Comparison of a conventional antimicrobial susceptibility assay to an oligonucleotide chip system for detection of drug resistance in Mycobacterium tuberculosis isolates

An oligonucleotide chip (Combichip Mycobacteria chip) detecting specific mutations in the rpoB, katG, and inhA genes of Mycobacterium tuberculosis was compared with conventional antimicrobial susceptibility results. The probes detecting drug resistance were as follows: 7 wild-type and 13 mutant probes for rifampin and 2 wild-type and 3 mutant probes for isoniazid. Target DNA of M. tuberculosis was amplified by PCR, followed by hybridization and scanning. Direct sequencing was performed to verify the results of the oligonucleotide chip. One-hundred seven of 115 rifampin-resistant strains (93%) had mutations in the rpoB gene. Eighty-five of 119 isoniazid-resistant strains (71%) had mutations in the katG gene or inhA gene. The diagnostic oligonucleotide chip with mutation-specific probes is a reliable and useful tool for the rapid and accurate diagnosis of resistance against rifampin and isoniazid in M. tuberculosis isolates.     

Mutation detection and accurate diagnosis of extensively drug-resistant tuberculosis: report from a tertiary care center in India

We screened and spoligotyped 150 consecutive phenotypically confirmed extensively drug-resistant Mycobacterium tuberculosis (XDR-TB) isolates (January 2008 to March 2009) for rifampin, isoniazid, fluoroquinolone, and aminoglycoside resistance targeting rpoB, inhA, katG, gyrA, gyrB, and rrs. Mutations predominant among XDR-TB were S315T (katG) (100% of isolates), S531L (rpoB) (97% of isolates), D94G (gyrA) (53% of isolates), and A1401G (rrs) (71% of isolates). Spoligotyping revealed 62% of the isolates to be Beijing.     

Molecular analysis of isoniazid and rifampin resistance in Mycobacterium tuberculosis isolates recovered from Barcelona

We studied the presence of mutations in the whole katG gene and specific regions of the oxyR-ahpC and mabA-inhA regulatory region in 61 Mycobacterium tuberculosis isoniazid-resistant isolates. An 81-bp region of the rpoB gene was also sequenced in 17 rifampin-resistant strains. Alterations in the katG gene were detected in 55% of the isolates. Mutation in codon 315 was the most prevalent (32%). Strains showed a high level of resistance, and most maintained a substantial catalase-peroxidase activity. Three strains with an isoniazid MIC of >or=32 microg/ml lacked catalase-peroxidase activity. Two of them had deletions in the catalytic domain of the KatG protein. One strain with deletion and three strains with mutations in the C-terminal domain showed low-level resistance and conserved the catalase-peroxidase activity. Mutations in the mabA-inhA regulatory region were identified in 32% of the isolates. All had low-level resistance, and the vast majority conserved catalase-peroxidase activity. Seventeen percent of the isoniazid-resistant isolates had no detectable alterations at the studied loci. Resistance to rifampin was associated with mutations in the 81-bp of the rpoB gene in all cases. IS6110 analysis indicated that recent transmission contributed substantially to the emergence of isoniazid- resistant tuberculosis in Barcelona through short transmission chains. A rapid genotypic assay, including the 315-katG codon and the -15 nucleotide of the mabA-inhA regulatory region, may cover 62% of isoniazid- resistant strains in Barcelona. In contrast, the targeting of the 81-bp region of rpoB would detect all our rifampin-resistant isolates.     

Molecular characterization of rifampin- and isoniazid-resistant Mycobacterium tuberculosis strains isolated in Poland

A total of 105 rifampin (RMP)- and/or isoniazid (INH)-resistant strains of Mycobacterium tuberculosis isolated from different parts of Poland in 2000 were screened for mutations associated with resistance to these drugs by two molecular methods, namely sequence analysis and real-time PCR technology. Three loci associated with drug resistance were selected for characterization: they were rpoB (RMP), katG, and the regulatory region of inhA (INH). Nineteen different mutations were identified in 64 RMP-resistant strains, and five new alleles were described. The most common point mutations were in codons 531 (41%), 516 (16%), and 526 (9%) of the rpoB gene. Mutations were not found in two (3%) of the isolates. In the case of resistance to INH, six different mutations in the katG gene of 83 resistant strains were detected. Fifty-seven (69%) isolates exhibited nucleotide substitutions at codon 315. One strain harbored a mutation affecting codon 279 (Gly279Thr). Twelve of 26 INH-resistant strains with the wild-type codon 315 (14.5% of all strains tested) had the mutation -15C-->T in the regulatory region of inhA. A full correlation between the DNA sequence analysis and real-time PCR data was obtained. We conclude that the real-time PCR method is fast and reliable for the detection of RMP and INH resistance-associated mutations in M. tuberculosis clinical isolates.     

