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.     

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。     

青岛地区结核分枝杆菌embB306基因突变特征与乙胺丁醇耐药的关系

目的：探讨青岛地区结核分枝杆菌 embB306突变特征与乙胺丁醇耐药的关系。方法应用聚合酶链反应（PCR）-DNA 测序法对来自青岛地区36株乙胺丁醇耐药和48株乙胺丁醇敏感的结核分枝杆菌的 embB 基因片段进行分析。结果48株乙胺丁醇敏感菌株中均未发生 embB306突变,而36株乙胺丁醇耐药菌株中有20株发生了embB306突变,突变率为55．6％;embB306突变在乙胺丁醇敏感菌株和乙胺丁醇耐药菌株中的分布差异具有统计学意义（χ2＝35．000,P ＜0．01）。结论在青岛地区,embB306的突变是青岛地区结核分枝杆菌对乙胺丁醇耐药的主要机制。     

合肥地区耐多药结核分枝杆菌异烟肼、链霉素及 乙胺丁醇耐药相关基因位点表达研究

目的研究耐多药结核分枝杆菌异烟肼(INH)、链霉素(SM)及乙胺丁醇(EMB)耐药相关基因位点的表达情况。方法选取该院2013年1月至2017年12月临床分离的101株耐多药结核分枝杆菌,利用测序法和基因芯片法分别检测INH、SM和EMB耐药相关基因及突变位点。结果 101株耐多药结核分枝杆菌中对SM耐药率69.3%;对EMB耐药率51.4%;对SM和EMB二者均耐药的耐药率44.5%。基因芯片法检出95例INH耐药相关基因突变,其中katG基因315M位点突变率为77.8%;inhA基因-15M位点突变率为89.4%。检出64例rpsL SM耐药相关基因,其中43M位点突变率为89.0%;88M位点突变率为11.0%。检出47例embB EMB耐药相关基因,以306M2位点突变为主,突变率为68.0%。测序法检出98例INH耐药相关基因突变,katG基因突变以315位点为主突变率为73.4%;inhA基因突变以-15位点为主突变率为87.7%;oxyR-ahpC基因-12和-10位点突变率分别为10.2%和8.2%。检出68例SM耐药相关基因突变,rpsL基因以43位点为主突变率为86.8%;rrs基因512、513、516位点突变率分别为5.9%、8.8%、8.8%。检出50例EMB耐药相关基因embB以306位点为主突变率为88.0%。结论耐多药结核分枝杆菌INH、SM及EMB耐药基因位点表达可作为结核分枝杆菌临床检测耐药性的有效依据,为提高耐多药结核分枝杆菌耐药性检测的敏感度,应将INH耐药相关oxyR-ahpC基因和SM耐药相关rrs基因纳入我国快速分子诊断产品中。     

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...     

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。     

结核杆菌耐药性调查与基因突变分析

目的了解结核分支杆菌耐药性与基因突变情况,分析耐药表型与基因突变的相关性。方法从临床标本中分离培养、鉴定结核分支杆菌;应用药敏试验、单链探针反向杂交试验技术(LiPA)和基因测序分析结核分支杆菌耐药性与基因突变情况。结果50株结核分支杆菌中,12株耐异烟肼(INH),7株耐利福平(RFP),15株耐链霉素(SM),2株耐乙胺丁醇(EMB)。LiPA检测耐RFP菌株,4株rpoB基因531位TCG→TTG突变;耐INH菌株中5株KatG基因315位AGC→ACC突变;耐SM菌株中6株、SM敏感株1株rpsl基因43位AAG→AGG突变,1株耐SM菌株rpsl基因88位AAG→AGG突变;rrs基因未检测到突变;1株耐EMB菌株与1株EMB敏感株的embB基因306位发生ATG→GTG突变。结论本市结核分支杆菌的耐药性增加的趋势显著,加强监测与综合干预非常必要。     

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。     

Phenotypic and molecular characterization of Mycobacterium tuberculosis isolates resistant to both isoniazid and ethambutol

In performing radiometric susceptibility testing on over 2,000 patient isolates of Mycobacterium tuberculosis during the past 6 years, we found that resistance to 7.5 microg/ml ethambutol (EMB) occurred only in isolates that are also resistant to 0.4 microg/ml isoniazid (INH). Using 157 selected isolates in the present study, we performed radiometric and agar proportion susceptibility tests and DNA sequencing of genetic regions associated with resistance to these two drugs. The goal was to study the occurrence of the common mutations associated with resistance to each drug and also to determine whether any particular INH-resistance-associated mutation occurred more often in combination with any particular EMB-resistance-associated mutation. In an analysis of 128 isolates resistant to 0.4 microg/ml INH, we found that a mutation at katG Ser315 was more common in isolates also resistant to 7.5 microg/ml EMB (61 of 67=91.0%) than in isolates either susceptible to EMB or resistant to 2.5 microg/ml EMB (39 of 60=65.0%). These observations suggest that INH-resistant strains with a mutation at katG Ser315 are more likely to acquire resistance to 7.5 microg/ml EMB than are isolates with INH-resistance-associated mutations at other sites. In addition, we found that 64 of 67 (95.5%) isolates resistant to 7.5 microg/ml EMB contained a mutation in either codon 306 or codon 406 of embB. Met306Val was the most common embB mutation, present in 52 (77.6%) of the 67 isolates. Most occurrences of this mutation (49 of 52=94.2%) were found in isolates that also contained the katG Ser315Thr mutation. Finally, sequencing this region of embB appears to be sufficiently sensitive for use as a rapid screening tool for detection of high-level resistance to EMB.     

