DNA测序技术在不同药物压力下大肠埃希菌耐药性产生机理研究中的应用  

Application of DNA sequencing technology in study on mechanism of resistance of Escherichia coli under different drug pressure

作　　者：王槐栋[1] 张晓天[1] 张超[1] 关世慧 董君雪 牟佳[1] 王放[1] 郑敬彤[1] 

机构地区：[1]吉林大学基础医学院病原生物学教研室,吉林长春130021

出　　处：《吉林大学学报（医学版）》2014年第1期109-112,共4页Journal of Jilin University：Medicine Edition

基　　金：吉林省科技厅科技发展计划项目资助课题(YYZX2012;20130415016ZX)

摘　　要：目的:对比长时间传代前后细菌对抗生素和抑菌中药敏感性的改变情况,检测某些耐药基因是否发生基因突变。方法:分别在含有左氧氟沙星和银花泌炎灵片的M-H肉汤培养基中对大肠埃希菌ATCC25922进行200代传代培养,测定最小抑菌浓度(MIC)并与给药前MIC进行对比,以PCR法扩增细菌解旋酶基因(gyrA)和拓扑异构酶Ⅳ基因(parC),进行测序。结果:与给药前比较,经含左氧氟沙星的培养基中连续传代的大肠埃希菌对左氧氟沙星的MIC明显上升(P<0.01),而经含银花泌炎灵片培养基连续传代的大肠埃希菌对银花泌炎灵片的MIC未出现明显差异(P>0.05)。基因测序结果,只有经左氧氟沙星培养基中连续传代的大肠埃希菌ATCC25922编码parC基因第381位发生突变,腺嘌呤(A)→胸腺嘧啶(T),其编码的赖氨酸(127)→天冬酰胺,并在第386位碱基插入胸腺嘧啶(T);而gyrA基因未见有碱基突变或插入。结论:在特定的环境压力下左氧氟沙星比银花泌炎灵片更容易诱导大肠埃希菌耐药性的产生,而parC基因突变可能是细菌耐药性产生的原因之一。Objective To compare the changes of drug-susceptibility in antibiotic and antimicrobial Chinese herbs before and after long time passaged, and to detect the genetic mutation of some resistance genes. Methods The Escherichia coli （E. coli） ATCC25922 was cultured in the M-H broth culture including Levofloxacin or Yinhuamiyanling by 200 passages. The minimum inhibitory concentration （MIC） of E. coli ATCC25922 of these two medinices was detected and compared with the MIC of untreated E. coli ATCC25922. The bacterial of the genes encoding gyrase （gyrA） and topoisomerase 1V were amplified by PCR. Results The MIC of the bacterial cultured in the M-H broth culture including Levofloxacin was increased compared with the MIC of untreated E. coll. There was no significant difference in the MIC of the bacteria cultured in the M-H broth culture including Yinhuamiyanling between before and after treatment （P〉0.05）. Several mutations were identified in gene of parC. The adenine （A） at the site 381 of parC was converted to thymine （T）, and a thymine （T） was inserted in at the site 386. All isolates harbored parC amino acid subctitutions at position 127 （Lys127-Asn）. The gene gyrA had no mutation. Oonclusion Levofloxacin is easier than Yinhuamiyanling to produce the resistance of E. coli ATCC25922, and the mutation of parC gene may be one of reseason of bacterial resistance.

关 键 词：诱导耐药 基因突变 左氧氟沙星 银花泌炎灵片 

分 类 号：R378.21[医药卫生—病原生物学] Q503[医药卫生—基础医学]