The inhibitory effects of polypyrrole on the biofilm formation of Streptococcus mutans

Autoři: Hidenobu Senpuku aff001;  Elif Bahar Tuna aff002;  Ryo Nagasawa aff001;  Ryoma Nakao aff001;  Makoto Ohnishi aff001
Působiště autorů: Department of Bacteriology I, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan aff001;  Department of Pediatric Dentistry, Faculty of Dentistry, Istanbul University, Istanbul, Turky aff002;  Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan aff003
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: 10.1371/journal.pone.0225584


Streptococcus mutans primary thrives on the biofilm formation on the tooth surface in sticky biofilms and under certain conditions can lead to carious lesions on the tooth surface. To search for a new preventive material for oral biofilm-associated diseases, including dental caries, we investigated the effects of polypyrrole, which contains an electrochemical polymer and causes protonation and incorporation of anion under low pH condition, on the biofilm formation of S. mutans and other streptococci. In this study, polypyrrole was applied in biofilm formation assays with the S. mutans strains UA159 and its gtfB and gtfC double mutant (gtfBC mutant), S. sanguinis, S. mitis and S. gordonii on human saliva and bovine serum albumin-coated 96-well microtiter plates in tryptic soy broth supplemented with 0.25% sucrose. The effects of polypyrrole on biofilm formation were quantitatively and qualitatively observed. High concentrations of polypyrrole significantly inhibited the biofilm formation of S. mutans UA159 and S. sanguinis. As an inhibition mechanism, polypyrrole attached to the surface of bacterial cells, increased chains and aggregates, and incorporated proteins involving GTF-I and GTF-SI produced by S. mutans. In contrast, the biofilm formation of gtfBC mutant, S. sanguinis, S. mitis and S. gordonii was temporarily induced by the addition of low polypyrrole concentrations on human saliva-coated plate but not on the uncoated and bovine serum albumin-coated plates. Moreover, biofilm formation depended on live cells and, likewise, specific interaction between cells and binding components in saliva. However, these biofilms were easily removed by increased frequency of water washing. In this regard, the physical and electrochemical properties in polypyrrole worked effectively in the removal of streptococci biofilms. Polypyrrole may have the potential to alter the development of biofilms associated with dental diseases.

Klíčová slova:

Anions – Bacterial biofilms – Biofilms – Glucans – Polysaccharides – Saliva – Streptococcus mutans – Sucrose


1. Burne RA. Oral streptococci products of their environment. J Dent Res. 1998; 77: 445–452. doi: 10.1177/00220345980770030301 9496917

2. Loesche WJ. Role of Streptococcus mutans in human dental decay. Microbiol Rev. 1986;50: 353–380. 3540569

3. Nyvad B, Kilian M. Comparison of the initial streptococcal microflora on dental enamel in caries-active and in caries-inactive individuals. Caries Res. 1990;24:267–272. doi: 10.1159/000261281 2276164

4. Aas JA, Paster BJ, Stokes LN, Olsen I, Dewhirst FE. Defining the normal bacterial flora of the oral cavity. J Clin Microbiol. 2005;43:5721–5732. doi: 10.1128/JCM.43.11.5721-5732.2005 16272510

5. Kroes I, Lepp PW, Relman DA. Bacterial diversity within the human subgingival crevice. Proc Natl Acad Sci USA. 1999;96:14547–14552. doi: 10.1073/pnas.96.25.14547 10588742

6. Paster BJ, Boches SK, Galvin JL, Ericson RE, Lau CN, Levanos VA, et al. Bacterial diversity in human subgingival plaque. J Bacteriol. 2001;183:3770–3783. doi: 10.1128/JB.183.12.3770-3783.2001 11371542

7. Hamada S, Slade HD. Biology, immunology, and cariogenicity of Streptococcus mutans. Microbiol Rev. 1980;44:331–384. 6446023

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

9. Banas JA. Virulence properties of Streptococcus mutans. Front Biosci. 2004;9:1267–1277. doi: 10.2741/1305 14977543

10. Kuramitsu HK. Molecular genetic analysis of the virulence of oral bacterial pathogens: an historical perspective. Crit Rev Oral Biol Med. 2003;14: 331–344. doi: 10.1177/154411130301400504 14530302

11. Das T, Sharma PK, Busscher HJ, van der Mei HC, Krom BP. Role of extracellular DNA in initial bacterial adhesion and surface aggregation. Appl Environ Microbiol. 2010;76:3405–3408. doi: 10.1128/AEM.03119-09 20363802

