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.     

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.     

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.     

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.     

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.     

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.     

Ethambutol resistance in Mycobacterium tuberculosis: critical role of embB mutations.

Ethambutol [(S,S')-2,2'-(ethylenediimino)di-1-butanol; EMB], is a first-line drug used to treat tuberculosis. To gain insight into the molecular basis of EMB resistance, we characterized the 10-kb embCAB locus in 16 EMB-resistant and 3 EMB-susceptible genetically distinct Mycobacterium tuberculosis strains from diverse localities by automated DNA sequencing and single-stranded conformation polymorphism analysis. All 19 organisms had virtually identical sequences for the entire 10-kb region. Eight EMB-resistant organisms had mutations located in codon 306 of embB that resulted in the replacement of the wild-type Met residue with Ile or Val. Automated sequence analysis of the 5' region (1,892 bp) of embB in an additional 69 EMB-resistant and 30 EMB-susceptible M. tuberculosis isolates from diverse geographic localities and representing 70 distinct IS6110 fingerprints confirmed the unique association of substitutions in amino acid residue 306 of EmbB with EMB resistance. Six other embB nucleotide substitutions resulting in four amino acid replacements were uniquely found in resistant strains. Sixty-nine percent of epidemiologically unassociated EMB-resistant organisms had an amino acid substitution not found in susceptible strains, and most (89%) replacements occurred at amino acid residue 306 of EmbB. For strains with the Met306Leu or Met306Val replacements EMB MICs were generally higher (40 microg/ml) than those for organisms with Met306Ile substitutions (20 microg/ml). The data are consistent with the idea that amino acid substitutions in EmbB alter the drug-protein interaction and thereby cause EMB resistance.     

Role of embB in natural and acquired resistance to ethambutol in mycobacteria.

The mycobacterial embCAB operon encodes arabinosyl transferases, putative targets of the antimycobacterial agent ethambutol (EMB). Mutations in embB lead to resistance to EMB in Mycobacterium tuberculosis. The basis for natural, intrinsic resistance to EMB in nontuberculous mycobacteria (NTM) is not known; neither is the practical implication of resistance to EMB in the absence of embB mutations in M. tuberculosis well understood. The conserved embB resistance-determining region (ERDR) of a collection of 13 strains of NTM and 12 EMB-resistant strains of M. tuberculosis was investigated. Genotypes were correlated with drug susceptibility phenotypes. High-level natural resistance to EMB (MIC, . or =64 microg/ml) was associated with a variant amino acid motif in the ERDR of M. abscessus, M. chelonae, and M. leprae. Transfer of the M. abscessus emb allele to M. smegmatis resulted in a 500-fold increase in the MICs. In M. tuberculosis, embB mutations were associated with MICs of > or =20 microg/ml while resistance not associated with an ERDR mutation generally resulted in MICs of < or =10 microg/ml. These data further support the notion that the emb region determines intrinsic and acquired resistance to EMB and might help in the reassessment of the current recommendations for the screening and treatment of infections with EMB-resistant M. tuberculosis and NTM.     

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。     

Lack of correlation between embB mutation and ethambutol MIC in Mycobacterium tuberculosis clinical isolates from China

Seventy-four Mycobacterium tuberculosis clinical isolates from China were subjected to drug susceptibility testing using ethambutol, isoniazid, rifampin, and ofloxacin. The results revealed that the presence of embB mutations did not correlate with ethambutol resistance but was associated with multiple-drug resistance, especially resistance to both ethambutol and rifampin.     

耐多药结核分枝杆菌耐药相关基因突变特征分析

目的了解中国耐多药结核分枝杆菌耐药相关基因的分子特征。方法对138株耐多药结核分枝杆菌和45株敏感菌的耐药相关基因inhA、katG和oxyR-ahpC间隔区(异烟肼)、rpob(利福平)、gyrA(氧氟沙星)和rrs(卡那霉素)进行序列测定,分析其基因突变特点。结果 138株耐多药结核分枝杆菌中,14.4%的菌株inhA基因发生突变,72.5%菌株的katG基因发生突变,15.9%菌株的oxyR-ahpC基因发生突变,同时考虑这3种基因,异烟肼耐药相关基因突变检出率可达90.6%;94.2%菌株的rpoB基因发生突变,74.5%菌株的gyrA基因发生突变,61.1%菌株的rrs基因发生突变,主要的突变位点为katG315(66.7%),inhA-15(9.4%),oxyR-ahpC-10(5.1%),rpoB516(13.8%),526(26.1%)和531(49.3%),gyrA90(21.6%)和94(51%),rrs1401(61.1%)。结论我国耐多药结核菌异烟肼、利福平、氧氟沙星和卡那霉素耐药相关基因最常见突变为katG315、inhA-15,rpoB531、526和516,gyrA94和90,rrs1401。     

