RESUMO
Objective To detect the influence of marOR mutations on antibiotic resistance in Shigella spp. Methods The marOR gene with four-base deletion was amplified by overlap PCR, then inserted in a T-vector and transformed into DH5α. The clone of marOR gene with four-base deletion and three point mutations was prepared from the strain having these mutations. Electrophoresis and sequencing were preformed to certify the correction of the cloned genes. Drug susceptibility tests were preformed for the strains harbouring the different clones [DH5α, DH5α (T), DH5α (marOR), DH5α (marOR-CATT), DH5α(marOR-CATT + 3m)]. Results Compared with the control strain (DH5α-T), the antibiotic resistances of marOR with four-base deletion [DH5α (marOR-CATT)] were higher to streptomycin, tobramycin, cefazolin and cefalexin, and the antibiotic resistances of marOR with four-base deletion and three point mutations [DH5α (marOR-CATT + 3m)] were higher to streptomycin and to tetracycline. The antibiotic resistances of DH5α (marOR-CATT) and DH5α (marOR-CATT +3m) to streptomycin, tetracycline, chloramphenicol, cefazolin, levofloxacin, ciprofloxacin and norfloxacin were higher than DH5α (marOR). The diameters of the antibiotics except the trimethoprim between DH5α (marOR-CATT) and DH5α (marORCATT +3m) had not significant disparity. Conclusion The four-base deletion in 1376-1379 sites of the marOR gene increased the resistance of Shigella spp to some antibiotics. The point mutations in 1411, 1417,1435 sites of the marOR gene have little influence on the antibiotic resistance of Shigella spp.
RESUMO
Objective To detect the mutations of the marOR gene and study the relations with the expressing level of the acrAB-tolC efflux pump in Shigella. Methods marOR genes were amplified by PCR for 100 clinical isolates and 5 reference strains of Shigella. The PCR products were digested by restriction endonuclease Taq Ⅰ , then analyzed by single strand conformation polymorphism(SSCP). The marOR genes of the mutated strains and sensitive strain were sequenced and the expressing leveLs of acrA, acrB and talC were determined by RT-PCR. Susceptibility tests of tetracycline (TE), chloramphenicol (C), ampicillin (Am) , gentamycin (GM), norfloxacin (NOR) and selectrin (SMZ-TMP) were performed in sequenced strains. Results marOR genes were found in all strains detected. SSCP analysis found the rate of mutations in marOR genes was 23%. Among 11 marOR gene-mutated strains which were sequenced, there were 9 strains having a four-base absence and three single-base mutations in different loci. The expressing levels of the acrAB-tolC efflux pump in the 11 strains were higher than those in sensitive strains and reference strain. Furthermore the 11 strains were multi-drug resistance. Conclusion The mutation rate of marOR gene in Shigella was high and the acrAB-tolC efflux pump genes were over-expressive in marOR gene-mutated strains which were multi-antibiotic resistance in the study.