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.     

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.     

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.     

Molecular characterization of drug-resistant and -susceptible Mycobacterium tuberculosis isolated from patients with tuberculosis in Korea

We investigated the causal relationship between genotype and phenotype of drug-resistant Mycobacterium tuberculosis isolates obtained from patients with pulmonary tuberculosis (TB) in Korea. Of 80 isolates tested, 17, 20, 1, and 7 isolates were mono-resistant to ethambutol (EMB), isoniazid (INH), pyrazinamide (PZA), and rifampicin (RFP), respectively, and 31 isolates (38.8%) were multidrug-resistant (MDR). Sequencing analysis showed that 78% (32/41) of RFP-resistant strains had mutations in the rifampicin resistance-determining region (RRDR) of rpoB, and the mutation at rpoB531 (59.4%) was most abundant. In 52 INH-resistant strains, mutations were found mostly at C-15T (n = 21, 40.4%) in the inhA promoter region as well as at katG315 (n = 12, 23.1%). Mutations at embB306 were mostly found in 26.7% (12/45) of EMB-resistant isolates. New mutations found here in MDR isolates include rpoB523 (Gly523Glu) and embB319 (Tyr319Ser). Consequently, mutations in the rpoB531, C-15T in the inhA promoter region, embB306, and katG315 would be a useful marker for rapid detection of MDR M. tuberculosis isolates in Korea.     

Characterization of mutations in multi- and extensive drug resistance among strains of Mycobacterium tuberculosis clinical isolates in Republic of Korea

In order to characterize molecular mechanisms of first- and second-line drug resistance in Mycobacterium tuberculosis and to evaluate the use of molecular markers of resistance, we analyzed 62 multidrug-resistant, 100 extensively drug-resistant, and 30 pan-susceptible isolates from Korean tuberculosis patients. Twelve genome regions associated with drug resistance, including katG, ahpC, and inhA promoter for isoniazid (INH); embB for ethambutol (EMB), rpoB for rifampin (RIF), pncA for pyrazinamide (PZA), gyrA for fluoroquinolones; rpsL, gidB, and rrs for streptomycin; rrs and eis for kanamycin (KM); rrs and tylA for capreomycin (CAP); and rrs for amikacin (AMK) were amplified simultaneously by polymerase chain reaction, and the DNA sequences were determined. We found mutations in 140 of 160 INH-resistant isolates (87.5%), 159 of 162 RIF-resistant isolates (98.15%), 127 of 143 EMB-resistant isolates (88.8%), 108 of 123 ofloxacin-resistant isolates (87.8%), and 107 of 122 PZA-resistant isolates (87.7%); 43 of 51 STM-resistant isolates (84.3%), 15 of 17 KM-resistant isolates (88.2%), and 14 of 15 (AMK and CAP)-resistant isolates (93.3%) had mutations related to specific drug resistance. In addition, the sequence analyses of the study revealed many novel mutations involving these loci. This result suggests that mutations in the rpoB531, katGSer315Thr, and C-15T in the inhA promoter region, and gyrA94, embB306, pncA159, rpsL43, and A1401G in the rrs gene could serve as useful markers for rapid detection of resistance profile in the clinical isolates of M. tuberculosis in Korea, with potentials for the new therapeutic benefits in actual clinical practice.     

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.     

Prevalence and Molecular Characteristics of Drug-Resistant Mycobacterium tuberculosis in Hunan,China

