katG mutations in isoniazid-resistant Mycobacterium tuberculosis isolates recovered from Finnish patients.

katG and inhA genes from isoniazid-resistant Mycobacterium tuberculosis strains isolated in Finland were examined by PCR or sequencing. By PCR, katG was not detected in 3 of 54 strains. Sequencing of katG from 13 strains showed small point mutations or insertions; a previously described mutation causing a Ser-to-Thr change at position 315 was found in 4 strains, and there were nine new missense mutations of katG. A 209-bp segment of inhA from 17 strains was sequenced, but no mutations were observed. This result indicates that different mutations prevail in different geographical areas.     

Characterization of the katG and inhA genes of isoniazid-resistant clinical isolates of Mycobacterium tuberculosis.

Resistance to isoniazid in Mycobacterium tuberculosis has been associated with mutations in genes encoding the mycobacterial catalase-peroxidase (katG) and the InhA protein (inhA). Among the 26 isoniazid-resistant clinical isolates evaluated in this study, mutations in putative inhA regulatory sequences were identified in 2 catalase-positive isolates, katG gene alterations were detected in 20 strains, and 4 isolates had wild-type katG and inhA genes. Mutations in the katG gene were detected in all 11 catalase-negative isolates: one frameshift insertion, two partial gene deletions, and nine different missense mutations were identified. An arginine-to-leucine substitution at position 463 was detected in nine catalase-positive isolates. However, site-directed mutagenesis experiments demonstrated that the presence of a leucine at codon 463 did not alter the activity of the M. tuberculosis catalase-peroxidase and did not affect the capacity of this enzyme to restore isoniazid susceptibility to isoniazid-resistant, KatG-defective Mycobacterium smegmatis BH1 cells. These studies further support the association between katG and inhA gene mutations and isoniazid resistance in M. tuberculosis, while also suggesting that other undefined mechanisms of isoniazid resistance exist.     

Analysis of ahpC gene mutations in isoniazid-resistant clinical isolates of Mycobacterium tuberculosis.

The ahpC genes of 57 clinical isolates and one in vitro mutant of Mycobacterium tuberculosis were evaluated by nucleotide sequence analyses. Although compensatory ahpC promoter mutations were identified in 8 catalase-negative, katG-defective strains, the ahpC genes of 25 catalase-positive, isoniazid-resistant isolates and 25 drug-sensitive strains were not altered.     

Molecular characterization of isoniazid-resistant Mycobacterium tuberculosis clinical isolates in Lithuania

Mutations at codon 315 of the katG gene were detected in 312 of 364 (85.7%) isoniazid-resistant Mycobacterium tuberculosis isolates. Seven of 52 (13.5%) isoniazid-resistant isolates with the wild-type Ser315 codon and 10 of 52 (19.2%) isoniazid-resistant isolates with a mutated katG allele had mutation -15C-->T in the promoter of the mabA-inhA operon.     

New multiplex PCR for rapid detection of isoniazid-resistant Mycobacterium tuberculosis clinical isolates

In this study, we describe a multiplex PCR to detect a AGC-->ACC (serine to threonine) mutation in the katG gene and a -15 C-to-T substitution (inhA(C-15T)) at the 5' end of a presumed ribosome binding site in the promoter of the mabA-inhA operon. These mutations have been reported in the majority of previous studies as the most frequent mutations involved in the resistance to isoniazid (INH) of Mycobacterium tuberculosis clinical strains with high levels of resistance. The method was optimized and validated after an analysis of 30 M. tuberculosis clinical isolates with known sequences of the relevant part of the katG gene and the regulatory region of the mabA-inhA operon. We analyzed 297 INH-resistant M. tuberculosis isolates collected in Spain from 1996 to 2003 by PCR-restriction fragment length polymorphism (using the katG gene), DNA sequencing, and the newly developed multiplex PCR. The results were concordant for all 297 isolates tested. The analysis revealed that 204 (68.7%) of the isolates carried one or both of the mutations. This finding suggests that with further development this multiplex PCR will be able to detect the majority of the INH-resistant M. tuberculosis clinical isolates from Spain and other countries where a high frequency of similar mutations occur.     

