Objectives: Most in-vitro studies on the antibacterial effects of antiseptics are performed on planktonic bacteria in monocultures, so far. However, this does not reflect the situation in the oral cavity with different bacteria symbiotically living in biofilms. Therefore, the aim of this study was to establish an in-vitro polymicrobial biofilm model which better simulates the oral conditions but keeps the advantage of in-vitro testing, namely the well-controlled experimental conditions.
Methods: three bacterial strains, which are involved in the different phases of oral biofim formation (Actinomyces naeslundi (An; T14V), Fusobactrium nucleatum (Fn; ATCC 10953) and Enteroccus faecalis (Ef; ATCC 29212),) have been selected. Biofilm formation was studied on a polystyrol substrate from 96-well tissue culture plates under static anaerobic conditions using two nutrient media (BHI medium (BD, USA) and artificial saliva (Pratten et al., 2000) with 1g/l sucrose. Growth was determined separately for each bacterial strain after incubation periods of up to 72 hours using quantitative real-time PCR (Light Cycler, Roche) in n=12 independent experiments. The amount of living bacteria was determined using a live/dead stain (Biofilm Viability Kit, Invitrogen). The extracellular polymeric matrix (EPS) was visualized by Concanavalin A in a confocal laser microscope.
Results: No bacterial adhesion was observed with BHI medium. However, adhesion and propagation of the bacterial strains with increasing incubation time was observed with the artificial saliva formulation. An and Ef showed significantly higher growth rates than Fn. Live/dead stain revealed a median of 49,9% (range: 46%-53%) live bacteria after 72 hours incubation and confocal laser microscopy showed a three-dimensional EPS containing structure.
Conclusions: Selecting three relevant bacteria for intraoral biofilm formation we could establish a three-dimensional in-vitro biofilm model with adhering bacteria and extracellular matrix formation. The suitability of this model must be further evaluated testing antimicrobials with known activities.