耐多药结核分枝杆菌中embB基因突变与乙胺丁醇耐药的相关性研究

目的 研究耐多药结核分枝菌中embB基因突变与乙胺丁醇耐药的相关性. 方法 比例法检测84株耐多药结核分枝杆菌的乙胺丁醇(EMB)耐药性,基因测序检测embB基因的突变,2检验分析二者之间的相关性. 结果 84株耐多药结核分枝杆菌中有43株(51.2%)对EMB耐药,41株(48.8%)对EMB敏感,57株耐多药菌株(67.9%)的embB基因发生突变.在43株EMB耐药菌株中,embB基因突变的菌株为40株(93.0%),而41株EMB敏感菌株中,embB基因突变的菌株为17株(41.5%),embB基因在耐药菌株中的突变频率远高于敏感菌株(2=25.58,P=0.00).embB306是最常见的突变位点,其在耐药菌株的突变率也高于敏感菌株(2=12.37,P=0.00),embB基因和embB306位点检测EMB耐药的敏感度、特异度和准确性分别为93.0%和65.1%,58.5%和73.2%,76.2%和69.0%. 结论 EMB耐药的产生与embB基因和embB306突变有关,二者用于检测EMB耐药有一定的参考意义.     

结核分枝杆菌乙胺丁醇耐药的分子机制及其快速鉴定

目的：了解结核分枝杆菌乙胺丁醇(EMB)耐药的临床分离株embB基因突变的情况，并探索快速检测结核分枝杆菌EMB耐药性的实验方法。方法：应用聚合酶链反应-单链构象多态性(PCR-SSCP)技术和PCR扩增产物测序方法。分析结核分枝杆菌EMB耐药和敏感各30株的embB基因。结果：以结核分枝杆菌H37RV标准株为对照。发现30株EMB耐药株中有11株(36．7％)embB基因扩增产物SSCP泳动异常，部分耐药菌株测序分析证实为306位密码子突变；而30株敏感株的embB基因扩增产物SSCP泳动均无异常。结论：embB基因突变是结核分枝杆菌EMB耐药的重要机制之一；PCR-SSCP技术可能成为一种检测结核分枝杆菌EMB耐药性的准确和快速的实验方法。     

TDI-FP技术检测耐乙胺丁醇结核分支杆菌embB306点突变

目的 应用TDI-FP技术分析结核分支杆菌embB 306点突变与乙胺丁醇耐药性的关系，并建立一种准确、快速的诊断结核分支杆菌乙胺丁醇耐药的新方法。方法 对临床82例耐多药结核病患者所感染的结核分支杆菌菌株进行常规培养和药敏实验；提取结核分支杆菌DNA，并PCR扩增205bp的embB基因片段，消化降解掉PCR产物中残余的dNTPs和引物，进行模板指导的荧光标记终止碱基掺入反应；应用Victor2测定反应产物的荧光偏振值，分析所有样品的embB基因306位点的基因型；对分析结果进行测序验证。结果 5例乙胺丁醇高度耐药患者中有3例所感染的结核分支杆菌发生embB 306的ATG→GTG突变；47例乙胺丁醇低度耐药患者中有15例为embB 306突变和未突变的混合感染；30例乙胺丁醇敏感患者的结核分支杆菌未发现embB 306突变。测序结果与实验相符合。结论 embB 306突变与结核分支杆菌对乙胺丁醇产生耐药性密切有关；应用TDI-FP技术可以准确、快速简便检测embB 306突变，在结核分支杆菌耐乙胺丁醇的临床诊断中具有潜在的应用前景。     

