The Regulation Of Two Component Signal Transduction System CpxAR On MDR Of S.gallinarium

Salmonella is a common zoonotic pathogen and brings a serious threat to the health of human and animal.With the widespread use of antimicrobial agents in recent decades,the resistance of salmonella to almost all commonly used antimicrobials become more and more serious.The problem of multiple drug resistance has become a outstanding threat to public health,food safety,and breeding of livestock and poultry.TCS is one of widespread signal transmission systems among bacterias that closely relate to bacterial?s resistance to acid,chemotaxis,hunger,resistance and pathogenicity.Therefore,it is an attractive new target for antibacterial activity.This experiment selected cpxAR system as the research object,which is a common TCS in gram-negative bacteria.Complete cpxR ORF was amplified using the DNA of salmonella typhimurium standard strains CVCC541 from chicken origin as template.The purified PCR product and p BAD/His A plasmid vector were digested by Xhol ? and Hind ?.The digested products were purified with T4 DNA ligase and the resulting products were put into the compotent DH5a cells with chemical transformation.The plasmids of positive clones which were screened through blue white spot were extracted after gene sequencing and transformed into compotent cells JS?JS?cpxR?JS?acrB?JS?cpxR?acrB.The over-expression cpx R strains were successfully constructed in the above strains.The minimal inhibitory concertrations(MICs)of 12 kinds of commonly used representative antibacterial agents on S.enterica Serovar Typhimurium strain(JS)and related engineering bacteria were determined with broth microdilution method.The results showed that cpxR gene deletion strain JS?cpxR showed 4-,4-,and 4-fold decreases in the MICs of Gentamicin,Amikacin and Ceftiofur,respectively,compared with that of the parent strain JS,while the complemental strain showed 16-,8-,4-,16-and 8-fold increases in the MICs of Gentamicin,Amikacin,Florfenicol Ceftriaxone and Ceftiofur compared with that of the mutant JS?cpxR.The acrB gene deletion strain showed 4-fold to 12-fold higher sensitivity to Doxycycline,Gentamycin,Amikacin,Ciprofloxacin,Enrofloxacin,Colistin,and Ceftiofur,compared with the parent strain JS.But the overexpression of cpxR in the acrB gene deletion strain caused 4-fold increases in the MICs of Doxycycline and Colistin,and the MICs of Doxycycline,Amikacin,Olaquindox,Ciprofloxacin,Enrofloxacin and Ceftiofur were 1/8,1/4,1/8,1/8,1/4,and 1/16 folds of those of JS.These data indicated that cpxAR can decrease susceptibilities of S.enterica Serovar Typhimurium to Tetracyclines,Aminoglycosides,Cephalosporins,Chloramphenicols.Moreover,when absence of acrB,the regulation of cpxAR on multidrug resistance is more significant.The mRNA expression levels of active efflux genes acrB?acrD?acrF?TolC?mdtA and regulatory genes mar A?sox S in JS?JS?cpxR?JS?acrB?JS?cpxR?acrB and the related complemental strain,and the relationship between these gene and CpxAR system were investigated by Real-time PCR.The results revealed that the expression of soxS and TolC showed different degrees of decreases when cpx R was overexpressed JS strain,and the expression of the active efflux gene acrB in cpxR overexpression strain of JS and the complemental strain JS?cpxR showed 2.22% and 19% decreases,respectively.These data indicated that cpxAR can down-regulated the expression of soxS,TolC and acrB,and up-regulated the expression of acrD.But there was an important link between the up-regulated function and acrB.