Effect Of Efflux Pump-inhibitors On Photodynamic Therapy Inhibition Of The Cariogenic Bacteria In Dental Plaque Biofilms

objective:Recently, a new therapy that using laster to activate photosensitizer and make photochemical effect inhibit pathogenic microorganisms named photodynamic therapy was found. PDT is effective in the treatment of bacterial disease without generating antibiotic resistance. Because of its unique antibacterial advantage PDT is getting more and more attention of all scholars.Recent studies have found that the structure of photosensitizer is similar with antibiotics. Photosensitizer may combind with bacterial efflux pump-specific binding sites to be its substrate. Through this pathway it changed concentration of the photosensitizer in bacteria and affected PDT effects.In this study we established the dental plaque biofilm model using mixture of bacterial suspension containing proteusbacillus vulgaris, Streptococcus sanguis, lactobacillus acidophilus and Actinomyces viscosus. We designed experiments to study how the microbial efflux pump inhibitor (EPI) affected PDT inhibition of cariogenic bacteria in dental plaque biofilm.Methods:1、Plate counting test mixture of bacterial suspension contained streptococcus mutans, streptococcus, eosinophilic lactobacillus and blood viscosity actinomycetes strains. to establish the dental plaque biofilm model.2、Set up dental plaque biofilm model Experiments are performed using streptococcus mutans, streptococcus, eosinophilic lactobacillus and blood viscosity actinomycetes strains.3、We observed the changes of biofilm structure with confocal laser microscope after staining with FITC-ConA and propyl iodide organism (propidine iodide, PI).Results:1、The results of ionizing mixed bacteria suspension experiment plate colony count displayed that the optimal concentration for verapamil was25μg/ml. Experimental conditions (HMME:40μg/ml; wavelength:532nm, the power density:0.14W/cm2, semiconductor laser continuous illumination of the90s).2、The results of mixed bacteria plaque biofilm experiment in vitro were as follows.The bacteriostatic effect of PDT was obvious. Compared to saline-treated group, in artificial caries model of only PDT group, the number of living cariogenic bacteria (CFU/mL) in plaque biofilms reduced significantly (P<0.05). The bacteriostatic rate was95.26%. CLMS showed that the intensity of green fluorescence in this group decreased significantly. The living bacteria in plaque biofilms were in low level and isolated and scattered dispersed state.Verapamil inhibited PDT bacteriostatic effets. Comperesd to PDT treated group, there was a significant upward trend in the number of living cariogenic bacteria in PDT combined verapamil treated group. Bacterial population significantly increased (P<0.05), and the sterilization rate was80.92%. The resluts of laser confocal microscopy showed the intensity of green fluorescence in this group increased significantly, it indicated bacteria activity increased significantly. In the group treated with verapamil before the photosensitizer, the intensity of green fluorescent increased obviously. And it looked cloud-like indicating that the bacterium aggregate together. The amount and activity of living bacterium increased significantly.Conclusions:1、PDT effectively inhibited the growth of cariogenic bacteria to make a role in anti-caries.2、Verapamil which was bacteria efflux pump inhibitor inhibited the effects of PDT. But the mechanism of photosensitizer which enter the bacteria bacterial efflux pump may be complicated. It still needs further study.