium iodide as a cell viability indicator for environmental bacteria. Cytometry A 71:592–598

    PubMed  Article  Google Scholar 

  50. 50.

    Biggerstaff JP, Le Puil M, Weidow BL, Prater J, Glass K, Radosevich M, White DC (2006) New methodology for viability testing in environmental samples. Mol Cell Probes 20:141–146

    PubMed  Article  Google Scholar 

  51. 51.

    Davey HM, Hexley P (2011) Red but not dead? Membranes of stressed Saccharomyces cerevisiae are permeable to propidium iodide. Environ Microbiol 13:163–171

    PubMed  Article  Google Scholar 

  52. 52.

    Karthikeyan R, Amaechi BT, Rawls HR, Lee VA (2011) Antimicrobial activity of nanoemulsion on cariogenic Streptococcus mutans. Arch Oral Biol 56:437–445

    PubMed  Article  Google Scholar 

  53. 53.

    Stocks SM (2004) Mechanism and use of the commercially avairable viability stain, BacLight. Cytometry A 61:189–195

    PubMed  Article  Google Scholar 

  54. 54.

    Takenaka S, Ohshima H, Ohsumi T, Okiji T (2012) Current and future strategies for the control of mature oral biofilms—shift from a bacteria-targeting to a matrix-targeting approach. J Oral Biosci 54:173–179

    Article  Google Scholar 

  55. 55.

    Herles S, Olsen S, Afflitto J, Gaffar A (1994) Chemostat flow cell system: an in vitro model for the evaluation of antiplaque agents. J Dent Res 73:1748–1755

    PubMed  Google Scholar 

  56. 56.

    Auschill TM, Hein N, Hellwig E, Follo M, Sculean A, Arweiler NB (2005) Effect of two antimicrobial agents on early in situ biofilm formation. J Clin Periodontol 32:147–152

    PubMed  Article  Google Scholar 

  57. 57.

    Arweiler NB, Lenz R, Sculean A, Al-Ahmad A, Hellwig E, Auschill TM (2008) Effect of food preservatives on in situ biofilm formation. Clin Oral Invest 12:203–208

    Article  Google Scholar 

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This investigation was supported in part by a Grant-in-Aid for Scientific Research (C) (no. 23592795) from the Japan Society for the Promotion of Science (JSPS), a Grant-in-Aid for Young Scientists (B) (no. 22791830) from the JSPS, and the JSPS Institutional Program for Young Researcher Overseas Visits.

Conflict of interest

The authors declare that they have no conflicts of interest.

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Correspondence to Shoji Takenaka.

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Wakamatsu, R., Takenaka, S., Ohsumi, T. et al. Penetration kinetics of four mouthrinses into Streptococcus mutans biofilms analyzed by direct time-lapse visualization. Clin Oral Invest 18, 625–634 (2014).

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  • Biofilm
  • Time-lapse observation
  • Penetration kinetics
  • Mouthrinse