结核分枝杆菌embB基因突变检测的研究进展

乙胺丁醇（EMB）是具有广谱抗分枝杆菌活性的一线抗结核合成药物。被广泛应用于结核分枝杆菌和鸟分枝杆菌、堪萨斯分枝杆菌等多种非结核分枝杆菌感染的治疗。近年来。结核病疫情呈上升趋势．其耐药问题也日趋严峻，乙胺丁醇（EMB）作为主要的抗结核药物，在原发和复发结核病患者中的耐药率有上升趋势．据报道在一些国家获得性EMB耐药率已达13．7％。有研究表明MTB耐乙胺丁醇与阿拉伯糖基转移酶的编码基因EMB操纵子突变或EMB蛋白过度表达有关。该操纵子由embA、embB、embC3个基因组成．其中embB基因（尤其是306位密码子）突变是EMB耐药的主要原因。现就EMB的药理作用、耐药机制、检测方法做一综述。     

embB 306位点作为耐多药结核分枝杆菌分子检测标记初探

目的 研究embB基因306位点(embB 306)突变在耐多药结核分枝杆菌中的分布,探讨以该位点作为耐多药分子检测标记的应用前景.方法 从上海疾病预防控制中心获得其保存的已知耐药表型的结核分枝杆菌291株,使用错配PCR及DNA测序两种方法检测其embB 306位点的突变,分析耐药表型与结核分枝杆菌embB 306位点的突变的相关性.结果 74株耐多药菌株中有38株(51.4%)该位点有突变(X2=93.8,P<0.01),而其他24株多耐药菌株中有9株(37.5%)为embB 306突变株(X2=60.1,P<0.01);41株耐单药菌株中仅有2株(4.9%)存在该位点的突变(X2=6.8,P=0.0093),而152株全敏感菌株中无一存在该位点突变.以embB 306位点作为耐多药结核分枝杆菌(MDR-TB)分子检测标记的特异度达94.9%(206/217).结论 以embB 306作为耐药突变位点,特别是耐多药结核分枝杆菌的分子检测标记,具有一定的灵敏度和较高的特异度,而且检测方法简单易行,可用于耐多药结核分枝杆菌的临床快速检测.     

结核分枝杆菌耐乙胺丁醇分离株embB基因突变的研究

目的 了解结核分枝杆菌耐乙胺丁醇（EMB）分离株embB基因突变情况,研究其应用价值。方法 通过聚合酶链反应－单链构象多态性（PCR－SSCP)、CPR－限制性片段长度多态性（RFLP）和PCR－直接测序技术分析102株结核分枝杆菌临床分离株embB基因。结果 以H37Rv标准株为对照,102株结核分枝杆菌临床分离株的162rDNA SSCP电泳图谱均与结核分枝杆菌标准株相同。41株药物敏感株的embB基因SSCP均泳动正常,RFLP和测序分析与对照株相同。61株耐EMB分离株可,23株（37．7％）embB基因SSCP泳动异常;8株RFLP分析异常;测序分析23株均为306位密码子突变,其EMB MICs均≥20μg/ml,其中8株为ATG→ATA或ATT突变,13株为ATG→GTG或CTG突变,后者EMB MICs均≥30μg/ml。结论 部分结核分枝杆菌耐EMB是由于其embB基因突变所致,PCR－SSCP技术可能成为测定部分结核分枝杆菌EMB耐药基因型,简便、快速的方法。     

embB基因突变与乙胺丁醇药敏表型及耐多药关系的研究

目的 研究结核分枝杆菌(Mycobacterium tuberculosis,MTB)embB306位点及其他突变位点与乙胺丁醇(ethambutol,EMB)的耐药表型及耐多药(multidrug resistant,MDR)的关系;分析embB基因突变与EMB药敏表型及MDR的关系. 方法 对临床分离的MTB采用BD MGIT 960 SIRE试剂比例法进行药敏试验,取48株EMB耐药、46株EMB敏感但耐其他药及7株四药均敏感MTB提取核酸并扩增embB基因全序列,对扩增产物进行测序分析embB基因序列,与H37Rv标准株序列比对,分析embB基因各突变位点、形式及频率. 结果 101株MTB发现embB基因序列上有17个不同位点突变形式.53株MTB在embB基因序列上发生突变,其中46株为EMB耐药,7株为EMB敏感;embB基因野生型的MTB有48株,其中2株为EMB耐药,46株为EMB敏感;embB突变型与embB野生型的MTB之间EMB耐药率有显著性差异(x2=68.95,P＜0.01).101株MTB中MDR有51株,其中有42株发生embB突变,9株为embB基因野生型,embB基因突变率在MDR和非MDR之间有显著性差异(x2=36.9,P＜0.01). 结论 embB306位点与EMB耐药及MDR中度相关,embB基因突变与EMB耐药及耐多药结核菌(multidrug resistant-Mycobacteriumtuberculosis,MDR-TB)高度相关,embB基因突变可作为MDR-TB的检测分子标记物,指导临床用药.     

