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

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-CI-EGS cat1 cat2重组质粒对16株临床分离的Cma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Cm100～200μg／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。

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

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 CaCl(2) 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 A(600). 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-C1-EGS + cat1 + 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 approximately 200 micro g/ml of Cm. They were sensitive to Cm on LB-agar plates containing 100 approximately 200 micro g/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.