Department of Biology, Faculty of Science, King Mongkut's Institute of Technology Ladkrabang, Bangkok, 10520, Thailand.
Tuberculosis Research Laboratory, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathum Thani, 12120, Thailand.
Ann Clin Microbiol Antimicrob. 2018 Jul 16;17(1):33. doi: 10.1186/s12941-018-0285-6.
Aminoglycosides such as amikacin and kanamycin are effective injectable second-line drugs for treatment of multidrug-resistant tuberculosis. Molecular mechanisms underlying aminoglycoside resistance are not well understood. We have previously identified the amikacin- and kanamycin-resistant M. tuberculosis MT433 clinical strain, of which all known mutations related to resistance have not been found. Drug efflux pump is one of reported resistance mechanisms that might play a role in aminoglycoside resistance.
The expression levels of sixteen putative efflux pump genes, including eis and one regulator gene, whiB7, of MT433 in the presence of kanamycin were determined using the reverse transcription-quantitative PCR method. The effects of upregulated genes on amikacin and kanamycin resistance were investigated by overexpression in M. tuberculosis H37Ra strain.
Upon kanamycin exposure, other than whiB7 and eis that were found extremely overexpressed, two drug efflux pump genes, namely Rv1877 and Rv2846c, showed specifically high-level of expression in M. tuberculosis MT433 strain. However, direct effect of overexpressed Rv1877 and Rv2846c on amikacin and kanamycin resistance could not be demonstrated in M. tuberculosis H37Ra overexpressed strain.
Our finding demonstrated that overexpression of eis could occur without any mutations in the promoter region and be detectable in clinical isolate. This might be a consequence of overexpressed whiB7, resulting in amikacin and kanamycin resistance in M. tuberculosis MT433 strain.
阿米卡星和卡那霉素等氨基糖苷类药物是治疗耐多药结核病的有效二线注射药物。氨基糖苷类药物耐药的分子机制尚不清楚。我们之前已经鉴定出耐阿米卡星和卡那霉素的结核分枝杆菌 MT433 临床株，其中尚未发现与耐药性相关的所有已知突变。药物外排泵是已报道的耐药机制之一，可能在氨基糖苷类耐药中发挥作用。
采用反转录定量 PCR 法检测 MT433 中 16 个推定外排泵基因（包括 eis 和一个调节剂基因 whiB7）在卡那霉素存在下的表达水平。通过在结核分枝杆菌 H37Ra 株中过表达来研究上调基因对阿米卡星和卡那霉素耐药性的影响。
在卡那霉素暴露下，除了发现极度过表达的 whiB7 和 eis 外，两个药物外排泵基因 Rv1877 和 Rv2846c 在结核分枝杆菌 MT433 株中表现出特异性高水平表达。然而，在过表达的结核分枝杆菌 H37Ra 株中，过表达的 Rv1877 和 Rv2846c 对阿米卡星和卡那霉素耐药性的直接影响无法证明。
我们的发现表明，在没有启动子区域突变的情况下，eis 的过表达可以发生，并在临床分离株中检测到。这可能是过表达 whiB7 的结果，导致结核分枝杆菌 MT433 株对阿米卡星和卡那霉素产生耐药性。
