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Bioreactors: How to Study Biofilms In Vitro

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Oral Biofilms and Modern Dental Materials

Abstract

The interactions taking place between a dental (bio)material, the surrounding tissues of the host, and the biofilm that grows to permanently colonize this microenvironment are amazingly complex when analyzed in detail yet contribute to a crucial factor: the balance between health and disease conditions. From a microbiological point of view, this has a dramatic impact on the longevity of dental treatments. Researchers have long since tried to recreate, even if in parts, this complexity on a bench, both using a reductionistic approach as often performed in research and, more recently, by trying to create models approaching the most realistic behavior. These efforts yielded a wide range of bioreactor systems currently available. We hope that in a future not too far, bioreactor models will be able to reliably reproduce most clinical conditions, dramatically reducing the need for animal and clinical studies. Unfortunately, a universal bioreactor able to mimic any clinical situation still does not exist. Each model comes entwined with its advantages and limitations that must be acknowledged when choosing which model best fits a distinct experimental design. This situation, together with a reduced overall level of standardization, makes the comparison of the obtained results very difficult. This chapter presents an overview of the microbial communities and the bioreactor models that are most significant for studying the microbiological performances of dental materials.

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References

  1. Simões M, Pereira MO, Sillankorva S, Azeredo J, Vieira MJ. The effect of hydrodynamic conditions on the phenotype of Pseudomonas fluorescens biofilms. Biofouling. 2007;23(3–4):249–58.

    Article  PubMed  Google Scholar 

  2. Marsh PD, Zaura E. Dental biofilm: ecological interactions in health and disease. J Clin Periodontol. 2017;44(S18):S12–22.

    Article  PubMed  Google Scholar 

  3. Peterson SN, Meissner T, Su AI, Snesrud E, Ong AC, Schork NJ, Bretz WA. Functional expression of dental plaque microbiota. Front Cell Infect Microbiol. 2014;4:108.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Senneby A, Davies J, Svensäter G, Neilands J. Acid tolerance properties of dental biofilms in vivo. BMC Microbiol. 2017;17(1):165.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Köves B, Magyar A, Tenke P. Spectrum and antibiotic resistance of catheter-associated urinary tract infections. GMS Infect Dis. 2017;5:Doc06.

    PubMed  PubMed Central  Google Scholar 

  6. Tenke P, Köves B, Nagy K, Hultgren SJ, Mendling W, Wullt B, Grabe M, Wagenlehner FM, Cek M, Pickard R, Botto H. Update on biofilm infections in the urinary tract. World J Urol. 2012;30(1):51–7.

    Article  PubMed  Google Scholar 

  7. Gomes IB, Meireles A, Gonçalves AL, Goeres DM, Sjollema J, Simões LC, et al. Standardized reactors for the study of medical biofilms: a review of the principles and latest modifications. Crit Rev Biotechnol. 2018;38(5):657–70.

    Article  PubMed  Google Scholar 

  8. Vickery K, Hu H, Jacombs AS, Bradshaw DA, Deva AK. A review of bacterial biofilms and their role in device-associated infection. Healthc Infect. 2013;18(2):61–6.

    Article  Google Scholar 

  9. Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: a common cause of persistent infections. Science. 1999;284(5418):1318–22.

    Article  PubMed  Google Scholar 

  10. Koo H, Allan RN, Howlin RP, Stoodley P, Hall-Stoodley L. Targeting microbial biofilms: current and prospective therapeutic strategies. Nat Rev Microbiol. 2017;15(12):740–55.

    Article  PubMed  PubMed Central  Google Scholar 

  11. McBain AJ. Chapter 4: In vitro biofilm models: an overview. Adv Appl Microbiol. 2009;69:99–132.

    Article  PubMed  Google Scholar 

  12. Azeredo J, Azevedo N, Briandet R, Cerca N, Coenye T, Costa AR, Desvaux M, Di Bonaventura G, Hébraud M, Jaglic Z, Kačániová M. Critical review on biofilm methods. Crit Rev Microbiol. 2016;43(3):313–51.

    Article  PubMed  Google Scholar 

  13. Cazzaniga G. Resin-based composites modulate oral biofilm formation, PhD thesis. University of Milan, 2017. https://doi.org/10.13130/g-cazzaniga_phd2017-02-2314.

