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.     

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.     

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.     

Molecular analysis of isoniazid-resistant Mycobacterium tuberculosis isolates from England and Wales reveals the phylogenetic significance of the ahpC -46A polymorphism

The present study investigated the prevalence and diagnostic potential of the most commonly reported mutations associated with isoniazid resistance, katG 315Thr, katG 315Asn, inhA -15T, inhA -8A, and the oxyR-ahpC intergenic region, in a population sample of 202 isoniazid-resistant Mycobacterium tuberculosis isolates and 176 randomly selected fully sensitive isolates from England and Wales identified by using a directed oligonucleotide array and limited DNA sequencing. The strains were recovered from patients originating from 29 countries; 41 isolates were multidrug resistant. Mutations affecting katG 315, the inhA promoter, and the oxyR-ahpC intergenic region were found in 62.7, 21.9, and 30% of 169 genotypically distinct isoniazid-resistant isolates, respectively, whereas they were found in 0, 0, and 8% of susceptible strains, respectively. The frequency of mutation at each locus was unrelated to the resistance profile or previous antituberculous drug therapy. The commonest mutation in the oxyR-ahpC intergenic region, ahpC -46A, was present in 23.7% of isoniazid-resistant isolates and 7.5% of susceptible isolates. This proved to be a phylogenetic marker for a subgroup of M. tuberculosis strains originating on the Indian subcontinent, which shared IS6110-based restriction fragment length polymorphism and spoligotype features with the Delhi strain and Central Asian strain CAS1; and this marker is strongly associated with isoniazid resistance and the katG 315Thr mutation. In total, 82.8% of unrelated isoniazid-resistant isolates could be identified by analysis of just two loci: katG 315 and the inhA promoter. Analysis of the oxyR-ahpC intergenic region, although phylogenetically interesting, does not contribute significantly to further identification of isoniazid-resistant isolates.     

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.     

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.     

95例耐多药结核菌株rpoB基因突变的分子特征

目的:探讨广东地区MDR-TB菌株rpoB基因突变的分子特征.方法:对95例MDR-TB菌株rpoB基因453-564位密码子片段进行PCR-直接测序.结果:95例MDR-TB菌株rpoB基因突变率91.58％.86例为点突变,1例插入突变,未发现缺失.常见位点为531 (63.22％)、526(20.69％)、516(9.20％).其中:单位点突变69例(80.23％),双位点突变16例(18.60％),三位点突变1例(1.17％).511位点突变常同时伴有其他位点突变(57.14％).结论:主要突变位点与国内外报道基本相同,但各位点所占比例具有地域差异;联合突变率较高,占19.54％.512位点插入(AGGAGC)突变可能为新突变类型.     

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.     

Mutations in katG gene sequences in isoniazid-resistant clinical isolates of Mycobacterium tuberculosis are rare.

In this study, a battery of oligonucleotides was directed toward the katG gene and PCR-single-stranded conformation polymorphism (SSCP) analysis was used to search for katG gene deviations in clinical isolates of Mycobacterium tuberculosis from different geographical regions. Since a complete deletion of the katG gene was not found, it is suggested that deletion is not a major mechanism of isoniazid (isonicotinic acid hydrazide; INH) resistance in these isolates. However, 7 of 39 isolates (4 of 25 from South Africa and 3 of 14 from other geographical regions) showed mobility shifts by SSCP analysis, suggesting aberrations in the katG gene. Direct sequence analysis confirmed that the mobility shifts were due to Thr-275-->Ala (Thr275Ala), Arg409Ala, Arg463Leu, and Asp695Ala mutations and a 12-bp deletion in the 5' region of the katG gene. Mutations at codons 275, 463, and 695 created altered restriction sites for HhaI, MspI, and HaeIII, respectively, and sequence results, supported by restriction fragment length polymorphism analysis, suggested that the PCR-SSCP procedure is a good indicator of mutations in PCR-amplified fragments. Identical mutations at codons 463 and 275 were found in isolates from different geographical regions. This may suggest a common evolutionary event, but one of the control isolates (susceptible to INH [3%; n = 30]) also had a mutation at codon 463. The results suggest that variations in the katG coding gene sequences of INH-resistant isolates of M. tuberculosis are infrequent and that defects in other regions of the M. tuberculosis genome are of equal or greater importance in contributing to the acquisition of resistance to INH.     

Molecular characterization of fluoroquinolone-resistant Mycobacterium tuberculosis clinical isolates from Shanghai, China

China is one of the countries with the highest prevalence of fluoroquinolone-resistant (FQ(r)) Mycobacterium tuberculosis. Nevertheless, knowledge on the molecular characterization of the FQ(r)M. tuberculosis strains of this region remains very limited. This study was performed to investigate the frequencies and types of mutations present in FQ(r)M. tuberculosis clinical isolates collected in Shanghai, China. A total of 206 FQ(r)M. tuberculosis strains and 21 ofloxacin-sensitive (FQ(s)) M. tuberculosis strains were isolated from patients with pulmonary tuberculosis in Shanghai. The phenotypic drug susceptibilities were determined by the proportion method, and the mutations inside quinolone resistance-determining region (QRDR) of gyrA and gyrB genes were identified by DNA sequence analyses. Among 206 FQ(r)M. tuberculosis strains, 44% (90/206) were multidrug-resistant isolates and 39% (81/206) were extensively drug-resistant isolates. Only 9% (19/206) were monoresistant to ofloxacin. In total, 79.1% (163/206) of FQ(r) isolates harboured mutations in either gyrA or gyrB QRDR. Mutations in gyrA QRDR were found in 75.7% (156/206) of FQ(r) clinical isolates. Among those gyrA mutants, a majority (75.6%) harboured mutations at amino acid position 94, with D94G being the most frequent amino acid substitution. Mutations in gyrA QRDR showed 100% positive predictive value for FQ(r)M. tuberculosis in China. Mutations in gyrB were observed in 15.5% (32/206) of FQ(r) clinical isolates. Ten novel mutations were identified in gyrB. However, most of them also harboured mutations in gyrA, limiting their contribution to FQ(r) resistance in M. tuberculosis. Our findings indicated that, similar to other geographic regions, mutations in gyrA were shown to be the major mechanism of FQ(r) resistance in M. tuberculosis isolates. The mutations in gyrA QRDR can be a good molecular surrogate marker for detecting FQ(r)M. tuberculosis in China.     

