异烟肼诱导致单耐异烟肼结核分枝杆菌假定药物外排泵基因表达量变化的初步研究

A primary investigation on the isoniazid-induced alterations in efflux gene expression among the isoniazid resistant Mycobacterium tuberculosis clinical isolates

目的检测单耐异烟肼结核分枝杆菌经异烟肼诱导培养前后假定药物外排泵基因表达量的变化，探讨可能导致结核分枝杆菌对异烟肼耐药的外排泵基因及导致基因高表达的原因。方法选取35株单耐异烟肼结核分枝杆菌临床分离株、10株全敏结核分枝杆菌临床分离株和H37Rv（标准对照），对各菌株进行无异烟肼和亚抑菌异烟肼浓度（1／4MIC）的7H9液体培养基诱导培养，提取RNA后反转录并采用real—timePCR技术分别检测27个假定外排泵基因表达量的变化，用公式2^-△△CT计算每个假定基因相对表达量的差异倍数。结果耐药株中27个假定外排泵基因有13个基因高表达，高表达RV1258c基因的菌株数最多为6株，其次是高表达RV2265和RV0849的菌株各有5株。35株菌中14株菌（40．00％）有高表达的外排泵基因，6株（17．14％）菌株均只有1个假定外排泵基因高表达；8株（22．86％）菌株有两种及以上基因高表达，其中4、2、1、1株菌株分别有2、4、5、7个基因高表达；10株全敏菌株和H37Rv 27个假定外排泵基因均无高表达。结论Rv1258c、Rv2265和Rv0849等基因可能是导致结核分枝杆菌对异烟肼耐药的外排泵基因，异烟肼可能为结核分枝杆菌部分假定外排泵基因的诱导物而导致其激活并高表达。

Objective To detect the changes on the expression of putative drug effiux genes caused by isoniazid-inducement in single resistance to the isoniazid Mycobacterium tuberculosis （M. tuberculosis） clinical isolates, for exploring the putative effiux genes which causing M. tuberculosis isoniazid resistance as well as the mechanism related to high expression of the putative effiux genes. Methods We selected 35 M. tuberculosis clinical isolates which were only resistant to isoniazid as well as 10 sensitive M. tuberculosis clinical isolates and using H37Rv as control. Each strain was cultured in 7H9 liquid medium without isoniazid and with subinhibitory isoniazid concentration （ 1/4 MIC） induction. After RNA extraction and reverse transcription,real-time PCR was conducted to assess the expression changes of 27 putative drug effiux pump genes with formula 2 ^-△△CT to calculate the expression of each putative drug effiux pump genes. Results Of the 27 putative genes, 13 of them were expressed at high level. High expression of Rv1258c gene had the maximum number of 6 strains, followed by high expression of Rv0849 and Rv2265 which both had 5 strains. Fourteen strains （40.00%） out of the 35 strains had high expression pump genes. Six strains （17.14%） had only one highly expressed putative effiux pump gene. Eight strains （22.86%） had two or more highly expressed putative effiux pump genes, including two, four, five, seven genes that highly expressed in 4, 2, 1,1 strains respectively. For the 27 putative genes, ten sensitive strains and H37Rv did not show highly expressed genes. Conclusion Rv1258c, Rv2265, Rv0849, etc. genes might be the putative effiux pumps genes of M. tuberculosis resistant to isoniazid. Isoniazid might serve as the inducer of M. tuberculosis part putative effiux pump genes, inducing activation and causing high expression of these putative effiux pump genes.