Experimental Studies Of Enterococcus Faecalis Biofilm In Refractory Periapical Periodontitis:Molecular Mechanisms And Laser Treatment

Objective:Identification and detection of Enterococcus faecalis isolated from extraradicular biofilm in refractory periapical periodontitis, and to analyze the relationship between the occurrence of E. faecalis and clinical symptom.To elucidate the biofilm structure of during the Enterococcus faecalis biofilm formation.To Study the relationship between the expression of the genes about quorum-sensing system and Enterococcus faecalis biofilm formation.To observe the influence of photodynamic therapy and laser diode on Enterococcus faecalis biofilm, and to evaluate the feasibility of laser treatment.Methods:1.42single rooted teeth which need periapical surgery were collected, and the clinical symptoms and physical signs were recorded. Bacterium samples from extraradicular biofilm were taken. The occurrence of E.faecalis by means of culture and polymerase chain reaction was investigated. The presence of E. faecalis and clinical signs was assessed to evaluate a possible relationship between clinical findings.2. Enterococcus faecalis biofilms formed on cover glasses in6,12,24and48hours respectively. Bacteria were stained with STTO9and PI and then visualized by confocal laser scanning microscope. Quantification of biofilms was achieved by staining with Crystal Violet. 3. Enterococcus faecalis biofilms formed on cover glasses in6,12,24and48hours respectively. Real-time fluorescence quantitative PCR method detected the expression of fsrB, gelE and sprE genes in the process of Enterococcus faecalis biofilm formation.4. Establish forty-eight Enterococcus faecalis biofilms model, and divide them into four groups:PDT group, DL group, NaClO group, normal saline group.The influence of Enterococcus faecalis in biofilms was observed according to CLSM and plate counting of bacteria method.Results:1. Culture and PCR detected E. faecalis in22of42and30of42of the teeth, the detection rates was52.4%and71.4%, respectively, there was statistical significance (P<0.05); The detection rates of E.faecalis by means of PCR between the pain and the painless group demonstrated statistical significance (P<0.05).2. Enterococcus faecalis could attach to glass surface and form a structural biofilm. It was shown that a lot of vital and dead bacteria gathered densely in Enterococcus faecalis biofilm. The nature and quantity of biofilm changed within24h has statistically significant difference (P<0.05).3. The expression of fsrB、gelE and sprE about quorum-sensing system change over time during Enterococcus faecalis biofilm formation within24h, which is statistically significant (P<0.05). 4. Compared to normal saline group, PDT, DL and NaClO made the number of Enterococcus faecalis in biofilms significantly reduced (P<0.05), No difference was observed between the three groups.Conclusions:1. PCR was more sensitive than culture in detecting E. faecalis in root apex of refractory periapical periodontitis. The occurrence of E. faecalis in cases of refractory periapical periodontitis correlates with pain.2. Assess of nature and quantity,24h is the period at which the Enterococcus faecalis biofilm attains maturity. The fsrB, gelE and sprE genes are closely related to the biofilm formation in Enterococcus faecalis, which probably promote the early biofilm formation during the6-24h.3. It was demonstrated that PDT and975nm diode laser are efficient at killing Enterococcus faecalis in biofilms, and its bactericidal efficacy can comparable with NaClO.