To determine the prevalence and molecular characteristics of drug-resistant tuberculosis in Hunan province, drug susceptibility testing and spoligotyping methods were performed among 171 M. tuberculosis isolates. In addition, the mutated characteristics of 12 loci, including katG, inhA, rpoB, rpsL, nucleotides 388 to 1084 of the rrs gene [rrs(388–1084)], embB, pncA, tlyA, eis, nucleotides 1158 to 1674 of the rrs gene [rrs(1158–1674)], gyrA, and gyrB, among drug-resistant isolates were also analyzed by DNA sequencing. Our results indicated that the prevalences of isoniazid (INH), rifampin (RIF), streptomycin (SM), ethambutol (EMB), pyrazinamide (PZA), capreomycin (CAP), kanamycin (KAN), amikacin (AKM), and ofloxacin (OFX) resistance in Hunan province were 35.7%, 26.9%, 20.5%, 9.9% 15.2%, 2.3%, 1.8%, 1.2%, and 10.5%, respectively. The previously treated patients presented significantly increased risks for developing drug resistance. The majority of M. tuberculosis isolates belonged to the Beijing family. Almost all the drug resistance results demonstrated no association with genotype. The most frequent mutations of drug-resistant isolates were katG codon 315 (katG₃₁₅), inhA15, rpoB₅₃₁, rpoB₅₂₆, rpoB₅₁₆, rpsL₄₃, rrs₅₁₄, embB₃₀₆, pncA₉₆, rrs₁₄₀₁, gyrA₉₄, and gyrA₉₀. These results contribute to the knowledge of the prevalence of drug resistance in Hunan province and also expand the molecular characteristics of drug resistance in China.     

High resolution melting curve assay for rapid detection of drug-resistant Mycobacterium tuberculosis

We developed and evaluated a high resolution melting (HRM) curve assay by using real-time PCR for the detection of the most frequent mutations of Mycobacterium tuberculosis, which are responsible for the resistance of four anti-TB drugs: rifampicin, isoniazid, ethambutol, and streptomycin. The HRM assay was successfully used for the detection of dominant mutations: A516V, H526A, H526T, S531L, L533P, and A516G/S531L in rpoB; S315T, and S315A in katG; ?15C/T, and ?8T/C in mab-inhA; M306I in embB; K88Q and K43R in rpsL; and 513A/C in rrs. We were able to discriminate the mutant from the wild type by analyzing the melting-curve shape in 40 clinical M. tuberculosis isolates, and the results of the HRM assay were completely consistent with those of DNA sequencing. This HRM assay is a simple, rapid, and cost-effective method that can be performed in a closed tube. Therefore, our assay is a potentially useful tool for the rapid detection of drug-resistant M. tuberculosis.     

Molecular Analysis of Codon 548 in the rpoB Gene Involved in Mycobacterium tuberculosis Resistance to Rifampin

Most Mycobacterium tuberculosis rifampin-resistant strains have been associated with mutations in an 81-bp rifampin resistance-determining region (RRDR) in the gene rpoB. However, if this region alone were targeted, rifampin-resistant strains with mutations outside the RRDR would not be detected. In this study, among 51 rifampin-resistant clinical isolates analyzed by sequencing 1,681-bp-long DNA fragments containing the RRDR, 47 isolates contained mutations within the RRDR, three isolates contained mutations both within and outside the RRDR, and only one isolate had a single missense mutation (Arg548His) located outside the RRDR. A drug susceptibility test of recombinant Mycobacterium smegmatis and M. tuberculosis isolates carrying mutated rpoB (Arg548His) showed an increased MIC for rifampin compared to that of the control strains. Modeling of the Arg548His mutant RpoB-DNA complex revealed that the His548 side chain formed a more stable hydrogen bond structure than did Arg548, reducing the flexibility of the rifampin-resistant cluster II region of RpoB, suggesting that the RpoB Arg548His mutant does not effectively interact with rifampin and results in bacterial resistance to the drug. This is the first report on the relationship between the mutation in codon 548 of RpoB and rifampin resistance in tuberculosis. The novel mutational profile of the rpoB gene described here will contribute to the comprehensive understanding of rifampin resistance patterns and to the development of a useful tool for simple and rapid drug susceptibility tests.     

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.     

