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.     

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.     

Evaluation of mycobacteria growth indicator tubes for susceptibility testing of Mycobacterium tuberculosis to isoniazid and rifampin

The reliability of mycobacteria growth indicator tubes (Mgit) for determining the susceptibility of Mycobacterium tuberculosis to isoniazid (0.1 μg/ml) and rifampin (2.0 μg/ml) was evaluated by comparing Mgit results to those obtained by the radiometric Bactec TB method. We tested 29 isolates, many selected for resistance. The turnaround times were 3–8 days (median 6) for Mgit and 4–10 days (median 6) for Bactec. Isoniazid results by both methods agreed for all isolates tested: 20 resistant and nine susceptible. Rifampin results agreed for 28 isolates: 10 resistant and 18 susceptible. One isolate yielded discrepant results: resistant to rifampin by Bactec, but susceptible by Mgit. This isolate was also rifampin-resistant by the traditional Method of Proportion and when tested by the Mgit method using 1.0 μg/ml of rifampin. The Mgit system is a nonradiometric alternative to the Bactec for rapid susceptibility testing of M. tuberculosis to isoniazid and rifampin; however, additional testing is needed to determine the optimal concentration of rifampin.     

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.     

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.     

Determinants of rifampin, isoniazid, pyrazinamide, and ethambutol pharmacokinetics in a cohort of tuberculosis patients

Evaluation of sources of pharmacokinetic variation can facilitate optimization of tuberculosis treatment regimens by identification of avoidable sources of variation and of risk factors for low or high drug concentrations in patients. Our objective was to describe the pharmacokinetics of rifampin, isoniazid, pyrazinamide, and ethambutol in a cohort of tuberculosis patients established on first-line treatment regimens and to evaluate the determinants of pharmacokinetic variation. Plasma concentration-time profiles were determined for each of the drugs in 142 patients with drug-sensitive pulmonary tuberculosis after 2 months of daily treatment in hospital. Pharmacokinetic measures were described by noncompartmental analysis. Multiple linear regression was used to evaluate the patient and the treatment factors associated with variation of the area under the concentration-time curve from 0 to 8 h. Several factors independently associated with variations in antituberculosis drug concentrations were identified: human immunodeficiency virus infection was associated with 39% and 27% reductions for rifampin and ethambutol, respectively; formulation factors were determinants of rifampin and isoniazid bioavailability; female patients had increased rifampin and isoniazid concentrations but reduced ethambutol concentrations; older patients had higher levels of isoniazid and ethambutol; patients with a history of previous antituberculosis treatment had lower ethambutol concentrations; and the dose per kilogram of body weight was associated with the concentrations of all four agents. Further studies are required to assess the implications of variations in antituberculosis drug concentrations for efficacy and safety before decisions are made to change the dosing strategy in patients at risk.     

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.     

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.     

Oxidative stress increases susceptibility of Mycobacterium tuberculosis to isoniazid

Isoniazid is a first-line antibiotic used in the treatment of infections caused by Mycobacterium tuberculosis. Isoniazid is a prodrug requiring oxidative activation by the catalase-peroxidase hemoprotein, KatG. Resistance to isoniazid can be obtained by point mutations in the katG gene, with one of the most common being a threonine-for-serine substitution at position 315 (S315T). The S315T mutation is found in more than 50% of isoniazid-resistant clinical isolates and results in an approximately 200-fold increase in the MIC of isoniazid compared to that for M. tuberculosis H37Rv. In the present study we investigated the hypothesis that superoxide plays a role in KatG-mediated isoniazid activation. Plumbagin and clofazimine, compounds capable of generating superoxide anion, resulted in a lower MIC of isoniazid for M. tuberculosis H37Rv and a strain carrying the S315T mutation. These agents did not cause as great of an increase in isoniazid susceptibility in the mutant strain when the susceptibilities were assessed by using the inhibitory concentration that causes a 50% decrease in growth. These results provide evidence that superoxide can play a role in isoniazid activation. Since clofazimine alone has antitubercular activity, the observation of synergism between clofazimine and isoniazid raises the interesting possibility of using both drugs in combination to treat M. tuberculosis infections.     

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.     