Detection of multidrug resistance in Mycobacterium tuberculosis

We developed a DNA sequencing-based method to detect mutations in the genome of drug-resistant Mycobacterium tuberculosis. Drug resistance in M. tuberculosis is caused by mutations in restricted regions of the genome. Eight genome regions associated with drug resistance, including rpoB for rifampin (RIF), katG and the mabA (fabG1)-inhA promoter for isoniazid (INH), embB for ethambutol (EMB), pncA for pyrazinamide (PZA), rpsL and rrs for streptomycin (STR), and gyrA for levofloxacin, were amplified simultaneously by PCR, and the DNA sequences were determined. It took 6.5 h to complete all procedures. Among the 138 clinical isolates tested, 55 were resistant to at least one drug. Thirty-four of 38 INH-resistant isolates (89.5%), 28 of 28 RIF-resistant isolates (100%), 15 of 18 EMB-resistant isolates (83.3%), 18 of 30 STR-resistant isolates (60%), and 17 of 17 PZA-resistant isolates (100%) had mutations related to specific drug resistance. Eighteen of these mutations had not been reported previously. These novel mutations include one in rpoB, eight in katG, one in the mabA-inhA regulatory region, two in embB, five in pncA, and one in rrs. Escherichia coli isolates expressing individually five of the eight katG mutations showed loss of catalase and INH oxidation activities, and isolates carrying any of the five pncA mutations showed no pyrazinamidase activity, indicating that these mutations are associated with INH and PZA resistance, respectively. Our sequencing-based method was also useful for testing sputa from tuberculosis patients and for screening of mutations in Mycobacterium bovis. In conclusion, our new method is useful for rapid detection of multiple-drug-resistant M. tuberculosis and for identifying novel mutations in drug-resistant M. tuberculosis.     

Direct detection of rifampin- and isoniazid-resistant Mycobacterium tuberculosis in auramine-rhodamine-positive sputum specimens by real-time PCR

Our objective was to evaluate the feasibility of a molecular assay based on a real-time PCR technique, carried out with a LightCycler instrument (Roche Biochemicals), to identify Mycobacterium tuberculosis bacilli and to detect rifampin and isoniazid resistance in DNA extracts from sputum samples. We studied three genes: rpoB, which is associated with rifampin resistance, and katG and inhA, which are associated with isoniazid resistance. A total of 205 sputum samples collected from 108 patients diagnosed with pulmonary tuberculosis with positive auramine-rhodamine-staining (AR) sputum samples, were tested. The sensitivities of the LightCycler PCR assay for the positive AR specimens was 97.5% (200 of 205) for rpoB and inhA genes and 96.5% (198 of 205) for the katG gene. For the total number of patients tested, the sensitivity was 100% (108 of 108 patients) for rifampin, whereas the sensitivity was 98.1% (106 of 108 patients) for isoniazid. Full agreement was found with the Bactec MGIT 960 method and the genotype inferred from the LightCycler data for rifampin. The phenotypic method for isoniazid reported 13 resistant strains (> or = 0.1 microg/ml). In seven (53.8%) strains there was a concordance between both methods, but we found that six (46.2%) strains reported as resistant by the phenotypic method were determined to be susceptible by real-time PCR. For the 75 strains reported as susceptible by the phenotypic method, the concordance with the LightCycler data was 100%. Our results demonstrate that rifampin-resistant M. tuberculosis could be detected in DNA extracted from auramine-rhodamine-positive sputum samples in a single-tube assay that took less than 3 h to perform for a collection of auramine-rhodamine-positive specimens obtained from patients with culture-documented pulmonary tuberculosis. Similarly, this occurs in half of the isoniazid-resistant M. tuberculosis DNA extracted from auramine-rhodamine-positive specimens.     

