体外诱导获取对利奈唑胺耐药的结核分枝杆菌菌株及其稳定性研究  

Induction in vitro and stability of Mycobacterium tuberculosis resistance to Linezolid

作　　者：胡明豪[1] 王彬[1] 付雷[1] 徐建[1] 陆宇[1] 

机构地区：[1]首都医科大学附属北京胸科医院耐药结核病研究北京市重点实验室北京市结核病胸部肿瘤研究所药物研究室,101149

出　　处：《中国防痨杂志》2017年第4期397-401,共5页Chinese Journal of Antituberculosis

基　　金：“十二五”国家科技重大专项（2015ZX09102007-015）;北京市医院管理局“登峰”计划（DFL20151501）

摘　　要：目的利用浓度2倍递增的方法体外诱导获得对利奈唑胺（linezolid，Lzd）耐药的结核分枝杆菌（Mycobacterium tuberculosis，MTB）菌株，考察其耐药稳定性及用于筛选新化合物的抗结核活性。方法将MTB标准株H37Rv（ATCC27294）在Lzd浓度2倍递增的含药固体培养基上进行传代培养，逐步诱导菌株对Lzd耐药，运用微孔板阿尔玛蓝（microplate alamar blueassay，MABA）法测定部分抗结核药物对体外诱导Lzd耐药株的最低抑菌浓度（minimal inhibitory concentration，MIC），对体外诱导获得的Lzd耐药株测序鉴定rpfa基因的突变情况。将诱导Lzd耐药菌株在无药固体培养基上连续传代培养10代以观察耐药稳定性变化，包括MIC变化和rjbfG基因突变情况。结果MTB标准株诱导后得到7株单克隆MTB菌株，其对Lzd的MIC值范围是5．99～18．28μg／ml，为诱导前（0．25μg／m1）8倍以上，成功获得Lzd耐药株，且对Sutezolid（PNU-100480）同时发生交叉耐药，7株菌株均检测到rplC基因中T460C的突变。体外诱导Lzd耐药株在无药固体培养基上连续传代10代，MIC值有所下降（范围为4．71～7．47μg／ml），但均稳定在诱导前8倍以上。结论通过体外诱导实验可以获得对2种恶唑烷酮类（Oxazolidinones）药物均耐药的MTB耐药菌株；rplC基因突变与MTB对恶唑烷酮类药物的耐药相关；MTB对恶唑烷酮类药物的耐药稳定性较强，较难复敏。Objective To gain strains of Mycobacterium tuberculosis （MTB） resistance with Linezolid （Lzd） utilizing twice increasing concentration induction in vitro, investigating its stability of drug resistance and using which to screen for anti-tuberculosis （anti-TB） activity of new compounds. Methods MTB standard strains H37Rv （ATCC27294） were sub-cultured on solid culture medium containing twice increasing concentration of Lzd and induced the drug resistance to Lzcl. The minimal inhibitory concentrations （MIC） of partial anti-TB drugs against induced drug resistant strains in vitro were detected through micro-plate alamar blue assay （MABA）, rplC genes of drug resistant strains obtained from induction in vitro were sequenced and identified for mutations. Induced drug resistant strains were continuously subeultured 10 generations on solid medium without drug to observe the resistance stability, including varieties of MIC and rplC gene mutations. Results Seven MTB monoclonal strains were obtained after induction with the MTB standard strains, the ranges of MIC values of Lzd against these strains were 5.99-18. 28μg/ml, which were eight times higher than before （0. 25 μg/ml）, drug resistant strains were successfully acquired and were resistant to Sutezolid （PNU-100480） simultaneously. T460C mutation in gene rplC was detected in all 7 strains by sequencing. After 10 serially generations on solid culture medium without drug, MIC values of induced drug resistant strains were slightly declined （4. 71- 7. 47 μg/ml）, but all were stable for more than eight times than before. Conclusion MTB strains resistant to two kinds of Oxazolidinones antibiotic were obtained by induction in vitro. Mutation of rplC gene was related to Oxazolidinones antibiotic drug resistance of MTB. Drug resistant stability of MTB to Oxazolidinones antibiotic was strong, and it is difficult for MTB to resume susceptibility.

关 键 词：分枝杆菌 结核 恶唑烷酮类 体外研究 抗药性 细菌 连续传代 

分 类 号：R378.911[医药卫生—病原生物学]