The Mechanism Of Gegen Qinlian Decoction Intervening The Drug Resistance Of Escherichia Coli

Objective:In this research,we collected stool samples from children with clinical damp-heat diarrhea,and used drug-resistant Escherichia coli as the research object to analyze the antibiotic resistance spectrum of children's stool-derived Escherichia coli.In this study,Gegen Qinlian Decoction,a classic traditional Chinese medicine prescription,was mainly used for quorum sensing(QS)and biofilm(bacterial biofilm,BBF)to analyze the intervention effect of Gegen Qinlian Decoction on drug-resistant Escherichia coli biofilm and the correlation between biofilm and bacterial quorum sensing system,to explain the mechanism of action of Gegen Qinlian Decoction on drug-resistant Escherichia coli at the molecular level.Methods:1 Isolation,identification and drug sensitivity test of drug resistant Escherichia coliChildren's stool-derived Escherichia coli was isolated,purified,identified and tested for drug resistance spectrum.The minimum inhibitory concentration(MIC)was determined by the micro broth dilution method,the minimum bactericidal concentration(MBC)was determined,and the Gram stain was determined.(Quick method).2 Morphological observation of drug resistant Escherichia coli biofilm(BBF)To observe the morphological changes of Gegen Qinlian Decoction on children's fecal-derived drug-resistant Escherichia coli biofilm formation,by screening of strong film-forming strains by crystal violet staining method,by using fluorescein isothiocyanate-labeled concanavalin A(FITC-Con A)and pyridine iodide(PI)fluorescent staining,by using laser scanning confocal microscope(LSCM)to observe the experimental method3 Effect of Gegen Qinlian Decoction on quorum sensing system(QS)of drug resistant Escherichia coliReal-time quantitative PCR(RT-PCR)was used to analyze whether the effect of Gegen Qinlian Decoction on the biofilm of fecal-derived drug-resistant Escherichia coli in children is related to the quorum sensing system.Results:1 Susceptibility testing of children's stool-derived Escherichia coli isolates was carried out,including,aminoglycosides,carbapenems,cephalosporins,penicillins,?-lactam/?-lactama se inhibitors There are 21 kinds of antibiotics in 11 categories including complexes,quinolones and tetracyclines.The resistance rate of the isolated strains to 21 antibiotics was6%?86%,and the proportion of multidrug resistant bacteria strains was as high as 88%.The resistance rate of cephalosporin antibiotics was 56.5%(CAZ% 18%,CZ% 74%,FEP%66%,CTX% 68%),and the resistance rate of quinolones antibiotics was about 46.7%(CIP% 44%,LVX 46%,MXF 50%).The isolated strains have low resistance rates to amikacin and carbapenem antibiotics(0% and 8%).The highest MIC values of cefazolin,cefepime and ampicillin against ESBL phenotype Escherichia coli are all >16?g/ml,and the highest MIC value of cefotaxime against ESBL phenotype Escherichia coli is >32?g/ml,The highest MIC values of gentamicin,tetracycline and amoxicillin-clavulanic acid for ESBL phenotype Escherichia coli are all >8?g/ml,and trimethoprim/sulfamethoxazole for ESBL phenotype Escherichia coli The highest MIC value of Helminthosporium is >32?g/ml,the highest MIC values of ciprofloxacin and moxifloxacin are >2?g/ml and >4?g/ml,respectively.The highest MIC value of colistin is 1?g/ml,all showing high Horizontal resistance.2 The morphological changes of children's fecal-derived drug-resistant Escherichia coli biofilm were observed under a laser confocal microscope.The green fluorescence gradually increased after 48 hours of drug action,and the red signal began to gather in small clusters,indicating biological The membrane has gradually increased and is relatively stable.After clinically isolated drug-resistant Escherichia coli were treated with different concentrations of Coptis and Scutellaria,their adhesion was reduced and the morphology of the bacteria changed.3 After the clinical isolates were treated with 1/4MIC(400?g/ml)of Scutellaria baicalensis,the mRNA expression of 5 genes hflX,LuxS,motA,pfS,and sdiA was down-regulated.Combined with the results of confocal microscope observation and RT-PCR,1/4MIC(400?g/ml)Scutellaria baicalensis is the optimal concentration to inhibit biofilm.After the strain was treated with 1/4MIC(400?g/ml)Coptis,the mRNA expression of pfS,motA,and hfl A were all down-regulated,and the expression of fitsQ,hflX,omp A,and sdiA were up-regulated to varying degrees..After 1/8MIC(200?g/ml)Scutellaria baicalensis Georgi and Coptis chinensis treated,the mRNA expression of genes was up-regulated.However,under laser confocal microscopy,1/8MIC(200?g/ml)Scutellaria baicalensis Georgi and Coptis Coptidis can inhibit the formation of biofilms.Conclusion:1 Clinical isolation and identification of Escherichia coli showed a high proportion of multi-drug resistant bacteria strains.Among them,the resistance rate of cephalosporin antibiotics and quinolone antibiotics is relatively high,56.5% and 46.7%,respectively.Children's antibiotic resistance is severe.2 Coptis and Scutellaria have an inhibitory effect on the biological formation of fecal-derived drug-resistant Escherichia coli in children.Scutellaria baicalensis has a stronger inhibitory effect on biofilm formation than Coptis at the same concentration.Scutellaria baicalensis can inhibit biofilm formation under the same drug.Effect,the greater the concentration of the drug,the stronger its effect.Combined with laser confocal microscopy and RT-PCR,1/4MIC(400?g/ml)Scutellaria baicalensis was the best inhibitory concentration for biofilm formation.3 1/4MIC(400?g/ml)Scutellaria baicalensis inhibits the biofilm formation of drug-resistant Escherichia coli by down-regulating the mRNA expression of 5 genes hflX,LuxS,motA,pfS,and sdiA to inhibit LuxS/AI-2 mediated the synthesis of AI-2 signal molecules in the interspecific QS system.1/4MIC(400?g/ml)Rhizoma Coptidis inhibits the biofilm formation of drug-resistant Escherichia coli.Its mechanism is to inhibit the two-component system qse BC closely linked to the flagella drive of Escherichia coli,thereby reducing AI-2 production.1/8MIC(200?g/ml)Scutellaria baicalensis Georgi and Coptidis scutellariae have up-regulated mRNA expression levels,but laser confocal microscopy shows that 1/8MIC(200?g/ml)Scutellaria baicalensis Georgi and Coptidis can inhibit the formation of biofilms.After RT-PCR,it is considered that it may be related to other mechanisms,and its possible mechanism of action can be studied from other angles in the future.