12. Zhang K, Oi M, Ling J. Effects of quorum sensing on cell viability in Streptococcus mutans biofilm formation. Biochem Biophys Res Commun. 2009;379:933–938. doi: 10.1016/j.bbrc.2008.12.175 19138664

13. Petersen FC, Tao L, Scheie AA. DNA binding-uptake system: a link between cell-to-cell communication and biofilm formation. J Bacteriol. 2005;187: 4392–4400. doi: 10.1128/JB.187.13.4392-4400.2005 15968048

14. Rice KC, Mann EE, Endres JL, Weiss EC, Cassat JE, Smeltzer MS, et al. The cidA murein hydrolase regulator contributes to DNA release and biofilm development in Staphylococcus aureus. Proc Nat Acad Sci USA. 2007;104:8113–8118. doi: 10.1073/pnas.0610226104 17452642

15. Liu Y, Burne RA. The major autolysin of Streptococcus gordonii is subject to complex regulation and modulates stress tolerance, biofilm formation, and extracellular-DNA release. J Bacteriol. 2001;193:2826–2837.

16. Guiton PS, Hung CS, Kline KA, Roth R, Kau AL, Hayes E, et al. Contribution of autolysin and sortase A during Enterococcus faecalis DNA-dependent biofilm development. Infect Immun. 2009;77:3626–3638. doi: 10.1128/IAI.00219-09 19528211

17. Reck M, Rutz K, Kunze B, Tomasch J, Surapaneni SK, Schulz S, et al. The biofilm inhibitor carolacton disturbs membrane integrity and cell division of Streptococcus mutans through the serine/threonine protein kinase PknB. J Bacteriol. 2011;193:5692–5706. doi: 10.1128/JB.05424-11 21840978

18. Liao S, Klein MI, Heim KP, Fan Y, Bitoun JP, Ahn SJ, et al. Streptococcus mutans extracellular DNA is upregulated during growth in biofilms, actively released via membrane vesicles, and influenced by components of the protein secretion machinery. J Bacteril. 2014;196:2355–2366.

19. Kawarai T, Narisawa N, Suzuki Y, Nagasawa R, Senpuku H. Streptococcus mutans biofilm formation is dependent on extracellular DNA in primary low pH conditions. J Oral Biosc. 2016;58:55–61.

20. Das T, Sehar S, Manefield M. The roles of extracellular DNA in the structural integrity of extracellular polymeric substance and bacterial biofilm development. Environ Microbiol Rep. 2013;5:778–786. doi: 10.1111/1758-2229.12085 24249286

21. Skotheim TA, Elsenbaumer RL, Reynolds JR. eds, Handbook of Conducting Polymers, 2nd edn, Marcel Dekker, New York, 1998. (b) Nalwa H. S., ed., Handbook of Organic Conductive Molecules and Polymers: Vol. 2. Conductive Polymers: Synthesis and Electrical Properties, Wiley, 1997.

22. Jang J, Yoon H. Multigram-scale fabrication of monodisperse conducting polymer and magnetic carbon nanoparticles. Small. 2005;1:1195–1199. doi: 10.1002/smll.200500237 17193418

23. Bidan G. 1995. in: Polymer Films in Sensor Applications, Harsányi G., ed., Technomic Publishing Co. Inc., Lancaster, chap. 3, pp. 206–260.

24. Li Y, Neoh KG, Cen L, Kang ET. Porous and electrically conductive polypyrrole-poly (vinyl alcohol) composite and its applications as a biomaterial. Langmuir. 2005;21:10702–10709. doi: 10.1021/la0514314 16262340

25. Abel ML, Chehimi M. M., Fricker F., et al. Adsorption of poly(methyl methacrylate) and poly(vinyl chloride) blends onto polypyrrole. Study by X-ray photoelectron spectroscopy, time-of-flight static secondary ion mass spectroscopy, and inverse gas chromatography. J Chromatography. 2002;969:273–285.

26. Skotheim TA, Elsenbaumer RL, Reynolds JR., eds, Handbook of Conducting Polymers, 2nd edn, Marcel Dekker,New York, 1998. (b) Nalwa H. S., ed., Handbook of Organic Conductive Molecules and Polymers: Vol. 2. Conductive Polymers: Synthesis and Electrical Properties, Wiley, 1997.

27. Ravichandran R, Sundarrajan S, Venugopal JR, Mukherjee S, Ramakrishna S. Applications of conducting polymers and their issues in biomedical engineering. J Royal Soc Inter. 2010;5:S559–579.