ethA, inhA, and katG loci of ethionamide-resistant clinical Mycobacterium tuberculosis isolates

Ethionamide (ETH) is a structural analog of the antituberculosis drug isoniazid (INH). Both of these drugs target InhA, an enzyme involved in mycolic acid biosynthesis. INH requires catalase-peroxidase (KatG) activation, and mutations in katG are a major INH resistance mechanism. Recently an enzyme (EthA) capable of activating ETH has been identified. We sequenced the entire ethA structural gene of 41 ETH-resistant Mycobacterium tuberculosis isolates. We also sequenced two regions of inhA and all or part of katG. The MICs of ETH and INH were determined in order to associate the mutations identified with a resistance phenotype. Fifteen isolates were found to possess ethA mutations, for all of which the ETH MICs were > or =50 microg/ml. The ethA mutations were all different, previously unreported, and distributed throughout the gene. In eight of the isolates, a missense mutation in the inhA structural gene occurred. The ETH MICs for seven of the InhA mutants were > or =100 microg/ml, and these isolates were also resistant to > or =8 microg of INH per ml. Only a single point mutation in the inhA promoter was identified in 14 isolates. A katG mutation occurred in 15 isolates, for which the INH MICs for all but 1 were > or =32 microg/ml. As expected, we found no association between katG mutation and the level of ETH resistance. Mutations within the ethA and inhA structural genes were associated with relatively high levels of ETH resistance. Approximately 76% of isolates resistant to > or =50 microg of ETH per ml had such mutations.     

Molecular characterization of isoniazid-resistant Mycobacterium tuberculosis isolates collected in Australia

Elucidation of the molecular basis of isoniazid (INH) resistance in Mycobacterium tuberculosis has led to the development of different genotypic approaches for the rapid detection of INH resistance in clinical isolates. Mutations in katG, in particular the S315T substitution, are responsible for INH resistance in a large proportion of tuberculosis cases. However, the frequency of the katG S315T substitution varies with population samples. In this study, 52 epidemiologically unrelated clinical INH-resistant M. tuberculosis isolates collected in Australia were screened for mutations at katG codon 315 and the fabG1-inhA regulatory region. Importantly, 52 INH-sensitive isolates, selected to reflect the geographic and genotypic diversity of the isolates, were also included for comparison. The katG S315T substitution and fabG1-inhA -15 C-to-T mutation were identified in 34 and 13 of the 52 INH-resistant isolates, respectively, and none of the INH-sensitive isolates. Three novel katG mutations, D117A, M257I, and G491C, were identified in three INH-resistant strains with a wild-type katG codon 315, fabG1-inhA regulatory region, and inhA structural gene. When analyzed for possible associations between resistance mechanisms, resistance phenotype, and genotypic groups, it was found that neither the katG S315T nor fabG1-inhA -15 C-to-T mutation clustered with any one genotypic group, but that the -15 C-to-T substitution was associated with isolates with intermediate INH resistance and isolates coresistant to ethionamide. In total, 90.4% of unrelated INH-resistant isolates could be identified by analysis of just two loci: katG315 and the fabG1-inhA regulatory region.     

An Altered Mycobacterium tuberculosis Metabolome Induced by katG Mutations Resulting in Isoniazid Resistance

