| Objective:Enterococcus faecalis generally have the ability to form biofilm. Enterococcusfaecalis in root canal mainly exists in the form of biofilm,Which have strongerresistance to the antibiotics. Ethylenediamine tetraacetic acid (EDTA) is a kind ofmetal-chelator.EDTA is used in root canal rinses as commonly. Latest research provethat EDTA has certain antibacterial activity and the capability to enhance theantimicrobial activity of several antibiotics against planktonic and biofilm cultures[1-5]. Chlorhexidine is a broad-spectrum antibiotic and also commonly used asclinical root canal rinses. This study is designed to investigate the effect of the EDTAin combination with CHX on the Enterococcus faecalis grown in suspension andbiofilm in vitro.The purpose of the present study is to provide a theoretical basis forimproving the success ratio of root canal therapy and looking for and ideal root canalirrigation in clinical.Methods:1. Minimum inhibitory concentrations (MIC) and minimum bactericidalconcentrations (MBC) of EDTA and chlorhexidine (CHX) against Enterococcusfaecalis grown in suspension were determined using double dilution methodrespectively.Chequerboard assays[6]were used to determine synergistic,indifferent orantagonistic interactions between EDTA and CHX. To evaluate the effect of thecombination, fractional inhibitory concentration (FICI) was calculated. The followingformulae were used to calculate the FICI index. FICI=MIC of the tested drug incombination/MIC of the tested drug alone.FICI index=FICI (CHX)+FICI(EDTA).Synergy was defined as FIC index of≤0.5, while indifference was defined asan FICI index of>0.5≤4and antagonism was defined as an FICI index of>4. FICIindex was an average of three independent experiments.2. Using96-well culture plate to establish the model of bactrial biofilm.17%EDTA solution was diluted with BHI culture medium to various concentrations. Thedifferent concentrations of EDTA were divided into six groups randomly. That was1MIC EDTA for group A1,2MIC EDTA for group B1,4MIC EDTA forgroup C1,8MIC EDTA for group D1,16MIC EDTA for group E1,32MIC EDTAfor group F1, Set the G1group (BHI)as the negative control group. MTT assaywas then used to compare the antimicrobial activities of different concentration ofEDTA. The combination of different concentration of EDTA-chlorhexidine weredivided into six groups randomly too. That was1MICEDTA+0.002%CHX forgroup A2,2MIC EDTA+0.002%CHX for group B2,4MIC EDTA+0.002%CHXfor group C2,8MIC EDTA+0.002%CHX for group D2,16MICEDTA+0.002%CHX for group E2,32MIC EDTA+0.002%CHX for group F2.Setthe G2group (BHI+0.002%CHX) as the positive control group and H2group(BHI+BHI) as the negative control group. MTT assay was then used tocompare the antimicrobial activities of the combination of different concentration ofEDTA-CHX.3. Mature biofilm of Enterococcus faecalis formed on glass slides for24h.Enterococcus faecalis biofilm then was treated by EDTA、CHX or an EDTA–CHXcombination for24h. The treated biofilm was stained by SYT09／PI and examinedthrough used of CLSM to observe the bacteria staining and the bacterial activity.Results:1. In this study, EDTA’s MIC value was0.53125%,5.3125mg/ml. The valueof MBC was not detected. CHX’s MIC value was0.00125mg/ml, MBC value was0.005mg/ml. The F1hole concentration was the minimal inhibitory concentration.According to the formula, EDTA (joint) concentration was1.328125mg/ml,chlorhexidine (joint) concentration was4.88x10-7mg/ml, FICI was0.250976.2. Enterococcus faecalis biofilm absorbance between groups was compared aftertreatment of drugs for24hours. There was no statistically difference betweem sevengroups A1, B1, C1, D1, E1,F1andG1group (P>0.05)； Combination groups of A2,B2, C2,D2, E2, F2compared with G2group (positive control group) had significantdifference (P <0.05)and compared with H2(negative control group) had significantdifference (P <0.05), and Comparisons between groups failed to find a significantdifference between A2, B2, C2and D2, E2, F21(p>0.05). 3. The images of the biofilm treated by EDTA group show that the biofilm wasdense, the most bacterial was stained green fluorescence,just a little bacterial wasstained red fluorescence and the superposition graph was green (Figure10).Chlorhexidine group can be seen that the number of bacteria in the biofilm wasdecreased,the proportion of green fluorescent was decreased and the red fluorescencewas increased in the proportion,The superposition image was yellow or orange,partof the region was dark red (Figure11). EDTA-0.002%CHX combination groupimages show that the number of bacteria in the biofilm was decreased, the vastmajority of the microscope field of view was red staining bacteria, only a smallamount of green fluorescent,bacteria were scattered distribution and thesuperposition graph was red(Figure12). Negative control group can be seen that themature biofilm bacteria was dense, layered. The most bacterial was stained green andsuperposition graph was green.(Figure9).Conclusion:1. EDTA’s MIC results demonstrated weaker bacteriostatic activity on thegrowth of the Enterococcus faecalis in the planktonic culture.2. FIC index values showed a synergistic antimicrobial activity of EDTA-CHXagainst Enterococcus faecalis in the planktonic culture.3. The antibiofilm effect of EDTA was not achieved under the experimentalconditions.4. The combination of EDTA-0.002%CHX had significant activity to removeEnterococcus faecalis biofilm and was better than0.002%CHX．... |
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