RNAi And Micromolecules Inhibit The Multidrug Efflux Pump Of Escherichia Coli

This research work used RNA interference technology and synthesized Micromolecules of Indole derivatives as our two major methods,study on their influence in AcrAB-TolC directed and efflux-mediated multidrug resistance in Escherichia coli.We tested the Drug Sensitivity in 480 clinical strains of E.coli from pigs,chose the multiresistant ones.then,use three kind of antibiotic(Chloramphenicol, Tetracycline,Aminoglycosidc) resistant gene PCR Kit to check,and select efflux-mediated multidrug resistance strain:Fj307.we also use Chloramphenicol, artificially induced ATCC25922 to mutate,and gain a multiresistant strain:YD02. these two strains as our major material,have been checked there mufidrug sensitivity and there resistance gene.We primarily believed that they are AcrAB-TolC directed multidrug resistance Escherichia coli.After study the AcrAB-TolC efflux system of E.coli,we choose the membrane fusion protein AcrA as target,to design and synthesized 4 siRNAs:siRNA₃5,siRNA₁64,siRNA₉29,siRNA_control.After the inhibit experiment while culture, we know the activity of siRNAs,rusults:7h～10h of experiment data reflect that, siRNA group have great difference with control group in each chloramphenicol concentration(128ug/mL,64ug/mL,32ug/mL),the siRNA can inhibit the acrA expression and reverse the drug-resistance in E.coli.we examined 32ug/mL concentration groups statistical,the absorbency data in 5 different treatment have great difference totally(P＜0.01),siRNA₁64 can decrease the MIC 4 times compare to other siRNA group.We can certainly infer that siRNA₁64 have interference ability.We studied the space structure of outer membrane factor TolC,based on the amino acid Asp¹⁵³ and Tyr362 in the protein export site,we designed and synthesized indole derivatives as efflux pump inhibitors,using methyl 1 H-indole-6-carboxylate and 1H-indole-6-amine.Use Fj307,YD02 as positive control,ATCC25922,Tw1b as negative control,The activity of the compounds was evaluated.The result showed that compounds could increase the susceptibility of Escherich.coli to tetracycline,chloramphenicol,erythromycin and ciprofloxacin up to 32 times.Research on the efflux pump,not only help to elucidate the essence of antibiotic resistance,it has great value on developing new antibiotic drugs,these two parts work can provide new ideas to solve the serious problem of antibiotic resistance in stockbreeding industry,further research on this pare have a great applicative potential on preventing and cure E.coli diseases,to reduce the drug residues,and improve the safety in food product.