焦磷酸测序技术检测结核分枝杆菌四种药物耐药性

目的 利用焦磷酸测序技术检测结核分枝杆菌对利福平、异烟肼、氧氟沙星、阿米卡星的耐药性,并对其临床应用进行评价.方法 收集上海市肺科医院2008-2009年临床确诊的肺结核患者的痰标本培养阳性结核分枝杆菌89株.所有菌株按<结核病诊断实验室检验规程>进行分枝杆菌培养、菌种鉴定.另外10株已知药敏结果的结核分枝杆菌来自上海市肺科医院菌株库.利用焦磷酸测序技术,以10株已知药敏结果和经直接测序已知耐药基因突变情况的结核分枝杆菌为试验菌株,探索焦磷酸测序检测结核分枝杆菌rpoB、katG、gyrA、rrs耐药基因的最佳模式,并用该条件检测89株结核分枝杆菌临床分离株,检测结果与Bactec 960药敏法进行比较.结果 rpoB、gyrA基因检测宜采用焦磷酸测序序列分析模式,katG、rrs基因检测宜采用焦磷酸测序单核苷酸多态性模式.以Bactec 960药敏法测定结果为判断标准,则焦磷酸测序法检测89株结核分枝杆菌临床分离株对利福平、异烟肼、氧氟沙星、阿米卡星耐药性的敏感度分别为98.0%、64.1%、79.5%、78.3%,特异度分别为97.5%、100.0%、90.0%、100.0%,准确性分别为97.8%、77.5%、85.4%、94.4%,该法检测利福平、氧氟沙星、阿米卡星的检测结果与Bactec 960药敏检测结果一致性较好,Kappa值均≥0.7.结论 焦磷酸测序技术检测结核分枝杆菌对利福平、异烟肼、氧氟沙星、阿米卡星耐药性具有快速、准确、高通量的优点,是一种可对结核分枝杆菌多种药物耐药性进行快速检测的方法.

Abstract：

Objective To develop an assay to determine Mycobacterium tuberculosis resistance to rifampin, isoniazid, ofloxacin and amikacin by pyrosequencing and evaluate the value of this method in clinical application. Methods Eighty-nine clinical isolates of Mycobacterium tuberculosis from tuberculosis patients were collected from Shanghai Pulmonary Hospital during 2008 to 2009. All strains were isolated from decontaminated sputum, cultured on Lowenstein-Jensen media and identified by traditional biochemical tests with standard methods. Ten Mycobacterium tuberculosis were selected from the strain bank of Shanghai Pulmonary Hospital. The optimal conditions of detection of rpoB, katG, gyrA and rrs gene by pyroseuencing were determined, using the 10 Mycobacterium tuberculosis strains whose drug susceptibility of Bactec 960 and sequence of rpoB, katG, gyrA, rrs gene were known. Then the drug susceptibility of 89 Mycobacterium tuberculosis clinical isolate strains were detected by pyrosequencing using this conditions and the results were compared with that of the Bactec 960 methods. Results The pyrosequencing program of sequence analysis was suitable for the detection of the mutations of rpoB and gyrA genes, and the program of single nucleotide polymorphism was suitable for katG and rrs genes. Among the 89 Mycobacterium tuberculosis clinical isolates,when using the drug susceptibility of Bactec 960 as the standard, the sensitivity of rifampin, isoniazid,ofloxacin and amikacin is 98.0%, 64. 1%, 79.5%, 78. 3% respectively, the specificity is 97.5%,100. 0%, 90. 0%, 100. 0% respectively, the accuracy is 97.8%, 77. 5%, 85.4%, 94. 4% respectively,tested by pyrosequencing. The results of the detection of resistance to rifampin, isoniazid, ofloxacin and amikacin in Mycobacterium tuberculosis using pyrosequencing technique were almost the same with that of Bactec 960, and Kappa ≥0. 7 in each detection. Conclusion Pyrosequencing is thus a rapid, accurate and high throughput method for detecting Mycobacterium tuberculosis resistance to these four drugs.     

In vitro synergistic activity of ethambutol, isoniazid, kanamycin, rifampin, and streptomycin against Mycobacterium avium-intracellulare complex.

Strains of Mycobacterium avium-intracellulare complex often exhibit in vitro resistance to common antimycobacterial agents. Combinations of etambutol, isoniazid, kanamycin, rifampin, and streptomycin were tested to determine if synergism occurred. Ninety-six percent of the strains were susceptible to a combination of ethambutol and rifampin at concentrations attainable clinically. Other combinations of antimycobacterial agents inhibited 4 to 82% of the isolates tested.     

Improved real-time PCR for rapid detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis clinical isolates

In this study we designed two pairs of probes for the detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis with real-time PCR procedures. One pair of probes spans the region between codon 510 and 528 of the rpoB gene, and the other one screens for mutation at the regulatory region of the inhA gene. We have evaluated these probes in combination with two other pairs of probes previously described to detect mutations in 20 susceptible and 53 unique resistant M. tuberculosis clinical isolates. We were able to detect nine different mutations affecting five codons of the rpoB gene, two different mutations at codon 315 of the katG gene and a nucleotide substitution (C209T) in the regulatory region of the inhA gene within two hours turnaround.