Effect of the Source of Biofilm Bacteria, Level of Biofilm Maturation, and Type of Disinfecting Agent on the Susceptibility of Biofilm Bacteria to Antibacterial Agents

Introduction: Biofilm development is a dynamic process that begins with the initial attachment of planktonic bacteria to a surface, eventually leading through different stages to a mature, structurally complex biofilm. The aim of this study was to assess the effect of the source of biofilm bacteria, the level of biofilm maturation, and the type of disinfecting agent on the susceptibility of biofilm bacteria to antibacterial agents. Methods: Multispecies biofilms from plaque bacteria of 6 donors were grown for up to 8 weeks on collagen-coated hydroxyapatite disks. After 1, 2, 3, 4, and 8 weeks of growth the biofilms were exposed to 1% sodium hypochlorite, 0.2/0.4% iodine-potassium iodide, and 2% chlorhexidine for 1 and 3 minutes. The percentage of killed biofilm bacteria was determined by using LIVE/DEAD viability staining and confocal laser scanning microscopy. One-way analysis of variance was used for statistical analysis. Results: One- and 2-week-old biofilms were moderately or very sensitive to the tested disinfecting agents, which killed 20%-100% of the biofilm bacteria. After 3 weeks of growth the biofilm bacteria were more resistant to the same agents, and only 10%-30% of the bacteria were killed (P < .001). The same pattern of the effect of biofilm age (maturation) on the resistance of bacteria was observed in all 6 biofilms and with all 3 disinfecting agents. Conclusion: The change of biofilm bacteria from sensitive to resistant against disinfecting agents occurred between 2 and 3 weeks of biofilm maturation. This development took place simultaneously in all biofilms grown from plaque bacteria from 6 different donors and was independent of the type of disinfecting agent used. The results emphasize the importance of knowing the maturation timeline of each biofilm model used to test the effectiveness of endodontic disinfecting agents against biofilm bacteria.