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. 2012 Oct 5:12:227.
doi: 10.1186/1471-2180-12-227.

Advancement of the 10-species subgingival Zurich biofilm model by examining different nutritional conditions and defining the structure of the in vitro biofilms

Thomas W Ammann  1 , Rudolf Gmür, Thomas Thurnheer
Affiliations

Advancement of the 10-species subgingival Zurich biofilm model by examining different nutritional conditions and defining the structure of the in vitro biofilms

Thomas W Ammann et al. BMC Microbiol. .

Abstract

Background: Periodontitis is caused by a highly complex consortium of bacteria that establishes as biofilms in subgingival pockets. It is a disease that occurs worldwide and its consequences are a major health concern. Investigations in situ are not possible and the bacterial community varies greatly between patients and even within different loci. Due to the high complexity of the consortium and the availability of samples, a clear definition of the pathogenic bacteria and their mechanisms of pathogenicity are still not available. In the current study we addressed the need of a defined model system by advancing our previously described subgingival biofilm model towards a bacterial composition that reflects the one observed in diseased sites of patients and analysed the structure of these biofilms.

Results: We further developed the growth media by systematic variation of key components resulting in improved stability and the firm establishment of spirochetes in the 10-species subgingival Zurich biofilm model. A high concentration of heat-inactivated human serum allowed the best proliferation of the used species. Therefore we further investigated these biofilms by analysing their structure by confocal laser scanning microscopy following fluorescence in situ hybridisation. The species showed mutual interactions as expected from other studies. The abundances of all organisms present in this model were determined by microscopic counting following species-specific identification by both fluorescence in situ hybridisation and immunofluorescence. The newly integrated treponemes were the most abundant organisms.

Conclusions: The use of 50% of heat-inactivated human serum used in the improved growth medium resulted in significantly thicker and more stable biofilms, and the quantitative representation of the used species represents the in vivo community of periodontitis patients much closer than in biofilms grown in the two media with less or no human serum. The appearance of T. denticola, P. gingivalis, and T. forsythia in the top layer of the biofilms, and the high abundance of T. denticola, reflects well the microbial situation observed at diseased sites. The improved model biofilms will allow further investigations of interactions between individual species and of the effects of atmospheric or nutritional changes, as well as interactions with tissue cells.

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Figures

Figure 1
Figure 1
Time course of biofilm growth comparing SAL, mFUM4, and iHS as growth media. Total counts determined by plating on CBA agar plates (T. denticola and T. forsythia are not cultivable on CBA). Each box represents N = 9 independent biofilms from three independent triplicate experiments. The boxes represent the inter quartile range of the data points, the bar indicates the median. The whiskers cover the data points within the 1.5x inter quartile range. Dots are outliers within 1.5 and 3 box lengths outside the interquartile range.
Figure 2
Figure 2
Bacterial attachment to the disc surface under different nutritional conditions 4 h after inoculation. Comparison of the growth media mFUM4 (A), iHS (B) and SAL (C). green: DNA staining using YoPro-1 + Sytox. The disc surface is visualized in grey colour. The images show representative areas of one disc each. Scale bars: 15 μm (A/B) and 10 μm (C).
Figure 3
Figure 3
Thickness (A) and total counts (FISH/IF) (B) of biofilms grown for 64.5 h in SAL, mFUM4, and iHS growth medium. Thickness was determined by CLSM, total counts were calculated from the species specific quantification by visual microscopic counting following FISH- or IF from N=9 independent biofilms from three independent experiments. Thickness was determined at N=44 (iHS), N=61 (mFUM4) and N=57 (SAL) randomly selected measurement spots on the discs. The boxes represent the inter quartile range of the data points, the bar indicates the median. The whiskers cover the data points within the 1.5x inter quartile range. Dots are outliers within 1.5 and 3 box lengths outside the interquartile range. ** indicates the significantly higher thickness (p≤0.001) of iHS biofilms compared to biofilms of both SAL and mFUM4.
Figure 4
Figure 4
Quantification of bacteria in biofilms grown for 64.5 h in SAL, mFUM4, and iHS growth medium. Bacteria were quantified by visual microscopic counting. Each box represents N=9 independent biofilms from three independent experiments. The boxes represent the inter quartile range of the data points, the bar indicates the median. The whiskers cover the data points within the 1.5x inter quartile range. Dots are outliers within 1.5 and 3 box lengths outside the interquartile range, and colored stars are extremes that are more than 3 boxlengths outside the interquartile range. * indicate significant differences with p≤0.05 between a pair of boxes, as indicated by the brackets.
Figure 5
Figure 5
Biofilms grown for 64.5 h in or mFUM4- (A) or iHS medium(B). FISH staining of a fixed biofilm; the biofilm base in the side views is directed towards the top view. (A) red: F. nucleatum, white: V. dispar, green: non-hybridised cells, DNA staining (YoPro-1 + Sytox), blue: EPS. (B) cyan: streptococci, red: F. nucleatum, green: non-hybridised cells, DNA staining (YoPro-1 + Sytox). Figures show a representative area of one disc. Scale bars: 20 μm.
Figure 6
Figure 6
Biofilms grown for 64.5 h in iHS medium. FISH staining of a fixed biofilm; the biofilm base in the side views is directed towards the top view. Cyan: V. dispar, green: non-hybridised cells, DNA staining (YoPro-1 + Sytox). Arrows: Microcolonies of V. dispar. Shown is a representative area of one disc. Scale bar: 30 μm.
Figure 7
Figure 7
3D-reconstructions of a 146 x 146 μm section of biofilms grown for 64.5 h in iHS medium. FISH staining of a fixed biofilm. P. gingivalis and T. forsythia are shown schematically as dots (fluorescence maxima of the cells). (A) cyan: T. forsythia, red: P. intermedia, green: non-hybridised cells, DNA staining (YoPro-1 + Sytox). (B) cyan: T. denticola, red: P. gingivalis, green: non-hybridised cells, DNA staining (YoPro-1 + Sytox). Figures show a representative area of one disc.
Figure 8
Figure 8
Biofilms grown for 64.5 h in iHS Medium. FISH staining of a fixed biofilm; the biofilm base in the side views is directed towards the top view. C. rectus is shown schematically as dots (fluorescence maxima of the cells). (A) red: A. oris, green: non-hybridised cells, DNA staining (YoPro-1 + Sytox), blue: EPS. (B) red: C. rectus, green: non-hybridised cells, DNA staining (YoPro-1 + Sytox). The red dots appear yellowish due to the transparency of the green channel. Figures show a representative area of one disc. Scale bars: 20 μm.
Figure 9
Figure 9
Schematic structure of the 10-species in vitro biofilms after 64 h of incubation in iHS medium. Distribution of the 10 species and EPS as observed by CLSM. The scale is not representative
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