Development and Experimental Validation of a Model for Oral Multispecies Biofilm Recovery after Chlorhexidine Treatment IMI PREPRINT SERIES

Short title: Biofilm resistance to chlorhexidine ¤ These authors contributed equally to this work. Abstract We combined experiments and mathematical modeling to study the recovery of oral multispecies biofilms following antimicrobial treatment, and further utilized mathematical modelling to explore the dynamics of the tolerance mechanisms of biofilms. Specifically, we investigated the proportion of viable bacteria in multispecies biofilms over time after exposure to chlorhexidine gluconate (CHX) or CHX with surface modifiers (CHX-Plus). The oral multispecies biofilms were grown for three weeks and then treated with 2% CHX or CHX-Plus for up to 10 minutes. Confocal laser scanning microscopy showed that CHX-Plus killed bacteria in biofilms more effectively than the regular 2% CHX. Cell death continued to increase for up to one week after exposure to the two CHX solutions. Two weeks after the CHX treatment, the biofilms had started to recover. Five weeks later, the proportion of the viable bacteria had recovered in the 1 and 3 minutes treatment groups but not after the 10 minutes treatment. The number of viable bacteria in all biofilms treated with the two CHX solutions returned to the pretreatment level eight weeks after exposure. To elucidate the mechanism, a new mathematical model for multiple bacterial phenotypes was developed to monitor the live and dead bacterial populations as well as the volume fraction of extracellular polymeric substances. The model adopted the notion that bacterial persisters exist in biofilm, which can survive CHX treatment. The model was then used to predict the viable bacterial population present after CHX treatment, which revealed the crucial role of not only by quorum sensing (QS) but also by persister cells in bringing about biofilm recovery. The present study indicates that the formation of recalcitrant oral biofilms probably leads to difficult-to-treat root canal infection, and provides insight into the kinetics of the bacterial persisters and the behavior of QS molecules in multispecies biofilms after antimicrobial treatment.