Mutation at embB Codon 306, a Potential Marker for the Identification of Multidrug Resistance Associated with Ethambutol in Mycobacterium tuberculosis

Ethambutol inhibits arabinogalactan and lipoarabinomannan biosynthesis in mycobacteria. The occurrence of mutations in embB codon 306 in ethambutol-susceptible isolates and their absence in resistant isolates has raised questions regarding the utility of this codon as a potential marker for resistance against ethambutol. The characterization of mutations on embB 306 will contribute to a better understanding of the mechanisms of resistance to this drug; therefore, the purpose of this study was to investigate the association between embB 306 mutations and first-line drug resistance profiles in tuberculosis isolates. We sequenced the region surrounding the embB 306 codon in 175 tuberculosis clinical isolates, divided according to drug sensitivity, in three groups: 110 were resistant to at least one first-line drug, of which 61 were resistant to ethambutol (EMB^r), 49 were sensitive to ethambutol (EMB^s) but were resistant to another drug, and 65 were pansensitive isolates (P^s). The associations between embB 306 mutations and phenotypic resistance to all first-line drugs were determined, and their validity and safety as a diagnostic marker were assessed. One of the P^s isolates (1/65), one of the EMB^s isolates (1/49), and 20 of the EMB^r isolates (20/61) presented with an embB 306 mutation. Four different single-nucleotide polymorphisms (SNPs) at embB 306 were associated with simultaneous resistance to ethambutol, isoniazid, and rifampin (odds ratio [OR], 17.7; confidence interval [CI], 5.6 to 56.1) and showed a positive predictive value of 82%, with a specificity of 97% for diagnosing multidrug resistance associated with ethambutol, indicating its potential as a molecular marker for several drugs.     

Convergent evolutionary analysis identifies significant mutations in drug resistance targets of Mycobacterium tuberculosis

Mycobacterium tuberculosis adapts to the environment by selecting for advantageous single-nucleotide polymorphisms (SNPs). We studied whether advantageous SNPs could be distinguished from neutral mutations within genes associated with drug resistance. A total of 1,003 clinical isolates of M. tuberculosis were related phylogenetically and tested for the distribution of SNPs in putative drug resistance genes. Drug resistance-associated versus non-drug-resistance-associated SNPs in putative drug resistance genes were compared for associations with single versus multiple-branch outcomes using the chi-square and Fisher exact tests. All 286 (100%) isolates containing isoniazid (INH) resistance-associated SNPs had multibranch distributions, suggestive of multiple ancestry and convergent evolution. In contrast, all 327 (100%) isolates containing non-drug-resistance-associated SNPs were monophyletic and thus showed no evidence of convergent evolution (P < 0.001). Convergence testing was then applied to SNPs at position 481 of the iniA (Rv0342) gene and position 306 of the embB gene, both potential drug resistance targets for INH and/or ethambutol. Mutant embB306 alleles showed multibranch distributions, suggestive of convergent evolution; however, all 44 iniA(H481Q) mutations were monophyletic. In conclusion, this study validates convergence analysis as a tool for identifying mutations that cause INH resistance and explores mutations in other genes. Our results suggest that embB306 mutations are likely to confer drug resistance, while iniA(H481Q) mutations are not. This approach may be applied on a genome-wide scale to identify SNPs that impact antibiotic resistance and other types of biological fitness.     

IS6110: conservation of sequence in the Mycobacterium tuberculosis complex and its utilization in DNA fingerprinting.

Multiple copies of an insertion sequence, IS6110, were shown to be present in the genome of members of the Mycobacterium tuberculosis complex (M. tuberculosis and M. bovis). Ten to 12 copies are present in various strains of M. tuberculosis, while strains of M. bovis contain only one to three copies. IS6110 was not detected in the DNA of other species of mycobacteria. Restriction endonuclease analysis indicated that the sequence of IS6110 is conserved across strain and species lines. Hybridization to the insertion sequence can be used to detect restriction fragment length polymorphism reflecting divergence in the sequence of regions flanking the various copies of IS6110. These differences were used to fingerprint various strains of the M. tuberculosis complex.     

