Relationship Between Multi-Drug Resistance And Expression Level Of AcrAB Efflux Pump In Escherichia Coli From Animal

AcrAB efflux pump plays a major role in the antibiotics resistance phenotype of Esherichia coli mulitiple antiobiotics resistance mutant.AcrAB-TolC system can efflux out an extraordinarily wide variety of antibiotics, chemotherapeutic agent,detergent and dyes.The coordinated operation of the inner membrane transporter AcrB and outer channel TolC is thought to be mediatd by AcrA.Here the level of mRNA of AcrA and AcrB and level of AcrA protein exprssion were determinated .132 pathogenic E.coli were isolated.Results of drug resistance test with 22 antibiotics by drug sensitive slips showed all strains were resistant to one or more antiobiotics,most of them were resistant 13. 14 and 15 antibiotics. Resistance specterum of them is wide. The minimal inhabitory concentration(MIC) of 6 antibiotics to 40 selected strains .tested with plat double dilution method,showed that level of resistance was high. 13 multiple resistant E.coli was selected ,which was isolated different animal and resistance level was different.8 primers were designed and synthesized.A 250 bp Exogenous DNA was inserted in cloned AcrA and AcrB by PCR.constructing a internal standard DNA respectively.Total RNA of E.coli was extracted.A sensitive reverse transcription-ploymerase chain reaction (RT-PCR) was performed ,it was found that the expression of mRNAs were detected in all selected E.coli.PCR. was performed using identical volum of product of reverse transcription and internal standard of different concentration .Concentration of internal standard while brightness of two special band in a lane was same was compared between resistant and sensitive E.coli,showed that mRNA level of 11 mulitiple resistant clinical isolates of E.coli (MIC of enrofloxacin>32 u g-ml-1 )was higher than that of ATCC25922. mRNA level of 2 mulitiple resistant clinical isolates of E.coli and sensitive E.coli H10407(MIC of enrofloxacin > 16 u- gml-1 )was no significant higher than that of ATCC25922.Contrasting to ATCC25922 , mRNA relative level of AcrA and AcrB of were similar.A pair of primers was designed spanning segment coding sequence of AcrA without the coding sequence of its signal pepdide. A PCR was performed using this pair of primerand genome DNA of seventh E.coli..The PCR product was cloned into pMD-18T vector and subsequently subjected to restriction enzyme anlysis and sequencing..The result showed that the cloned AcrA gene fragment is 696bp and 99.8% homogenous to published data with replacement of 1 nucleotide causing none of amino acid changed.This contruct was digested with BamH I and Hind Eland ligated the Pet28a(+) vector digested with the same enzymes using T4 DNA ligase.And a construct Pet-28a(+)-AcrA was generated by transforming the competent cell of BL21(DE3) of E.co/f.Induced the expression of AcrA protein with IPTG at a final concentration of ImM.Expresion of AcrA was detected on SDS-PAGE.Rabbit was immuned with purified AcrA protein. Antibody against AcrA was obtained by detetermination of ELASA and Western-blotting. Western-blotting with antibody against AcrA showed that AcrA protein was overpressed in 4 of 4 clinical isolates of E.coli with high level enrofloxacin resistance(MIC32ug-ml-1 ),and not in sensitive E.coli. Contrasting to ATCC25922 , mRNA relative level of AcrA and protein expression relative level of AcrA were similar.It showed that mRNA relative level of AcrA and AcrB and mRNA relative level of AcrA and protein expression relative level of AcrA of E.coli were similar.The level of expression of AcrA and AcrB of E.coli may related to resistance level of fluoroquinolone (enrofloxacin),but the ralation between level of expression of AcrA and AcrB and the resistance level of other drugs were not found.