| Shigella spp. is the causative agent of bacillary dysentery (referred to as bacillary dysentery), it has been more than one hundred years since it’s discovery. Shigella infection is still the important issue of global public health. Annual incidence of bacillary dysentery in China nearly50million, the morbidity of the first several of37kinds of monitoring infectious diseases, poses a serious threat to public health [1].Since the treatment of antibiotic used in bacillary dysentery,the resistance rates increasing every year. Seventies of last century,quinolones began to used in clinical application and has become the preferred drug for treatment of bacillary dysentery. However, with the extensive use of these drugs in recent years, drug resistance is becoming increasingly serious.Objective:In order to invest antimicrobial resistance of shigella and to explore the mechanism of resistance to fluoroquinolones in Tianjin the past three years. this subject selected Shigella clinic isolates from intestinal outpatient separation of clinical isolates from2009to2011in three hospitals,they were ananlysed by serotype test and antibiotic susceptibility test. Explore the mechanism of fluoroquinolone-resistant. strain from the chromosomal target gene mutation and the plasmid-mediated.Materials and methods:A totle of139shigella clinical isolates were collected from three hospitals in Tianjin from2009to2012, using the serum agglutination test to identify the serological group and sub-type, clarify the popular serotypes. K-B disk diffusion method with17kinds of commonly used antibiotics are used for their antimicrobial susceptibility, analyze and compare the different antimicrobial susceptibility of different serogroups of Shigella. Four fluoroquinolone antibiotics such as ciprofloxacin, levofloxacin are used to selected fluoroquinolone-resistant isolates.The gyrA, parC, qnrA, qnrB, qnrS, aac (6’)-Ib-cr and qepA genes were was amplified by PCR and the sequence results is contrast in Genebank. Conjuction test was performed between PMQR gene positive strains and Escherichia coli Series J53AZR, using the broth microdilution method to detect the change of the antimicrobial susceptibility of fluoroquinolone. Extract the plasmid of donor strain, recipient strain and the zygomycetes, analyze the plasmid profile.Use the PCR to confirm the PMQR gene is successfully imported the into the recipient strain.Results:1, A totle of139Shigella are collected by conventional biochemical and serum agglutination test, included17S.flexneri (including1a,2a,2b,4,5b,6and X, Y variants)121S.sonnei. The main Shigella serogroups is S.sonnei in Tianjin area nearly three years. Second is S.flexneri subtypes F2a. Antimicrobial susceptibility results showed that: the susceptibility rate of the139shigella to nalidixic is lowest(1.44%), followed were ampicillin, sulfamethoxazole and streptomycin, the sensitive rate are2.22%,3.59%and4.32%respectively. The rate of multiple-antibiotic resistant strains that resistant to three kinds and more than three kinds antibiotic is close to98%. The susceptibility to many antibiotics between S. flexneri and S.sonnei are different. The susceptibility rate of S. flexneri resistant to fluoroquinolones and third generation cephalosporin is significantly higher than S.sonnei.2, In this study, a total of5fluoroquinolone-resistant Shigella are sellected, all of them are S. flexneri, the resistance rate is3.59%(5/139). There is no S. Sonnei resistant to fluoroquinolone, but the nalidixic-acid resistance rate is high. The degree of the fluoroquinolone-resistant strains are high, and two strains in which are resistant to3,4-generation fluoroquinolone. All of the fluoroquinolone-resistant strains have mutations both in gyrA83(Serâ†'Leu) and parC80(Serâ†'Ile). Compared with the remaining4strains,N8resistant to levofloxacin lacks the87point mutations of the gyrA gene, and the other4strains exist gyrA87(Aspâ†'Gly, Asn) point mutation.4PMQR gene positve strains are detected in139Shigella, included one qurS1-positive S. flexneri N8,3aac(6’)-Ib-cr gene-positive bacteria are S.sonnei. that sensitive to fluoroquinolone. Three joint strains by plasmid conjugation experiments are collected, there is about the size of10kb plasmid transfer from the donor strain into the recipient strain. All conjugactive strain DNA template were amplified for the corresponding PMQR gene, susceptibility results show that before and after the plasmid conjugation, the MIC of ciprofloxacin and norfloxacin were increased by16,8and4,4dilution with qnrSl and aac (6’)-Ib-cr genes respectively, and the MIC of levofloxacin and ofloxacin increased16,8and1,4 dilution, but no one can achieve the antimicrobial resistant level. Further confirm that PMQR gene mediated low-level resistant, and prompts that the qnr-mediated quinolone resistance is higher than the aac (6’)-Ib-cr.Conclusion:S.sonnei is the main serotype in Tianjin nearly three years, second is S.flexneri subtypes F2a. Multidrug resistance rate of shigella is high. three kinds antibiotic is close to98%. The susceptibility to many antibiotics between S.flexneri and S.sonnei are different. The susceptibility rate of S.flexneri resistant to fluoroquinolones and third-generation cephalosporin is significantly higher than S.sonnei. Fluoroquinolone resistant rates is lower than5%, and all of them are S. flexneri. The plasmid-mediated quinolone resistance genes are discovered in shigella clinical isolates in Tianjin region, it has not been reported previously. The fluoroquinolone-resistance mechanisms in shigella exist the target enzyme mutations at chromosome DNA and harboring PMQR gene, but major in mutations at target allozyme DNA. PMQR gene can brocast between different serotypes of bacteria, cause the horizontal transmission of quinolone-resistance. |
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