Characterization of mutations conferring extensive drug resistance to Mycobacterium tuberculosis isolates in Pakistan

The increasing incidence of extensively drug-resistant (XDR) Mycobacterium tuberculosis in high-tuberculosis-burden countries further highlights the need for improved rapid diagnostic assays. An increasing incidence of XDR M. tuberculosis strains in Pakistan has been reported, but drug resistance-associated mutations in these strains have not been evaluated previously. We sequenced the "hot-spot" regions of rpoB, katG, inhA, ahpC, gyrA, gyrB, and rrs genes in 50 XDR M. tuberculosis strains. It was observed that 2% of rifampin, 6% of isoniazid, 24% of fluoroquinolone, and 32% of aminoglycoside/capreomycin resistance in XDR M. tuberculosis strains would be undetected if only these common hot-spot regions were tested. The frequencies of resistance-conferring mutations were found to be comparable among all XDR M. tuberculosis strain families present, including the Central Asian Strain, Beijing, and East African Indian genogroups and the Unique isolates. Additional genetic loci need to be tested for detection of mutations conferring fluoroquinolone, aminoglycoside, and capreomycin resistance in order to improve molecular diagnosis of regional XDR M. tuberculosis strains.     

Molecular detection of mutations associated with first- and second-line drug resistance compared with conventional drug susceptibility testing of Mycobacterium tuberculosis

The emergence of multi- and extensively drug-resistant tuberculosis is a significant impediment to the control of this disease because treatment becomes more complex and costly. Reliable and timely drug susceptibility testing is critical to ensure that patients receive effective treatment and become noninfectious. Molecular methods can provide accurate and rapid drug susceptibility results. We used DNA sequencing to detect resistance to the first-line antituberculosis drugs isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and ethambutol (EMB) and the second-line drugs amikacin (AMK), capreomycin (CAP), kanamycin (KAN), ciprofloxacin (CIP), and ofloxacin (OFX). Nine loci were sequenced: rpoB (for resistance to RIF), katG and inhA (INH), pncA (PZA), embB (EMB), gyrA (CIP and OFX), and rrs, eis, and tlyA (KAN, AMK, and CAP). A total of 314 clinical Mycobacterium tuberculosis complex isolates representing a variety of antibiotic resistance patterns, genotypes, and geographical origins were analyzed. The molecular data were compared to the phenotypic data and the accuracy values were calculated. Sensitivity and specificity values for the first-line drug loci were 97.1% and 93.6% for rpoB, 85.4% and 100% for katG, 16.5% and 100% for inhA, 90.6% and 100% for katG and inhA together, 84.6% and 85.8% for pncA, and 78.6% and 93.1% for embB. The values for the second-line drugs were also calculated. The size and scope of this study, in numbers of loci and isolates examined, and the phenotypic diversity of those isolates support the use of DNA sequencing to detect drug resistance in the M. tuberculosis complex. Further, the results can be used to design diagnostic tests utilizing other mutation detection technologies.     

IS6110限制性片段长度多态性和间隔区寡核苷酸分型技术在结核分枝杆菌菌株鉴定中的应用

目的 评价IB6110-限制性片段长度多态性（IS6110-RFLP）和间隔区寡核苷酸分型技术（spoligotyping）两种方法在结核病流行病学上的应用，探讨我国各地区的结核分枝杆菌菌株特点。方法 收集158株结核分枝杆菌临床分离株，分别应用IS6110-RFLP和Spoligotyping两种方法进行鉴定。结果 （1）IS6110-RFLP的分辨力大于spoligotyping分型。（2）将本次试验结果与国际spoligotype数据库进行比较。结果 有14个类型属于共有类型，其中类型1为流行的类型，分布广泛，即所说的北京基因型。（3）广东地区与其他地区成簇率和北京基因型所占比例差异有统计学意义（P〈0．05）。广东地区成簇率和北京基因型所占比例均显著低于其他地区。结论 同时应用IS6110-RFLP分型和Spoligotyping两种方法进行结核病流行病学调查研究非常有效，在中国不同地区的菌株具有不同的特点。