| The development of antibiotics and other antimicrobial agents can be regarded as the greatest achievement of modern medicine,which increases the average life expectancy of human beings.However,overuse and abuse of antibiotics has led to the resistance of microorganisms.Quinolone antibacterial agents play an important role in the clinical use of human beings and animals.But,the drug resistance of pathogens to quinolones has become increasingly serious,Especially,the resistance rate of Escherichia coli to quinolones increased year by year.Nowadays,many problems exist in the clinical treatment of quinolones,such as the large dosage and the high cost of treatment.If we can control or solve the problem of bacterial drug resistance,is undoubtedly a major achievement in the medical field,greatly improving the antibacterial effect of existing antibiotics.At present,there are many studies on bacterial resistance to bacteria,such as reserpine,verapamil,carbonyl cyanide,and so on.However,it is difficult to use in clinic because of its side effects.In recent years,it has been proved that the effective components of natural plant,such as berberine and piperine,can inhibit the activity of efflux pump.The effective ingredients of Chinese medicine is relatively safe,no resistance,small residual harm,has obvious advantages in clinical application and product development.In this study,combined with the previous laboratory studies,the main effective component of chinese tetrandrine-tetrandrine,was selected to study whether it has the effect of inhibiting the efflux of quinolones,lay the foundation for later confirmation of efflux pump inhibitors,at the same time through the preliminary inquiry of drug-resistant reversal mechanism for tetrandrine the development of triptolide efflux inhibitors provide a theoretical basis for.1?Reversal effect of tetrandrine on Escherichia coli isolated from clinical isolates(1)The effect of tetrandrine on the antibacterial activity of ciprofloxacin lactate was determined by the combined drug sensitivity test;(2)Using different concentrations of ciprofloxacin lactate HM broth to carry out the bactericidal test of drug-resistant bacteria 73-2,and to draw the bactericidal curve of ciprofloxacin lactate and the combination of tetrandrine and CCCP to 73-2;(3)The mutation rate of Escherichia coli ATCC25922 in the presence of tetrandrine was measured by using MH agar plate containing tetrandrine and ciprofloxacin lactate of different concentrations;(4)Drug resistant bacteria were detected 73-2 and Escherichia coli ATCC25922 in tetrandrine conditions by HPLC,the volume of bacteria body ciprofloxacin lactate,with CCCP as the drug control;(5)The antibiotic resistant strain 73-2 was repeatedly cultured with a LB broth containing tetrandrine to obtain a reversal of the susceptibility recovery.Results: tetrandrine combined with ciprofloxacin lactate FIC index is 0.1875,has a strong synergistic effect,the effects of powder using 160 ?g/m L tetrandrine and 10?g/ml CCCP and ciprofloxacin combined with ciprofloxacin lactate by the United Front 320 ?g/m L reduced to 10 ?g/mL on drug resistant bacteria 73-2 MIC value of Escherichia coli the MIC value had no effect on bacteria ATCC25922;Tetrandrine can improve the killing effect of ciprofloxacin lactate on 73-2,which can inhibit the growth of bacteria by 1/2MIC concentration;Under the condition of ciprofloxacin lactate concentration in 4MIC,the mutation of Escherichia coli ATCC25922 could be reduced effectively,and the mutation rate of 10-7 was reduced to 10-9;In the presence of tetrandrine,accumulation of drugresistant 73-2 cells in vivo of ciprofloxacin lactate is more significant than the normal increase,is about 4.2 times,and Escherichia coli ATCC25922 basic did not change,in the condition of CCCP,also appeared in the same volume,73-2 lactic acid ciprofloxacin resistant bacteria is about 3.9 times under normal conditions;The strain B(Tet)was obtained by lactic acid bacteria(ciprofloxacin)against the MIC strain by using the broth containing tetrandrine.Conclusion: tetrandrine can enhance the effect of ciprofloxacin to kill bacteria and inhibit the mutation of bacteria,which can increase the accumulation of ciprofloxacin in the cells of drug-resistant bacteria.Tetrandrine has the efflux inhibitor activity.2?Study on the reversal mechanism of tetrandrine against clinical isolates of Escherichia coliDetection of antibiotic resistant bacteria in normal growth and 73-2 growth in tetrandrine under the condition of drug resistant bacteria 73-2(Tet)A and the reversal of drug resistance bacteria in normal growth(Tet)expression changes of efflux pump genes acrA,acrB,tol C,acrZ and marA,rob,gene regulation acrR.Results: in the presence of tetrandrine resistant bacteria growth 73-2(Tet)and the reversal of drug resistance bacteria B(Tet)with respect to the expression of drug-resistant 73-2 efflux pump gene acrA/B/Z mRNA were significantly decreased,while the expression of B(Tet)efflux pump gene mRNA decreased more than 73-2(Tet).The expression of MarA and rob mRNA played a positive role in the regulation of efflux pump was significantly reduced.B(Tet)showed a significant increase in the expression of acrR,a negative regulator of mRNA,while the expression of 73-2(Tet)acrR was only increased in mRNA.No mutation was found in the nucleotide sequence of the B(Tet)and the 73-2 of resistant bacteria.Conclusion: tetrandrine can inhibit the expression of efflux pump gene acrA/B/Z,tolC and positive regulatory gene marA and rob in Escherichia coli,which can promote the expression of negative regulatory gene acrR.The nucleotide sequence of the efflux pump gene and the regulatory gene can not be mutated again.Tetrandrine can reverse the resistance of bacteria by affecting the expression of efflux pump.3?Effect of tetrandrine on efflux pump of AcrAB-TolC in vitroIn vitro 1/2MIC induced by ciprofloxacin resistant Escherichia coli ATCC25922 by using multistep method;To determine whether tetrandrine could reverse the drug resistance of the induced bacteria by combining tetrandrine with ciprofloxacin lactate;To detect the changes of mRNA expression in A(Cip)efflux pump gene acrA,acrB,acrZ and tolC under the condition of tetrandrine.Results: the 1/2MIC induced by A(Cip)can obtain drug-resistant genetic stability as a starting point;tetrandrine can make lactic acid ciprofloxacin on A(Cip)MIC decreased from 640 ?g/m L to 80 ?g/m L;the amount of efflux pump genes acrA,acrB,acrZ,tolC,mRNA expression in the condition of tetrandrine was reduced significantly.Conclusion: tetrandrine on drug resistant bacteria in vitro can also carry out good resistance reversal,and the main way is to reduce the expression of efflux pump AcrABTolC. |
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