摘要
目的:利用浓度倍增方法体外诱导对利福平(rifampicin,Rfp)耐药的结核分枝杆菌(mycobacteriurn tuberculosis,MTB)菌株,研究耐药机制演变过程及遗传稳定性。方法:将MTB标准菌株H37Rv在Rfp浓度2倍递增的7HIO液体培养基中进行传代培养,逐步诱导MTB菌株对Rfp耐药。利用Xpert MTB/RIF及荧光PCR熔解曲线法检测不同浓度Rfp诱导菌株rpoB基因的突变情况,实时荧光定量PCR方法检测外排泵基因Rv1457c、Rv1458c mRNA表达量,测算与标准菌株Rv1457c、Rv1458c相对表达量比值,将Rfp耐药菌株在无药罗氏培养基上连续培养10代,观察耐药性的变化。结果:MTB标准菌株诱导到Rfp 32μg/mL,获得rpoB基因突变菌株;Rv1457c和Rv1458c基因表达量随Rfp诱导浓度递增而逐渐上升,当浓度达到0.5μg/mL及以上时,与标准菌株的比值超过2,提示诱导菌株Rv1457c和Rv1458c基因已处于高表达状态。在无药培养基上连续传代10代后,rpoB基因发生突变前各浓度Rfp诱导菌株MIC明显降低,而rpoB基因突变菌株MIC值基本稳定。结论:通过体外诱导实验可以获得Rfp耐药MTB菌株,药物外排泵基因表达与MTB菌株诱导浓度有关,rpoB基因突变后的MTB菌株对Rfp耐药性能获得稳定遗传。
Objective:To gain strains of mycobacterium tuberculosis(MTB)resistance with rifampicin(Rfp)utilizing twice increasing concentration induction in vitro,to study the drug resistance mechanism,evolution and genetic stability.Methods:MTB standard strains H37Rv(ATCC27294)were sub-cultured on liquid culture medium containing twice increasing of Rfp and induced drug resistant strains to Rfp.Using Xpert MTB/RIF and fluorescence PCR melting curve method to respectively detect the Rfp resistance gene(rpoB gene)with induced strain to obtain different concentrations;Using the method of real-time fluorescent quantitative PCR to detect mRNA expression of drug efflux pump gene Rv1457c,Rv1458c with induced strain to obtain different concentrations;The relative expression ratios of rv1457c and rv1458c between induced strains and standard strains were further measured.Analyze drug efflux pump gene expression in the induction of different concentrations of MTB resistant phenotypes,rpoB gene mutation type differences in MTB strains;The induced strains with different concentrations were subcultured on drug-free Roche medium for 10 generations to observe the changes of drug resistance stability.Results:rpoB gene of standard MTB strains induced to 32μg/mL was mutationed;Rv1457c and Rv1458c gene expression were gradually raised with concentration increasing Rfp induction.MIC value of Rfp induced MTB strains werw decreased after consecutive batches 10 generations in the different no medicine concentration liquid medium,But MIC value of rpoB gene mutations MTB strains was stable and more nine times than before induction.Conclusion:Rfp resistant MTB strains can be obtained by in vitro induction experiments,drug efflux pump gene expression was correlated with induced concentration of MTB strains.But Rfp resistance MTB strains of rpoB gene mutations can secure a stable inheritance.
作者
司晓燕
陈俊林
施慧慧
瞿梅
马娟
顾德林
SI Xiaoyan;CHEN Junlin;SHI Huihui;QU Mei;MA Juan;GU Delin(Department of Clinical Laboratory,Nantong Sixth People’s Hospital,Jiangsu 226011)
出处
《交通医学》
2022年第1期10-14,18,共6页
Medical Journal of Communications
基金
江苏省卫生健康委员会科研课题(Z2018028)。
关键词
结核分枝杆菌
利福平
体外诱导
耐药机制
外排泵基因
RPOB基因
mycobacteriurn tuberculosis(MTB)
Rifampicin(Rfp)
in vitro induction
drug resistance mechanism
efflux pump gene
rpoB gene
