| Objectives:Enterococcus faecalis is a commensal opportunistic pathogen found in the mouth,intestine and vaginal tract of humans.As an invasive pathogen in the oral cavity,E.faecalis is one of the leading causes of periapical endodontic lesions.However,due to the strong biofilm-forming capacity and tolerance of E.faecalis to conventional antibiotics and treatments,limited therapeutic options are available.In this experiment,we studied the antibacterial and biofilm removal activity of ClyR against Enterococcus faecalis,in order to explore whether ClyR can be used as a new antimicrobial for treating E.faecalis infection in oral cavity.Methods:(1)The ClyR was iuduced and expressed in vitro in E.coli BL21 by His Trap FF column affinity chromatography;(2)The influence of ClyR with time and concentration on Enterococcus faecalis was quantitatively determined by gradient dilution plate counting method;(3)By monitoring the decrease of turbidity,the bactericidal stability of ClyR in different EDTA,p H and bactericidal spectrum were determined by the Synergy H1 microplate reader;(4)The bactericidal pattern of ClyR was visualized by transmission electron microscope(TEM)section;(5)The degradation of biofilms by ClyR was quantitatively and qualitatively evaluated by crystal violet test and dilution plate counting method;(6)Scanning electron microscopy and confocal microscopy was used to characterize the morphologic and structural changes of ClyR against Enterococcus faecalis biofilms;(7)Removal effect of ClyR against Enterococcus faecalis biofilms was conducted by dental model in vitro;(8)Cytotoxicity of ClyR to periodontal ligament stem cells was determined by Cytotoxicity Test(CCK-8).Results:(1)ClyR protein with high concentration and purity can be expressed by the purification of nickel column and the endotoxin content was less than 1EU/ml.(2)ClyR had a robust and rapid cracking activity against Enterococcus faecalis in a time-and dose-dependent manner.(3)The enzymatic activity of ClyR showed that even EDTA up to 800 μM,ClyR still kept high activity;ClyR showed optimum p H of 5-11;ClyR was active against standard strain and multiple isolates of Enterococcus faecalis.(4)TEM results showed that the ratio of the number of cells with intact cell wall to the number of ruptured cells declined along with increasing concentration of ClyR,and the observation “ghost cells” is quite consistent with the phenomenon of lysin-mediated osmotic cell lysis reported elsewhere.(5)ClyR could effectively degrade E.faecalis biofilms in a dose-dependent manner.(6)The microscopic results show that ClyR leads to the degradation of mature biofilm by direct cracking of bacteria in biofilm.(7)In vitro tooth model,the removal effect of ClyR on biofilm was remarkable,and the killing rate of living bacteria in biofilm was more than 90% at low dose of 50 μg/m L,which was obviously superior to ampicillin.The effect is similar to that of calcium hydroxide,which is commonly used in the treatment of dental pulp disease.(8)Cytotoxicity test(CCK-8)showed that ClyR,as high as 1000 ug/ml,had no obvious cytotoxicity after incubation with periodontal ligament stem cells for 24,72 hours.Conclusion:By detecting the bactericidal activity of ClyR on Enterococcus faecalis,ClyR has the potential to treat dental pulp infection caused by Enterococcus faecalis. |
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