| Objective:Oleanolic acid(OA)is an oleanane-type pentacyclic triterpene compound whose basic structure is isopropylene diene with 30 carbon atoms and its chemical formula is C30H48O3.Can exist in free form,can also exist in the form of glycosides bound to glycosides,with a safe pharmacological effects,low toxicity,side effects and fewer adverse reactions.It has been suggested that the mechanism of action of oleanolic acid on Enterococcus faecalis may be related to the autolysis of Enterococcus faecalis.However,the effect of oleanolic acid on the enzyme activity and autolytic enzyme synthesis of Enterococcus faecalis is still unclear;and whether oleanolic acid can induce the formation of resistant strains of Enterococcus faecalis and the resistance gene in bacteria will occur What kind of change is not yet clear.In this experiment,Enterococcus faecalis was used as a research object to study the effect of oleanolic acid on the growth of Enterococcus faecalis over time,and the effect of changes in the autolysis curve caused by changes in the autolytic enzymes of Enterococcus faecalis,using oleanolic acid.Screening for Enterococcus faecalis-resistant strains,and detecting changes in major drug-resistance genes in drug-resistant strains,exploring the antibiotic resistance mechanism of Enteroboccus faecalis,in order to provide oleanolic acid as a root canal disinfectant drug The theoretical basis provides a reference for clinical rational use of root canal irrigation fluid and rational drug use.Methods:1.10 groups of different concentrations of oleanolic acid DMSO solution according to the volume ratio of 5% DMSO was added to the BHI solution to prepare drug-containing BHI culture medium,the concentration of oleanolic acid in the medium were: 256 mg / m L,128 mg / m L,64 mg / m L,32 mg / m L,16 mg / m L,8 mg / m L,4 mg / m L,2 mg / m L,1 mg / m L,0.5 mg / m L.The drug-containing BHI broth in accordance with the concentration gradient in decreasing order added 24-well plates,each well by adding 3m L drug-containing broth,and then inoculated with equal concentrations of recovered Enterococcus faecalis bacteria,anaerobic culture for 24 hours,And the concentration of the drug corresponding to the last clear culture hole visible to the naked eye was taken as the minimum inhibitory concentration(MIC)of oleanolic acid to Enterococcus faecalis.2.Based on the minimum inhibitory concentration(MIC)of the oleanolic acid obtained in Experiment 1 on Enterococcus faecalis,five groups were set up:(1)1 / 2MIC + Enterococcus faecalis group,(2)MIC + Enterococcus faecalis Liquid group,(3)MIC + Enterococcus faecalis group,(4)DMSO + BHI + Enterococcus faecalis group(solvent control group),(5)BHI + Enterococcus faecalis group(blank group).In accordance with the above five groups preparation of drug-containing BHI culture medium,and then inoculated with Enterococcus faecalis 37 ℃ anaerobic box culture,the use of microplate reader measured OD600 wavelength OD value every 1 hour for a total of 16 hours,recording each group of bacteria OD value,using spss17 statistical analysis of data,calculate the mean ± standard deviation,drawn into a three-line table,and depicts the growth curve of 16 hours Enterococcus faecalis.3.The design experiment was divided into three groups,the first group: 1/4 MIC group(1 / 4MIC + DMSO + BHI + Enterococcus faecalis),the second group: solvent control group(DMSO + BHI + Enterococcus faecalis)Blank control group(BHI + Enterococcus faecalis).Bacteria were cultured for 5 hours to reach the logarithmic phase according to the above experiment 2 method.The autolysis of E.faecalis was induced by using 0.1% Triton X-100 and Tris-Cl with p H = 7.5 to induce enterococcus faecalis autolysis.The OD value was measured every hour.The autolysis rate was calculated according to the autolysis rate formula.After analysis,draw the autolysis curve.Enterococcus faecalis resistant oleanolic acid bacteria strains.Enterococcus faecium obtained from experiment 1 MIC value of oleanolic acid preparation of drug-sensitive solid medium,the use of 1 / 4MIC oleanolic acid bacteria BHI solid Enterococcus faecalis strains,once a day,a total of 4 days after entering the next A concentration of 1 / 2MIC oleanolic acid bacteria BHI solid culture E.faecalis,once a day,a total of 4 times after entering the MIC of oleanic acid bacteria BHI Enterococcus faecalis strain culture.And so on,each increase in a concentration gradient in case of bacteria is difficult to grow or reproduce the