J Periodontal Implant Sci.  2018 Feb;48(1):12-21. 10.5051/jpis.2018.48.1.12.

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

• Tavares LJ
• Klein MI
• Panariello BHD
• Dorigatti de Avila E
• Pavarina A

• KMID: 2405406
• DOI: http://doi.org/10.5051/jpis.2018.48.1.12

Abstract

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.

Keyword

Bacterial adhesion; Biofilms; Fusobacterium nucleatum; Porphyromonas gingivalis

MeSH Terms

Bacterial Adhesion
Biofilms*
Biomass
Fusobacterium nucleatum*
Fusobacterium*
Gentian Violet
In Vitro Techniques*
Microscopy, Electron, Scanning
Polystyrenes
Porphyromonas gingivalis*
Porphyromonas*
Gentian Violet
Polystyrenes

Figure

• 

  Figure 1 Growth curves represented by OD at 600 nm and CFU/mL for F. nucleatum NCTC 11326 (OD, 0.4±0.01; 8.2±0.007 CFU/mL) (A) and P. gingivalis ATCC 33277 (OD, 0.7±0.01; 9.5±0.1 CFU/mL) (B) at the mid-log phase. OD: optical density, CFU: colony-forming units.

• 

  Figure 2 Schematic presentation of the sequence of experiments. CV: crystal violet, SEM: scanning electron microscopy.

• 

  Figure 3 The adhesion phase was evaluated via quantitative measurements of crystal violet staining as an indicator of biomass accumulation after incubation for 24 hours (blue bar) in comparison to 48 hours (green bar). OD: optical density. a)Indicates a statistically significant difference (P<0.05).

• 

  Figure 4 Biofilm formation was evaluated via quantitative measurements of crystal violet staining as an indicator of biomass accumulation after incubation for 3 days (blue bar) in comparison to 5 (green bar) and 7 days (navy bar). OD: optical density. a)Indicates a statistically significant difference (P<0.05).

• 

  Figure 5 SEM images of single- and dual-species biofilms: F. nucleatum: (A) 3 days, (B) 5 days, (C) 7 days; P. gingivalis: (D) 3 days, (E) 5 days, (F) 7 days; and dual-species (G) 3 days, (H) 5 days, (I) 7 days (blue arrow, F. nucleatum; yellow arrow, P. gingivalis; red arrow, biomass; bar=5 µm). SEM: scanning electron microscopy.

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