Contribution of rpoB Mutations to Development of Rifamycin Cross-Resistance in Mycobacterium tuberculosis

The contributions of 23 insertion, deletion, or missense mutations within an 81-bp fragment of rpoB, the gene encoding the β-subunit of the DNA-dependent RNA polymerase of Mycobacterium tuberculosis, to the development of resistance to rifamycins (rifampin, rifabutin, rifapentine, and KRM-1648) in 29 rifampin-resistant clinical isolates were defined. Specific mutant rpoB alleles led to the development of cross-resistance to all rifamycins tested, while a subset of mutations were associated with resistance to rifampin and rifapentine but not to KRM-1648 or rifabutin. To further study the impact of specific rpoB mutant alleles on the development of rifamycin resistance, mutations were incorporated into the rpoB gene of M. tuberculosis H37Rv, contained on a mycobacterial shuttle plasmid, by in vitro mutagenesis. Recombinant M. tuberculosis clones containing plasmids with specific mutations in either codon 531 or 526 of rpoB exhibited high-level resistance to all rifamycins tested, whereas clones containing a plasmid with a mutation in codon 516 exhibited high-level resistance to rifampin and rifapentine but were susceptible to both rifabutin and KRM-1648. These results provided additional proof of the association of specific rpoB mutations with the development of rifamycin resistance and corroborate previous reports of the usefulness of rpoB genotyping for predicting rifamycin-resistant phenotypes.     

Epidemiology of Isoniazid Resistance Mutations and Their Effect on Tuberculosis Treatment Outcomes

Isoniazid resistance is highly prevalent in Vietnam. We investigated the molecular and epidemiological characteristics and the association with first-line treatment outcomes of the main isoniazid resistance mutations in Mycobacterium tuberculosis in codon 315 of the katG and in the promoter region of the inhA gene. Mycobacterium tuberculosis strains with phenotypic resistance to isoniazid from consecutively diagnosed smear-positive tuberculosis patients in rural Vietnam were subjected to Genotype MTBDRplus testing to identify katG and inhA mutations. Treatment failure and relapse were determined by sputum culture. In total, 227 of 251 isoniazid-resistant strains (90.4%) had detectable mutations: 75.3% in katG codon 315 (katG₃₁₅) and 28.2% in the inhA promoter region. katG₃₁₅ mutations were significantly associated with pretreatment resistance to streptomycin, rifampin, and ethambutol but not with the Beijing genotype and predicted both unfavorable treatment outcome (treatment failure or death) and relapse; inhA promoter region mutations were only associated with resistance to streptomycin and relapse. In tuberculosis patients, M. tuberculosis katG₃₁₅ mutations but not inhA mutations are associated with unfavorable treatment outcome. inhA mutations do, however, increase the risk of relapse, at least with treatment regimens that contain only isoniazid and ethambutol in the continuation phase.     

Selection of Mutations To Detect Multidrug-Resistant Mycobacterium tuberculosis Strains in Shanghai,China