广西结核分枝杆菌乙胺丁醇耐药基因突变特征

目的 了解广西壮族自治区结核分枝杆菌乙胺丁醇(EMB)耐药基因突变特征及其影响因素，为结核病的分子诊断和临床治疗用药提供依据。方法 对2018-2019年广西30个结核病耐药监测点连续不间断收集的655株结核分枝杆菌(其中52株乙胺丁醇耐药菌株，603株乙胺丁醇敏感菌株)进行全基因组测序，分析乙胺丁醇耐药基因突变特征及其影响因素。结果 655株结核分枝杆菌中，54株发生乙胺丁醇耐药基因突变，突变率为8.24%(54/655)。在比例法检测EMB耐药的52株菌株中，21株发生基因突变，突变率为40.38%(21/52);603株EMB敏感株中，33株发生基因突变，突变率为5.47%(33/603)，耐药株中的基因突变率高于敏感株(χ²=77.133,P=0.000)。EMB耐药表型与基因突变的符合率为40.38%(21/52)，二者检测结果吻合度不高(Kappa值=0.343,P <0.001)。54株发生基因突变的菌株，突变基因为emb A、emb B和emb C，突变形式有20种，其中单位点突变29株，占53.70%(29/54)，联合位点突变25株，占4...     

耐药结核分枝杆菌基因突变分析

目的 探讨结核分枝杆菌耐药表型与基因突变位点之间的相互关系.方法 采用序列特异性引物分别扩增92株结核分枝杆菌利福平耐药基因rpoB,异烟肼耐药基因katG、inhA、ahpC,链霉素耐药基因rrs、rpsL,乙胺丁醇耐药基因embB及喹诺酮耐药基因gyrA,SSCP筛选出突变序列,DNA测序分析突变性质.结果 59株利福平耐药株rpoB基因突变检出率94.9%(56/59),以Ser450Trp突变最多;90株异烟肼耐药株中,katG基因突变检出率38.9%(35/90),以Ser315Thr最多,3株检出inhA基因突变,ahpC基因无突变检出;34株喹诺酮耐药株中gyrA基因突变检出率82.4%(28/34),主要为Asp94Gly,其次为Ala90Val;31株链霉素耐药株中,15株检出rrs突变,最常见为A514C和A1041G,10株发生rpsL Lys88Arg突变,总的链霉素基因突变检出率为77.4%(24/31);31株乙胺丁醇耐药株中embB 基因突变检出率19.4%(6/31),主要为Met306Val.结论 耐药结核分枝杆菌耐药情况较为严重,以DNA测序为基础的基因突变分析能快速有效地检测结核分枝杆菌的rpoB、katG、gyrA、rrs、rpsL、embB 等耐药分子标识,显示了西安地区耐药性结核分枝杆菌的突变特点,为结核病的临床诊断和合理用药提供了实验依据.     

探针熔解分析法快速检测结核分枝杆菌乙胺丁醇耐药突变

目的 评价探针熔解分析法快速检测结核分枝杆菌乙胺丁醇耐药突变的应用价值,为临床检测提供指导依据.方法 613份痰标本于2009年9月至2010年4月收集自厦门市疾病预防控制中心、厦门市第一医院和漳州市疾病预防控制中心.结核分枝杆菌H37Rv标准株来源于国家结核病参比实验室.37株包含结核与非结核分枝杆菌的标准盘由中国药品生物制品检定所提供.首先采用梯度稀释的野生型标准株H37Rv DNA考察探针熔解曲线分析法的分析灵敏度,然后用标准盘验证其检测特异性,最后以测序法为对照方法,采用613份结核分枝杆菌的标本评价该方法的临床应用价值.结果 探针熔解分析法可检测到3拷贝/反应,且能特异检测结核分枝杆菌.613份结核分枝杆菌的检测结果显示,符合要求的583份标本的试剂盒检测结果与测序结果完全一致,其中embB306突变菌株数为34株,embB 378-380突变菌株数为23株,embB 406突变菌株数为3株,embB 497突变菌株数为3株.结论 探针熔解分析法检测结核分枝杆菌乙胺丁醇耐药突变特异性好,灵敏度高,能有效地检测临床标本常见乙胺丁醇耐药突变位点,是值得推广的快速检测方法.