  14. Marsh PD. In sickness and in health - what does the oral microbiome mean to us? An ecological perspective. Adv Dent Res. 2018;29(1):60–5.

    Article  PubMed  Google Scholar 

  15. Kroes I, Lepp PW, Relman DA. Bacterial diversity within the human subgingival crevice. Proc Natl Acad Sci U S A. 1999;96:14547–52.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Kawamura Y, Kamiya Y. Metagenomic analysis permitting identification of the minority bacterial populations in the oral microbiota. J Oral Biosci. 2012;54(3):132–7.

    Article  Google Scholar 

  17. Ionescu A, Brambilla E, Hahnel S. Does recharging dental restorative materials with fluoride influence biofilm formation? Dent Mater. 2019;35(10):1450–63.

    Article  PubMed  Google Scholar 

  18. Hahnel S, Wastl DS, Schneider-Feyrer S, Giessibl FJ, Brambilla E, Cazzaniga G, Ionescu A. Streptococcus mutans biofilm formation and release of fluoride from experimental resin-based composites depending on surface treatment and S-PRG filler particle fraction. J Adhes Dent. 2014;16(4):313–21.

    PubMed  Google Scholar 

  19. Nassar HM, Gregory RL. Biofilm sensitivity of seven Streptococcus mutans strains to different fluoride levels. J Oral Microbiol. 2017;9(1):1328265.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Chow J, Lee SM, Shen Y, Khosravi A, Mazmanian SK. Host–bacterial symbiosis in health and disease. Adv Immunol. 2010;107:243–74.

    Article  PubMed  PubMed Central  Google Scholar 

  21. Kim Y-S, Kang S-M, Lee E-S, Lee JH, Kim B-R, Kim B-I. Ecological changes in oral microcosm biofilm during maturation. J Biomed Opt. 2016;21(10):101409.

    Article  PubMed  Google Scholar 

  22. Rudney JD, Chen R, Lenton P, Li J, Li Y, Jones RS, Reilly C, Fok AS, Aparicio C. A reproducible oral microcosm biofilm model for testing dental materials. J Appl Microbiol. 2012;113(6):1540–53.

    Article  PubMed  PubMed Central  Google Scholar 

  23. Ionescu AC, Cazzaniga G, Ottobelli M, Garcia-Godoy F, Brambilla E. Substituted nano-hydroxyapatite toothpastes reduce biofilm formation on enamel and resin-based composite surfaces. J Funct Biomater. 2020;11(2):36.

    Article  PubMed Central  Google Scholar 

  24. Ledder RG, Gilbert P, Pluen A, Sreenivasan PK, Vizio WD, McBain AJ. Individual microflora beget unique oral microcosms. J Appl Microbiol. 2006;100(5):1123–31.

    Article  PubMed  Google Scholar 

  25. Robinson CJ, Bohannan BJM, Young VB. From structure to function: the ecology of host-associated microbial communities. Microbiol Mol Biol Rev. 2010;74(3):453–76.

    Article  PubMed  PubMed Central  Google Scholar 

  26. Kuramitsu HK, He X, Lux R, Anderson MH, Shi W. Interspecies interactions within oral microbial communities. Microbiol Mol Biol Rev. 2007;71(4):653–70.

    Article  PubMed  PubMed Central  Google Scholar 

  27. Sim CPC, Dashper SG, Reynolds EC. Oral microbial biofilm models and their application to the testing of anticariogenic agents. J Dent. 2016;50:1–11.

    Article  PubMed  Google Scholar 

  28. ten Cate JM. Models and role models. Caries Res. 2015;49(S1):3–10.

    Article  PubMed  Google Scholar 

  29. Rickard AH, Gilbert P, High NJ, Kolenbrander PE, Handley PS. Bacterial coaggregation: an integral process in the development of multispecies biofilms. Trends Microbiol. 2003;11(2):94–100.

    Article  PubMed  Google Scholar 

  30. Hajishengallis G, Liang S, Payne MA, Hashim A, Jotwani R, Eskan MA, McIntosh ML, Alsam A, Kirkwood KL, Lambris JD, Darveau RPA. Low-abundance biofilm species orchestrates inflammatory periodontal disease through the commensal microbiota and the complement pathway. Cell Host Microbe. 2011;10(5):497–506.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Nibali L, Henderson B. The human microbiota and chronic disease: dysbiosis as a cause of human pathology. Hoboken, NJ: John Wiley & Sons; 2016. p. 560.

