Effects Of Matrine On The Regulation Of AcrAB-TolC In Drug Resistance

Objective: To study the effect of matrine on thebiofilm formation of multiple resistant Escherichia coli and to explore the molecular regulation mechanism of matrine on multiple resistant Escherichia coli,then investigates whether matrine would regulate the efflux pump AcrAB-TolC of Escherichia coli,so as to provide new ideas for clinical treatment of multidrug-resistant bacteriaMethods: Escherichia coli ATCC25922 and multipledrug resistant Escherichia coli(levofloxacin,penicillin,tetracycline,cefixime,etc.)obtained from the laboratory of the First Affiliated Hospital of Guangxi Medical University.The minimum inhibitory concentration(MIC)of matrine and levofloxacin in multipledrug resistant E.coli and E.coli ATCC25922 was measured by two times dilution method.Crystal violet technology was used to compare the formation of biofilms and the morphological characteristics of biofilms under scanning electron microscope(SEM).The expression levels of AcrB,MarA,AcrR and RamA genes in different groups were measured by fluorescence quantitative PCR.Results:(1)The results obtained by the two culture dilution method were as follows: In multiple drug-resistant E.coli the MIC of levofloxacin is 64mg/ml and the MIC of matrine is 4.096mg/ml;In E.coli ATCC25922 the MIC of levofloxacin is 0.16ug/ml and MIC of matrine is 4.096mg/ml.The effect of matrine on the growth of E.coli showed that 1/16 MIC had no effect on the growth of multipie drug-resistant E.coli(P>0.05).(2)Under the effect of matrine in 1/16 MIC.the MIC of levofloxacin of multiple resistant E.coli was 0.5mg/ml;Under the scanning electron microscope,it was found that the biofilms formed by 1/2MIC matrine,1/4MIC matrine and 1/8MIC matrine were scattered and fragmented in the multi resistant E.coli,and the membrane formed was less.The blank group,1/16 MIC matrine,1/2MIC levofloxacin combined with 1/16 MIC matrine group,1/4MIC levofloxacin combined with 1/16 MIC matrine group,1/8MIC levofloxacin combined with 1/16 MIC matrine group,1/16 MICmatrine group,1/2MIC levofloxacin group,1/4MIC levofloxacin group,and 1/8MIC levofloxacin group could see the biofilm in the cloud,large and thick membrane.(3)After adding different concentrations of levofloxacin in multiple resistant E.coli,the formation of biofilms could be induced compared with the blank group or 1/16 MIC matrine,and there was a positive correlation with the concentration of levofloxacin;However,after the combination of levofloxacin and 1/16 MIC matrine,the formation of biofilm was less than that of the same concentration of levofloxacin(P<0.05).The results were found in fluorescence quantitative PCR:The blank group of multiple drug-resistant E.coli was compared with the blank group of E.coli ATCC25922,the expression of AcrB,MarA and RamA increased,and the expression of AcrR decreased.Multiple drug-resistant E.coli,1/2MIC matrine,1/4MIC matrine and 1/8MIC matrine could inhibit the expression of AcrB?MarA and RamA,and promote the expression of AcrR.In multiple drug-resistant E.coli,the expression of AcrB,MarA and RamA increased with the addition of different concentrations of levofloxacin,and the expression of AcrR decreased,which was positively correlated with the concentration of levofloxacin in a certain range.However,different concentrations of levofloxacin combined with 1/16 MIC decreased the level of AcrB,MarA and RamA,and the expression of AcrR increased.Conclusions:(1)The appearance of multidrug-resistant bacteria is related to the expression level of AcrAB-TolC.(2)The active efflux pump AcrAB-TolC is related to the formation of the biofilm of bacteria,in addition to expelled the substances that enter the bacteria,the up-regulated expression of AcrAB-TolC is more likely to form biofilm that further produce drug resistance.(3)Matrine increased the AcrR,inhibited MarA and RamA,whichreduced the expression of Gram-negative bacteria AcrAB-TolC and decreased the function of the drug efflux.(4)AcrAB-TolC is not a major factor affecting matrine entry into E.coli.(5)In multiple drug-resistant bacteria,the expression of the AcrAB-TolC efflux pump can be induced when when given a certain concentration of levofloxacin.When levofloxacin is combined with low dose matrine,both of them have synergistic effects,the expression of the AcrAB-TolC efflux pump is inhibited,the biofilm formation is reduced,and the multidrug-resistant bacteria are sensitive to the antibiotics,a new treatment for the clinical treatment of drug-resistant bacteria is proposed.