Journal of Periodontal and Implant Science

  • 제48권1호
  • /
  • Pages.12-21
  • /
  • 2018
  • /
  • 2093-2278(pISSN)
  • /
  • 2093-2286(eISSN)
대한치주과학회 (Korean Academy of Periodontology)
권호 표지
DOI QR코드

DOI QR Code

An in vitro model of Fusobacterium nucleatum and Porphyromonas gingivalis in single- and dual-species biofilms

  • Tavares, Livia Jacovassi (Department of Dental Materials and Prosthodontics, Sao Paulo State University - UNESP School of Dentistry at Araraquara) ;
  • Klein, Marlise Inez (Department of Dental Materials and Prosthodontics, Sao Paulo State University - UNESP School of Dentistry at Araraquara) ;
  • Panariello, Beatriz Helena Dias (Department of Dental Materials and Prosthodontics, Sao Paulo State University - UNESP School of Dentistry at Araraquara) ;
  • de Avila, Erica Dorigatti (Department of Dental Materials and Prosthodontics, Sao Paulo State University - UNESP School of Dentistry at Araraquara) ;
  • Pavarina, Ana Claudia (Department of Dental Materials and Prosthodontics, Sao Paulo State University - UNESP School of Dentistry at Araraquara)
  • 투고 : 2017.12.12
  • 심사 : 2018.02.10
  • 발행 : 2018.02.28
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

Purpose: The goal of this study was to develop and validate a standardized in vitro pathogenic biofilm attached onto saliva-coated surfaces. Methods: Fusobacterium nucleatum (F. nucleatum) and Porphyromonas gingivalis (P. gingivalis) strains were grown under anaerobic conditions as single species and in dual-species cultures. Initially, the bacterial biomass was evaluated at 24 and 48 hours to determine the optimal timing for the adhesion phase onto saliva-coated polystyrene surfaces. Thereafter, biofilm development was assessed over time by crystal violet staining and scanning electron microscopy. Results: The data showed no significant difference in the overall biomass after 48 hours for P. gingivalis in single- and dual-species conditions. After adhesion, P. gingivalis in single- and dual-species biofilms accumulated a substantially higher biomass after 7 days of incubation than after 3 days, but no significant difference was found between 5 and 7 days. Although the biomass of the F. nucleatum biofilm was higher at 3 days, no difference was found at 3, 5, or 7 days of incubation. Conclusions: Polystyrene substrates from well plates work as a standard surface and provide reproducible results for in vitro biofilm models. Our biofilm model could serve as a reference point for studies investigating biofilms on different surfaces.