Abstract：

Objective To evaluate the potential use of a probe melting analysis (PMA) assay in detecting the embB mutations which confer resistance against ethambutol in Mycobacterium tuberculosis. Methods The analysis sensitivity and specificity of PMA were investigated by detecting a serially diluted H37 Rv DNA and a reference panel from National Institute for the Control of Pharmaceutical and Biological Product. Six hundred and thirteen sputum samples were collected from the Xiamen Center for Disease Control and Prevention, Xiamen First Hospital and Center for Zhangzhou Disease Control and Prevention from September 2009 to April 2010. The PMA assay was then evaluated by detecting 613 clinical isolates and the results were compared with the sequencing results. Results The PMA assay could specifically detect Mycobacterium tuberculosis and had a limit of detection of 3 copies per reaction. The assay results with 613 clinical isolates showed that PMA gave a 100% concordance with sequencing in the 583 qualified samples, among which 34 were mutations at embB 306,23 at embB 378-380, 3 at embB 406 and 3 at embB 497. Conclusions PMA assay is a sensitive and specific method enabling efficient detection of common embB mutations causing ethambutol-resistance. The rapidness of this method together with its reliability would facilitate its use in routine testing.     

四川德阳地区结核分枝杆菌耐药基因突变位点的分布特点及基因型耐药的分析

目的探讨四川德阳地区结核分枝杆菌耐药基因突变位点的分布特点及基因型耐药的分析。方法收集在2010年2月-2013年3月检出的257例肺结核病患者结核分枝杆菌DNA阳性的痰液标本,采用聚合酶链式反应（PCR）和反向点杂交（RDB）相结合的基因芯片技术对结核分枝杆菌耐药基因突变位点进行检测和分析。结果 257例肺结核患者中检测出49例存在结核分枝杆菌耐药基因位点突变,其中发生katG 315、rpsL43、embB 306和rpoB 531丝氨酸（S531L）位点突变的分别为30例（11.67%）、18例（7.00%）、11例（4.28%）和10例（3.89%）。234例初治肺结核患者对异烟肼、利福平、乙胺丁醇及链霉素的基因型耐药率分别为9.83%、4.27%、3.42%和5.13%,耐多药率为2.99%;23例复治肺结核患者对上述药物的基因型耐药率分别为52.17%、26.09%、13.04%和43.48%,耐多药率为13.04%。初治患者的基因型耐药率及耐多药率均明显低于复治患者。结论四川德阳地区结核分枝杆菌常见耐药基因突变位点分别为katG 315、rpsL 43、embB 306和rpoB 531丝氨酸（S531L）突变位点;肺结核初治患者的基因型耐药率及耐多药率均明显低于复治患者;该地区肺结核的耐多药率相对较低。     

Association between embB codon 306 mutations and drug resistance in Mycobacterium tuberculosis

embB306 mutants were detected in both ethambutol (EMB)-resistant and EMB-susceptible strains of Mycobacterium tuberculosis. Multidrug-resistant (MDR) strains had a higher proportion of embB306 mutants than non-MDR strains (odds ratio, 6.78; P < 0.001). The embB306 locus is a candidate marker for rapid detection of MDR and extremely drug resistant tuberculosis.     

Significance of mutations in embB codon 306 for prediction of ethambutol resistance in clinical Mycobacterium tuberculosis isolates

We analyzed 159 Mycobacterium tuberculosis isolates (101 ethambutol [EMB]-resistant strains, 33 multidrug-resistant but not EMB-resistant strains, and 25 fully susceptible strains) for the presence of mutations in embB codon 306 (embB306). Mutations were detected only in EMB-resistant strains (n = 69; 68%), thus confirming the significance of embB306 mutations for the prediction of resistance to EMB.     

A single point mutation in the embB gene is responsible for resistance to ethambutol in Mycobacterium smegmatis.

