Limits...
In vitro modeling of host-parasite interactions: the 'subgingival' biofilm challenge of primary human epithelial cells.

Guggenheim B, Gmür R, Galicia JC, Stathopoulou PG, Benakanakere MR, Meier A, Thurnheer T, Kinane DF - BMC Microbiol. (2009)

Bottom Line: The new model takes into account that the microbial challenge derives from a biofilm community and not from planktonically cultured bacterial strains.It will facilitate easily the introduction of additional host cells such as neutrophils for future biofilm:host cell challenge studies.Our methodology may generate particular interest, as it should be widely applicable to other biofilm-related chronic inflammatory diseases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Oral Biology, Section for Oral Microbiology and General Immunology, University of Zürich, Plattenstrasse 11, CH-8032 Zürich, Switzerland. bernie@zzmk.uzh.ch

ABSTRACT

Background: Microbial biofilms are known to cause an increasing number of chronic inflammatory and infectious conditions. A classical example is chronic periodontal disease, a condition initiated by the subgingival dental plaque biofilm on gingival epithelial tissues. We describe here a new model that permits the examination of interactions between the bacterial biofilm and host cells in general. We use primary human gingival epithelial cells (HGEC) and an in vitro grown biofilm, comprising nine frequently studied and representative subgingival plaque bacteria.

Results: We describe the growth of a mature 'subgingival' in vitro biofilm, its composition during development, its ability to adapt to aerobic conditions and how we expose in vitro a HGEC monolayer to this biofilm. Challenging the host derived HGEC with the biofilm invoked apoptosis in the epithelial cells, triggered release of pro-inflammatory cytokines and in parallel induced rapid degradation of the cytokines by biofilm-generated enzymes.

Conclusion: We developed an experimental in vitro model to study processes taking place in the gingival crevice during the initiation of inflammation. The new model takes into account that the microbial challenge derives from a biofilm community and not from planktonically cultured bacterial strains. It will facilitate easily the introduction of additional host cells such as neutrophils for future biofilm:host cell challenge studies. Our methodology may generate particular interest, as it should be widely applicable to other biofilm-related chronic inflammatory diseases.

Show MeSH

Related in: MedlinePlus

Characterization of 9-species 'subgingival' in vitro biofilm model. (A) Time flow of biofilm formation. Data are means from triplicate biofilms and gained comparatively by culture analysis (left panel), FISH with taxa-specific 16S rRNA probes (central panel), or indirect IF with species-specific mAb (right panel, only four taxa). (B) Total bacteria and all individual taxa of 64.5 h biofilms enumerated by culture (upper panel) and FISH/IF(lower panel), assessing four completely independent experiments, each performed with triplicate biofilms. Culture data are expressed as CFU per biofilm, FISH/IF data as number of bacteria per biofilm. Box colors indicate Gram-negative (red) or Gram-positive (blue) bacteria. Abbreviations: TCC, total cell count; Crec, Campylobacter rectus; Fnuc, F. nucleatum subsp. vincentii; Pgin, Porphyromonas gingivalis; Pint, Prevotella intermedia; Tfor, Tannerella forsythia; Vdis, Veillonella dispar; Anae, Actinomyces naeslundii; Sint, Streptococcus intermedius; Sora, Streptococcus oralis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2818713&req=5

Figure 1: Characterization of 9-species 'subgingival' in vitro biofilm model. (A) Time flow of biofilm formation. Data are means from triplicate biofilms and gained comparatively by culture analysis (left panel), FISH with taxa-specific 16S rRNA probes (central panel), or indirect IF with species-specific mAb (right panel, only four taxa). (B) Total bacteria and all individual taxa of 64.5 h biofilms enumerated by culture (upper panel) and FISH/IF(lower panel), assessing four completely independent experiments, each performed with triplicate biofilms. Culture data are expressed as CFU per biofilm, FISH/IF data as number of bacteria per biofilm. Box colors indicate Gram-negative (red) or Gram-positive (blue) bacteria. Abbreviations: TCC, total cell count; Crec, Campylobacter rectus; Fnuc, F. nucleatum subsp. vincentii; Pgin, Porphyromonas gingivalis; Pint, Prevotella intermedia; Tfor, Tannerella forsythia; Vdis, Veillonella dispar; Anae, Actinomyces naeslundii; Sint, Streptococcus intermedius; Sora, Streptococcus oralis.

Mentions: We generated in vitro 'subgingival' biofilms containing nine different bacterial species representative of marginal and subgingival plaque. We determined the kinetics of biofilm formation using three independent bacteria detection and enumeration assays to quantitate all biofilm members (Fig. 1A). Scraped from the surface of the hydoxyapatite discs after only 20 min of anaerobic incubation to evaluate initial adherence, the bacteria showed large quantitative inter-species differences. Campylobacter rectus accounted for nearly 90% of the cells at this time point. V. dispar, A. naeslundii, and S. oralis were in 1-10% range, whereas the other five organisms were all below 0.1% of the total CFU. FISH and IF, as optical single cell identification techniques, were not sufficiently sensitive to reliably enumerate all nine species at 20 min.


