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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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。     

Molecular genetic analysis of nucleotide polymorphisms associated with ethambutol resistance in human isolates of Mycobacterium tuberculosis

Ethambutol (EMB) is a central component of drug regimens used worldwide for the treatment of tuberculosis. To gain insight into the molecular genetic basis of EMB resistance, approximately 2 Mb of five chromosomal regions with 12 genes in 75 epidemiologically unassociated EMB-resistant and 33 EMB-susceptible Mycobacterium tuberculosis strains isolated from human patients were sequenced. Seventy-six percent of EMB-resistant organisms had an amino acid replacement or other molecular change not found in EMB-susceptible strains. Thirty-eight (51%) EMB-resistant isolates had a resistance-associated mutation in only 1 of the 12 genes sequenced. Nineteen EMB-resistant isolates had resistance-associated nucleotide changes that conferred amino acid replacements or upstream potential regulatory region mutations in two or more genes. Most isolates (68%) with resistance-associated mutations in a single gene had nucleotide changes in embB, a gene encoding an arabinosyltransferase involved in cell wall biosynthesis. The majority of these mutations resulted in amino acid replacements at position 306 or 406 of EmbB. Resistance-associated mutations were also identified in several genes recently shown to be upregulated in response to exposure of M. tuberculosis to EMB in vitro, including genes in the iniA operon. Approximately one-fourth of the organisms studied lacked mutations inferred to participate in EMB resistance, a result indicating that one or more genes that mediate resistance to this drug remain to be discovered. Taken together, the results indicate that there are multiple molecular pathways to the EMB resistance phenotype.     

Allelic Exchange and Mutant Selection Demonstrate that Common Clinical embCAB Gene Mutations Only Modestly Increase Resistance to Ethambutol in Mycobacterium tuberculosis

Mutations within codon 306 of the Mycobacterium tuberculosis embB gene modestly increase ethambutol (EMB) MICs. To identify other causes of EMB resistance and to identify causes of high-level resistance, we generated EMB-resistant M. tuberculosis isolates in vitro and performed allelic exchange studies of embB codon 406 (embB406) and embB497 mutations. In vitro selection produced mutations already identified clinically in embB306, embB397, embB497, embB1024, and embC13, which result in EMB MICs of 8 or 14 μg/ml, 5 μg/ml, 12 μg/ml, 3 μg/ml, and 4 μg/ml, respectively, and mutations at embB320, embB324, and embB445, which have not been identified in clinical M. tuberculosis isolates and which result in EMB MICs of 8 μg/ml, 8 μg/ml, and 2 to 8 μg/ml, respectively. To definitively identify the effect of the common clinical embB497 and embB406 mutations on EMB susceptibility, we created a series of isogenic mutants, exchanging the wild-type embB497 CAG codon in EMB-susceptible M. tuberculosis strain 210 for the embB497 CGG codon and the wild-type embB406 GGC codon for either the embB406 GCC, embB406 TGC, embB406 TCC, or embB406 GAC codon. These new mutants showed 6-fold and 3- to 3.5-fold increases in the EMB MICs, respectively. In contrast to the embB306 mutants, the isogenic embB497 and embB406 mutants did not have preferential growth in the presence of isoniazid or rifampin (rifampicin) at their MICs. These results demonstrate that individual embCAB mutations confer low to moderate increases in EMB MICs. Discrepancies between the EMB MICs of laboratory mutants and clinical M. tuberculosis strains with identical mutations suggest that clinical EMB resistance is multigenic and that high-level EMB resistance requires mutations in currently unknown loci.Ethambutol (EMB) is a first-line antituberculosis drug that is often used in combination with other drugs to treat tuberculosis and to prevent the emergence of drug resistance. EMB also has a place in the treatment of drug-resistant and multidrug-resistant tuberculosis (2). The recent global increase in the incidence of drug-resistant tuberculosis has produced many strains that are resistant to EMB. Therefore, it is prudent to test isolates from all tuberculosis patients for their EMB susceptibility, especially when EMB is used to treat multidrug-resistant tuberculosis. Unfortunately, conventional culture-based EMB susceptibility test methods have poor intertest and interlaboratory reproducibilities (8, 21). This has made it difficult to firmly rule out the presence of EMB resistance by the use of conventional assays. Culture-based Mycobacterium tuberculosis drug susceptibility tests are also quite slow (12, 20).Genetic tests for EMB resistance are potentially more rapid and more accurate than conventional culture-based resistance testing. Genetic assays identify resistance by detecting mutations that encode EMB resistance on the M. tuberculosis chromosome, principally within the embB gene (5, 17, 25). The results of genetic assays can be available within hours; they have high interassay reproducibilities and have the potential to have high sensitivities (5, 25). However, genetic testing for EMB resistance has been hindered by a persistent uncertainty concerning the role of specific mutations in EMB resistance. Initially, the role of mutations within codon 306 of the embB gene (embB306) was questioned. Although embB306 mutations were present in 30 to 68% of EMB-resistant clinical isolates (1, 13, 22), some studies had noted a widespread presence of embB306 mutations in EMB-susceptible isolates (1, 7, 9). The role of embB306 mutations was firmly established to be a cause of low- and moderate-level (two to seven times the MIC for the wild type) EMB resistance in a recent allelic exchange study (19). However, that study also demonstrated that embB306 mutations do not in themselves cause high-level (MICs > 20 μg/ml) EMB resistance. Furthermore, the cause of EMB resistance in the 32 to 70% of clinical EMB-resistant M. tuberculosis isolates that did not have embB306 mutations remained an open question.Several clinical studies have suggested that other mutations in the embCAB operon are responsible for at least some of the remaining EMB-resistant tuberculosis cases. The most commonly occurring embCAB mutations other than embB306 have been found in embB406 and embB497. Importantly, these two mutations have been detected in clinical isolates with high-level EMB resistance (11, 14). However, other studies identified embB406 mutations in EMB-susceptible clinical isolates (7, 15, 23). Other mutations in the embB and embC genes have also been identified in EMB-resistant clinical M. tuberculosis isolates (6, 22, 23), but at low frequencies, making it difficult to firmly establish associations with EMB resistance. Thus, the actual role of non-embB306 mutations in EMB resistance has not been proven.In the study described here, we examined the role of embB mutations outside of the embB306 codon in EMB resistance. Using in vitro-selected mutants and allelic exchange techniques, our results demonstrate that non-embB306 mutations in the embCAB operon play an important role in EMB resistance, but like mutations in embB306, these mutations confer only a low to a moderate increase in EMB MICs. Our study strongly suggests that unrecognized mycobacterial gene targets for EMB resistance and high-level resistance remain to be discovered.     

