Construction Of The AcrB Gene Deletion Mutant From Avian Escherichia Coli And Some Related Biological Functions

The AcrB mutant of duck E. coli standard strain was constructed by using λ Red recombination system.The mRNA expression level of acrA, acrB, acrD, acrE, acrF, mar A, soxS and robA were determined by real time fluorescence quantitative PCR in different E.coli. strains including duck E. coli standard strain, the AcrB mutant of duck E. coli standard strain and their drug-induced strains. To investigate the producement of multi-drug resistant and it’s regulatory mechanism.Thit study established the new simple and highly efficient method to disrupt chromosomal genes in duck E. coli, provided the methodological basis for the further disrupting of more chromosomal genes at the same time.We designed a pair of primers based on the original sequences of AcrB gene clusters in the GenBank.we generated PCR products by using primers with the homologies extension of AcrB gene to be deleted and templated plasmid pKD4carrying selectable antibiotic Kanamycin resistance(kan) gene firstly, which is flanted by FRT(FLPrecognition target) sites, and then transformed the pkD46plasmid into the duck E. coli standard strains, these strains with pkD46plasmid were selected on ampicillin resistant plate, named O73/pkD46.The PCR products were introduced into duck E. coli standard strain with the help of λ Red-mediated recombination system, AcrB gene was replaced by the kan gene, these strains expressing Kanamycin resistance (kan) gene were selected by Kanamycin agar. Using this system, AcrB gene in chromosome of E. coli was deleted.The mutant was obtained and further conformed by PCR aplification and sequencing, and was named O73△acrB. We got the mutant by long and short homologous extension,the long homologous extension was acquiered difficultly,but was electroporated easily.073and073△acrB strains were induced in broth containing gentamicin,ceftiofur, florfenicol, enrofloxacin, doxycycline with1/2MIC subinhibitory concentrations to the30th generation.The MIC profiles of different strains were determined using the microbroth dilution method as the CLSI recommended secondly.We found that the MIC profiles of O73△acrB declined significantly compaired to073. The MICs of different drugs against073was2、8、16、2、8μg/mL, and the MIC of different drugs against O73△acrB was1、0.06、2、0.03、0.5μg/mL, the MIC of different drugs against drug-induced strains of073were128μg/mL, the MIC of different drugs against drug-induced strains of O73△acrB was4、2、4、2、4μg/mL respectively. The results showed that acrB gene plays an important role in active efflux mechanism.The mRNA expression level of acrA, tolC, acrD, acrE, acrF, mar A, soxS and rob A were determined by real time fluorescence quantitative PCR in different E.coli strains including duck E. coli standard strain, the AcrB mutant of duck E. coli standard strain and their drug-induced strains to investated the role of AcrB gene reverselyFor073△acrB,the mRNA expression level of acrA mar A, soxS and robA genes were higher than that of073. The results showed that other active efflux genes and regulative genes play more positive role after acrB gene was disrupted.For the drug-induced strains of073, the mRNA expression level of acrA and tolC genes were higher than that of073. the mRNA expression level of soxS and acrF genes were lower than that of073. the mRNA expression level of mar A was higher for enrofloxacin drug-induced strain than that of073. the mRNA expression level of acrD and acrE was higher for gentamicin drug-induced strain than that of O73.the mRNA expression level of robA was higher for doxycycline drug-induced strain than that of073.For the drug-induced strains of073△acrB, the mRNA expression level of marA, soxS and robA genes were higher than that of073,and the margin of increase is larger than the drug-induced strains of073. the mRNA expression level of acrD was higher for florfenicol, enrofloxac than that of073. the mRNA expression level of acrE was higher for gentamicin than that of073.