In vitro modeling of host-parasite interactions: the 'subgingival' biofilm challenge of primary human epithelial cells.

Guggenheim B, Gmür R, Galicia JC, Stathopoulou PG, Benakanakere MR, Meier A, Thurnheer T, Kinane DF - BMC Microbiol. (2009)

Characterization of 9-species 'subgingival' in vitro biofilm model. (A) Time flow of biofilm formation. Data are means from triplicate biofilms and gained comparatively by culture analysis (left panel), FISH with taxa-specific 16S rRNA probes (central panel), or indirect IF with species-specific mAb (right panel, only four taxa). (B) Total bacteria and all individual taxa of 64.5 h biofilms enumerated by culture (upper panel) and FISH/IF(lower panel), assessing four completely independent experiments, each performed with triplicate biofilms. Culture data are expressed as CFU per biofilm, FISH/IF data as number of bacteria per biofilm. Box colors indicate Gram-negative (red) or Gram-positive (blue) bacteria. Abbreviations: TCC, total cell count; Crec, Campylobacter rectus; Fnuc, F. nucleatum subsp. vincentii; Pgin, Porphyromonas gingivalis; Pint, Prevotella intermedia; Tfor, Tannerella forsythia; Vdis, Veillonella dispar; Anae, Actinomyces naeslundii; Sint, Streptococcus intermedius; Sora, Streptococcus oralis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2818713&req=5

Figure 1: Characterization of 9-species 'subgingival' in vitro biofilm model. (A) Time flow of biofilm formation. Data are means from triplicate biofilms and gained comparatively by culture analysis (left panel), FISH with taxa-specific 16S rRNA probes (central panel), or indirect IF with species-specific mAb (right panel, only four taxa). (B) Total bacteria and all individual taxa of 64.5 h biofilms enumerated by culture (upper panel) and FISH/IF(lower panel), assessing four completely independent experiments, each performed with triplicate biofilms. Culture data are expressed as CFU per biofilm, FISH/IF data as number of bacteria per biofilm. Box colors indicate Gram-negative (red) or Gram-positive (blue) bacteria. Abbreviations: TCC, total cell count; Crec, Campylobacter rectus; Fnuc, F. nucleatum subsp. vincentii; Pgin, Porphyromonas gingivalis; Pint, Prevotella intermedia; Tfor, Tannerella forsythia; Vdis, Veillonella dispar; Anae, Actinomyces naeslundii; Sint, Streptococcus intermedius; Sora, Streptococcus oralis.
Mentions: We generated in vitro 'subgingival' biofilms containing nine different bacterial species representative of marginal and subgingival plaque. We determined the kinetics of biofilm formation using three independent bacteria detection and enumeration assays to quantitate all biofilm members (Fig. 1A). Scraped from the surface of the hydoxyapatite discs after only 20 min of anaerobic incubation to evaluate initial adherence, the bacteria showed large quantitative inter-species differences. Campylobacter rectus accounted for nearly 90% of the cells at this time point. V. dispar, A. naeslundii, and S. oralis were in 1-10% range, whereas the other five organisms were all below 0.1% of the total CFU. FISH and IF, as optical single cell identification techniques, were not sufficiently sensitive to reliably enumerate all nine species at 20 min.

Bottom Line: The new model takes into account that the microbial challenge derives from a biofilm community and not from planktonically cultured bacterial strains.It will facilitate easily the introduction of additional host cells such as neutrophils for future biofilm:host cell challenge studies.Our methodology may generate particular interest, as it should be widely applicable to other biofilm-related chronic inflammatory diseases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Oral Biology, Section for Oral Microbiology and General Immunology, University of Zürich, Plattenstrasse 11, CH-8032 Zürich, Switzerland. bernie@zzmk.uzh.ch

ABSTRACT

Background: Microbial biofilms are known to cause an increasing number of chronic inflammatory and infectious conditions. A classical example is chronic periodontal disease, a condition initiated by the subgingival dental plaque biofilm on gingival epithelial tissues. We describe here a new model that permits the examination of interactions between the bacterial biofilm and host cells in general. We use primary human gingival epithelial cells (HGEC) and an in vitro grown biofilm, comprising nine frequently studied and representative subgingival plaque bacteria.

Results: We describe the growth of a mature 'subgingival' in vitro biofilm, its composition during development, its ability to adapt to aerobic conditions and how we expose in vitro a HGEC monolayer to this biofilm. Challenging the host derived HGEC with the biofilm invoked apoptosis in the epithelial cells, triggered release of pro-inflammatory cytokines and in parallel induced rapid degradation of the cytokines by biofilm-generated enzymes.

Conclusion: We developed an experimental in vitro model to study processes taking place in the gingival crevice during the initiation of inflammation. The new model takes into account that the microbial challenge derives from a biofilm community and not from planktonically cultured bacterial strains. It will facilitate easily the introduction of additional host cells such as neutrophils for future biofilm:host cell challenge studies. Our methodology may generate particular interest, as it should be widely applicable to other biofilm-related chronic inflammatory diseases.

Show MeSH
Related in: MedlinePlus