Novel tools are urgently needed for the rapid, reliable detection of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis. To develop such tools, we need information about the frequency and distribution of the mycobacterial mutations and genotypes that are associated with phenotypic drug resistance. In a population-based study, we sequenced specific genes of M. tuberculosis that were associated with resistance to rifampin and isoniazid in 242 phenotypically MDR isolates and 50 phenotypically pan-susceptible isolates from tuberculosis (TB) cases in Shanghai, China. We estimated the sensitivity and specificity of the mutations, using the results of conventional, culture-based phenotypic drug susceptibility testing as the standard. We detected mutations within the 81-bp core region of rpoB in 96.3% of phenotypically MDR isolates. Mutations in two structural genes (katG and inhA) and two regulatory regions (the promoter of mabA-inhA and the intergenic region of oxyR-ahpC) were found in 89.3% of the MDR isolates. In total, 88.0% (213/242 strains) of the phenotypic MDR strains were confirmed by mutations in the sequenced regions. Mutations in embB306 were also considered a marker for MDR and significantly increased the sensitivity of the approach. Based on our findings, an approach that prospectively screens for mutations in 11 sites of the M. tuberculosis genome (rpoB531, rpoB526, rpoB516, rpoB533, and rpoB513, katG315, inhA-15, ahpC-10, ahpC-6, and ahpC-12, and embB306) could detect 86.8% of MDR strains in Shanghai. This study lays the foundation for the development of a rapid, reliable molecular genetic test to detect MDR strains of M. tuberculosis in China.Multidrug-resistant (MDR) tuberculosis (TB), defined as resistance to at least rifampin (RIF) and isoniazid (INH), and extensively drug-resistant (XDR) TB, defined as additional resistance to any fluoroquinolone and one injectable second-line drug, are among the most serious health threats of the 21st century. The epidemic of MDR TB is especially severe in China, a nation with the world''s second largest number of TB cases and the largest number of MDR TB cases (39). A recent study reported that 9.3% of all TB cases in China are MDR, almost twice the worldwide MDR prevalence (4.8%) (14). While a lot of attention has been focused on acquired drug resistance among TB patients who receive an inadequate treatment regimen or who cannot adhere to their treatment regimen, several studies also showed that a large number of MDR TB cases are likely caused by transmission of MDR strains of Mycobacterium tuberculosis (2, 19). Therefore, there is an urgent need for new tools and approaches that will provide a rapid, reliable, and cost-effective diagnosis of MDR TB, particularly in resource-limited settings. This will help to prevent transmission of MDR strains and to optimize treatment regimens for MDR cases.Drug susceptibility testing by the conventional solid medium culture method is highly sensitive and specific but extremely slow, due to the slow growth of M. tuberculosis. Liquid culture methods can reduce the turnaround time but require specialized instrumentation and reagents and are not feasible in most resource-limited settings. New molecular diagnostic methods represent a potentially rapid and sensitive alternative to conventional diagnostics. The molecular basis for phenotypic rifampin resistance is linked to mutations in the 81-bp core region of rpoB. Phenotypic isoniazid resistance has been associated with mutations in katG, particularly at codon 315, as well as with mutations in inhA, the promoter of mabA-inhA, and the intergenic region of oxyR-ahpC (3, 33, 34, 37, 43). Recently, a database of tuberculosis drug resistance mutations (TBDReaMDB) was established (27), and several genotypic diagnostic methods based on specific drug resistance-conferring mutations were developed. Two line probe assays, the INNO-LiPARif.TB assay (Innogenetics, Belgium) and the GenoType MTBDR Plus assay (Hain Lifescience, Nehren, Germany), have been approved by the World Health Organization (WHO) as tools for the rapid diagnosis of MDR TB (11, 20, 22). These tools are rapid and reproducible, but performance varies by geographic location, depending on the prevalent strains of M. tuberculosis and the type and frequency of drug resistance-conferring mutations in the population being tested (20, 22). Therefore, a thorough understanding of the diversity of the mycobacterial genetic mutations will form the foundation for new diagnostic methods.Despite the large number of MDR TB cases in China, relatively few studies have determined the prevalence of different drug resistance-conferring mutations among MDR clinical isolates. In this study, we investigated the type and frequency of drug resistance-conferring mutations that occurred among M. tuberculosis clinical isolates that were phenotypically MDR. Our goal was to identify and select a limited, parsimonious number of mutation sites that can be used to prospectively and rapidly screen isolates to detect MDR TB in Shanghai.     

Detection of gyrA and gyrB mutations in Clostridium difficile isolates by real-time PCR

Fluoroquinolone (FQ)-resistance in Clostridium difficile has been associated with mutations in the quinolone-resistance determining region (QRDR) of gyr genes. In particular, the majority of resistant clinical isolates show mutations in codon 82 of gyrA or in codon 426 of gyrB. A real-time PCR method was developed to identify these mutations in FQ-resistant C. difficile strains. Twenty-one clinical isolates, selected as representative of the different gyr alleles known up to date, and 20 clinical isolates with unknown behavior towards FQs were used to validate the method. Each mutation was detected by real-time amplification followed by hybridization with two fluorescent probes designed with the sequence complementary to the wild-type sequences of gyr genes. The melting peak analysis of the probe-PCR product hybrid was performed on a LightCycler (Roche Diagnostic). Single and multiplex assays were performed with the same reaction conditions. In both cases, isolates showing mutations in gyr sequences had a well distinguished T_m compared to that of isolates showing wild-type genes or silent mutated codons in the nucleotide region covered by probes. The results obtained indicate that this real-time PCR assay is a rapid, reproducible and accurate screening method of the predominant mutations determining FQ-resistance in C. difficile strains.     