키워드

참고문헌

  1. Malcolm J, Millington O, Millhouse E, Campbell L, Adrados Planell A, Butcher JP, et al. Mast cells contribute to Porphyromonas gingivalis-induced bone loss. J Dent Res 2016;95:704-10. https://doi.org/10.1177/0022034516634630
  2. Feng X, Zhu L, Xu L, Meng H, Zhang L, Ren X, et al. Distribution of 8 periodontal microorganisms in family members of Chinese patients with aggressive periodontitis. Arch Oral Biol 2015;60:400-7. https://doi.org/10.1016/j.archoralbio.2014.11.015
  3. Eke PI, Dye BA, Wei L, Thornton-Evans GO, Genco RJ. Prevalence of periodontitis in adults in the United States: 2009 and 2010. J Dent Res 2012;91:914-20. https://doi.org/10.1177/0022034512457373
  4. Marcenes W, Kassebaum NJ, Bernabe E, Flaxman A, Naghavi M, Lopez A, et al. Global burden of oral conditions in 1990-2010: a systematic analysis. J Dent Res 2013;92:592-7. https://doi.org/10.1177/0022034513490168
  5. Zitzmann NU, Berglundh T. Definition and prevalence of peri-implant diseases. J Clin Periodontol 2008;35:286-91. https://doi.org/10.1111/j.1600-051X.2008.01274.x
  6. Socransky SS, Haffajee AD. Periodontal microbial ecology. Periodontol 2000 2005;38:135-87. https://doi.org/10.1111/j.1600-0757.2005.00107.x
  7. How KY, Song KP, Chan KG. Porphyromonas gingivalis: an overview of periodontopathic pathogen below the gum line. Front Microbiol 2016;7:53.
  8. van Winkelhoff AJ, Loos BG, van der Reijden WA, van der Velden U. Porphyromonas gingivalis, Bacteroides forsythus and other putative periodontal pathogens in subjects with and without periodontal destruction. J Clin Periodontol 2002;29:1023-8. https://doi.org/10.1034/j.1600-051X.2002.291107.x
  9. Periasamy S, Kolenbrander PE. Mutualistic biofilm communities develop with Porphyromonas gingivalis and initial, early, and late colonizers of enamel. J Bacteriol 2009;191:6804-11. https://doi.org/10.1128/JB.01006-09
  10. Tribble GD, Kerr JE, Wang BY. Genetic diversity in the oral pathogen Porphyromonas gingivalis: molecular mechanisms and biological consequences. Future Microbiol 2013;8:607-20. https://doi.org/10.2217/fmb.13.30
  11. Kirschbaum M, Schultze-Mosgau S, Pfister W, Eick S. Mixture of periodontopathogenic bacteria influences interaction with KB cells. Anaerobe 2010;16:461-8. https://doi.org/10.1016/j.anaerobe.2010.03.009
  12. Saito A, Kokubu E, Inagaki S, Imamura K, Kita D, Lamont RJ, et al. Porphyromonas gingivalis entry into gingival epithelial cells modulated by Fusobacterium nucleatum is dependent on lipid rafts. Microb Pathog 2012;53:234-42. https://doi.org/10.1016/j.micpath.2012.08.005
  13. Feuille F, Ebersole JL, Kesavalu L, Stepfen MJ, Holt SC. Mixed infection with Porphyromonas gingivalis and Fusobacterium nucleatum in a murine lesion model: potential synergistic effects on virulence. Infect Immun 1996;64:2094-100.
  14. Metzger Z, Lin YY, Dimeo F, Ambrose WW, Trope M, Arnold RR. Synergistic pathogenicity of Porphyromonas gingivalis and Fusobacterium nucleatum in the mouse subcutaneous chamber model. J Endod 2009;35:86-94. https://doi.org/10.1016/j.joen.2008.10.015
  15. Diaz PI, Zilm PS, Rogers AH. Fusobacterium nucleatum supports the growth of Porphyromonas gingivalis in oxygenated and carbon-dioxide-depleted environments. Microbiology 2002;148:467-72. https://doi.org/10.1099/00221287-148-2-467
  16. Ahn SH, Song JE, Kim S, Cho SH, Lim YK, Kook JK, et al. NOX1/2 activation in human gingival fibroblasts by Fusobacterium nucleatum facilitates attachment of Porphyromonas gingivalis. Arch Microbiol 2016;198:573-83. https://doi.org/10.1007/s00203-016-1223-7
  17. Periasamy S, Chalmers NI, Du-Thumm L, Kolenbrander PE. Fusobacterium nucleatum ATCC 10953 requires Actinomyces naeslundii ATCC 43146 for growth on saliva in a three-species community that includes Streptococcus oralis 34. Appl Environ Microbiol 2009;75:3250-7. https://doi.org/10.1128/AEM.02901-08
  18. Ebersole JL, Feuille F, Kesavalu L, Holt SC. Host modulation of tissue destruction caused by periodontopathogens: effects on a mixed microbial infection composed of Porphyromonas gingivalis and Fusobacterium nucleatum. Microb Pathog 1997;23:23-32. https://doi.org/10.1006/mpat.1996.0129
  19. Kumada Y, Benson DR, Hillemann D, Hosted TJ, Rochefort DA, Thompson CJ, et al. Evolution of the glutamine synthetase gene, one of the oldest existing and functioning genes. Proc Natl Acad Sci U S A 1993;90:3009-13. https://doi.org/10.1073/pnas.90.7.3009
  20. Amoroso PF, Adams RJ, Waters MG, Williams DW. Titanium surface modification and its effect on the adherence of Porphyromonas gingivalis: an in vitro study. Clin Oral Implants Res 2006;17:633-7. https://doi.org/10.1111/j.1600-0501.2006.01274.x
  21. de Avila ED, Avila-Campos MJ, Vergani CE, Spolidorio DM, Mollo Fde A Jr. Structural and quantitative analysis of a mature anaerobic biofilm on different implant abutment surfaces. J Prosthet Dent 2016;115:428-36. https://doi.org/10.1016/j.prosdent.2015.09.016
