Root Canal Treatment Again Dung Enterococcus Isolation And Identification Of Stable Fluorescent Protein Markers Carrier Construction

Dental periapical periodontitis is an oral common disease, while root canaltreatment is the preferred treatment method at present. In principle perfect rootcanal therapy can promote the periapical healing, but still there is a rate of thefailure in clinical cases, the study have found that the main reason is thepersistence of the bacteria in the root canal, especially the Enterococcus faecaliswith high relevance ratio.Enterococcus faecalis is one of the Streptococcus, usually exist in humanoral cavity and enteric canal. It can invade in the dentinal tubules, tolerantcommon antibiotics such as calcium hydroxide, resist hunger and form biofilmseparately. With these special abilities, enterococcus faecalis plays an importantrole in root canal reinfection as its relevance ratio is much larger than that in primary infections. But whether the high relevance ratio has connection with thepatient’s individual factors, the teeth situation, or the duration of treatment? Isthere a different biological characteristic through different isolates? It’s worth tofind out the answer.This experiment is a research on the biological characteristicof E.f, which isolated form the root canal reinfection teeth of different patients,so that we can offer reference in order to eliminate the root canal reinfectioncaused by E.f.At present,a lot of genes was studied have a relatianshipe with biofilmformation,such as ESP、GelE、 SprE、frs. In this work, we found that bacteriabiofilm formation was significantly reduced when the bfrg gene was mutated.For better study looked at biofilm dynamic formation process, we built a stablegreen fluorescent protein expression vector to analysis the formation of E.fbiofilm. Here is the four parts of the experiment:Isolation and identification of E.fIn this study, we use high specificity E.f culture medium and agar to isolateE.f form54clinical cases and identify all E.f by16SrRNA sequencingtechnology which can prove them in gene level，then calculate relevance ratiounder the different host, different micro-environment. Among all54cases,18cases have been proved to be E.f, the relevance ratio is33%. After statisticalanalysis, there is no statistical differences in sex, age, periapical lesion in X-ray,the time since last treatment, and the length owed to fill in root canal(P>0.05).On the contrary, there is a statistical difference in the E.f relevance ratio betweenperfect root canal filling and defective one(P<0.05). All data demonstrate E.f inroot canal reinfection have a higher detection rate, especially in the defectiveroot canal filling, which remind us a good root canal filling can prevent rootcanal reinfection. Biological characteristic of E.fIn this part, we compare the bio-characteristic between the standard E.fATCC29212(control group) and the18E.f identified in test1(test group) inseveral ways:1. Test the growth curve by agar planking count method andturbidimetry method;2.Test the biofilm formation ability of each E.f byturbidimetric titration;3.Test the drug tolerance against some commonly usedantibiotics by double dilute turbidimetry method. The growth curves show eachE.f has the same growth characteristic:0-4h is the recovery phase,4-12h is theexponential phase, after12h comes the plateau phase(P>0.05),and at each timepoint bacteria concentration have no statistical differences between the twogroups by OD600(P>0.05). The test group and the control group both can formthe biofilm while there is no statistical difference between each other by OD570(P>0.05). All E.f are drug-sensitive against ampicillin, kanamycin andvancomycin and drug-tolerance against metronidazole and streptomycin, butthere is no statistical difference between each bacterium (P>0.05). All showsthat E.f ATCC29212and the18E.f have no significant difference in growthcharacteristic.Discovery of BFRG, biofilm formation related geneIn this experiment, tnptransposome transposon of EZ-Tn5(DHFR-1) wastransformed into E. faecalis to construct a mutant library of E. faecalis. Threedifferent staining methods, crystal violet (CV), Syto9and FDA, were used toscreen biofilm formation defects mutant strain. In addition, we could clone themutant gene that significantly reduced biofilm formation. We found that bacteriabiofilm formation was significantly reduced when an unknown function genewas mutated. We named the gene as biofilm formation gene (bfrg-genes). Theresults showed that, the mass and living cells of biofilms were significantly lower (more than50%) than the control group (wild-type E. faecalis). After bfrggene was mutated, the green fluorescence intensity, biofilm volume, surface area,the maximum biofilm thickness and other indicators were reduced comparedwith wild-type bacteria. However, the biofilm porosity and roughness wereincreased significantly. These results suggested that the volume and thethickness of E. faecalis biofilm formation were significantly reduced, the voidswas larger. In addition, the bfrg gene may promote biofilm formation.Building stable green fluorescent expression vector of E.fIn this part,we choose conserved genes lactate dehydrogenase in E.f astarget spot, use the commonly used suicide carrier pFW5as skeleton to buildexpression vector, and design two pairs of primer for pud, pds，then put thefluorescence clips gfp*with the promoter into pud-pds, finally build the stablegreen fluorescent expression vector with promoter pFW5-pus-pds-gfp*. Verifiedthe vector by PCR, double-enzyme cleavage method and sequencing..Preparefor the building of stable green fluorescent expression of E.f.