Özgür Didem, Ersoy Leyla, Ülger Mahmut, Tezcan Ülger Seda, Aslan Gönül
Kafkas University Faculty of Medicine, Department of Medical Microbiology, Kars, Türkiye.
Mersin University Faculty of Medicine, Department of Medical Microbiology, Mersin, Türkiye.
Mikrobiyol Bul. 2023 Apr;57(2):207-219. doi: 10.5578/mb.20239916.
Tuberculosis (TB) is caused by Mycobacterium tuberculosis, still one of the most common life-threatening infectious diseases worldwide. Although drug resistance in M.tuberculosis is mainly due to spontaneous chromosomal mutations in genes encoding drug target or drug activating enzymes, the resistance cannot be explained only by these mutations. Low permeability of the cell wall, drug inactivating enzymes and especially efflux pumps (EPs) are other mechanisms of drug resistance in mycobacteria. Efflux pump inhibitors (EPIs) binding to M.tuberculosis EPs were shown to inhibit efflux of anti-TB drugs, to enhance M.tuberculosis killing, to reduce drug resistance and to produce synergistic effects with first line anti-TB drugs. In this study, we aimed to determine the minimum inhibitory concentration (MIC) of first-line anti-TB drugs in the presence of verapamil (VER) and the expression of 21 putative EP genes belonged to the ATP-binding cassette (ABC), major facilitator superfamily (MFS) and resistance-nodulation-division (RND) families which might have caused the resistance in nine M.tuberculosis complex clinical isolates resistant to all of the first line anti-TB drugs. MIC values of the isolates were determined in 96-well U-bottom plates by the resazurin microtiter test (REMA) method based on the color change principle. According to the determined MIC values of each isolate, freshly grown cultures in Middlebrook 7H9 broth were exposed to first-line anti-TB drugs and MIC of first-line anti-TB drugs in the presence of VER (½ MIC) at 37°C for 48 hours for RNA extraction. The non-drug exposed cultures were used as control. Total RNA was extracted using the RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) and then treated with DNase I (Thermo Fischer Scientific Inc., Waltham, MA). Complementary DNA (cDNA) from the extracted RNAs was synthesized with the "First strand cDNA synthesis kit" (Thermo Fischer Scientific Inc., Waltham, MA) using oligo primers. The expression levels of efflux pump genes by quantitative realtime polymerase chain reaction (qRt-PCR) were performed using the QuantiTect SYBR Green Rt-PCR Kit (Qiagen, Germany). The housekeeping sigma factor gene sigA (Rv2703) was used as internal control in qRt‑PCR assays. Relative quantification of the clinical isolates was determined by the 2-∆∆Ct method by comparing the expression levels of efflux genes in cultures exposed to primary anti-TB drugs and VER with those of non-drug exposed cultures. MIC values of nine isolates by REMA method was determined between 32-512 μg/mL, 1-128 μg/mL, 2-32 μg/mL, 4-16 μg/mL and 15.62-250 μg/mL for streptomycin (SM), isoniazid (INH), rifampicin (RIF), ethambutol (EMB) and VER, respectively. In the presence of ½ MIC VER, it was determined that the MIC of SM decreased 2-32 fold in eight isolates, the MIC of INH decreased by 2-8 fold in nine isolates, the MIC of RIF decreased by 2-16 fold in eight isolates, and the MIC of EMB decreased 2-4 fold in only five isolates. There was an increase in the expression of Rv1273c, Rv1456c, Rv1457 and Rv1819 efflux pump genes from the ABC family, Rv1634 and Rv0842 from the MFS family and Rv3823 efflux from the RND family in isolates exposed to ½ MIC of first-line anti-TB drugs stress. Rv1456c and Rv1819 were found to be associated with SM resistance, Rv1273c with EMB resistance, Rv1457, Rv0842 and Rv3823 with both RIF and EMB resistance, and Rv1634 with INH, RIF and EMB resistance. It was determined that there was a decrease in the expression levels of eight efflux pump genes from the ABC family (Rv1456c, Rv1457c, Rv1458c, Rv0194, Rv1272c, Rv1686c, Rv1687c, Rv1819c), six from MFS family (Rv0842, Rv0849, Rv1634, Rv2265, Rv2456c, Rv0876c) and two from RND family (Rv0507, Rv0676c) in isolates exposed to MIC of first-line anti-TB drugs in the presence of VER (½ MIC). Further studies with clinical isolates are needed to investigate the EPIs that can be used in alternative therapy and to determine the contribution of EPs to the development of resistance due to the increasing TB resistance.
