Study on the mechanism of Shuanghuanglian injection agent effect on multi drug resistant strains of Escherichia coli resistance
|School||Beijing University of Traditional Chinese Medicine|
|Course||Traditional Chinese Medicine|
|Keywords||Multi-drug resistance Escherichia coli Shuanghuanlian Powder-Injection R plasmid β-lactamase Drug accumulation level|
In recent years, with the widely use of antibiotic in clinical, the problem of bacterial resistance became more and more serious, the spread of multi-drug resistance bacteria and extensive drug-resistant bacteria becomes a worldwide major clinical problem, it causes the increase of mortality rates and treatment costs, result in significant social and economic burden. Escherichia coli is a common clinical pathogens, the date of Ministry of Health National Antimiocrobial Resistance Investigation Net (Mohnarin) from2006to2011show that the top five were Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli was the top one and the drug-resistant strains was increasing, the date of CHINET from2006to2011show that the ESBLs producing E.coli was always greater than50%, it brings great difficulties for clinical antimicrobial therapy. Therefore,to study the countermeasures against multi-drug resistance Escherichia coli has very important significance.Chinese medicine is a great treasure, some studies show that Chinese medicine has effect to inhibite bacterial, and has been widely used in clinical treatment of infectious diseases, some traditional Chinese medicine also has the potential to reduce bacterial resistance,but the study of its mechanism was not yet in-depth.So this study want to research the action mechanism of Shuanghuanglian on multi-drug resistant Escherichia coli,and it will provide a theoretical basis and new research ideas for Chinese medicine used on resistant infections.This paper includes literature review and experimental studyThe literature review:The literature review introduced the research progress of traditional Chinese medicine antibacterial function and resistance mechanisms of Escherichia coli.The experimental Study:Objective:To explore the mechanism of Shuanghuanlian Powder-Injection on resistance of multi-drug resistant Escherichia coli by study the effect of eliminate the R plasmids, restrain the β-lactamase activity, increase the accumulation of daunomycin within multi-drug resistant Escherichia coli. and lay the foundation for the further development of anti-drug-resistant Chinese medicine.Methods:1.To take multi-drug resistant Escherichia coli from the clinical laboratory which identified by VITEK-32.2.Determine the R plasmids elimination rate of Shuanghuanlian Powder-Injection on the multidrug-resistant Escherichia coli using photolithography, then extracted the plasmids according to the plasmid extracted box operation. analysed the plasmids eliminated situation by agarose gel electrophoresis,UV gel imager.3.Extracted β-lactamase by Ultrasonic cell crushing device, quantitated protein content with the Bradford method,took the Nitrocefin as the acted substrate to determine the β-lactamase activity.4. The accumulation of daunorubicin within the bacteria treated with ShuangHuangLian powder-Injection was observed using fluorospectrophotometry.5.The effect of ShuangHuangLian powder-Injection on bacterial membrance permeability structure was observed using the transmission electron microscope.Results:1After treated with Shuanghuanglian Powder-Injection (1/2MIC12.5mg/ml、1/4MIC6.25mg/ml、1/8MIC3.125mg/ml), The eliminate rate on R plasmids of the multi-drug resistance Escherichia coli were20.4%,5.4%,0.3%.After the intervention of Shuanghuanglian Powder-Injection (1/2MIC), plasmid map have lost a plasmid bando2The β-lactamase activity of the multi-drug resistance Escherichia coli cultured by meat soup、sulbactan、Shuanghuanglian Powder-Injection(1/2MIC12.5mg/ml,1/4MIC6.25mg/ml、1/8MIC3.125mg/ml) were9.52±0.71、3.55±0.30、3.81±0.36、7.54±0.49、9.08±0.61, the result showed that the β-lactamase activity was decreased after the effect of Shuanghuanglian Powder-Injection (1/2MIC12.5mg/ml,1/4MIC6.25mg/ml, the difference was statistically significant(.P<0.05), this show that Shuanghuanglian could decrease the β-lactamase activity in a dose-dependent,the effect of Shuanghuanglian Powder-Injection (1/2MIC) was similar with sulbactam.3After treated with Shuanghuanglian Powder-Injection (1/2MIC)4,6,8h, the degree of daunorubicin accumulated in multi-drug resistance Escherichia coli was increased than control group (P<0.05), especially after6h-8h. After treated with Shuanghuanglian Powder-Injection (1/2MIC,1/4MIC,1/8MIC)6h, the degree of daunorubicin accumulated in Escherichia coli was increased than control group (P<0.05), and the degree of daunorubicin accumulated in Escherichia coli was different after treated with different concentrations of Shuanghuanglian Powder-Injection (P<0.05).4.After observed by transmission electron microscopy,multi-drug resistance Escherichia coli appeared that the split was reduced and the cytoplasm was lost after treated with Shuanghuanglian Powder-Injection.Conclusion:1. Shuanghuanlian Powder-Injection have a significant effect of eliminate the plasmids of multi-drug resistance Escherichia coli at high concentrations.2.Shuanghuanlian Powder-Injection could restrain the P-lactamase activity of multi-drug resistance Escherichia coli at high and moderate concentrations,and it has the same effect at a high concentration with sulbactam. 3. Shuanghuanlian Powder-Injection could increase the accumulation of daunomycin within multi-drug resistant Escherichia coli in a dose-dependent and time-dependent manners;4. Shuanghuanlian Powder-Injection could destroy the integrity of E.coli cell membrance.