| The development of antimicrobial resistance in clinical isolates has paralleled with the antimicrobial usage, and antimicrobial resistance may be caused in many ways, in which increasing efflux activity is the most important mechanism. Acr AB-Tol C efflux pump, extensively existing among Gram-negative bacterias, can expel a panel of structure unrelated chemicals out of the cell, leading multi-drug resistance. The purpose of the present study was to explore the resistance development mechanism in Acr AB-Tol C or regulatory genes deletion mutants derivated from E.coli K12 under the increasing ciprofloxacin pressure.A set of single-gene(including efflux pump or regualtor genes) deletion mutants were derivated from Escherichia coli K12 by Red recombination. Firstly, PCR products, with 36-to 50-nt extensions that are homologous to the target genes acr A, acr B, tol C, mar A, mar R, sox S and sox R, were generated using plasmids p KD3 and p KD4 carrying antibiotic resistance genes that are flanked by FRT sites as templates. Then each PCR product was introduced into E. coli K12 to inactivate the function of the targeted gene. Subsequently we measured the growth ability and motility of each mutants. The results showed that the growth ability of mutants were not impaired comparing with parent strain.K12 and single-gene deletion mutants were grown at 37 ? in the presence of increasing ciprofloxacin pressure to induce ciprofloxacin resistant mutants. The selected ciprofloxacin resistant mutants from each step were subjected to determine the MIC of tested drugs by agar dilution method. Mutations of gyr A, gyr B, par C and par E within the quinolone resistance determining regions(QRDRs) were detected by PCR amplification and sequencing. The expression of efflux pump genes, regulator genes and outer membrane protein genes were all detected by RT-PCR. The results showed that once the gene tol C was knocked out, resistant strains were difficult to be selected, suggesting that the intergrity of tripartite efflux pump Acr AB-Tol C has severe importance on the development of resistance. Diverse mutations of target genes within QRDR were found, and resistance related amino acid alterations were Ser83 Leu, Asp87 Gly, Asp87 His in gyr A and Gly78 Asp in par C gene. Regarding the acr A or acr B deletion mutants, low level resistance to ciprofloxacin were acquried from target gene mutation, and the high level of resistance was due to the additional activation of redundant efflux pump Acr EF, which enhence the efflux of drugs. Besides, the decreased expression of Omp F also contributed to the resistance development,.Once the positive regulator genes mar A or sox S was deleted, mutants with desreased susceptibility to ciprofloxacin were selected. While the repressor genes mar R or sox R was interrupted, resistant strains could be selected. All the mutants had a single mutation in the gene gyr A.The above results showed that mar A and sox S can activate the expression of Acr AB-Tol C by removing the function of repressor of mar R and sox R.We concluded that the tripartite multi-drug efflux pump system Acr AB-Tol C plays an important role in the development of resistance in the bacteria. Once this system is impaired, the associated redundant efflux pump Acr EF would play an alternative role. |
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