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.     

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.     

Mycobacterium tuberculosis embB codon 306 mutations confer moderately increased resistance to ethambutol in vitro and in vivo

Ethambutol (EMB) is a major component of the first-line therapy of tuberculosis. Mutations in codon 306 of embB (embB306) were suggested as a major resistance mechanism in clinical isolates. To directly analyze the impact of individual embB306 mutations on EMB resistance, we used allelic exchange experiments to generate embB306 mutants of M. tuberculosis H37Rv. The level of EMB resistance conferred by particular mutations was measured in vitro and in vivo after EMB therapy by daily gavage in a mouse model of aerogenic tuberculosis. The wild-type embB306 ATG codon was replaced by embB306 ATC, ATA, or GTG, respectively. All of the obtained embB306 mutants exhibited a 2- to 4-fold increase in EMB MIC compared to the wild-type H37Rv. In vivo, the one selected embB306 GTG mutant required a higher dose of ethambutol to restrict its growth in the lung compared to wild-type H37Rv. These experiments demonstrate that embB306 point mutations enhance the EMB MIC in vitro to a moderate, but significant extent, and reduce the efficacy of EMB treatment in the animal model. We propose that conventional EMB susceptibility testing, in combination with embB306 genotyping, may guide dose adjustment to avoid clinical treatment failure in these low-level resistant strains.     

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.     

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.     

Rapid detection of ethambutol-resistant Mycobacterium tuberculosis strains by PCR-RFLP targeting embB codons 306 and 497 and iniA codon 501 mutations

Mutations at embB gene codons 306 and 497 and iniA gene codon 501 occur frequently in ethambutol (EMB)-resistant Mycobacterium tuberculosis strains worldwide. The identification of these mutations in resistant strains has been achieved by labor-intensive DNA sequencing or by tedious amplification protocols followed by restriction endonuclease digestion. In this report, we describe PCR-restriction fragment length polymorphism (RFLP)-based methods for determining substitutions at embB codons 306 and 497 and iniA codon 501 directly in BACTEC cultures of M. tuberculosis isolates. The wild-type and mutant alleles are revealed by easily interpretable and different RFLP patterns. The methods optimized initially on reference strains were tested directly on BACTEC cultures of 25 randomly selected clinical M. tuberculosis isolates, seven of which were determined to contain EMB-resistant strains by phenotypic drug susceptibility testing. The PCR-RFLP methods identified mutations in four of seven EMB-resistant strains with three isolates containing mutated embB codon 306 and one isolate containing mutated embB codon 497. The results of PCR-RFLP were confirmed by DNA sequencing. The worldwide prevalence figures for mutations at embB codons 306 and 497 and iniA codon 501 suggest that nearly half of EMB-resistant M. tuberculosis strains could be identified within one working day even in developing countries equipped with simple PCR technology instead of weeks required for phenotypic drug susceptibility testing. Further, since EMB resistance is also associated with multiple-drug resistance from some geographical locations, detection of EMB resistance may also lead to rapid identification of multidrug-resistant strains of M. tuberculosis.     

Role of embB codon 306 mutations in Mycobacterium tuberculosis revisited: a novel association with broad drug resistance and IS6110 clustering rather than ethambutol resistance

Mutations at position 306 of embB (embB306) have been proposed as a marker for ethambutol resistance in Mycobacterium tuberculosis; however, recent reports of embB306 mutations in ethambutol-susceptible isolates caused us to question the biological role of this mutation. We tested 1,020 clinical M. tuberculosis isolates with different drug susceptibility patterns and of different geographical origins for associations between embB306 mutations, drug resistance patterns, and major genetic group. One hundred isolates (10%) contained a mutation in embB306; however, only 55 of these mutants were ethambutol resistant. Mutations in embB306 could not be uniquely associated with any particular type of drug resistance and were found in all three major genetic groups. A striking association was observed between these mutations and resistance to any drug (P < 0.001), and the association between embB306 mutations and resistance to increasing numbers of drugs was highly significant (P < 0.001 for trend). We examined the association between embB306 mutations and IS6110 clustering (as a proxy for transmission) among all drug-resistant isolates. Mutations in embB306 were significantly associated with clustering by univariate analysis (odds ratio, 2.44; P = 0.004). In a multivariate model that also included mutations in katG315, katG463, gyrA95, and kasA269, only mutations in embB306 (odds ratio, 2.14; P = 0.008) and katG315 (odds ratio, 1.99; P = 0.015) were found to be independently associated with clustering. In conclusion, embB306 mutations do not cause classical ethambutol resistance but may predispose M. tuberculosis isolates to the development of resistance to increasing numbers of antibiotics and may increase the ability of drug-resistant isolates to be transmitted between subjects.