结核病（TB）由结核分枝杆菌引起，它仍是全球最常见的危及生命的传染病之一。尽管结核分枝杆菌的耐药性主要归因于编码药物靶点或药物激活酶的基因发生自发染色体突变，但这种耐药性不能仅用这些突变来解释。细胞壁通透性低、药物失活酶尤其是外排泵（EPs）是分枝杆菌耐药的其他机制。已证明与结核分枝杆菌外排泵结合的外排泵抑制剂（EPIs）可抑制抗结核药物的外排，增强对结核分枝杆菌的杀灭作用，降低耐药性，并与一线抗结核药物产生协同作用。在本研究中，我们旨在确定在维拉帕米（VER）存在的情况下一线抗结核药物的最低抑菌浓度（MIC），以及属于ATP结合盒（ABC）、主要易化子超家族（MFS）和耐药-固氮-细胞分裂（RND）家族的21个假定EP基因的表达情况，这些基因可能导致了9株对所有一线抗结核药物耐药的结核分枝杆菌复合群临床分离株产生耐药性。通过基于颜色变化原理的刃天青微量滴定试验（REMA）方法，在96孔U型底平板中测定分离株的MIC值。根据每个分离株测定的MIC值，将在Middlebrook 7H9肉汤中新鲜培养的培养物暴露于一线抗结核药物，并在37℃下将一线抗结核药物在VER存在下（½ MIC）的MIC暴露48小时以提取RNA。未暴露于药物的培养物用作对照。使用RNeasy Mini试剂盒（德国希尔德市Qiagen公司）提取总RNA，然后用DNase I（美国马萨诸塞州沃尔瑟姆市Thermo Fischer Scientific公司）处理。使用寡核苷酸引物，用“第一链cDNA合成试剂盒”（美国马萨诸塞州沃尔瑟姆市Thermo Fischer Scientific公司）从提取的RNA合成互补DNA（cDNA）。使用QuantiTect SYBR Green Rt-PCR试剂盒（德国Qiagen公司）通过定量实时聚合酶链反应（qRt-PCR）进行外排泵基因的表达水平检测。在qRt-PCR分析中，持家sigma因子基因sigA（Rv2703）用作内对照。通过2-∆∆Ct方法，比较暴露于一线抗结核药物和VER的培养物中与未暴露于药物的培养物中外排基因的表达水平，确定临床分离株的相对定量。通过REMA方法测定的9株分离株对链霉素（SM）、异烟肼（INH）、利福平（RIF）、乙胺丁醇（EMB）和VER的MIC值分别在32 - 512μg/mL、1 - 128μg/mL、2 - 32μg/mL、4 - 16μg/mL和15.62 - 250μg/mL之间。在½ MIC VER存在的情况下，确定8株分离株中SM的MIC降低了2 - 32倍，9株分离株中INH的MIC降低了2 - 8倍，8株分离株中RIF的MIC降低了2 - 16倍，仅5株分离株中EMB的MIC降低了2 - 4倍。在暴露于½ MIC一线抗结核药物应激的分离株中，ABC家族的Rv1273c、Rv1456c、Rv1457和Rv1819外排泵基因、MFS家族的Rv1634和Rv0842以及RND家族的Rv3823外排基因的表达增加。发现Rv1456c和Rv1819与SM耐药相关，Rv1273c与EMB耐药相关，Rv1457、Rv0842和Rv3823与RIF和EMB耐药均相关，Rv1634与INH、RIF和EMB耐药相关。确定在VER（½ MIC）存在的情况下，暴露于一线抗结核药物MIC的分离株中，ABC家族的8个外排泵基因（Rv1456c、Rv1457c、Rv1458c、Rv0194、Rv1272c、Rv1686c、Rv1687c、Rv1819c）、MFS家族的6个外排泵基因（Rv0842、Rv0849、Rv1634、Rv2265、Rv2456c、Rv0876c）和RND家族的2个外排泵基因（Rv0507, Rv0676c）的表达水平降低。由于结核病耐药性增加，需要对临床分离株进行进一步研究，以研究可用于替代疗法的EPIs，并确定外排泵对耐药性发展的贡献。
