Experimental and Theoretical Investigation of Multispecies Oral Biofilm Resistance to Chlorhexidine Treatment

We investigate recovery of multispecies oral biofilms following chlorhexidine gluconate (CHX) and CHX with surface modifiers (CHX-Plus) treatment. Specifically, we examine the percentage of viable bacteria in the biofilms following their exposure to CHX and CHX-Plus for 1, 3, and 10 minutes, respectively. Before antimicrobial treatment, the biofilms are allowed to grow for three weeks. We find that (a). CHX-Plus kills bacteria in biofilms more effectively than the regular 2% CHX does, (b). cell continues to be killed for up to one week after exposure to the CHX solutions, (c). the biofilms start to recover after two weeks, the percentage of the viable bacteria recovers in the 1 and 3 minutes treatment groups but not in the 10 minutes treatment group after five weeks, and the biofilms fully return to the pretreatment levels after eight weeks. To understand the mechanism, a mathematical model for multiple bacterial phenotypes is developed, adopting the notion that bacterial persisters exist in the biofilms together with regulatory quorum sensing molecules and growth factor proteins. The model reveals the crucial role played by the persisters, quorum sensing molecules, and growth factors in biofilm recovery, accurately predicting the viable bacterial population after CHX treatment.