phenomenon of return to a concentration gradient again repeated until a new higher concentration gradient in E.faecalis can survive,and can produce a single Colonies consistent form up,but not more than 10 generations of training.According to the above method,the concentration of each group gradually increased,respectively: 1 / 4MIC,1 / 2MIC,MIC,2MIC,4MIC,8MIC,16 MIC,32MIC until 32 MIC medium was cultured to grow a single colony,using BHI liquid Cultivation of resistant strains in culture medium-80 ℃ freezer kept frozen.4.According to GENBANK,the reference sequences of seven resistant strains of Enterococcus faecalis were searched,Primer Premier 5 sequence analysis software was used to design the primers,and the best primer sequences were selected to synthesize the primers.The trizol kit was used to extract the Enterococcus faecalis group and Enterococcus faecium aldose-resistant group DNA.The target gene was amplified using primers and the amplified target DNA fragment was sequenced.The two bacterial resistance genes were compared Change.Results:1.The minimum inhibitory concentration(MIC)of oleanolic acid in inhibiting E.faecalis growth and reproduction in this experiment was 16 μg / m L.2.In this study,E.faecalis strains were found to have no bacterial growth within 1 to 16 hours in the oleanolic acid MIC group and the 2MIC group,reaching logarithmic phase in the low concentration group(1 / 2MIC group)9h after inoculation,solvent control group(DMSO + Bacterial liquid group)and blank control group(BHI + Bacterial liquid group)began to logarithmic phase 4h after bacterial inoculation,reached stable phase in 7h.Note 1 / 2MIC oleanolic acid concentration of oleanolic acid can still grow,but containing 1 / 2MIC oleanolic acid BHI solution will make Enterococcus faecalis need more time to reserve the required breeding enzyme,energy and Metabolic intermediates,therefore,can inhibit the proliferation of bacteria in a short period of time,thus delaying the logarithmic time,but no antibacterial effect.In addition,solvent control compared with the blank control group we can see: DMSO on bacterial growth has little effect.3.Oleanolic acid on Enterococcus faecalis autolysis curve significantly improve the autolysis rate of Enterococcus faecalis more than doubled.In the BHI agar medium containing 32 times MIC oleanolic acid,Enterococcus faecalis can grow a single colonies in consistent form,and E.coli strains resistant to Enterococcus faecalis can be screened successfully.4.Oleanolic acid had a significant effect on the autolysis curve of Enterococcus faecalis,and the autolysis rate of Enterococcus faecalis could be increased more than one time after 1.5 hours of autolysis induction,reaching the plateau within 1.5-3 hours.5.Enterococcus faecalis five resistance genes,including tet M,erm B,aac(6 ’)-aph(2’),ant(6)-1,aph(3 ’)-III occur gene mutations.A frameshift mutation occurred at the bases of the first site and the 459 th and 460 th sites of the Tet M gene.The gene aac(6’)-aph(2’)undergoes a base substitution mutation at the second position and a base shift pattern mutation occurs between the 6,7,and 192 positions.The erm B gene was frame-shifted at positions 9,12,13,and 20,and base substitutions occurred at positions 2,5,and 6.An insertion mutation occurred from position 593,and a total of 12 base pairs were inserted.The gene ant(6’)-I had point mutations at the 2nd and 4th sites,and base substitution mutations occurred at the 7th,8th,and 9th sites,at the 10 th and 25 th and 26 th sites.A frameshift mutation occurred in the base,and an insertion mutation occurred after the 556 site and a total of 12 bases were inserted.The aph3’-III gene has undergone frameshift mutations before the 1st,3rd,and 16 th sites.There are many peaks at different positions in the peak map of TEM gene sequencing,which makes it difficult to determine the base.The detection results are inaccurate and have no reference value.Conclusion: Oleanolic acid has a good antibacterial effect on Enterococcus faecalis,and the oleanolic acid below the minimum inhibitory concentration can also inhibit the growth of Enterococcus faecalis for a short time,causing the growth curve of Enterococcus faecalis to move to the right.Oleanolic acid can significantly increase the autolysis rate of Enterococcus faecalis The five drug resistance genes of Enterococcus faecalis including oleosin,tet M,erm B,AAC(6 ’)-APH(2’)-1,APH(3 ’)-III all have gene mutations. |
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