The most common form of drug resistance found in tuberculosis (TB)-positive clinical samples is monoresistance to isoniazid. Various genomics and proteomics studies to date have investigated this phenomenon; however, the exact mechanisms relating to how this occurs, as well as the implications of this on the TB-causing organisms function and structure, are only partly understood. Considering this, we followed a metabolomics research approach to identify potential new metabolic pathways and metabolite markers, which when interpreted in context would give a holistic explanation for many of the phenotypic characteristics associated with a katG mutation and the resulting isoniazid resistance in Mycobacterium tuberculosis. In order to achieve these objectives, gas chromatography-time of flight mass spectrometry (GCxGC-TOFMS)-generated metabolite profiles from two isoniazid-resistant strains were compared to a wild-type parent strain. Principal component analyses showed clear differentiation between the groups, and the metabolites best describing the separation between these groups were identified. It is clear from the data that due to a mutation in the katG gene encoding catalase, the isoniazid-resistant strains experience increased susceptibility to oxidative stress and have consequently adapted to this by upregulating the synthesis of a number of compounds involved in (i) increased uptake and use of alkanes and fatty acids as a source of carbon and energy and (ii) the synthesis of a number of compounds directly involved in reducing oxidative stress, including an ascorbic acid degradation pathway, which to date hasn''t been proposed to exist in these organisms.     

Screening and characterization of mutations in isoniazid-resistant Mycobacterium tuberculosis isolates obtained in Brazil

We investigated mutations in the genes katG, inhA (regulatory and structural regions), and kasA and the oxyR-ahpC intergenic region of 97 isoniazid (INH)-resistant and 60 INH-susceptible Mycobacterium tuberculosis isolates obtained in two states in Brazil: São Paulo and Paraná. PCR-single-strand conformational polymorphism (PCR-SSCP) was evaluated for screening mutations in regions of prevalence, including codons 315 and 463 of katG, the regulatory region and codons 16 and 94 of inhA, kasA, and the oxyR-ahpC intergenic region. DNA sequencing of PCR amplicons was performed for all isolates with altered PCR-SSCP profiles. Mutations in katG were found in 83 (85.6%) of the 97 INH-resistant isolates, including mutations in codon 315 that occurred in 60 (61.9%) of the INH-resistant isolates and 23 previously unreported katG mutations. Mutations in the inhA promoter region occurred in 25 (25.8%) of the INH-resistant isolates; 6.2% of the isolates had inhA structural gene mutations, and 10.3% had mutations in the oxyR-ahpC intergenic region (one, nucleotide −48, previously unreported). Polymorphisms in the kasA gene occurred in both INH-resistant and INH-susceptible isolates. The most frequent polymorphism encoded a G₂₆₉A substitution. Although KatG₃₁₅ substitutions are predominant, novel mutations also appear to be responsible for INH resistance in the two states in Brazil. Since ca. 90.7% of the INH-resistant isolates had mutations identified by SSCP electrophoresis, this method may be a useful genotypic screen for INH resistance.Isoniazid (INH), a first-line antituberculosis drug, is bactericidal and has a simple chemical structure consisting of a pyridine ring and a hydrazide group. INH is a prodrug that enters actively growing tubercle bacilli by passive diffusion (2). The bifunctional bacterial enzyme catalase-peroxidase (KatG) converts INH to a range of oxygenated and organic toxic radicals that attack multiple targets in the mycobacterial cell (35, 36, 48). The best-characterized target of these radicals is the cell wall mycolic acid, but DNA, carbohydrates, lipids, and NAD metabolism may be targeted as well (16, 36, 50).The tuberculosis case rate in Brazil is the 15th highest in the world, with an estimated prevalence of 64 cases per 100,000 population; moreover, ∼0.9% of the new cases are multidrug resistant (45). A recent nationwide investigation of primary INH resistance found a national frequency of 3.8% (29); however, the percentages varied greatly between geographic regions of the country. The incidence of tuberculosis cases in Brazil also varies widely among geographic regions, with 18,112 new reported cases in São Paulo State (51.40 cases per 100,000 population) in 1998 (38) and 2,684 new cases in Paraná State (28.99 cases per 100,000 population) in the same year (37).Molecular studies of the mechanisms of resistance to INH in Mycobacterium tuberculosis demonstrated that a significant number of drug-resistant strains have mutations in the katG gene, which encodes the KatG enzyme. Initial investigations of katG found large deletions in resistant strains (48, 49), but subsequent studies showed this to be rare. Mutations reduce the ability of KatG to activate the prodrug INH, thus leading to resistance (11, 17, 24, 42). In addition, mutations in other genes, including inhA and kasA, and in the oxyR-ahpC intergenic region have been associated with INH resistance but in much lower percentages of strains (26, 32, 33, 50).An activated INH radical appears to inhibit the InhA enzyme by reacting with the NAD(H) cofactor bound to the InhA active site, which compromises the mycolic acid synthesis (23). Mutation at the InhA enzyme''s site of interaction can reduce its affinity for NAD(H) and confer INH and ethionamide resistance to strains (1). The overexpression of InhA because of an upregulation mutation in the promoter region of inhA (preceding the mabA-inhA operon) can also cause resistance to INH by a titration mechanism (1, 2, 3, 8, 16, 23). Mutations in the oxyR-ahpC intergenic region, where the putative promoter of ahpC is located, are considered to be a compensatory mechanism for the loss of KatG function in resistant strains (18, 33, 35, 46, 47). These mutations may be used as surrogate markers for the detection of INH resistance in M. tuberculosis (33, 39, 41, 50).Mdluli et al. (25) reported that the ketoacyl acyl carrier protein synthase (KasA), encoded by the kasA gene, which is involved in the biosynthesis of mycolic acids, is a likely target for INH. They found an association between mutations in the kasA gene and resistance to INH in M. tuberculosis. However, Lee et al. (22) observed mutations in the kasA gene in resistant and in susceptible M. tuberculosis strains from Singapore. Recently, Larsen et al. (21) demonstrated no correlation between resistance to INH and overexpression of KasA.A variety of methods have been used to facilitate the rapid detection of mutations in mycobacteria. One widely used method is PCR-single-strand conformational polymorphism (PCR-SSCP) (7, 28, 43). If any two single strands of DNA differ by one or more nucleotides, differences in the secondary structure of these strands may be identified by their electrophoretic mobilities in nondenaturing polyacrylamide gels (9), offering a convenient and cost-efficient method for analyzing mutations in PCR products. The PCR-SSCP method has been demonstrated to be useful for screening mutations associated with antituberculosis drug resistance (7, 10, 15, 30, 46).We investigated the prevalence of mutations in the genes, katG, kasA, and inhA (regulatory and structural regions) and in the oxyR-ahpC intergenic region. We evaluated the usefulness of SSCP electrophoresis for the detection of those mutations among INH-resistant isolates from São Paulo and Paraná, Brazil.     

