Study On The Relations Of AcrAB-toIC Efflux System And Shigella Sonnei Multigrug Resistance

ObjectiveTo study the multidrug resistance phenotype of Shigella sonnei isolates and explorethe relation between AcrAB-TolC efflux pump and multidrug resistance. To elucidate themolecular mechanism of multidrug resistant, to guide the clinical rational use ofantimicrobial agents, and provide a theoretical basis to the resistant strains as well as theresearch and development of new drugs.Methods1The collection, identification and drug sensitivity of Shigella sonnei: A total of41strains of Shigella sonnei were isolated from Ji’nan City Center for Disease Control and theFourth People’s Hospital of Ji’nan city. All strains were identified by biochemical andserological reaction. The susceptibility to7antibiotics (ampicillin, cefotaxime,ciprofloxacin, gentamicin, chloramphenicol, tetracycline, sulfamethoxazole) was detectedby the agar disk diffusion method and E-test, and analyze its resistance patterns andresistance ratio.2Establishment of Shigella sonnei efflux model and Interference testing of effluxpump inhibitors: Two wild Shigella sonnei strains, wereinduced by continuous inductionculture tomake them into multiple resistant strains, and test them drug susceptibility byE-test. comparing the MIC value differenc of wild strains and induced strains. Addingactive efflux pump inhibitors of PAβN to the induced strains and detect the MIC ofciprofloxacin before and after the efflux inhibitor.3Detection of AcrAB-TolC efflux pump gene acrA,acrB,tolC,acrR and marR and thechanges of expression level of mRNA: Using polymerase chain reaction (PCR) to amplifythe acrA,acrB,tolC,acrR and marR genes of all tested strains, electrophoresis andphotographed, to observe the changes of mRNA levels of acrR and marR between the wildstrains and induced strains. Result1The drug susceptibility results:the resistance rate of41S.sonnei strains to7antimicrobial drugs:41isolates were all sensitive to ciprofloxacin and chloramphenicol.Theresistance rate of ampicillin,cefotaxime and gentamicin were41.5%,12.2%and46.3%respectively.The resistance rate of tetracycline and sulfamethoxazole were all85.4%.among these strains,36srrains were resistant., the resistant rate was87.8%(36/41),16S.sonnei strains were resistant to more than4kinds of antimicrobial, it means that39%(16/41) were multi-drug resistance. There were6kinds of resistance patterns, with TS themost, accounting for41.5%. Only5strains were susceptible to all antimicrobials.2Shigella sonnei induction results and the changes of phenotypes in the presence ofthe efflux pump inhibitor: A total of69passages except of drug-resistance to ciprofloxacin,and several other kinds of antibiotics also have different changes., after induction, thewildstrains were gradually transformed into multiple resistant strains. Adding active effluxpump inhibitors of PAβN to the induced strains, The MIC value to ciprofloxacindecreasedsignificantly, but did not reach the sensitive level before induction.3Active efflux pump control gene detection and mRNA expression of the situation:All strains were amplified respectively for1194bp,3150bp,1582bp,512bp and426bpamplification, respectively acrA, acrB, tolC, acrR and marR genes. Before and after druginduced cells were detected the expression of acrR and marR, but induced resistant strainsacrR and marR expression levels were significantly lower than the sensitive strains.Conclusion1The41Shigella sonnei strains to ampicillin resistance,gentamicin, tetracycline,sulfamethoxazole compound rate of more than40%, but the cephalosporins and sulfa drugsensitivity, clinical can choose higher sensitivity in the treatment of Shigella sonneiinfection causes diarrhea.2After establishment the ciprofloxacin-resistant models, several other antimicrobialagents which different from ciprofloxacin in the construction and mechanism also haveresistance, these drugs are substrates of efflux pump AcrAB. addictionally,, after addingactive efflux pump inhibitor PAβN to the induced resistant strains, the MIC values changedin different degrees, which fully confirmed the activation of active efflux system is themain mechanism of inducing strains of multidrug resistance.3The pump genes and its regulatory genes were amplified in all strains, whichidentify all strains exist the acrAB-TolC system, the decreased expression of repressorgene regulate the efflux pump gene expression up which result to multiple antibioticresistance, only the repressor gene expression level decreased activation of efflux pump resulted in multiple antibiotic resistance.