28. Thompson BC, Moulton SE, Ding J, Richardson R, Cameron A, O'Leary S, et al. Optimising the incorporation and release of a neurotrophic factor using conducting polypyrrole. J Contr Rel. 2006;116:285–294.

29. Ke K, Lin L, Liang H, Chen X, Han C, Li J, et al. Polypyrrole nanoprobes with low non-specific protein adsorption for intracellular mRNA detection and photothermal therapy. Chem Commun. 2015;51:6800–6803.

30. Molino PJ, Zhang B, Wallace GG, Hanks TW. Surface modification of polypyrrole/biopolymer composites for controlled protein and cellular adhesion. Biofouling. 2013;29:1155–1167. doi: 10.1080/08927014.2013.830110 24063598

31. Miksa B, Slomkowski S. Adsorption and covalent immobilization of human serum albumin (HSA) and gamma globulins (gamma G) onto poly(styrene/acrolein) latexes with pyrene, dansyl, and 2,4-dinitrophenyl labels. J Biomater Sci. 1995;7:77–96.

32. Azioune A, Slimane AB, Hamou LA, Pleuvy A, Chehimi MM, Perruchot C, et al. Synthesis and characterization of active ester-functionalized polypyrrole-silica nanoparticles: application to the covalent attachment of proteins. Langmuir. 2004;20:3350–3356. doi: 10.1021/la030407s 15875868

33. Pande R, Ruben GC, Lim JO, Tripathy S, Marx KA. DNA bound to polypyrrole films: high-resolution imaging, DNA binding kinetics and internal migration. Biomaterials. 1998;19:1657–1667. doi: 10.1016/s0142-9612(98)00043-x 9840001

34. Saoudi B, Jammul N, Chehimi MM, McCarthy GP, Armes SP. Adsorption of DNA onto Polypyrrole-Silica Nanocomposites. J Colloid Inter Sci. 1997;192:269–273.

35. Ateh DD, Navsaria HA, Vadgama P. Polypyrrole-based conducting polymers and interactions with biological tissues. J Royal Soc Inter. 2006;3:741–752.

36. Motegi M, Takagi Y, Yonezawa H, Hanada N, Terajima J, Watanabe H, et al. Assessment of genes associated with Streptococcus mutans biofilm morphology. Appl Environ Microbiol. 2006;72:6277–6287. doi: 10.1128/AEM.00614-06 16957255

37. Suzuki Y, Nagasawa R, Senpuku H. Inhibiting effects of fructanase on competence-stimulating peptide-dependent quorum sensing system in Streptococcus mutans. J Infect Chemother. 2017;23:634–641. doi: 10.1016/j.jiac.2017.06.006 28729051

38. Ohta H, Kato H, Okahashi N, Takahashi I, Hamada S, Koga T. Characterization of a cell-surface protein antigen of hydrophilic Streptococcus mutans strain GS-5. J Gen Microbiol. 1989;135:981–988. doi: 10.1099/00221287-135-4-981 2600589

39. Senpuku H, Kato H, Takeuchi H, Noda A, Nisizawa T. Identification of core B cell epitope in the synthetic peptide inducing cross-inhibiting antibodies to a surface protein antigen of Streptococcus mutans. Immunol Invest. 1997;26:531–548. doi: 10.3109/08820139709088538 9399097

40. Sato Y, Senpuku H, Okamoto K, Hanada N, Kizaki H. Streptococcus mutans binding to solid phase dextran mediated by the glucan-binding protein C. Oral Microbiol Immunol. 2002;17:252–256. doi: 10.1034/j.1399-302x.2002.170408.x 12121476

41. Hamada S, Horikoshi T, Minami T, Okahashi N, Koga T. Purification and characterization of cell-associated glucosyltransferase synthesizing water-insoluble glucan from serotype c Streptococcus mutans. J Gen Microbiol. 1989;135:335–344. doi: 10.1099/00221287-135-2-335 2533242

42. Freedman ML, Coykendall AL, O'Neill EM. Physiology of "mutans-like" Streptococcus ferus from wild rats. Infect Immun. 1982;35:476–482. 6276304

43. Necas J, Bartosikova L, Brauner P, Kolar J. Hyaluronic acid (hyaluronan): a review. Vet Med. 2008;8:397–411.

44. Bitoun JP, Liao S, Yao X, Ahn SJ, Isoda R, Nguyen AH, et al. BrpA is involved in regulation of cell envelope stress responses in Streptococcus mutans. Appl Environ Microbiol. 2012;78:2914–2922. doi: 10.1128/AEM.07823-11 22327589