High Proportion of Heteroresistance in gyrA and gyrB in Fluoroquinolone-Resistant Mycobacterium tuberculosis Clinical Isolates

Heteroresistance is the coexistence of populations with differing nucleotides at a drug resistance locus within a sample of organisms. Although Sanger sequencing is the gold standard for sequencing, it may be less sensitive than deep sequencing for detecting fluoroquinolone heteroresistance in Mycobacterium tuberculosis. Twenty-seven fluoroquinolone monoresistant and 11 fluoroquinolone-susceptible M. tuberculosis isolates were analyzed by Sanger and Illumina deep sequencing. Individual sequencing reads were analyzed to detect heteroresistance in the gyrA and gyrB genes. Heteroresistance to fluoroquinolones was identified in 10/26 (38%) phenotypically fluoroquinolone-resistant samples and 0/11 (P = 0.02) fluoroquinolone-susceptible controls. One resistant sample was excluded because of contamination with the laboratory strain M. tuberculosis H37Rv. Sanger sequencing revealed resistance-conferring mutations in 15 isolates, while deep sequencing revealed mutations in 20 isolates. Isolates with fluoroquinolone resistance-conferring mutations by Sanger sequencing all had at least those same mutations identified by deep sequencing. By deep sequencing, 10 isolates had a single fixed (defined as >95% frequency) mutation, while 10 were heteroresistant, 5 of which had a single unfixed (defined as <95% frequency) mutation and 5 had multiple unfixed mutations. Illumina deep sequencing identified a higher proportion of fluoroquinolone-resistant M. tuberculosis isolates with heteroresistance than did Sanger sequencing. The heteroresistant isolates frequently demonstrated multiple mutations, but resistant isolates with fixed mutations each had only a single mutation.     

Mutations in gyrA and gyrB among Fluoroquinolone- and Multidrug-Resistant Mycobacterium tuberculosis Isolates

In order to correlate the mutations inside the entire gyrA and gyrB genes with the level of resistance to ofloxacin (OFX) and moxifloxacin (MFX) in isolates of multidrug-resistant Mycobacterium tuberculosis (MDR-TB), a total of 111 isolates were categorized into OFX-susceptible (MIC, ≤2 μg/ml) and low-level (MIC, 4 to 8 μg/ml) and high-level (MIC, ≥16 μg/ml) OFX-resistant isolates and MFX-susceptible (MIC, ≤0.5 μg/ml) and low-level (MIC, 1 to 2 μg/ml) and high-level (MIC, ≥4 μg/ml) MFX-resistant isolates. Resistance-associated mutations inside the gyrA gene were found in 30.2% of OFX-susceptible and 72.5% and 72.2% of low-level and high-level OFX-resistant isolates and in 28.6% of MFX-susceptible and 58.1% and 83.9% of low-level and high-level MFX-resistant isolates. Compared with OFX-susceptible isolates, low-level and high-level OFX-resistant isolates had a significantly higher prevalence of mutations at gyrA codons 88 to 94 (17.0%, 65.0%, and 72.2%, respectively; P < 0.001) and a higher prevalence of the gyrB G512R mutation (0.0%, 2.5%, and 16.7%, respectively; P = 0.006). Similarly, compared with MFX-susceptible isolates, low-level and high-level MFX-resistant isolates had a significantly higher prevalence of mutations at gyrA codons 88 to 94 (14.3%, 51.6%, and 80.6%, respectively; P < 0.001) as well as a higher prevalence of the gyrB G512R mutation (0.0%, 0.0%, and 12.9%, respectively; P = 0.011). D94G and D94N mutations in gyrA and the G512R mutation in gyrB were correlated with high-level MFX resistance, while the D94A mutation was associated with low-level MFX resistance. The prevalence of mutations at gyrA codons 88 to 94 and the gyrB G512R mutation were higher among fluoroquinolone (FQ)-susceptible East Asian (Beijing) and Indo-Oceanic strains than they were among Euro-American strains, implying that molecular techniques to detect FQ resistance may be less specific in areas with a high prevalence of East Asian (Beijing) and Indo-Oceanic strains.     