结核分枝杆菌临床分离株异烟肼耐药相关基因突变的分子特征

目的 阐明结核分枝杆菌异烟肼（INH）耐药相关基因突变特征.方法 对137株结核分枝杆菌临床分离株（耐异烟肼菌株87株,异烟肼敏感菌株90株）的9个结构基因furA、katG、inhA、kasA、Rv0340、iniB、iniA、iniC和efpA以及两个调控区oxyR-ahpC基因间隔区和mabA-inhA启动子进行DNA片段扩增及序列分析.结果 82株（94.3%）INH耐药分离株的katG基因存在突变,其中katGSer315Thr突变占优势（55.2%）.50株INH敏感的分离菌katG的463密码子没有突变.35株（40.2%）INH耐药的分离株katG的463有突变.87株INH耐药株中,20株（23.0%）的katG基因存在两重突变.13株（14.9%）分离菌inhA基因的启动子区存在突变,4.6%的分离菌有inhA结构基因突变,11.5%oxyR-ahpC基因间区存在突变.iniBAC区域和efpA中发现耐药性关联突变.结论 研究证实多个基因突变与异烟肼耐药之间的关系,并且为阐明结核分枝杆菌耐药机制提供线索.     

Genotypic assessment of isoniazid and rifampin resistance in Mycobacterium tuberculosis: a blind study at reference laboratory level.

Progress in understanding the basis of resistance to isoniazid (INH) and rifampin (RMP) has allowed molecular tests for the detection of drug-resistant tuberculosis to be developed. Consecutive isolates (n = 95) of Mycobacterium tuberculosis, from a Spanish reference laboratory investigating outbreaks of multidrug-resistant tuberculosis, were coded and sent to two external laboratories for genotypic analysis of INH and RMP resistance by PCR-single-strand conformation polymorphism (SSCP) analysis of specific regions of four genes: part of the coding sequence of katG and the promoter regions of inhA and ahpC for INH and the RMP resistance region of rpoB. After correction for the presence of outbreak strains and multiple isolates from single patients, RMP resistance was detected successfully by PCR-SSCP in > 96% of the RMP-resistant strains. PCR-SSCP had a sensitivity of 87% for INH resistance detection, and mutations in katG, inhA, katG-inhA, ahpC, and katG-ahpC were identified in 36.8, 31.6, 2.6, 13.2, and 2.6%, respectively, of the unique strains. Specificity was 100%. Molecular detection of resistance to the two main antituberculous drugs, INH and RMP, can be accomplished accurately by using a strategy which limits analysis to four genetic regions. This may allow the expedient analysis of drug resistance by reference laboratories.     

Rapid detection of isoniazid, rifampin, and ofloxacin resistance in Mycobacterium tuberculosis clinical isolates using high-resolution melting analysis

A high-resolution melting analysis (HRMA) assay was developed to detect isoniazid, rifampin, and ofloxacin resistance in Mycobacterium tuberculosis by targeting resistance-associated mutations in the katG, mabA-inhA promoter, rpoB, and gyrA genes. A set of 28 (17 drug-resistant and 11 fully susceptible) clinical M. tuberculosis isolates was selected for development and evaluation of HRMA. PCR amplicons from the katG, mabA-inhA promoter, rpoB, and gyrA genes of all 28 isolates were sequenced. HRMA results matched well with 18 mutations, identified by sequencing, in 17 drug-resistant isolates and the absence of mutations in 11 susceptible isolates. Among 87 additional isolates with known resistance phenotypes, HRMA identified katG and/or mabA-inhA promoter mutations in 66 of 69 (95.7%) isoniazid-resistant isolates, rpoB mutations in 51 of 54 (94.4%) rifampin-resistant isolates, and gyrA mutations in all of 41 (100%) ofloxacin-resistant isolates. All mutations within the HRMA primer target regions were detected as variant HRMA profiles. The corresponding specificities were 97.8%, 100%, and 98.6%, respectively. Most false-positive results were due to synonymous mutations, which did not affect susceptibility. HRMA is a rapid, sensitive method for detection of drug resistance in M. tuberculosis which could be used routinely for screening isolates in countries with a high prevalence of tuberculosis and drug resistance or in individual isolates when drug resistance is suspected.     

Rapid genotypic detection of rifampin- and isoniazid-resistant Mycobacterium tuberculosis directly in clinical specimens

A multiplex PCR DNA strip assay (Genotype MTBDR) designed to detect rifampin (rpoB) and high-level isoniazid (katG) resistance mutations in Mycobacterium tuberculosis isolates was optimized for clinical specimens. Successful genotypic results were achieved with 36 of 38 (95%) smear-positive respiratory specimens, allowing rapid therapeutic adjustments in transmittable drug-resistant tuberculosis.     