Phenotypic and genotypic characterization of drug-resistant Mycobacterium tuberculosis strains

Of 142 pulmonary tuberculosis patients, 76 were considered high risk for the development of resistance, and 24 were confirmed as resistant strain carriers. Resistant isoniazid strains presented a high frequency of katG and ahpC mutations (90%) correlated with an MIC >4 microg/mL (94%). inhA mutations were not seen. rpoB mutations were identified in 78.6% of rifampicin-resistant strains, usually in codon 531 (72.7%), and 75% had an MIC >16 microg/mL. katG and rpoB mutations recognized 88.2% of multidrug-resistant strains and proved more efficient than the katG and rpoB mutations alone. Seventy percent of resistant pyrazinamide strains had pncA mutations between genes 136 and 188, 62.5% of them with an MIC >900 microg/mL. Pyrazinamidase inactivity was not an efficient resistance marker because 60% of pncA-mutated strains maintained enzymatic activity despite displaying good correlation with high resistance levels. Resistant ethambutol strains had embB mutations in codon 306, with MIC >16 microg/mL.     

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.     

Novel mutations within the embB gene in ethambutol-susceptible clinical isolates of Mycobacterium tuberculosis

Genetic analysis of the embB gene revealed mutations in 17 (68%) of 25 ethambutol (EMB) resistant isolates (M306I, M306V, M306L, Q497R) but also in 4 (20%) of 20 EMB-susceptible isolates of Mycobacterium tuberculosis, namely, an ATG-->ATM substitution resulting in M306I, G406N, and the novel alterations M423I and A659T.     

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.     

Analysis of embCAB Mutations Associated with Ethambutol Resistance in Multidrug-Resistant Mycobacterium tuberculosis Isolates from China

Ethambutol (EMB) plays a pivotal role in the chemotherapy of drug-resistant tuberculosis (TB), including multidrug-resistant tuberculosis (MDR-TB). Resistance to EMB is considered to be caused by mutations in the embCAB operon (embC, embA, and embB). In this study, we analyzed the embCAB mutations among 139 MDR-TB isolates from China and found a possible association between embCAB operon mutation and EMB resistance. Our data indicate that 56.8% of MDR-TB isolates are resistant to EMB, and 82.2% of EMB-resistant isolates belong to the Beijing family. Overall, 110 (79.1%) MDR-TB isolates had at least one mutation in the embCAB operon. The majority of mutations were present in the embB gene and the embA upstream region, which also displayed significant correlations with EMB resistance. The most common mutations occurred at codon 306 in embB (embB306), followed by embB406, embA(−16), and embB497. Mutations at embB306 were associated with EMB resistance. DNA sequencing of embB306–497 was the best strategy for detecting EMB resistance, with 89.9% sensitivity, 58.3% specificity, and 76.3% accuracy. Additionally, embB306 had limited value as a candidate predictor for EMB resistance among MDR-TB infections in China.     

Identification of a new antitubercular drug candidate, SQ109, from a combinatorial library of 1,2-ethylenediamines

OBJECTIVES: The aim of this study was to identify a candidate drug for clinical development from a previously synthesized combinatorial library based on the 1,2-ethylenediamine structure of ethambutol. METHODS: Sixty-nine of the most potent hits against Mycobacterium tuberculosis from the original studies were subjected to a sequential set of tests in vitro and in vivo--determination of MIC for M. tuberculosis H37Rv, cytotoxicity, intracellular antimycobacterial activity, permeability evaluation and in vivo efficacy testing. RESULTS: Twenty-seven compounds with MICs of < or = 15.6 microM were tested on Vero cells to determine in vitro cytotoxicity (IC50) and to establish a selectivity index (SI) (SI = IC50/MIC). Ten compounds with acceptable SI were tested for activity against intracellular bacteria--all were equivalent (within 1%) or superior to ethambutol and several demonstrated cidal activity. Five of the most potent compounds were tested for in vivo efficacy in a murine model of chronic tuberculosis infection. CONCLUSION: Compound SQ109 with an MIC of 0.7-1.56 microM (H37Rv, Erdman and drug-resistant strains of M. tuberculosis), an SI of 16.7 and 99% inhibition activity against intracellular bacteria, demonstrated potency in vivo and limited toxicity in vitro and in vivo, and was selected for further development.