耐左氧氟沙星结核分枝杆菌gyrA基因上新的突变位点

目的:分析耐左氧氟沙星结核分枝杆菌临床菌株gyrA基因的突变情况及其耐药机制.方法:用聚合酶链反应(PER)和DNA直接测序技术(DS)测定64株耐左氧氟沙星结核分枝杆菌gyrA基因的QRDR(quinolone resistance-determining regions)序列.结果:64株耐药菌株有47株QRDR序列发生突变,其中45株为单位点突变,另2株为双位点突变;突变分布为70位突变2株、89位1株、90位12株、91位4株、94位30株,其中70位和89位为新发现的突变位点.结论:结核分枝杆菌喹诺酮类药物耐受现象与gyrA基因QRDR的突变有关,包括新发现的70位和89位突变.     

Molecular characteristics of rifampicin- and isoniazid-resistant Mycobacterium tuberculosis isolates from the Russian Federation

OBJECTIVES: Three Mycobacterium tuberculosis genetic loci--rpoB and katG genes and the fabG1(mabA)-inhA operon promoter region--were studied to reveal the mutations associated with rifampicin and isoniazid resistance. METHODS: Four hundred and twelve isolates of M. tuberculosis from different regions of the Russian Federation were collected during 1997-2005. A matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS)-based minisequencing method was used for the detection of mutations. RESULTS: Thirteen different variants of single mutations in codons 533, 531, 526, 516, 513 and 511 of the rifampicin resistance-determining region of the rpoB gene as well as the TTG insertion in the 514a position were found among the rifampicin-resistant isolates. Single nucleotide substitutions in codons 531, 526 and 516 (64.8%, 10.3% and 7.7%, respectively) were the most prevalent mutations. Codon 526 was shown to be the most variable of all. No mutations were detected in rpoB genes for 29 (10.7%) of the rifampicin-resistant isolates. 76.9% of the isoniazid-resistant isolates carried single mutations in codon 315 of the katG gene. For another 12.9% of them, double mutations in the katG gene and the fabG1(mabA)-inhA promoter region were revealed. No mutations were detected in 8.2% of the isoniazid-resistant isolates. CONCLUSIONS: Molecular analysis of the loci of rpoB and katG genes and the inhA promoter region of 412 M. tuberculosis clinical isolates from various parts of the Russian Federation was carried out. The new MALDI-TOF MS-based method may be used for rapid and accurate monitoring of the spread of drug resistance.     