45. Wen ZT, Burne RA. Functional genomics approach to identifying genes required for biofilm development by Streptococcus mutans. Appl Environ Microbiol. 2002;68:1196–1203. doi: 10.1128/AEM.68.3.1196-1203.2002 11872468

46. Biswas S, Biswas I. Role of HtrA in surface protein expression and biofilm formation by Streptococcus mutans. Infect Immun. 2005;73:6923–6934. doi: 10.1128/IAI.73.10.6923-6934.2005 16177372

47. Ouyang J, Tian XL, Versey J, Wishart A, Li YH. The BceABRS four-component system regulates the bacitracin-induced cell envelope stress response in Streptococcus mutans. Antimicrob Agents Chemother. 2010;54:3895–906. doi: 10.1128/AAC.01802-09 20606066

48. Arrigucci R, Pozzi G. Identification of the chain-dispersing peptidoglycan hydrolase LytB of Streptococcus gordonii. PLoS One. 2017;12:e0176117. doi: 10.1371/journal.pone.0176117 28414782

49. Mooser G, Hefta SA, Paxton RJ, Shively JE, Lee TD. Isolation and sequence of an active-site peptide containing a catalytic aspartic acid from two Streptococcus sobrinus alpha-glucosyltransferases. J Biol Chem. 1991;266:8916–8922. 1827439

50. Kato C, Nakano Y, Lis M, Kuramitsu HK. Molecular genetic analysis of the catalytic site of Streptococcus mutans glucosyltransferases. Biochem Biophys Res Commun. 1992;189:1184–1188. doi: 10.1016/0006-291x(92)92329-v 1472027

51. Scannapieco FA. Saliva-bacterium interactions in oral microbial ecology. Crit Rev Oral Biol Med. 1994;5:203–248. doi: 10.1177/10454411940050030201 7703323

52. Yao Y, Berg EA, Costello CE, Troxler RF, Oppenheim FG. Identification of protein components in human acquired enamel pellicle and whole saliva using novel proteomics approaches. J Biol Chem. 2003;278:5300–5308. doi: 10.1074/jbc.M206333200 12444093

53. Postollec F, Norde W, van der Mei HC, Busscher HJ. Enthalpy of interaction between coaggregating and non-coaggregating oral bacterial pairs—a microcalorimetric study. J Microbiol Methods. 2003;55:241–247. doi: 10.1016/s0167-7012(03)00145-3 14500015

54. Bos R, van der Mei HC, Busscher HJ. Physico-chemistry of initial microbial adhesive interactions—its mechanisms and methods for study. FEMS Microbiol Rev. 1999;23:179–230. doi: 10.1111/j.1574-6976.1999.tb00396.x 10234844

55. Hang JH, Pyo M. pH-dependent mass and volume changes of polypyrrole/poly (styrene sulfonate). Bull Kor Chem Soc. 2006;27:2067–2070.

56. Pei Q, Qian R. Protonation and deprotonation of polypyrrole chain in aqueous solution. Synth Met. 1991;45:35–48.

57. Li Y, Qian R. Study on the chemical compensation of conducting polypyrrole by NaOH solution. Synth Met. 1988;26:139–151

58. Lambert RJ, Stratford M. Weak-acid preservatives: Modelling microbial inhibition and response. J Appl Microbiol. 1999;86:157–164. doi: 10.1046/j.1365-2672.1999.00646.x 10030018

59. Russell JB. Another explanation for the toxicity of fermentation acids at low pH: Anion accumulation versus uncoupling. J Appl Microbiol. 1992;73:363–370.

60. Collier JH, Camp JP, Hudson TW, Schmidt CE. Synthesis and characterization of polypyrrole-hyaluronic acid composite biomaterials for tissue engineering applications. J Biomed Mater Res. 2000;50:574–84. doi: 10.1002/(sici)1097-4636(20000615)50:4<574::aid-jbm13>;2-i 10756316

61. Kim S, Jang Y, Jang M, Lim A, Hardy JG, Park HS, et al. Versatile biomimetic conductive polypyrrole films doped with hyaluronic acid of different molecular weights. Acta Biomater. 2018;80:258–268. doi: 10.1016/j.actbio.2018.09.035 30266636

62. Das T, Sehar S, Koop L, Wong YK, Ahmed S, Siddiqui KS, et al. Influence of calcium in extracellular DNA mediated bacterial aggregation and biofilm formation. PLoS One. 2014;9:e91935. doi: 10.1371/journal.pone.0091935 24651318

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2019 Číslo 11