Evaluation of Pyrosequencing for Detecting Extensively Drug-Resistant Mycobacterium tuberculosis among Clinical Isolates from Four High-Burden Countries

Reliable molecular diagnostics, which detect specific mutations associated with drug resistance, are promising technologies for the rapid identification and monitoring of drug resistance in Mycobacterium tuberculosis isolates. Pyrosequencing (PSQ) has the ability to detect mutations associated with first- and second-line anti-tuberculosis (TB) drugs, with the additional advantage of being rapidly adaptable for the identification of new mutations. The aim of this project was to evaluate the performance of PSQ in predicting phenotypic drug resistance in multidrug- and extensively drug-resistant tuberculosis (M/XDR-TB) clinical isolates from India, South Africa, Moldova, and the Philippines. A total of 187 archived isolates were run through a PSQ assay in order to identify M. tuberculosis (via the IS6110 marker), and to detect mutations associated with M/XDR-TB within small stretches of nucleotides in selected loci. The molecular targets included katG, the inhA promoter and the ahpC-oxyR intergenic region for isoniazid (INH) resistance; the rpoB core region for rifampin (RIF) resistance; gyrA for fluoroquinolone (FQ) resistance; and rrs for amikacin (AMK), capreomycin (CAP), and kanamycin (KAN) resistance. PSQ data were compared to phenotypic mycobacterial growth indicator tube (MGIT) 960 drug susceptibility testing results for performance analysis. The PSQ assay illustrated good sensitivity for the detection of resistance to INH (94%), RIF (96%), FQ (93%), AMK (84%), CAP (88%), and KAN (68%). The specificities of the assay were 96% for INH, 100% for RIF, FQ, AMK, and KAN, and 97% for CAP. PSQ is a highly efficient diagnostic tool that reveals specific nucleotide changes associated with resistance to the first- and second-line anti-TB drug medications. This methodology has the potential to be linked to mutation-specific clinical interpretation algorithms for rapid treatment decisions.     

The Arabinosyltransferase EmbC Is Inhibited by Ethambutol in Mycobacterium tuberculosis