基因型分析法快速检测耐药结核分枝杆菌

目的 探讨基因型分析法检测结核分枝杆菌异烟肼和利福平耐药性的价值.方法 采用多重PCR和线性探针反向膜杂交法来检测异烟肼耐药基因katG S315T和inhA C-15T突变以及利福平耐药基因rpoB D516V,H526Y,H526D,S531L突变来判断78株结核分枝杆菌临床分离株的耐药性,并与金标准L-J固体培养基药敏法以及BACTEC960液体培养药敏法进行对比分析.结果 基因型分析法在6h内可以完成;结核分枝杆菌常规药敏检测需要3个月.与后者相比,基因型分析法检测异烟肼耐药的敏感性和特异性分别为89％ （16/18）和99％ （77/78）;检测利福平耐药的敏感性和特异性均为100％（13/13,78/78）.结论 基因型分析法检测结核菌耐药性快速准确,对耐药结核病的早期诊断和治疗很有帮助,有望在临床实验室广泛开展.     

Correlating genetic mutations with isoniazid phenotypic levels of resistance in Mycobacterium tuberculosis isolates from patients with drug-resistant tuberculosis in a high burden setting

European Journal of Clinical Microbiology & Infectious Diseases - We analysed mutations in katG, inhA and rpoB genes, and isoniazid phenotypic resistance levels in Mycobacterium tuberculosis...     

Mutations prevalent among rifampin- and isoniazid-resistant Mycobacterium tuberculosis isolates from a hospital in Vietnam

Vietnam is ranked 13th among the WHO list of 22 high-burden countries, based upon estimated total number of tuberculosis cases. Despite having a model national tuberculosis program, consistently achieving and exceeding WHO targets for detection and cure, drug-resistant and multidrug-resistant tuberculosis cases continue to rise. Rapid multidrug-resistant tests applicable in this setting, coupled with effective treatment regimens, would be a useful tool in reversing this trend, allowing early identification of patients with multidrug-resistant tuberculosis and avoiding resistance-amplifying regimens. Sequencing of consecutive isolates identified by the National Tuberculosis Program showed 89% of isoniazid-resistant isolates could be detected by targeting just 2 codons, katG 315 and -15C-->T in the inhA promoter, while rifampin resistance will be more complex to detect, with many different mutation and insertion events in rpoB. The most prevalent rifampin resistance-conferring mutations, as in other countries, were in rpoB codons 531 (43%), 526 (31%), and 516 (15%). However, a hybridization-based resistance test with probes targeting the 5 most common mutations would only detect 78% of rifampin-resistant isolates. Overall, these data suggest that rifampin resistance may be used as a surrogate marker for multidrug-resistant tuberculosis and that a sensitivity of between 70 to 80% may be possible for rapid molecular detection of multidrug-resistant tuberculosis in this setting.     

Performance of the genotype MTBDR line probe assay for detection of resistance to rifampin and isoniazid in strains of Mycobacterium tuberculosis with low- and high-level resistance

We assessed the performance of the Genotype MTBDR line probe assay that offers the simultaneous identification of Mycobacterium tuberculosis and its resistance to rifampin (RIF) and isoniazid (INH) by detecting the most commonly found mutations in the rpoB and katG genes. One hundred thirteen M. tuberculosis isolates were tested. The nucleotide sequences of the katG and inhA genes and the mabA-inhA promoter region were also determined. The MTBDR assay detected 100% and 67% (n = 64) of the strains resistant to RIF and INH, respectively. Among the latter, 62 strains carried a Ser315Thr mutation in katG, 59 of them displaying a high level of resistance to INH. Two strains with a low level of INH resistance had a Ser315Asn mutation. No mutation was found by the MTBDR assay for 31 INH-resistant strains (33%), of which 24 showed a low level of resistance. By DNA sequencing, we found among them various mutations in the KatG protein for 7 strains, a C-->T mutation in position -15 of the mabA-inhA promoter in 17 strains, and a Ser94Ala mutation in InhA for 7 strains. In conclusion, the MTBDR assay, which fits easily in the workflow of a routine laboratory, enabled the detection of 100% of the RIF-resistant strains and 89% of the INH-resistant strains with a high level of resistance but only 17% of the strains characterized by a low level of INH resistance, indicating that the test can be used as a rapid method to detect in the same experiment the rifampin-resistant and the high-level isoniazid-resistant strains of M. tuberculosis.     