  22. de Avila ED, Lima BP, Sekiya T, Torii Y, Ogawa T, Shi W, et al. Effect of UV-photofunctionalization on oral bacterial attachment and biofilm formation to titanium implant material. Biomaterials 2015;67:84-92. https://doi.org/10.1016/j.biomaterials.2015.07.030
  23. Montelongo-Jauregui D, Srinivasan A, Ramasubramanian AK, Lopez-Ribot JL. An in vitro model for oral mixed biofilms of candida albicans and Streptococcus gordonii in synthetic saliva. Front Microbiol 2016;7:686.
  24. Barros J, Grenho L, Fontenente S, Manuel CM, Nunes OC, Melo LF, et al. Staphylococcus aureus and Escherichia coli dual-species biofilms on nanohydroxyapatite loaded with CHX or ZnO nanoparticles. J Biomed Mater Res A 2017;105:491-7. https://doi.org/10.1002/jbm.a.35925
  25. Liu Y, Busscher HJ, Zhao B, Li Y, Zhang Z, van der Mei HC, et al. Surface-adaptive, antimicrobially loaded, micellar nanocarriers with enhanced penetration and killing efficiency in staphylococcal biofilms. ACS Nano 2016;10:4779-89. https://doi.org/10.1021/acsnano.6b01370
  26. Malaikozhundan B, Vaseeharan B, Vijayakumar S, Pandiselvi K, Kalanjiam MA, Murugan K, et al. Biological therapeutics of Pongamia pinnata coated zinc oxide nanoparticles against clinically important pathogenic bacteria, fungi and MCF-7 breast cancer cells. Microb Pathog 2017;104:268-77. https://doi.org/10.1016/j.micpath.2017.01.029
  27. Badihi Hauslich L, Sela MN, Steinberg D, Rosen G, Kohavi D. The adhesion of oral bacteria to modified titanium surfaces: role of plasma proteins and electrostatic forces. Clin Oral Implants Res 2013;24 Suppl A100:49-56. https://doi.org/10.1111/j.1600-0501.2011.02364.x
  28. Kohavi D, Klinger A, Steinberg D, Mann E, Sela NM. Alpha-amylase and salivary albumin adsorption onto titanium, enamel and dentin: an in vivo study. Biomaterials 1997;18:903-6. https://doi.org/10.1016/S0142-9612(97)00026-4
  29. Moura JS, da Silva WJ, Pereira T, Del Bel Cury AA, Rodrigues Garcia RC. Influence of acrylic resin polymerization methods and saliva on the adherence of four Candida species. J Prosthet Dent 2006;96:205-11. https://doi.org/10.1016/j.prosdent.2006.07.004
  30. Pereira-Cenci T, Cury AA, Cenci MS, Rodrigues-Garcia RC. In vitro Candida colonization on acrylic resins and denture liners: influence of surface free energy, roughness, saliva, and adhering bacteria. Int J Prosthodont 2007;20:308-10.
  31. Sanchez MC, Llama-Palacios A, Blanc V, Leon R, Herrera D, Sanz M. Structure, viability and bacterial kinetics of an in vitro biofilm model using six bacteria from the subgingival microbiota. J Periodontal Res 2011;46:252-60. https://doi.org/10.1111/j.1600-0765.2010.01341.x
  32. Thein ZM, Samaranayake YH, Samaranayake LP. Characteristics of dual species Candida biofilms on denture acrylic surfaces. Arch Oral Biol 2007;52:1200-8. https://doi.org/10.1016/j.archoralbio.2007.06.007
  33. Ciandrini E, Campana R, Federici S, Manti A, Battistelli M, Falcieri E, et al. In vitro activity of Carvacrol against titanium-adherent oral biofilms and planktonic cultures. Clin Oral Investig 2014;18:2001-13. https://doi.org/10.1007/s00784-013-1179-9
  34. Navarro Llorens JM, Tormo A, Martinez-Garcia E. Stationary phase in gram-negative bacteria. FEMS Microbiol Rev 2010;34:476-95. https://doi.org/10.1111/j.1574-6976.2010.00213.x
  35. Rolfe MD, Rice CJ, Lucchini S, Pin C, Thompson A, Cameron AD, et al. Lag phase is a distinct growth phase that prepares bacteria for exponential growth and involves transient metal accumulation. J Bacteriol 2012;194:686-701. https://doi.org/10.1128/JB.06112-11
  36. Kim KS, Anthony BF. Importance of bacterial growth phase in determining minimal bactericidal concentrations of penicillin and methicillin. Antimicrob Agents Chemother 1981;19:1075-7. https://doi.org/10.1128/AAC.19.6.1075
  37. de Avila ED, de Molon R, Vergani CE, Mollo FA Jr, Salih V. The relationship between biofilm and physicalchemical properties of implant abutment materials for successful dental implants. Materials (Basel) 2014;7:3651-62. https://doi.org/10.3390/ma7053651
  38. de Avila ED, de Molon R, Spolidorio DM, Mollo FA Jr. Implications of surface and bulk properties of abutment implants and their degradation in the health of periodontal tissue. Materials (Basel) 2013;6:5951-66. https://doi.org/10.3390/ma6125951
  39. de Avila ED, Vergani CE, Mollo Junior FA, Junior MJ, Shi W, Lux R. Effect of titanium and zirconia dental implant abutments on a cultivable polymicrobial saliva community. J Prosthet Dent 2017;118:481-7. https://doi.org/10.1016/j.prosdent.2017.01.010
  40. de Avila ED, de Molon RS, Lima BP, Lux R, Shi W, Junior MJ, et al. Impact of physical chemical characteristics of abutment implant surfaces on bacteria adhesion. J Oral Implantol 2016;42:153-8. https://doi.org/10.1563/aaid-joi-D-14-00318

피인용 문헌

  1. Antibiofilm effect of ozonized physiological saline solution on peri‐implant-related biofilm vol.92, pp.8, 2021, https://doi.org/10.1002/jper.20-0333