The Regulatory Rule Of Glogal Regular Gene MarA On MDR In Escherichia Coil Isolated From Duck

After the knockout of multi-drug resistant regulation genes marA in E. coli standard strains (O73) originated from ducks with red recombination system, the deleting mutant of marA, O73△marA was constructed and the minimum inhibitory construction(MICs) of five antibiotics were analysed. The mRNA expression level of active efflux genes and regulation genes in these strains were determined by real time fluorescence quantitative PCR.When O73, O73△marA strains were induced in broth containing five different drugs. The aim of this study is to explore the mechanism of multi-resistance development and its regulation.The Kanamycin resistance(kan)gene flanked by homology extensions of marA gene was amplified by PCR. The PCR products were introducted into E. coli O73/pkD46by electroporatiom, When induced by L-arabinose, the plasmid pKD46could express three recombinant proteins of λ-prophage, with the help of λ Red-mediated recombination system, marA gene was replace by homology extensions connected with kan gene, the strains expressing kan resistance gene were selected by kan agar, Using this system,marA gene in chromosome of Escherichia coli was delected. O73△marA muntants was obtained and further confirned by PCR amplification and sequencing. The upstream and downstream long homologous sequences of marA, and kan resistance gene of pkD4were amplified by PCR, cloned into the pET-28vertor and pkD4. Homologous recombinant plasmids pET28-marA were the constructed. After the pET28-marA were transformed into E.coil DH5a and amplified,fragments of homologous recombinant plasminds were obtained by restricted enzyme reaction and electro-transformed into E. coli O73. which led to the replacement of target genes marA with Kan-resistant gene. The MDR profiles of E. coli O73and O73△marA mutants were determined using the microbroth dilution method.5antimicrobial agents consisted of Gentamycin、Ceftiofur、Florfeniol、Enrofloxacin and Doxycycline.Expecially,O73△marA muntants of MIC significantly reduced.compared with the O73strains induced with Gentamycin、Ceftiofur and Doxycycline. O73strains of MIC were2、2and4μg/mL,O73△marA muntants of MIC were0.125,0.125and0.5μg/mL.O73strains were induced in broth containing Gentamycin、Ceftiofur、Florfeniol、 Enrofloxacin and Doxycycline with MIC subinhibitory concentrations to40th generation,the expression level of active efflux system genes and regulation genes in different E.coli strains were determined by real time fluorescence quantitative PCR of The results showed that:When the standard strains O73and O73△marAmutant strains were uninduced,the expression level of acrA, acrB, TolC, acrD, acrE, acrF, SoxS, RobA genes were decreased (between0.04～0.74).The expression level of acrD gene was0.04, which was declined the highest;the expression level of acrE gene was0.74, in that strains were lower than the expression of active efflux genes and regulation genes. O73strains were induced with above the five antimicrobials. The expression level of the active efflux genes in most of induce-strains (O73)increased compared with the parent strain.,which were between0.12～2.73. Doxycycline-induced O73strain in40th generation, the expression level of tolC gene was2.43, which was2.30times compared with the parent strain. Ceftiofur-induced O73strain in40th generation, the expression level of acrE gene was0.12, in that strains were lower than the expression of active efflux genes and regulation genes.The expression level of acrA, acrB, TolC, acrD, acrE. acrF, SoxS, RohA genes were not significant changes compared with the uninduced dstandard strains.O73△marA strains were induced with above the five antimicrobials, the expression level of the active efflux genes were between1.50～55.33. O73△marA strains the expression level of acrF gene in Ceftiofur-induced was1.50, which was rised the lowest and was1.50times compared with the O73strain. O73△marA strains the expression level of acrF gene in Enrofloxacin-induced was3355. which rised55times compared with the O73strains.Compared with the O73△marA strains were induced with the five antimicrobials, the expression level of the active efflux genes in most of induced-O73strain increased. The expression level of acrA、acrB、TolC、acrD、acrE、 acrF、SoxS、RobA were55、20、16、43、16、25、17、19times of those of the uninduced standard strain.which were251、57、30、464、23、27、72、76times of those of the uninduced O73△marA strains.The expression level of eight kinds of active efflux genes In Gentamycin-induced and uninduced O73strains、O73△marA strains compared with two kinds regulation genes:O73strains were induced in the highest level (40th generation), the expression level of the active efflux genes were similar to that of the standard strains:O73△marA strains were induced to40th generation, the expression levels of the active efflux genes were much higher than those of uninduced O73△marA strains,other active efflux genes and regulation genes,such as soxS also had similar rule.