Biological and molecular characteristics of Mycobacterium tuberculosis clinical isolates with low-level resistance to isoniazid in Japan

We reevaluated the BACTEC MGIT 960 antimicrobial susceptibility testing system (MGIT 960 AST) by using 1,112 isolates of Mycobacterium tuberculosis. When the results of MGIT 960 AST were compared with that of the proportion method using Ogawa medium (Ogawa PM), discrepant results were obtained for 30 strains with isoniazid, all resistant by MGIT 960 AST but susceptible by Ogawa PM. For 93% of the strains that produced discrepant results, the MIC was 0.4 or 0.8 microg/ml, showing resistance by the proportion method using Middlebrook agar plates. Furthermore, it was also established by analyses of the katG and inhA genes that strains resistant only by MGIT 960 AST have a low level of isoniazid (INH) resistance, indicating that MGIT 960 AST is a reliable method. Ninety-six strains were resistant to 0.1 microg/ml INH by MGIT 960 AST. When they were divided into three groups, Low-S (susceptible at 0.2 microg/ml), Low-R (resistant at 0.2 microg/ml), and High-R (resistant at 1.0 microg/ml), by Ogawa PM, 43.3% of the Low-S strains had mutations in the promoter region of inhA and no mutations were detected in katG codon 315, while 61.7% of the High-R strains had katG codon 315 mutations or a gross deletion of katG. These results suggest that mutations in inhA are associated with low-level resistance to INH and katG codon 315 mutations are associated with high-level resistance to INH. In addition, the analyses demonstrated some relationship of mutations in the inhA gene with ethionamide resistance for the Low-S strains, but not for the High-R strains.     

Rifampin-isoniazid oligonucleotide typing: an alternative format for rapid detection of multidrug-resistant Mycobacterium tuberculosis

A reverse line blot DNA hybridization format for rapid detection of multidrug-resistant tuberculosis was developed. Simultaneous detection of rifampin and isoniazid resistance in clinical isolates of Mycobacterium tuberculosis was based on the same amplification/reverse hybridization principle of the widely used spoligotyping. The test involved probing nine DNA regions that are targets of common drug resistance-associated mutations in the genes rpoB, katG, and inhA. Addition of quaternary amine tetramethyl ammonium chloride to the hybridization buffer promoted multiple hybrid formations at a single annealing temperature irrespective of the different GC contents of probes. The assay was standardized using 20 well-documented strains from the Institute of Tropical Medicine (Belgium) and evaluated blindly in a central laboratory with 100 DNA samples that were obtained from cultured clinical isolates and shipped dried from three other countries. Compared with drug susceptibility testing, both sensitivity and specificity for rifampin resistance detection were 93.0% while for isoniazid the values were 87.7% and 97.7%, respectively. Compared with sequencing and GenoType MTBDRplus methods, sensitivity and specificity reached 96.4% and 95.5% for rifampin and 92.7% and 100% for isoniazid. Altogether, 40/45 (89%) multidrug-resistant isolates were correctly identified. Advantages of this in-house development include versatility, capacity to run up to 41 samples by triplicate in a single run, and reuse of the membrane at least 10 times. These features substantially reduce cost per reaction and make the assay an attractive tool for use in reference laboratories of countries that have a high burden of multidrug-resistant tuberculosis but that cannot afford expensive commercial tests because of limited resources.     

Evaluation of the Genotype MTBDR assay for rapid detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis isolates

A novel PCR-based reverse hybridization method Genotype MTBDR assay (Hain Lifescience GmbH, Nehren, Germany) was evaluated for rapid detection of rifampin (RIF) and isoniazid (INH) resistance in Turkish Mycobacterium tuberculosis isolates. The Genotype MTBDR assay is designed to detect mutations within the 81-bp hotspot region of rpoB and mutations at katG codon 315. A total of 41 RIF-resistant M. tuberculosis isolates with rpoB mutations that were previously tested by the INNO-LiPA Rif.TB kit and also characterized by DNA sequencing were included in the study. Thirty-seven of these isolates were also resistant to INH. RIF resistance was correctly identified in 39 of 41 isolates (95.1%) with the Genotype MTBDR assay probes specific for these mutations. One isolate with a Gln-490-His mutation and another one with a CGG insertion between codons 514 and 515 were identified as RIF sensitive by the Genotype MTBDR assay. While the INNO-LiPA Rif.TB kit was able to determine the CGG insertion between codons 514 and 515, the Gln-490-His mutation outside the 81-bp hotspot region was not detected by the INNO-LiPA Rif.TB kit. These isolates had MICs of >or=32 microg/ml for RIF. The Genotype MTBDR assay also correctly identified 27 of 37 INH-resistant isolates (73%) with mutations in katG codon 315. In conclusion, the Genotype MTBDR assay may be useful for the rapid diagnosis of the most common mutations found in multidrug-resistant M. tuberculosis strains. However, the test results should always be confirmed with phenotypic methods.     

