Ramasubban Gayathri, Therese Kulandai Lily, Lakshmipathy Dhanurekha, Sridhar R, Meenakshi N, Madhavan Hajib N
L&T Microbiology Research Centre, Vision Research Foundation, KNBIRVO Building, 41 College Road, Chennai 600006, India.
L&T Microbiology Research Centre, Vision Research Foundation, KNBIRVO Building, 41 College Road, Chennai 600006, India.
J Glob Antimicrob Resist. 2015 Mar;3(1):1-4. doi: 10.1016/j.jgar.2014.10.002. Epub 2014 Nov 8.
The objective of this study was to detect mutations associated with isoniazid (INH) and rifampicin (RIF) resistance in Mycobacterium tuberculosis isolates from newly diagnosed and previously treated tuberculosis patients using a PCR-based DNA sequencing technique. Phenotypic drug susceptibility testing was performed using a BACTEC™ MicroMGIT Culture System in 354 M. tuberculosis isolates. Among the 354 isolates, 18 were multidrug-resistant tuberculosis (MDR-TB). PCR-based DNA sequencing was performed targeting the rpoB gene for RIF and the whole of the katG gene and the promoter and coding region of the inhA gene for INH. Results were analysed using MultAlin analysis to identify the presence of polymorphisms or mutations by comparing with already available GenBank sequences. Only 37.5% of RIF-resistant isolates showed the presence of the most commonly reported mutation (Ser531Leu). The most commonly reported mutation (Ser531Leu) was detected in six MDR-TB isolates. The frequency of mutations associated with INH resistance was 31.5% (17/54) and 29.6% (16/54) for katG and inhA, respectively. Comparing the relative distribution of mutations in the two target loci revealed that 12 isolates (22.2%) had a mutation in both katG and inhA. Apart from previously reported mutations in the katG gene, there were three novel deletion and six novel substitution mutations. As reported in previous studies, Ser531Leu was the most common mutation detected in RIF-resistant isolates. The genetic mechanism of INH resistance in M. tuberculosis is highly complex involving several genes, and much remains to be explored to achieve a better understanding of this complex mechanism.
本研究的目的是使用基于聚合酶链反应（PCR）的DNA测序技术，检测新诊断和既往治疗过的结核病患者的结核分枝杆菌分离株中与异烟肼（INH）和利福平（RIF）耐药相关的突变。使用BACTEC™ MicroMGIT培养系统对354株结核分枝杆菌进行了表型药物敏感性测试。在这354株分离株中，18株为耐多药结核病（MDR-TB）。针对RIF的rpoB基因以及针对INH的整个katG基因、inhA基因的启动子和编码区进行了基于PCR的DNA测序。通过与已有的GenBank序列进行比较，使用MultAlin分析来鉴定多态性或突变的存在。仅37.5%的利福平耐药分离株显示存在最常报道的突变（Ser531Leu）。在6株MDR-TB分离株中检测到了最常报道的突变（Ser531Leu）。与INH耐药相关的katG和inhA突变频率分别为31.5%（17/54）和29.6%（16/54）。比较两个靶位点突变的相对分布发现，12株分离株（22.2%）在katG和inhA中均有突变。除了katG基因中先前报道的突变外，还有3个新的缺失突变和6个新的替代突变。如先前研究报道，Ser531Leu是在利福平耐药分离株中检测到的最常见突变。结核分枝杆菌中INH耐药的遗传机制高度复杂，涉及多个基因，要更好地理解这一复杂机制仍有许多有待探索之处。
