Development of a Multispecies Biofilm Community by Four Root Canal Bacteria

Introduction: The development of multispecies biofilm models are needed to explain the interactions that take place in root canal biofilnns during apical periodontitis. The aim of this study was to investigate the ability of 4 root canal bacteria to establish a multispecies biofilm community and to characterize the main structural, compositional, and physiological features of this community. Methods: Four clinical isolates isolated from infected root canals, Actinomyces naeslundii, Lactobacillus saliva rius, Streptococcus gordonii, and Enterococcus faecalis, were grown together in a miniflow cell system. Simultaneous detection of the 4 species in the biofilm communities was achieved by fluorescence in situ hybridization in combination with confocal microscopy at different time points. The LIVE/ DEAD Bac Light technique (Molecular Probes, Carlsbad, CA) was used to assess cell viability and to calculate 3dimensional architectural parameters such as biovolume (mu m(3)). Redox fluorescence dye 5-cyano-2,3-ditoly1 tetrazolium chloride was used to assess the metabolic activity of biofilm bacteria. Results: The 4 species tested were able to form stable and reproducible biofilm communities. The biofilms formed in rich medium generally showed continuous growth over time, however, in the absence of glucose biofilms showed significantly smaller biovolumes. A high proportion of viable cells (>90%) were generally observed, and biofilm growth was correlated with high metabolic activity of cells. The community structure of biofilms formed in rich medium did not change considerably over the 120-hour period, during which E. faecalis, L. salivarius, and S. gordonii were most abundant. Conclusions: The ability of 4 root canal bacteria to form multispecies biofilm communities shown in this study give insights into assessing the community lifestyle of these microorganisms in vivo. This multispecies model could be useful for further research simulating stresses representative of in vivo conditions. (J Endod 2012;38:318-323)