Oral bacteria possess the ability to form biofilms on solid surfaces. After the penetration of oral bacteria into the pulp, the contact between biofilms and pulp tissue may result in pulpitis, pulp necrosis and/or periapical lesion. Depending on the environmental conditions and the availability of nutrients in the pulp chamber and root canals, mainly Gram-negative anaerobic microorganisms predominate and form the intracanal endodontic biofilm. The objective of the present study was to investigate the role of different substrates on biofilm formation as well as the separate and collective incorporation of six endodontic pathogens, namely Enterococcus faecalis, Staphylococcus aureus, Prevotella nigrescens, Selenomonas sputigena, Parvimonas micra and Treponema denticola into a nine-species "basic biofilm". This biofilm was formed in vitro as a standard subgingival biofilm, comprising Actinomyces oris, Veillonella dispar, Fusobacterium nucleatum, Streptococcus anginosus, Streptococcus oralis, Prevotella intermedia, Campylobacter rectus, Porphyromonas gingivalis, and Tannerella forsythia. The resulting endodontic-like biofilms were grown 64 h under the same conditions on hydroxyapatite and dentin discs. After harvesting the endodontic-like biofilms, the bacterial growth was determined using quantitative real-time PCR, were labeled using fluorescence in situ hybridization (FISH) and analyzed by confocal laser scanning microscopy (CLSM). The addition of six endodontic pathogens to the "basic biofilm" induced a decrease in the cell number of the "basic" species. Interestingly, C. rectus counts increased in biofilms containing E. faecalis, S. aureus, P. nigrescens and S. sputigena, respectively, both on hydroxyapatite and on dentin discs, whereas P. intermedia counts increased only on dentin discs by addition of E. faecalis. The growth of E. faecalis on hydroxyapatite discs and of E. faecalis and S. aureus on dentin discs were significantly higher in the biofilm containing all species than in the "basic biofilm". Contrarily, the counts of P. nigrescens, S. sputigena and P. micra on hydroxyapatite discs as well as counts of P. micra and T. denticola on dentin discs decreased in the all-species biofilm. Overall, all bacterial species associated with endodontic infections were successfully incorporated into the standard multispecies biofilm model both on hydroxyapatite and dentin discs. Thus, future investigations on endodontic infections can rely on this newly established endodontic-like multispecies biofilm model.
Keywords: FISH; endodontic pathogens; endodontic-like multispecies biofilm; pulp necrosis; pulpitis; quantitative real-time PCR (qPCR), CLSM.