Study On AcrAB Eefflux-mediated Resistance In Salmonella Gallinarum

Salmonella was one of the most frequent pathogenic bacteria in clinical infection of the human and the veterinary medicine. Salmonella gallinarum was an important pathogen that did harm to the well developing of poultry industry in our country. Antibacterials were usually used to prevent and cure those diseases. Fluoroquinolones(FQNS) was chemosynthesis, high performance, harmfulless and generally utilized in clinic. Along with mis-used of FQNS drugs in long term, drug resistance was greatly widespread, and the ratio of drug resistance raised up gradually. The emergence of drug resistance had caused the failure of preventing and curing, which caused great loss to husbandry.Invest indicated that drug resistance had a close relation with efflux system (efflux pump) in Salmonella. AcrAB-TolC was main efflux pump, that contained drug proton transporter AcrB, periplasm fusion protein AcrA and membrane channel protein TolC. Since acrA gene and acrB gene were controlled by the same operon, AcrAB was unified functionally.5 salmonella gallinarum strains of different resistant levels had found through vitro inducing tests(16×MIC,32×MIC,64×MIC,128×MIC,256×MIC). The sensitivities of these strains to ten antibacterials were assessed by using controlled disk diffusion. The resistance of 5 isolated to fluoroquinolones ,cyclomycin, comb-amindan was 100%. The resistance of 128×MIC,256×MIC to ampicillin and ancef was als sensitive, but all strains were sensitive to amkin and cidomycin. The result illustrated that the resistant rate of Salmonella to most antibacterials was very high, and the fluoroquinolones also existed crossing resistance.CCCP could make the energy of strains low, and the intaking rate of ciprofloxacin was tested by ultraviolet spectrophotometry. Drug concentration in persister was lower than sensitive strains when CCCP absented; the concentration was increased following to time and the strains remained higher steady state concentration, when CCCP presented. Efflux pump was not the important affect in C79-13, so the steady state concentration kept highly. The result certified there was efflux pump in resistant strains initially.Salmonella had the resistance mechanisms mediated by AcrAB efflux pump. The activities of AcrAB efflux pump had correlation with the resistant level. The acrA genes were amplified by PCR from three salmonella gallinarum strains of different resistance levels induced by vitro showing resistance to Ciprofloxacin. Sequence comparison with C79-13 reference strain showed an amino acid substitution of Met→40→Thr, in strain 16×MIC and an amino acid substitution of Arg→131→Cys in strain 64×MIC. However no amino acid change was found in acrA of strain 128×MIC. These results indicated that mutations of acrA gene may play an insignificant role in drug resistance in salmonella gallinarum. In order to discuss the resistant mechanisms of Salmonella gallinarum to fluoroquinolones farther, the competitive RT-PCR was applied to detect the mRNA of AcrAB efflux pump. Compared with C79-13, the mRNA expression levels of acrA gene and acrB gene in all resistance strains increased significantly and had the corresponding relationship.