Sowajassatakul Angkanang, Prammananan Therdsak, Chaiprasert Angkana, Phunpruch Saranya
Department of Biology, Faculty of Science, King Mongkut's Institute of Technology Ladkrabang, Bangkok 10520, Thailand.
BMC Microbiol. 2014 Jun 22;14:165. doi: 10.1186/1471-2180-14-165.
The emergence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) makes the treatment and control of tuberculosis difficult. Rapid detection of drug-resistant strains is important for the successful treatment of drug-resistant tuberculosis; however, not all resistance mechanisms to the injectable second-line drugs such as amikacin (AK), kanamycin (KM), and capreomycin (CAP) are well understood. This study aims to validate the mechanisms associated with AK, KM, and CAP resistance in M. tuberculosis clinical strains isolated in Thailand.
A total of 15,124 M. tuberculosis clinical strains were isolated from 23,693 smear-positive sputum samples sent from 288 hospitals in 46 of 77 provinces of Thailand. Phenotypic analysis identified 1,294 strains as MDR-TB and second-line drugs susceptibility was performed in all MDR-TB strains and revealed 58 XDR-TB strains. Twenty-nine KM-resistant strains (26 XDR-TB and 3 MDR-TB) could be retrieved and their genes associated with AK, KM, and CAP resistance were investigated compared with 27 KM-susceptible strains. Mutation of the rrs (A1401G) was found in 21 out of 29 KM-resistant strains whereas mutations of eis either at C-14 T or at G-37 T were found in 5 strains. Three remaining KM-resistant strains did not contain any known mutations. Capreomycin resistance was determined in 28 of 29 KM-resistant strains. Analysis of tlyA revealed that the A33G mutation was found in all CAP-resistant strains and also in susceptible strains. In contrast, the recently identified tlyA mutation T539G and the novel Ins49GC were found in two and one CAP-resistant strains, respectively. In addition, our finding demonstrated the insertion of cytosine at position 581 of the tap, a putative drug efflux encoding gene, in both KM-resistant and KM-susceptible strains.
Our finding demonstrated that the majority of KM resistance mechanism in Thai M. tuberculosis clinical strains was rrs mutation at A1401G. Mutations of the eis promoter region either at C-14 T or G-37 T was found in 5 of 29 strains whereas three strains did not contain any known mutations. For CAP resistance, 3 of 28 CAP-resistant strains contained either T539G or Ins49GC mutations at tlyA that might be associated with the resistant phenotype.
耐多药结核病（MDR-TB）和广泛耐药结核病（XDR-TB）的出现使结核病的治疗和控制变得困难。快速检测耐药菌株对于耐药结核病的成功治疗很重要；然而，并非所有对阿米卡星（AK）、卡那霉素（KM）和卷曲霉素（CAP）等二线注射用药物的耐药机制都已完全了解。本研究旨在验证泰国分离的结核分枝杆菌临床菌株中与AK、KM和CAP耐药相关的机制。
从泰国77个省中46个省的288家医院送来的23,693份涂片阳性痰标本中，共分离出15,124株结核分枝杆菌临床菌株。表型分析确定1,294株为耐多药结核病，对所有耐多药结核菌株进行了二线药物敏感性检测，发现58株广泛耐药结核菌株。可以获取29株耐卡那霉素菌株（26株广泛耐药结核菌株和3株耐多药结核菌株），并与27株卡那霉素敏感菌株比较，研究它们与AK、KM和CAP耐药相关的基因。29株耐卡那霉素菌株中有21株发现rrs（A1401G）突变，5株发现eis基因在C-14 T或G-37 T处发生突变。其余3株耐卡那霉素菌株未发现任何已知突变。在29株耐卡那霉素菌株中的28株中检测了卷曲霉素耐药性。对tlyA的分析表明，所有卷曲霉素耐药菌株以及敏感菌株中均发现A33G突变。相比之下，最近发现的tlyA突变T539G和新发现的Ins49GC分别在2株和1株卷曲霉素耐药菌株中发现。此外，我们的研究结果表明，在耐卡那霉素菌株和卡那霉素敏感菌株中，假定的药物外排编码基因tap的第581位均插入了胞嘧啶。
我们的研究结果表明，泰国结核分枝杆菌临床菌株中大多数卡那霉素耐药机制是rrs基因在A1401G处发生突变。29株菌株中有5株发现eis启动子区域在C-14 T或G-37 T处发生突变，而3株菌株未发现任何已知突变。对于卷曲霉素耐药性，28株卷曲霉素耐药菌株中有3株在tlyA基因处含有T539G或Ins49GC突变，这可能与耐药表型有关。
