外源性指导序列体外转换临床大肠杆菌氯霉素抗性表型的实验研究  

Experimental study on phenotypic conversion of clinical chloromycetin-resistant strains of E. coli to drug-sensitive strains by using EGS technique in vitro

作　　者：高美英[1] 陈如[1] 刘守桂[1] 封江南[2] 

机构地区：[1]华中科技大学同济医学院附属同济医院感染科,武汉430030 [2]卫生部武汉生物制品研究所免疫研究室

出　　处：《中华医学杂志》2004年第15期1294-1298,共5页National Medical Journal of China

基　　金：国家自然科学基金资助项目(39570846)

摘　　要：目的 研究外源性指导序列(EGS)体外逆转临床分离大肠杆菌氯霉素抗性的能力。方法 构建针对氯霉素(Cm)乙酰转移酶(cat)并含卡那霉素(Km)抗性基因筛选指标的EGS重组质粒以及只含Km抗性基因但不含EGS的对照质粒。采用CaCl2方法将重组质粒导入临床分离的耐Cm大肠杆菌株中。通过质粒抽提、菌落PCR鉴定EGS阳性克隆子,分光光度计A600检测导入EGS质粒后耐Cm菌株在液体培养基中的生长率以及KB法检测在固体培养基中的药物敏感试验,探讨EGS分子逆转耐药菌的逆转效应。结果 以pEGFP-C1-EGS cat1+cat2重组质粒对16株临床分离的CmR大肠杆菌供试菌进行转化试验,EGS转化子在含Km的培养基上筛选。其中4株转化菌在Cm 100—200 μg/ml的液体培养基中生长受抑;在Cm 100-200μg/ml的固体培养基中药敏试验对Cm敏感;转化菌质粒抽提检测出特异EGS条带;菌落PCR扩增鉴定存在EGS。表明16株供试菌中4株临床分离株获得了耐药菌型的表型转换、耐Cm的大肠杆菌临床分离株恢复了对Cm的敏感性。结论EGS分子具有逆转临床耐药菌为敏感菌的能力。Objective To explore the possibility of phenotypic conversion of clinical chloromycetin (Cm)-resistant isolates of E. coli to drug-sensitive ones with external guide sequences (EGS) in vitro. Methods Recombinant EGS plasmids directed against Cm acetyl transferase ( cat) and containing kanamycin ( Km) drug-resistance gene and control plasmids only containing kanamycin-resistance gene without EGS were constructed. By using CaCl2 method, the recombinant plasmids were introduced into the clinically isolated Cm-resistant E. coli strains. Extraction of plasmids and PCR were applied to identify the EGS positive clones; The growth rate in liquid broth culture of Cm-resistant bacteria after EGS containing plasmid transformation was determined by spectrophotometer A600. Drug sensitivity was tested in solid culture by using KB method. Results Transformation studies were carried out on 16 clinically isolated Cm-resistant E. coli strains with pEGFP-Cl-EGS + catl + cat2 recombinant plasmid. Transformants were screened on LB-agar plates containing Km after transformation using EGS. In 4 tested strains of them, transformants with specific EGS plasmid showed growth inhibition when grown in liquid broth culture containing 100 - 200 μg/ml of Cm. They were sensitive to Cm on LB-agar plates containing 100 - 200 μg/ml of Cm in drug-sensitivity test. Extraction of plasmids showed the existence of EGS bands. PCR amplified products of EGS. The above facts indicated that the 4 strains out of the 16 clinical isolates had been converted to drug-sensitive phenotype, and Cm-resistant clinically isolated E. coli resumed sensitivity to Cm. Conclusion EGS has the capability of converting the phenotype of clinical drug-resistant isolates to drug sensitivity.

关 键 词：外源性指导序列 EGS 体外转换 大肠杆菌 氯霉素 抗性表型 抗性逆转 

分 类 号：R96[医药卫生—药理学]