| Active efflux system (efflux pump) is a major mechanism that causes multipleresistance in Escherichia coli. AcrAB-TolC is the most important one. AcrAB-TolCcomprises medicine proton transportor AcrB and periplasm fusion protein AcrA andouter membrane passage protein TolC. It has many substrates, its high standardexpression shows tolerance against many kinds of compounds, even high tolerance.Escherichia coli is a major pathogenic bacterium in fowls and livestocks, it exists inmany places and animals. Multiple resistance is produced and spreaded easily.Multidrug resistance mechanism mediated by two efflux pumps i.e. AcrA and AcrB inEscherichia coli was studied by detecting the changes on the resistance level in controlstrains induced by single drug, comparing with clinical isolated strains in threeexperiments.Sodium salicylate, choramphenicol, tetracycline and ciprofloxacin were used tobe single inducers, 2×MIC was taken. E.coli ATCC25922, which is quality control(QC) strain, was cultured in nutrition containing single inducer continuous, until thedensity of inducer raised no longer, to obtain midrange and high-range persisterrespectively;at the same time three multidrug resistance strains were isolated fromclinic. MICs of all strains were detected. The results showed that MICs of high-rangestrain isolated from clinic and strain induced by choramphenicol were high mutidrugpersister to Quinolone, aminoglycosides, sulfamido, macrolide, β-lactam, chlorocol,penicillins and so on, relatively high-range strain isolated from clinic had more highpersister to aminoglycosides, chlorocol and penicillins. Similar drug resistance existedin midrange and high-range persisters isolated from clinic and high-range straininduced by Sodium salicylate and tetracycline. But the ciprofloxacin inducing strainobtained higher resistance to Quinolone, chlorocol, jiemycin, sulfanilamide, MICs ofall strains reached and exceeded 4×, even 1000× in Escherichia coli. It concludedsurrounging pressures caused by Sodium salicylate, choramphenicol, Tetracycline andciprofloxacin may lead to the muti-drug resistance in Escherichia coli, and theresistance spectrums are consistent with the substrates of AcrAB-TolC, which may bethe result of activing the expression of AcrAB-TolC.Carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used to detect thechanges of fluoquinolones absorbed by ATCC25922 and multidrug resistanct strainswith or without CCCP, to judgement the existence of efflux pump on all strains. Theresults showed that density of ciprofloxacin in all strains with CCCP was higher thanstrains without CCCP. The density of ciprofloxacin in all strains was lower than that ofATCC25922 with or without CCCP (beside strain induced by ciprofloxacin instable),the time that all strains and ATCC25922 intaked ciprofloxacin and reachedstabilization were positive related to MIC. It showed that multi-drug resistance in allstrains was the result of efflux, which pumps drugs in E.coli.The sequencing analysis of acrA and acrB gene encoding AcrA and AcrBrespectively showed that there were basic group changes in the two genes, but noamino acids mutation. The change of resistance level of experimental strains hadhappened under acrA and acrB amino acids had't mutation. Using real time RT-PCRto detection of mRNA expression of acrA and acrB genes in E. coli drug resistantstrains induced and isolated from clinic.It concluded acrA copies/μL arrived 1015 inclinical high-range strain and chloramphenicol inducing strain;1014 in tetracyclineand Sodium Salicylate inducing strains, clinical mid-range strains and low-rangestrains;1013 in mid-range strains induced;1012 in ATCC. AcrB copies/μL arrived 1013in clinical high-range strain;1012 in mid-range strains;1011 in low-range strains andATCC. But acrB copies/μL in all strains was more than that of ATCC. Itdemonstrated that the expression quantities of acrA and acrB gene were different,approximate multidrug resistance may be obtained on ATCC25922 induced by singledrug and strains isolated from clinic, drug-resistant levels were positive correlationwith the level of expression of acrA and acrB gene.The experiment was systemically studied the mechanism of multiple resistancemediated by the expression system center of AcrAB-TolC in E. coli from many linksand points of views through molecular biology level. It provided the evidence for thesupervisation and control of multiple resistance mediated in E. coli, so it has academicand research value in some extent.
|
PDF Full Text Request