ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
Objective To study the association of multi-drug resistance with mutations in mar gene in clinical isolates of Shigella.Methods Fifty-four clinical isolates of Shigella were collected.Susceptibility tests of tetracycline(TE),chloramphenieol(C),ampicillin(AM),cipr ofloxacin(CIP),and norfloxacin(NOR)were performed in a11 isolates.Hexane and cyclohexane were used to study the organic solvent tolerance of all clinical isolates.marOR genes of these strains were analyzed by potymerase chain reaction-single strand conformation polymorphism(PCR-SSCP)and were sequenced. Results Thirty-five multi-drug resistant strains were identified,and the rate of multi-drug resistance was 64.8%.Thirty-eight of 54 strains were tolerant to organic solvent,including 33 multi-drug resistance strains,3 drug resistant strains and 2 sensitive strains. marOR gene was found in all strains by PCR, and 5 multi-drug resistant strains,(14.29%)carrying marOR gene mutations in multi-drug resistant strains were found by SSCP analysis.DNA sequence analysis of marOR gene revealed that there was a 4 base deletion from base 1376 to 1379 in marO gene that resulted in frame-shift mutation.However,this deletion mutation didn;t exist in the standard strain S51250 and sensitive strains.There were 10 point mutations of marR gene in multi-drug resistant strains,2 of them resulting in amino acid changes:1752(G→A)Gly→ser,1854(T→c)Tyr→His.The rest mutations were all nonsense,and some of them occurred in sensitive strains or in many other strains.Conclusion The marO gene mutation may play an important role in the regulation of multi-drug resistance in Shigella spp.