Study On Multi-drug Resistance Mediated By Active Efflux System AcrAB-TolC In Escherichia Coli

Escherichia coli is major pathogenic bacterium in fowls and livestocks. Active efflux system (efflux pump) is major mechanism that causes multiple resistance of Escherichia coli. AcrAB-TolC is the most important efflux pump. It has many substrates, this system can efflux out an extraordinarily wide variety of antibiotics, chemotherapeutic agent, detergent and dyes, its high standard expression shows tolerance against many kind of compounds, even high tolerance.176 strains of Escherichia coli were isolated from chicken farms in Shandong province to investigate their resistance susceptibility to antimicrobials. Result of antimicrobial disk susceptibility test showed that antimicrobial resistance of bacteria was serious and mainly multi-drug resistance. Most isolates are resistant to more than 9 kinds of antimicrobials. 6 multiple resistant Escherichia coli was selected, which resistance level was different. The minimal inhibitory concentration (MIC) of 7 antibiotics to 6 selected strains and standard strain, tested with cuvette double dilution method, showed that level of resistance was high.To identify if there was the effects of efflux pump inhibitors to counter of resistance, the influence of active efflux inhibitors carbonyl cyanide m-chlorophenylhydrazone(CCCP) on minimal inhibitory concentration(MIC) of both standard Escherichia coli and resistant Escherichia coli to Ciprofloxacin, Enrofloxacin and Tetracycline were measured by agar dilution method. Results indicated that three antimicrobials had no effect on standard strains MIC after addition to CCCP, CCCP had little effect on the most drug resistance strains to Tetracycline. CCCP had distinct effect on resistance strains MIC to Ciprofloxacin and Enrofloxacin, for example , CCCP decreased the drug resistance strain S26 MIC to Ciprofloxacin and Enrofloxacin to 1/8.The accumulation of ciprofloxacin and enrofloxacin in both clinical resistant Escherichia coli and standard Escherichia coli ATCC25922, and the changes that fluoquinolones obsorbed by ATCC25922 and multi-drug resistant strains were assayed. The results showed that CCCP had effect on the accumulation of ciprofloxacin and enrofloxacin in clinical resistant Escherichia coli and standard Escherichia coli .In ACTT25922, the density of fluoquinolones in bactria by intaking time, and the density of stabilization when CCCP was added or not; In resistant strains S9,S26and S124, the density of fluoquinolones in bactria increased by time when CCCP was added, and they showed stabler; However, after the addition of CCCP, the accumulation of antibiotic in S5 and S77 were increased slightly.A Quantitative detection assay of AcrA-mRNA was developed by Quantitative Competitive RT-PCR. The relationship of AcrA-mRNA and resistance level in the experimental strains was analyzed. The results showed that the level of AcrA-mRNA of clinical resistant Escherichia coli increased to a great extent. There was correlation between the resistance level of experimental strains and the level of AcrA-mRNA.The result shows that antimicrobial resistance of bacteria was serious and mainly multi-drug resistance. The change of active efflux inhibitors carbonyl cyanide m-chlorophenylhydrazone(CCCP) on the accumulation of antibiotics in Escherichia coli were measured; the level of AcrA-mRNA of Escherichia coli was assay. The result provides the evidence for the supervisation and control of Multi-drug Resistance Mediated in Escherichia coli, and it was the significance of economic value and academic research.