J Periodontal Implant Sci.  2014 Apr;44(2):79-84. 10.5051/jpis.2014.44.2.79.

A periodontitis-associated multispecies model of an oral biofilm

• Park JH
• Lee JK
• Um HS
• Chang BS
• Lee SY

• KMID: 1914508
• DOI: http://doi.org/10.5051/jpis.2014.44.2.79

Abstract

PURPOSE
While single-species biofilms have been studied extensively, we know notably little regarding multispecies biofilms and their interactions. The purpose of this study was to develop and evaluate an in vitro multispecies dental biofilm model that aimed to mimic the environment of chronic periodontitis.
METHODS
Streptococcus gordonii KN1, Fusobacterium nucleatum ATCC23726, Aggregatibacter actinomycetemcomitans ATCC33384, and Porphyromonas gingivalis ATCC33277 were used for this experiment. The biofilms were grown on 12-well plates with a round glass slip (12 mm in diameter) with a supply of fresh medium. Four different single-species biofilms and multispecies biofilms with the four bacterial strains listed above were prepared. The biofilms were examined with a confocal laser scanning microscope (CLSM) and scanning electron microscopy (SEM). The minimum inhibitory concentrations (MIC) for four different planktonic single-species and multispecies bacteria were determined. The MICs of doxycycline and chlorhexidine for four different single-species biofilms and a multispecies biofilm were also determined.
RESULTS
The CLSM and SEM examination revealed that the growth pattern of the multispecies biofilm was similar to those of single-species biofilms. However, the multispecies biofilm became thicker than the single-species biofilms, and networks between bacteria were formed. The MICs of doxycycline and chlorhexidine were higher in the biofilm state than in the planktonic bacteria. The MIC of doxycycline for the multispecies biofilm was higher than were those for the single-species biofilms of P. gingivalis, F. nucleatum, or A. actinomycetemcomitans. The MIC of chlorhexidine for the multispecies biofilm was higher than were those for the single-species biofilms of P. gingivalis or F. nucleatum.
CONCLUSIONS
To mimic the natural dental biofilm, a multispecies biofilm composed of four bacterial species was grown. The 24-hour multispecies biofilm may be useful as a laboratory dental biofilm model system.

Keyword

Biofilms; Microbial sensitivity tests; Microbiology; Periodontitis

MeSH Terms

Aggregatibacter actinomycetemcomitans
Bacteria
Biofilms*
Chlorhexidine
Chronic Periodontitis
Doxycycline
Fusobacterium nucleatum
Glass
Microbial Sensitivity Tests
Microscopy, Electron, Scanning
Periodontitis
Plankton
Porphyromonas gingivalis
Streptococcus gordonii
Chlorhexidine
Doxycycline

Figure

• 

  Figure 1 Biofilm growth curves. P. gingivalis: Porphyromonas gingivalis, F. nucleatum: Fusobacterium nucleatum, S. gordonii: Streptococcus gordonii, A. actinomycetemcomitans: Aggregatibacter actinomycetemcomitans, CFU: colony forming unit.

• 

  Figure 2 Confocal micrographs represent a two-dimensional maximum projection of the biofilms after 24 hours, from the biofilm surface (left image) to the deepest layer of the biofilm (right image) (×400): (A) Porphyromonas gingivalis, (B) Fusobacterium nucleatum, (C) Streptococcus gordonii, (D) Aggregatibacter actinomycetemcomitans, and (E) multispecies bacteria.

• 

  Figure 3 Representative scanning electron microscopy images of the biofilms after 24 hours (×30,000): (A) Porphyromonas gingivalis, (B) Fusobacterium nucleatum, (C) Streptococcus gordonii, (D) Aggregatibacter actinomycetemcomitans, and (E) multispecies bacteria. Bars=1 µm.

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