Ethambutol (EMB) is an antimycobacterial drug used extensively for the treatment of tuberculosis caused by Mycobacterium tuberculosis. EMB targets the biosynthesis of the cell wall, inhibiting the synthesis of both arabinogalactan and lipoarabinomannan (LAM), and is assumed to act via inhibition of three arabinosyltransferases: EmbA, EmbB, and EmbC. EmbA and EmbB are required for the synthesis of arabinogalactan, and at least one enzyme (M. tuberculosis EmbA [EmbA_Mt]) is essential in M. tuberculosis. EmbC_Mt is also essential for the viability of M. tuberculosis but is involved in the synthesis of LAM. We show that mutations in EmbC_Mt that reduce its arabinosyltransferase activity result in increased sensitivity to EMB and the production of smaller LAM species in M. tuberculosis. Overexpression of EmbC_Mt was not tolerated in M. tuberculosis, but overexpression of Mycobacterium smegmatis EmbC (EmbC_Ms) led to EMB resistance and the production of larger LAM species in M. tuberculosis. Treatment of wild-type M. tuberculosis strains with EMB led to inhibition of LAM synthesis, resulting in the production of smaller species of LAM. In contrast, no change in LAM production was seen in EMB-resistant strains. Overexpression of EmbB_Ms in M. tuberculosis also resulted in EMB resistance, but at a lower level than that caused by EmbC_Ms. Overexpression of EmbA_Mt in M. tuberculosis had no effect on EMB resistance. Thus, there is a direct correlation between EmbC activity and EMB resistance, as well as between EmbC activity and the size of the LAM species produced, confirming that EmbC is one of the cellular targets of EMB action.Tuberculosis (TB), one of the oldest diseases known to humans, remains a major public health threat. It is estimated that one-third of the world''s population are infected with the causative agent, Mycobacterium tuberculosis. The scale of the problem is increasing, and the disease is becoming deadlier as it intersects with the spread of human immunodeficiency virus. The emergence of multidrug-resistant strains establishes the urgent need to fully understand the mechanisms of drug resistance. Ethambutol (EMB) is a bacteriostatic, antimycobacterial drug first described in 1961 (31) and has been prescribed for TB treatment since 1966. EMB is used worldwide for TB therapy in combination with isoniazid, pyrazinamide, and rifampin (rifampicin). It is also effective in the treatment of opportunistic mycobacterial infections of patients with human immunodeficiency virus. Unfortunately, resistance to EMB has been reported in as many as 4% of clinical isolates of M. tuberculosis and is prevalent among multidrug-resistant strains (5).The effects of EMB are highly pleiotropic, and over the past 40 years, many efforts have been made to understand its intracellular target(s). Kilburn and Greenberg (12) were the first to indicate that EMB treatment of Mycobacterium smegmatis caused rapid bacterial declumping, suggesting cell wall changes. It was subsequently demonstrated that EMB inhibited the transfer of mycolic acids to the cell wall (29), resulting in the accumulation of trehalose monomycolate, trehalose dimycolate, and free mycolic acids in the medium (13). This was later related to inhibition of the biosynthesis of the mycobacterial cell wall core polymer, arabinogalactan (AG), leading to a lack of arabinan receptors for the mycolic acids (29). The mycolyl-AG-peptidoglycan complex (in which AG is linked to peptidoglycan and the mycolic acids) is a major structural component of the cell wall, providing a hydrophobic permeability barrier. It was noted subsequently that AG is not the only cell wall arabinan affected by EMB and that EMB also inhibits the synthesis of the arabinan core of lipoarabinomannan (LAM) (6, 17), a key surface molecule in the host-pathogen interaction (16). In M. smegmatis, EMB exposure leads to the accumulation of decaprenol phosphoarabinose (DPA), the arabinosyl donor for arabinan biosynthesis, confirming the effect of EMB on arabinosyltransferases involved in AG and LAM production (32).The genetic basis of resistance to EMB in mycobacteria has been the subject of much scrutiny. Initial work with M. smegmatis led to the identification of a cluster of genes from the related species Mycobacterium avium that conferred EMB resistance when overexpressed (3). The locus has been characterized; it consists of a regulator, EmbR, and two arabinosyltransferases, EmbA and EmbB (3). In M. tuberculosis and M. smegmatis, the emb locus encodes three arabinosyltransferases (EmbC, EmbA, and EmbB); the EmbR regulator is located elsewhere on the chromosome. The identification of point mutations in codons 289 and 292 of M. smegmatis embB that conferred EMB resistance (1, 15) led to a search for similar mutations in codons 303 and 306 of embB in EMB-resistant clinical isolates of M. tuberculosis (15, 30). While mutations have been associated with EMB resistance in several studies (1, 15, 26, 30), there is still some controversy about the role of these mutations, particularly those at codon 306, in mediating EMB resistance, since the most common mutations have been found in fully sensitive isolates (9, 14, 18, 25). Most recently, work using isogenic strains has once again suggested that these mutations are linked to resistance (23). While much attention has been focused on the role of EmbB in mediating resistance, studies have suggested that multiple molecular pathways to an EMB resistance phenotype exist and that these may be associated with mutations elsewhere in the genome (22, 27).The precise mode of action of EMB is still unclear, although given that EmbB has been implicated in EMB resistance, it has been considered to be the primary target of EMB. However, several lines of evidence suggest that EmbB is not the only target and that the target(s) may differ between mycobacterial species. In particular, inhibition of EmbB in M. smegmatis cannot be the sole antibacterial effect, since embB deletion mutants are viable, indicating that the gene product is not essential in culture (7). Thus, in the fast-growing nonpathogenic species, there must be another target of inhibition, and it seems likely that EMB must inhibit two or more of the Emb arabinosyltransferase activities to effect growth inhibition. In contrast, EmbA (2) and EmbC (8) are independently essential in M. tuberculosis, and the available data strongly suggest that EmbB is essential (24). Thus, in theory, inhibition of any one of these could lead to stasis.We investigated the role of EmbC as a target of EMB action. We constructed strains of M. tuberculosis carrying mutated alleles of embC with reduced arabinosyltransferase activity or strains overexpressing EmbC. Phenotypic analyses demonstrated a direct correlation between EmbC activity, the level of resistance to EMB, and the size of the LAM species produced.