    Book  Google Scholar 

  32. Marsh PD, Hunter JR, Bowden GH, Hamilton IR, McKee AS, Hardie JM, et al. The influence of growth rate and nutrient limitation on the microbial composition and biochemical properties of a mixed culture of oral bacteria grown in a chemostat. J Gen Microbiol. 1983;129(3):755–70.

    PubMed  Google Scholar 

  33. Shu M, Wong L, Miller JH, Sissons CH. Development of multispecies consortia biofilms of oral bacteria as an enamel and root caries model system. Arch Oral Biol. 2000;45(1):27–40.

    Article  PubMed  Google Scholar 

  34. McKee AS, McDermid AS, Ellwood DC, Marsh PD. The establishment of reproducible, complex communities of oral bacteria in the chemostat using defined inocula. J Appl Bacteriol. 1985;59(3):263–75.

    Article  PubMed  Google Scholar 

  35. Burmølle M, Webb JS, Rao D, Hansen LH, Sørensen SJ, Kjelleberg S. Enhanced biofilm formation and increased resistance to antimicrobial agents and bacterial invasion are caused by synergistic interactions in multispecies biofilms. Appl Environ Microbiol. 2006;72(6):3916–23.

    Article  PubMed  PubMed Central  Google Scholar 

  36. Lin NJ. Biofilm over teeth and restorations: what do we need to know? Dent Mater. 2017;33(6):667–80.

    Article  PubMed  Google Scholar 

  37. Zanin ICJ, Gonçalves RB, Junior AB, Hope CK, Pratten J. Susceptibility of Streptococcus mutans biofilms to photodynamic therapy: an in vitro study. J Antimicrob Chemother. 2005;56(2):324–30.

    Article  PubMed  Google Scholar 

  38. Fernández CE, Tenuta LMA, Cury JA. Validation of a cariogenic biofilm model to evaluate the effect of fluoride on enamel and root dentine demineralization. PLoS One. 2016;11(1):e0146478.

    Article  PubMed  PubMed Central  Google Scholar 

  39. Zhang A, Chen R, Aregawi W, He Y, Wang S, Aparicio C, et al. Development and calibration of biochemical models for testing dental restorations. Acta Biomater. 2020;109:132–41.

    Article  PubMed  PubMed Central  Google Scholar 

  40. Forssten SD, Björklund M, Ouwehand AC. Streptococcus mutans, caries and simulation models. Nutrients. 2010;2(3):290–8.

    Article  PubMed  PubMed Central  Google Scholar 

  41. Philip N, Suneja B, Walsh L. Beyond Streptococcus mutans: clinical implications of the evolving dental caries aetiological paradigms and its associated microbiome. Br Dent J. 2018;224(4):219.

    Article  PubMed  Google Scholar 

  42. Banas JA, Drake DR. Are the mutans streptococci still considered relevant to understanding the microbial etiology of dental caries? BMC Oral Health. 2018;18(1):129.

    Article  PubMed  PubMed Central  Google Scholar 

  43. Matsumoto-Nakano M. Role of Streptococcus mutans surface proteins for biofilm formation. Jpn Dent Sci Rev. 2018;54(1):22–9.

    Article  PubMed  Google Scholar 

  44. Childers NK, Momeni SS, Whiddon J, Cheon K, Cutter GR, Wiener HW, et al. Association between early childhood caries and colonization with Streptococcus mutans genotypes from mothers. Pediatr Dent. 2017;39(2):130–5.

    PubMed  PubMed Central  Google Scholar 

  45. Hajishengallis E, Parsaei Y, Klein MI, Koo H. Advances in the microbial etiology and pathogenesis of early childhood caries. Mol Oral Microbiol. 2017;32(1):24–34.

    Article  PubMed  Google Scholar 

  46. Yu OY, Zhao IS, Mei ML, Lo EC-M, Chu C-H. Dental biofilm and laboratory microbial culture models for cariology research. Dent J. 2017;5(2):21.

    Article  Google Scholar 

  47. Balouiri M, Sadiki M, Ibnsouda SK. Methods for in vitro evaluating antimicrobial activity: a review. J Pharm Anal. 2016;6(2):71–9.

    Article  PubMed  Google Scholar 

  48. Milho C, Andrade M, Boas DV, Alves D, Sillankorva S. Antimicrobial assessment of phage therapy using a porcine model of biofilm infection. Int J Pharm. 2019;557:112–23.

