Colonisation of gingival epithelia by subgingival biofilms in vitro: Role of red complex bacteria

Objectives: Biofilm formation on tooth surface results in colonisation and invasion of the juxtaposed gingival tissue, eliciting strong inflammatory responses that lead to periodontal disease. This in vitro study investigated the colonisation of human gingival multi-layered epithelium by multi-species subgingival biofilms, and evaluated the relative effects of the red complex species (Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola). Methods: The grown biofilm consisted of Fusobacterium nucleatum, Campylobacter rectus, Veillonella dispar, P. gingivalis, Prevotella intermedia, T. forsythia, T. denticola, Actinomyces oris, Streptococcus anginosus and Streptococcus oralis, or its variant lacking the red complex. After 48 h in co-culture with the gingival epithelia, the bacterial species in the biofilm were quantified, whereas their localisation on the cell surface was investigated by combining confocal-laser scanning microscopy (CLSM) and fluorescence in situ hybridisation (FISH), as well as by scanning electron microscopy (SEM). Results: Exclusion of the red complex quantitatively affected S. oralis, but not other species. The red-complex species were all able to colonise the gingival epithelial cells. A co-localisation trend was observed between P. gingivalis and T. denticola, as determined by FISH. However, in the absence of all three red complex bacteria from the biofilm, an immense colonisation of streptococci (potentially S. oralis) was observed on the gingival epithelia, as confirmed by both CLSM and SEM. Conclusions: While the red complex species synergise in colonizing gingival epithelia, their absence from the biofilm enhances streptococcal colonisation. This antagonism with streptococci reveals that the red complex may regulate biofilm virulence, with potential implications in periodontal pathogenesis. (C) 2014 Elsevier Ltd. All rights reserved.