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Objective To investigate the role of AcrAB-TolC efflux pump in fluoroquinolones resistance by Shigella. spp and to explore the significance of AcrAB-TolC efflux pump on mutation of acrR, soxS and marOR as well as on drug re?sistence. Methods Drug resistant bacteria were selected by Kirby-Bauer disk diffusion test. After addition of efflux pump inhibitor carbonylcyanide-m-chlorophenylhydrazone (CCCP), change of minimal inhibitory concentration (MIC)s of nilidixic acid, Levofloxacin, ofloxacin, ciprofloxacin and Norfloxacin were examined. The DNA binding region of acrA, acrB, soxS, acrR and marOR gene in these mutants were amplified by PCR and sequenced. Results Among the 159 clinical isolates of Shigella,11 strains are resistant to fluoroquinolone. After the addition of CCCP, MICs of 2 fluoroquinolone resistant strains decreased; the MICs of 7 fluoroquinolone resistant strains did not change; MICs of 2 fluoroquinolone resistant strains in?creased. The corresponding nucleotides C, A, T, T on the 36th to 39th of marOR gene were missing, showing by sequencing, in fluoroquinolone resistent strains which might be regulated by the efflux pump gene AcrAB-TolC. Conclusion Efflux pump inhibitor could restrain the activity of efflux partially. The mutations of marOR might play an important role in fluoroquino?lone resistent by shigella.
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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.
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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.
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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.
ABSTRACT
OBJECTIVE To study the mechanisms of quinolones resistance in Escherichia coli.METHODS Forty E.coli clinical isolates were randomly collected from clinical specimens at the Tianjin First Central Hospital from Mar 2004 to Dec 2005.Then we detected the susceptibility to antibiotics in 40 clinical isolates of E.coli by MICs and K-B disk diffusion method.In order to investigate the mutations in the target genes,we amplified the QRDR of gyrA and parC by PCR.Later we analyzed the PCR products by single strand conformation polymorphism analysis(SSCP).In the meantime,the PCR products of marOR region were sequenced to detect the possible gene changes which contributed to the increasing expression of MarA and then lead to the Mar phenotype.RESULTS The alterations in gyrA were found in all quinolones-resistante strains.Asp87→Asn and Ala84→Pro were found besides the common amino acid alteration.The alterations in parC were found in thirty-six strains resistant to quinolones.There were no parC alterations in ECO24 which was nalidixic acid-resistant and ofloxacin/gatifloxacin-susceptible.ECO11 Which was resistant to quinolones only had no gene changes in marOR region.Six gene changes in marOR region were found in ECO5 which was resistant to mutiple antibodies.The alteration in 1879 bp changed the terminator.CONCLUSIONS The alterations in gyrA and parC are responsible for the resistant phenotypes in E.coli.That is,the alterations in the gyrA are primarily responsible for resistance to quinolones,and the alterations in the parC may play a complemental role in enhancing resistance to fluoroquinolones.Moreover,the randomly collected strains resistant to quinolones,have found some mutations in marOR.It may be play certain roles in multiple antibiotic resistance of E.coli.