    Article  PubMed  Google Scholar 

  49. Morgan SJ, Lippman SI, Bautista GE, Harrison JJ, Harding CL, Gallagher LA, et al. Bacterial fitness in chronic wounds appears to be mediated by the capacity for high-density growth, not virulence or biofilm functions. PLoS Pathog. 2019;15(3):e1007511.

    Article  PubMed  PubMed Central  Google Scholar 

  50. Bahamondez-Canas TF, Heersema LA, Smyth HD. Current status of in vitro models and assays for susceptibility testing for wound biofilm infections. Biomedicine. 2019;7(2):34.

    Google Scholar 

  51. Pierce CG, Uppuluri P, Tummala S, Lopez-Ribot JL. A 96 well microtiter plate-based method for monitoring formation and antifungal susceptibility testing of Candida albicans biofilms. JoVE J Vis Exp. 2010;44:e2287.

    Google Scholar 

  52. Skogman ME, Vuorela PM, Fallarero A. A platform of anti-biofilm assays suited to the exploration of natural compound libraries. JoVE J Vis Exp. 2016;118:e54829.

    Google Scholar 

  53. Kampf G. Antiseptic stewardship for wound and mucous membrane antiseptics. In: Kampf G, editor. Antiseptic stewardship: biocide resistance and clinical implications. Cham: Springer International Publishing; 2018. p. 689–94.

    Chapter  Google Scholar 

  54. Ceri H, Olson ME, Stremick C, Read RR, Morck D, Buret A. The Calgary Biofilm Device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms. J Clin Microbiol. 1999;37(6):1771–6.

    Article  PubMed  PubMed Central  Google Scholar 

  55. Brown JL, Johnston W, Delaney C, Short B, Butcher MC, Young T, et al. Polymicrobial oral biofilm models: simplifying the complex. J Med Microbiol. 2019;68(11):1573–84.

    Article  PubMed  Google Scholar 

  56. Bradshaw DJ, Marsh PD, Schilling KM, Cummins D. A modified chemostat system to study the ecology of oral biofilms. J Appl Bacteriol. 1996;80(2):124–30.

    Article  PubMed  Google Scholar 

  57. Peters AC, Wimpenny JWT. A constant-depth laboratory model film fermentor. Biotechnol Bioeng. 1988;32(3):263–70.

    Article  PubMed  Google Scholar 

  58. Rozenbaum RT. Antimicrobial and nanoparticle penetration and killing in infectious biofilms, PhD thesis. Rijksuniversiteit Groningen, 2019. http://hdl.handle.net/11370/0f2d1f8e-8898-4fb3-af42-7e8fd68c58e5.

  59. Cotter JJ, O’Gara JP, Stewart PS, et al. Characterization of a modified rotating disk reactor for the cultivation of Staphylococcus epidermidis biofilm. J Appl Microbiol. 2010;109:2105–17.

    Article  PubMed  Google Scholar 

  60. Möhle RB, Langemann T, Haesner M, Augustin W, Scholl S, Neu TR, et al. Structure and shear strength of microbial biofilms as determined with confocal laser scanning microscopy and fluid dynamic gauging using a novel rotating disc biofilm reactor. Biotechnol Bioeng. 2007;98(4):747–55.

    Article  PubMed  Google Scholar 

  61. Kharazmi A, Giwercman B, Høiby N. Robbins device in biofilm research. In: Methods in enzymology. New York: Academic Press; 1999. p. 207–15.

    Google Scholar 

  62. Jass J, Costerton JW, Lappin-Scott HM. Assessment of a chemostat-coupled modified Robbins device to study biofilms. J Ind Microbiol. 1995;15(4):283–9.

    Article  Google Scholar 

  63. Goeres DM, Hamilton MA, Beck NA, Buckingham-Meyer K, Hilyard JD, Loetterle LR, et al. A method for growing a biofilm under low shear at the air–liquid interface using the drip flow biofilm reactor. Nat Protoc. 2009;4(5):783–8.

    Article  PubMed  Google Scholar 

  64. Ledder RG, McBain AJ. An in vitro comparison of dentifrice formulations in three distinct oral microbiotas. Arch Oral Biol. 2012;57(2):139–47.

