In Vitro Biofilm Formation Using Five Oral Bacterials Species

Background: Dental plaque is a biofilm formed by hundred of bacteria that adhere to tooth surfaces in the oral cavity microenvironment. The composition of this biofilm is very heterogeneous, but in general terms is divided into early and late colonizers, and its eventual composition will influence the development of caries, gingivitis or periodontitis. The utilization of in vitro models of biofilm formation will enable us to study the different bacterial interactions occurring within the biofilm and the expression of some of its virulence factors. Objective: The objective of this investigation is to develop a in vitro model that would enable the growth of a biofilm composed of oral bacteria. Materials and Methods: Actinomyces naeslundii, Streptococcus oralis, Veillonella parvula, Fusobacterium nucleatum y Porphyromonas gingivalis have been inoculated and allow to grow onto a closed anaerobic system of constant flow (artificial mouth device) and allow to adhere onto hydroxilapatite disks bathed in saliva. This growth has been assessed by means of fluorescence in situ hybridization, confocal laser microscopy and scanning electronic microscopy. Results: With confocal laser microscopy the formation of a biofilm of approximately 30 µm in thickness was evidenced after 72 hours of growth. All five species could be detected within the biofilm formation, being A. naeslundii then main species forming the biofilm base adhered to the salivary proteins. Conclusion: The formation of an in vitro biofilm composed of five oral bacteria in an anaerobic constant flow system has been demonstrated. To our knowledge this is the first report of an in vitro oral biofilm system including P. gingivalis and other four oral bacterial species.