Nachappa Somanna Ajjamada, Neelambike Sumana M, Amruthavalli Chokkanna, Ramachandra Nallur B
1 Genetics and Genomics Lab, Department of Studies in Genetics and Genomics, University of Mysore , Mysuru, India .
2 Department of Microbiology, JSS Medical College and Hospital , Mysuru, India .
Microb Drug Resist. 2018 May;24(4):377-385. doi: 10.1089/mdr.2017.0048. Epub 2017 Aug 16.
Diagnosis of drug-resistant tuberculosis predominantly relies on culture-based drug susceptibility testing, which take weeks to produce a result and a more time-efficient alternative method is multiplex allele-specific PCR (MAS-PCR). Also, understanding the role of mutations in causing resistance helps better drug designing.
To evaluate the ability of MAS-PCR in the detection of drug resistance and to understand the mechanism of interaction of drugs with mutant proteins in Mycobacterium tuberculosis.
Detection of drug-resistant mutations using MAS-PCR and validation through DNA sequencing. MAS-PCR targeted five loci on three genes, katG 315 and inhA -15 for the drug isoniazid (INH), and rpoB 516, 526, and 531 for rifampicin (RIF). Furthermore, the sequence data were analyzed to study the effect on interaction of the anti-TB drug molecule with the target protein using in silico docking.
We identified drug-resistant mutations in 8 out of 114 isolates with 2 of them as multidrug-resistant TB using MAS-PCR. DNA sequencing confirmed only six of these, recording a sensitivity of 85.7% and specificity of 99.3% for MAS-PCR. Molecular docking showed estimated free energy of binding (ΔG) being higher for RIF binding with RpoB S531L mutant. Codon 315 in KatG does not directly interact with INH but blocks the drug access to active site.
We propose DNA sequencing-based drug resistance detection for TB, which is more accurate than MAS-PCR. Understanding the action of resistant mutations in disrupting the normal drug-protein interaction aids in designing effective drug alternatives.
耐多药结核病的诊断主要依赖基于培养的药敏试验，该试验需要数周才能得出结果，而一种更具时效性的替代方法是多重等位基因特异性PCR（MAS-PCR）。此外，了解突变在耐药性产生中的作用有助于更好地进行药物设计。
评估MAS-PCR检测耐药性的能力，并了解药物与结核分枝杆菌突变蛋白的相互作用机制。
使用MAS-PCR检测耐药突变，并通过DNA测序进行验证。MAS-PCR针对三个基因上的五个位点，katG 315和inhA -15用于检测异烟肼（INH）耐药，rpoB 516、526和531用于检测利福平（RIF）耐药。此外，利用计算机对接分析序列数据，研究抗结核药物分子与靶蛋白相互作用的影响。
我们使用MAS-PCR在114株分离株中鉴定出8株耐药突变，其中2株为耐多药结核病。DNA测序仅证实了其中6株，MAS-PCR的灵敏度为85.7%，特异性为99.3%。分子对接显示利福平与RpoB S531L突变体结合的估计结合自由能（ΔG）更高。KatG中的315密码子不直接与INH相互作用，但会阻止药物进入活性位点。
我们建议对结核病进行基于DNA测序的耐药性检测，其比MAS-PCR更准确。了解耐药突变在破坏正常药物-蛋白质相互作用中的作用有助于设计有效的替代药物。