    Article  PubMed  Google Scholar 

  65. Ionescu A, Wutscher E, Brambilla E, Schneider-Feyrer S, Giessibl FJ, Hahnel S. Influence of surface properties of resin-based composites on in vitro Streptococcus mutans biofilm development. Eur J Oral Sci. 2012;120(5):458–65.

    Article  PubMed  Google Scholar 

  66. Brambilla E, Ionescu A, Cazzaniga G, Edefonti V. The influence of antibacterial toothpastes on in vitro Streptococcus mutans biofilm formation: a continuous culture study. Am J Dent. 2014;27(3):7.

    Google Scholar 

  67. Yoon HY, Lee SY. Establishing a laboratory model of dental unit waterlines bacterial biofilms using a CDC biofilm reactor. Biofouling. 2017;33(10):917–26.

    Article  PubMed  Google Scholar 

  68. Li Y, Carrera C, Chen R, Li J, Lenton P, Rudney JD, et al. Degradation in the dentin–composite interface subjected to multispecies biofilm challenges. Acta Biomater. 2014;10(1):375–83.

    Article  PubMed  Google Scholar 

  69. Yawata Y, Nguyen J, Stocker R, Rusconi R. Microfluidic studies of biofilm formation in dynamic environments. In: O’Toole GA, editor. J Bacteriol. 2016;198(19):2589–95.

    Google Scholar 

  70. Benoit MR, Conant CG, Ionescu-Zanetti C, Schwartz M, Matin A. New device for high-throughput viability screening of flow biofilms. Appl Environ Microbiol. 2010;76(13):4136–42.

    Article  PubMed  PubMed Central  Google Scholar 

  71. Groisman A, Lobo C, Cho H, Campbell JK, Dufour YS, Stevens AM, et al. A microfluidic chemostat for experiments with bacterial and yeast cells. Nat Methods. 2005;2(9):685–9.

    Article  PubMed  Google Scholar 

  72. Kim J, Hegde M, Kim SH, Wood TK, Jayaraman A. A microfluidic device for high throughput bacterial biofilm studies. Lab Chip. 2012;12(6):1157–63.

    Article  PubMed  Google Scholar 

  73. Busscher HJ, van der Mei HC. Microbial adhesion in flow displacement systems. Clin Microbiol Rev. 2006;19(1):127–41.

    Article  PubMed  PubMed Central  Google Scholar 

  74. Gellen LS, Wall-Manning GM, Sissons CH. Checkerboard DNA-DNA hybridization technology using digoxigenin detection. In: Hilario E, Mackay J, editors. Protocols for nucleic acid analysis by nonradioactive probes. Totowa, NJ: Humana Press; 2007. p. 39–67.

    Chapter  Google Scholar 

  75. Mougeot J-LC, Stevens CB, Cotton SL, Morton DS, Krishnan K, Brennan MT, et al. Concordance of HOMIM and HOMINGS technologies in the microbiome analysis of clinical samples. J Oral Microbiol. 2016;8(1):30379.

    Article  PubMed  Google Scholar 

  76. Adams SE, Arnold D, Murphy B, Carroll P, Green AK, Smith AM, et al. A randomised clinical study to determine the effect of a toothpaste containing enzymes and proteins on plaque oral microbiome ecology. Sci Rep. 2017;7(1):43344.

    Article  PubMed  PubMed Central  Google Scholar 

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Authors and Affiliations

  1. Oral Microbiology and Biomaterials Laboratory, Department of Biomedical, Surgical, and Dental Sciences, University of Milan, Milan, Italy

    Andrei Cristian Ionescu & Eugenio Brambilla

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  1. Andrei Cristian Ionescu
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  2. Eugenio Brambilla
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Correspondence to Andrei Cristian Ionescu .

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Editors and Affiliations

  1. Oral Microbiology and Biomaterials Laboratory, Department of Biomedical, Surgical, and Dental Sciences, University of Milan, Milan, Italy

    Andrei Cristian Ionescu

  2. Prosthetics and Dental Materials Clinic, Leipzig University, Leipzig, Germany

    Sebastian Hahnel

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Ionescu, A.C., Brambilla, E. (2021). Bioreactors: How to Study Biofilms In Vitro. In: Ionescu, A.C., Hahnel, S. (eds) Oral Biofilms and Modern Dental Materials . Springer, Cham. https://doi.org/10.1007/978-3-030-67388-8_4

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