PCR直接测序法分析结核分枝杆菌katG基因突变

为了了解我国结核分枝杆菌耐异烟肼（ＩＮＨ）分离株ｋａｔＧ基因突变情况，研究其临床意义。本研究通过聚合酶链反应（ＰＣＲ）、ＰＣＲ－单链构象多态性（ＳＳＣＰ）和ＰＣＲ直测序法分析９２株结核分枝杆菌临床分离株的ｋａｔＧ基因。以Ｈ３７Ｒｖ标准株为对照，２３株药物敏感体中，２１株ｋａｔＧ基因ＳＳＣＰ分析正常，２株异常。３６株耐非ＩＮＨ药物的分离株中，２０株ｋａｔＧ基因ＳＳＣ正常，１６株异常。３３株耐ＩＮＨ分     

Molecular analysis of katG gene mutations in strains of Mycobacterium tuberculosis complex from Africa.

A sample of 124 isoniazid (INH)-resistant and 88 susceptible strains of Mycobacterium tuberculosis complex from south, central, and west Africa was analyzed by direct sequence analysis and PCR-restriction fragment length polymorphism analysis of their catalase-peroxidase (katG) genes. Point mutations at codon 315 were found in the genomes of 64% of INH-resistant strains, but no complete deletions were identified. Mutations at codon 463 were independent of INH resistance and were linked to the geographic origins of the strains.     

First linezolid-resistant clinical isolates of Mycobacterium tuberculosis

Linezolid resistance was found in 4 (1.9%) of 210 multidrug-resistant Mycobacterium tuberculosis strains. The MICs of linezolid were 4 microg/ml (one strain) and 8 microg/ml (three strains). Since no mutations were detected in potential target genes, the mechanism of resistance remains unclear.     

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.     

新疆结核分枝杆菌临床分离株Spoligotyping基因型的初步研究

目的研究新疆结核分枝杆菌Spoligotyping基因分型,初步了解其基因型多态性状况及主要流行株。方法在新疆维吾尔自治区胸科医院收集一个连续时间段的结核分枝杆菌临床分离菌株,采用比例法检测进行耐药性检测、间隔寡核苷酸分型(Spoligotyp ing)方法进行分型研究。基因聚类分析采用B ioNum erics 5.0数据库软件,统计学分析采用χ2检验,P0.05为差异有统计学意义。结果共收集到结核分枝杆菌临床分离菌株175株,其中对利福平、异烟肼、链霉素和乙胺丁醇全敏感115株,耐药60株(包括单耐药31株和耐多药29株)。经Spoligotyp ing分型,这些菌株可分为4个基因群49种基因型,最大的1个基因群为北京家族,占68.57%(120/175)。北京家族中,敏感菌株63.87%(76/119),耐药菌株占36.13%(43/119);非北京家族中,敏感菌株68.52%(37/54),耐药菌株占31.48%(17/54),北京家族与非北京家族的耐药率之间差异无统计学意义(P=0.551)。结论新疆结核分枝杆菌临床菌株存在明显的基因多态性,主要流行菌株为北京基因型。北京基因型与耐药性无明显相关性。     

Characterization of novel Mycobacterium tuberculosis pncA gene mutations in clinical isolates from the Ukraine

Multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis cases in the Ukraine are increasing. Pyrazinamide (PZA) is critically important for first- and second-line tuberculosis (TB) treatment regimes. However, PZA drug susceptibility testing is time consuming and technically challenging. The present study utilized Next-generation sequencing (NGS) to identify mutations in the pncA gene from clinical isolates and to assess the prevalence of pncA gene mutations in MDR/XDR-TB patients. Clinical isolates were inactivated in molecular transport media and shipped from Kharkiv, Ukraine, to San Antonio, TX. Whole-genome and targeted pncA gene sequencing was carried out using Illumina MiSeq instrumentation. Mutations were noted in 67 of 91 (74%) clinical isolates comprising substitutions, insertions, and deletions in the pncA coding and upstream promoter region. Of 45 mutation types, there were 11 novel, i.e., to date unknown, pncA mutations identified of which 3 were confirmed PZA resistant. Seven isolates contained mixed base mutations, whereas 4 harbored doubled mutations. Data reported here further support use of NGS for pncA gene characterization and may contribute in significant fashion to PZA therapy, especially in MDR- and XDR-TB patients.     

Culture-independent prediction of isoniazid resistance in Mycobacterium tuberculosis by katG gene analysis directly from sputum samples.

BACKGROUND: The molecular prediction of isoniazid (INH) resistance in Mycobacterium tuberculosis is hampered by the need for specialized equipment, expertise, high costs, a limited range of detectable mutations, or several of these factors. The rationale for the study was to find a practical alternative and to demonstrate generally valid problems. METHODS AND RESULTS: DNA extracted from decontaminated sputum pellets was used to amplify a 0.26 kb target sequence within the katG gene. Mutations of codon 315, frequently found in isoniazid-resistant isolates, could be discriminated in a simple agarose minigel format following an AciI digest of the nested polymerase chain reaction (PCR) product. Within a panel of 22 sputum samples, INH resistance could be predicted in 5 of 10 samples containing isoniazid-resistant M. tuberculosis. The protocol is robust, requires little expertise and no specialized equipment, and provides the test results within 2 days. CONCLUSION: The results show the feasibility to rapidly and easily detect mutations highly predictive of isoniazid resistance. Nevertheless, this, like any other molecular resistance prediction test, is affected by often neglected factors, including mutation prevalences, the phenomenon of heteroresistance, and a possible bias toward one's own method.     

High prevalence of KatG Ser315Thr substitution among isoniazid-resistant Mycobacterium tuberculosis clinical isolates from northwestern Russia, 1996 to 2001

A total of 204 isoniazid (INH)-resistant strains of Mycobacterium tuberculosis isolated from different patients in the northwestern region of Russia from 1996 to 2001 were screened by a PCR-restriction fragment length polymorphism (RFLP) assay. This assay uses HapII cleavage of an amplified fragment of the katG gene to detect the transversion 315AGC-->ACC (Ser-->Thr), which is associated with INH resistance. This analysis revealed a 93.6% prevalence of the katG S315T mutation in strains from patients with both newly and previously diagnosed cases of tuberculosis (TB). This mutation was not found in any of 57 INH-susceptible isolates included in the study. The specificity of the assay was 100%; all isolates that contained the S315T mutation were classified as resistant by a culture-based susceptibility testing method. The Beijing genotype, defined by IS6110-RFLP analysis and the spacer oligonucleotide typing (spoligotyping) method, was found in 60.3% of the INH-resistant strains studied. The katG S315T shift was more prevalent among Beijing genotype strains than among non-Beijing genotype strains: 97.8 versus 84.6%, respectively, for all isolates, including those from patients with new and previously diagnosed cases, isolated from 1999 to 2001 and 100.0 versus 86.5%, respectively, for isolates from patients with new cases isolated from 1996 to 2001. The design of this PCR-RFLP assay allows the rapid and unambiguous identification of the katG 315ACC mutant allele. The simplicity of the assay permits its implementation into routine practice in clinical microbiology laboratories in regions with a high incidence of TB where this mutation is predominant, including northwestern Russia.