Detection of first- and second-line drug resistance in Mycobacterium tuberculosis clinical isolates by pyrosequencing

Conventional phenotypic drug susceptibility testing (DST) methods for Mycobacterium tuberculosis are laborious and very time-consuming. Early detection of drug-resistant tuberculosis (TB) is essential for prevention and control of TB transmission. We have developed a pyrosequencing method for simultaneous detection of mutations associated with resistance to rifampin, isoniazid, ethambutol, amikacin, kanamycin, capreomycin, and ofloxacin. Seven pyrosequencing assays were optimized for following loci: rpoB, katG, embB, rrs, gyrA, and the promoter regions of inhA and eis. The molecular method was evaluated on a panel of 290 clinical isolates of M. tuberculosis. In comparison to phenotypic DST, the pyrosequencing method demonstrated high specificity (100%) and sensitivity (94.6%) for detection of multidrug-resistant M. tuberculosis as well as high specificity (99.3%) and sensitivity (86.9%) for detection of extensively drug-resistant M. tuberculosis. The short turnaround time combined with multilocus sequencing of several isolates in parallel makes pyrosequencing an attractive method for drug resistance screening in M. tuberculosis.     

Molecular characteristics of rifampin- and isoniazid-resistant mycobacterium tuberculosis strains isolated in Vietnam

Molecular characterization of the drug resistance of Mycobacterium tuberculosis strains with different origins can generate information that is useful for developing molecular methods. These methods are widely applicable for rapid detection of drug resistance. A total of 166 rifampin (RIF)- and/or isoniazid (INH)-resistant strains of M. tuberculosis have been isolated from different parts of Vietnam; they were screened for mutations associated with resistance to these drugs by sequence analysis investigating genetic mutations associated with RIF and INH resistance. Seventeen different mutations were identified in 74 RIF-resistant strains, 56 of which (approximately 76%) had mutations in the so-called 81-bp "hot-spot" region of the rpoB gene. The most common point mutations were in codons 531 (37.8%), 526 (23%), and 516 (9.46%) of the rpoB gene. Mutations were not found in three strains (4.05%). In the case of INH resistance, five different mutations in the katG genes of 82 resistant strains were detected, among which the nucleotide substitution at codon 315 (76.83%) is the most common mutation. This study provided the first molecular characterization of INH and RIF resistance of M. tuberculosis strains from Vietnam, and detection of the katG and rpoB mutations of the INH and RIF-resistant strains should be useful for rapid detection of the INH- and RIF-resistant strains by molecular tests.     

Molecular characterization of isoniazid and rifampin resistance of Mycobacterium tuberculosis clinical isolates from Malatya, Turkey

Molecular characterization of drug resistance of Mycobacterium tuberculosis strains of different origins can generate information useful for developing molecular methods that are widely applicable for rapid drug resistance detection. Using DNA sequencing and allele-specific polymerase chain reaction (AS-PCR), we investigated genetic mutations associated with isoniazid (INH) and rifampin (RIF) resistance among 29 drug-resistant clinical isolates of M. tuberculosis collected from Malatya, Turkey, including 19 multi-drug-resistant (MDR) isolates. Point mutations were detected at codons 531, 516, 526, and 513 of the RNA polymerase beta- subunit gene (rpoB) in 10 (47.6%), five (23.8%), three (14.3%), and three (14.3%) of the 21 RIF-resistant isolates, respectively. Of the five isolates having mutations in codon 516, three also had mutations at codon 527; one had a concurrent mutation at codon 572. Mutations at codon 315 of the catalase-peroxidase-encoding gene (katG) were found in 17 (63.0%) of the 27 INH-resistant isolates. Interestingly, the katG codon 315 mutation was observed at a much higher frequency in MDR isolates than in INH-mono-resistant isolates ( approximately 79% vs. 25%). This study provided the first molecular characterization of INH and RIF resistance of M. tuberculosis clinical isolates from Eastern Turkey, and extended our knowledge of molecular basis of M. tuberculosis drug resistance.