Jagielski Tomasz, Bakuła Zofia, Roeske Katarzyna, Kamiński Michał, Napiórkowska Agnieszka, Augustynowicz-Kopeć Ewa, Zwolska Zofia, Bielecki Jacek
Department of Applied Microbiology, Institute of Microbiology, Faculty of Biology, University of Warsaw, I. Miecznikowa 1, 02-096 Warsaw, Poland
Department of Applied Microbiology, Institute of Microbiology, Faculty of Biology, University of Warsaw, I. Miecznikowa 1, 02-096 Warsaw, Poland.
J Antimicrob Chemother. 2015 Dec;70(12):3214-21. doi: 10.1093/jac/dkv253. Epub 2015 Aug 25.
Progress in the detection of drug-resistant TB has been underpinned by the development and implementation of new, reliable and rapid diagnostic tools. These rely mostly on the detection of specific mutations conferring resistance to anti-TB drugs. The aim of this study was to search for mutations associated with isoniazid resistance among Mycobacterium tuberculosis clinical isolates.
A collection of 150 M. tuberculosis strains, including 50 MDR, 50 isoniazid-monoresistant and 50 pan-susceptible strains, was used. For all the strains, seven structural genes (katG, inhA, ahpC, kasA, ndh, nat and mshA) and two regulatory regions (mabA-inhA promoter and oxyR-ahpC intergenic region) were PCR amplified and sequenced in their entirety.
Sixty-six distinct mutations were detected at all nine loci investigated, accounting for 109 (72.7%) of the strains tested. The number of strains with any mutation among the MDR, isoniazid-monoresistant and pan-susceptible groups was 49 (98%), 37 (74%) and 23 (46%), respectively. Mutations in the katG gene predominated, with 29 different types distributed among 46 (92%) MDR, 31 (62%) isoniazid-monoresistant and 2 (4%) pan-susceptible strains. Twenty-nine and 19 mutations were found exclusively in MDR and isoniazid-monoresistant strains, respectively.
This study revealed 17 mutations, previously unreported, that might be of potential use as new surrogate markers of isoniazid resistance. Their diagnostic accuracy needs to be confirmed on larger strain samples and from different geographical settings. For isoniazid resistance detection, molecular approaches should still be a complement to rather than a replacement for conventional drug susceptibility testing. This is supported by the lack of mutations in any of the nine genetic loci investigated in 18 isoniazid-resistant strains from this study.
新型、可靠且快速的诊断工具的开发与应用推动了耐药结核病检测的进展。这些工具大多依赖于对赋予耐抗结核药物能力的特定突变的检测。本研究的目的是在结核分枝杆菌临床分离株中寻找与异烟肼耐药相关的突变。
使用了一组150株结核分枝杆菌菌株，包括50株耐多药菌株、50株异烟肼单耐药菌株和50株全敏感菌株。对所有菌株，对7个结构基因（katG、inhA、ahpC、kasA、ndh、nat和mshA）和2个调控区域（mabA-inhA启动子和oxyR-ahpC基因间区域）进行PCR扩增并完整测序。
在所研究的所有9个位点共检测到66个不同的突变，占所测试菌株的109株（72.7%）。耐多药组、异烟肼单耐药组和全敏感组中出现任何突变的菌株数量分别为49株（98%）、37株（74%）和23株（46%）。katG基因中的突变占主导，有29种不同类型分布在46株（92%）耐多药菌株、31株（62%）异烟肼单耐药菌株和2株（4%）全敏感菌株中。分别在耐多药菌株和异烟肼单耐药菌株中仅发现29个和19个突变。
本研究揭示了17个以前未报道的突变，可能作为异烟肼耐药的新替代标志物具有潜在用途。它们的诊断准确性需要在更大的菌株样本和不同地理环境中得到证实。对于异烟肼耐药检测，分子方法仍应是传统药物敏感性试验的补充而非替代。本研究中18株异烟肼耐药菌株在所研究的9个基因位点中均未出现突变，这支持了上述观点。