Ethambutol [EMB; dextro-2,2'-(ethylenediimino)-di-1-butanol] is an effective drug when used in combination with isoniazid for the treatment of tuberculosis. It inhibits the polymerization of arabinan in the arabinogalactan and lipoarabinomannan of the mycobacterial cell wall. Recent studies have shown that arabinosyltransferases could be targets of EMB. These enzymes are encoded by the emb locus that was identified in Mycobacterium smegmatis, Mycobacterium leprae, Mycobacterium avium, and Mycobacterium tuberculosis. We demonstrate that a missense mutation in the M. smegmatis embB gene, one of the genes of the emb locus, confers resistance to EMB. The level of resistance is not dependent on the number of copies of the mutated embB gene, indicating that this is a true mechanism of resistance. The mutation is located in a region of the EmbB protein that is highly conserved among the different mycobacterial species. We also identified in this region two other independent mutations that confer EMB resistance. Furthermore, mutations have recently been described in the same region of the EmbB protein from clinical EMB-resistant M. tuberculosis isolates. Together, these data strongly suggest that one of the mechanisms of resistance to EMB consists of missense mutations in a particular region of the EmbB protein that could be directly involved in the interaction with the EMB molecule.     

Detection of embB codon 306 mutations in ethambutol resistant Mycobacterium tuberculosis directly from sputum samples: a low-cost, rapid approach

Substitutions of codon 306 in the gene embB are the most common mutations found in ethambutol resistant Mycobacterium tuberculosis. The characterization of these mutations has been hampered by the need for prior cultivation of the mycobacteria, or the need for DNA sequencing, or both. Here, we describe a simple and culture-independent technique to detect embB codon 306 mutations directly from sputum samples, requiring little more than a PCR machine and a simple agarose minigel. There is no need for labelled probes or DNA sequencing. In a preliminary test of feasibility, interpretable results were obtained from 21 of 24 selected sputum samples, 12 of which were determined to contain ethambutol resistant M. tuberculosis after culture. All of six samples with embB codon 306 mutations were correctly identified. Although an exact validation of this technique is beyond the scope of this technical report, we conclude from well-known embB codon 306 mutation prevalence figures that approximately one half of EMB resistant cases could already be predicted within 2 working days, with little equipment or hands-on time needed, instead of weeks required for conventional resistance testing.     

Transfer of embB codon 306 mutations into clinical Mycobacterium tuberculosis strains alters susceptibility to ethambutol, isoniazid, and rifampin

Implicated as a major mechanism of ethambutol (EMB) resistance in clinical studies of Mycobacterium tuberculosis, mutations in codon 306 of the embB gene (embB306) have also been detected in EMB-susceptible clinical isolates. Other studies have found strong associations between embB306 mutations and multidrug resistance, but not EMB resistance. We performed allelic exchange studies in EMB-susceptible and EMB-resistant clinical M. tuberculosis isolates to identify the role of embB306 mutations in any type of drug resistance. Replacing wild-type embB306 ATG from EMB-susceptible clinical M. tuberculosis strain 210 with embB306 ATA, ATC, CTG, or GTG increased the EMB MIC from 2 microg/ml to 7, 7, 8.5, and 14 microg/ml, respectively. Replacing embB306 ATC or GTG from two high-level EMB-resistant clinical strains with wild-type ATG lowered EMB MICs from 20 microg/ml or 28 microg/ml, respectively, to 3 microg/ml. All parental and isogenic mutant strains had identical isoniazid (INH) and rifampin (RIF) MICs. However, embB306 CTG mutants had growth advantages compared to strain 210 at sub-MICs of INH or RIF in monocultures and at sub-MICs of INH in competition assays. CTG mutants were also more resistant to the additive or synergistic activities of INH, RIF, or EMB used in different combinations. These results demonstrate that embB306 mutations cause an increase in the EMB MIC, a variable degree of EMB resistance, and are necessary but not sufficient for high-level EMB resistance. The unusual growth property of embB306 mutants in other antibiotics suggests that they may be amplified during treatment in humans and that a single mutation may affect antibiotic susceptibility against multiple first-line antibiotics.