Molecular analysis of isoniazid resistance in Mycobacterium tuberculosis isolates recovered from South Korea

The katG, inhA and ahpC genes, in 71 isoniazid (INH)-resistant and 26 INH-susceptible Mycobacterium tuberculosis isolates, from South Korea were examined by sequencing and MspI restriction enzyme analysis. Mutations in the katG 315 alone, katG 315 plus inhA, katG 315 plus ahpC, katG 309 alone, katG 309 plus inhA, inhA alone, and ahpC alone, were detected in 54.9, 2.8, 1.4, 1.4, 1.4, 19.7, and 5.6% of the 71 INH-resistant isolates, respectively. There was no statistically significant difference (p > 0.05) in the frequencies of these mutations for the INH-monoresistant compared with the multidrug-resistant isolates. Mutations in the katG codon 315 were associated with the high-level of INH resistance (MIC, >1 microg/ml), whereas the mutation in the inhA promoter region was associated with the low-level of INH resistance (MIC, >0.2 to 1 microg/ml). The previously undescribed GGT-->GAT (Gly-->Asp) mutation in the katG codon 309 was found in two rifampin, including-multidrug-resistant isolates, but we cannot assess if this is predictive of INH resistance. The sensitivity and specificity of molecular analysis of the katG codon 315 and/or the inhA promoter region were 80.3 and 100%, respectively. Therefore, mutations in these regions are highly predictive of INH resistance in South Korea.     

High-dose isoniazid therapy for isoniazid-resistant murine Mycobacterium tuberculosis infection

The use of isoniazid (INH) for the treatment of INH-resistant Mycobacterium tuberculosis infection has been controversial. The purpose of the present studies was to determine if there is a dose response with INH for INH-susceptible M. tuberculosis Erdman (ATCC 35801), and whether high-dose INH (100 mg/kg of body weight) was more effective than standard-dose INH (25 mg/kg) for therapy of tuberculosis infections caused by INH-resistant mutants of M. tuberculosis Erdman. Six-week-old CD-1 mice were infected with approximately 10(7) viable mycobacteria. Early control groups of infected but untreated mice were euthanized by CO(2) inhalation 1 week later when treatment was initiated. INH (25, 50, 75, and 100 mg/kg) was given by gavage 5 days/week for 4 weeks. Late control groups of untreated mice and treated mice were sacrificed 2 days after the last dose of drug. Spleens and right lungs were removed aseptically and homogenized, and viable cell counts were determined by titration on 7H10 agar plates. In the next study, INH at 100 mg/kg was compared to INH at 25 mg/kg against an isogenic mutant of M. tuberculosis Erdman (INH MIC, 2 microg/ml) and the parent strain. This mutant was found to have a mutation in the KatG protein (Phe to Leu at position 183). In the first study, there was no dose response with increasing doses of INH. In the second study, there was no significant difference between the reduction of viable cell counts for mice treated with INH at 100 mg/kg and that for mice treated with INH at 25 mg/kg (parent or INH-resistant mutant). These preliminary results suggest that INH may be useful in combination therapy of M. tuberculosis infections caused by low-level INH-resistant organisms (INH MICs, 0.2 to 5 microg/ml) and that higher doses of INH are unlikely to be more efficacious than the standard 300-mg/day dose.     

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.     

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.     

Molecular Characterization of Multidrug-Resistant Mycobacterium tuberculosis Isolates from China

To investigate the molecular characterization of multidrug-resistant tuberculosis (MDR-TB) isolates from China and the association of specific mutations conferring drug resistance with strains of different genotypes, we performed spoligotyping and sequenced nine loci (katG, inhA, the oxyR-ahpC intergenic region, rpoB, tlyA, eis, rrs, gyrA, and gyrB) for 128 MDR-TB isolates. Our results showed that 108 isolates (84.4%) were Beijing family strains, 64 (59.3%) of which were identified as modern Beijing strains. Compared with the phenotypic data, the sensitivity and specificity of DNA sequencing were 89.1% and 100.0%, respectively, for isoniazid (INH) resistance, 93.8% and 100.0% for rifampin (RIF) resistance, 60.0% and 99.4% for capreomycin (CAP) resistance, 84.6% and 99.4% for kanamycin (KAN) resistance, and 90.0% and 100.0% for ofloxacin (OFX) resistance. The most prevalent mutations among the MDR-TB isolates were katG315, inhA15, rpoB531, -526, and -516, rrs1401, eis-10, and gyrA94, -90, and -91. Furthermore, there was no association between specific resistance-conferring mutations and the strain genotype. These findings will be helpful for the establishment of rapid molecular diagnostic methods to be implemented in China.     

Molecular characterization of ofloxacin-resistant Mycobacterium tuberculosis strains from Russia

In this work, we studied the variation in the gyrA and gyrB genes in ofloxacin- and multidrug-resistant Mycobacterium tuberculosis strains circulating in northwest Russia. Comparison with spoligotyping data suggested that similar to the spread of multidrug-resistant tuberculosis, the spread of fluoroquinolone-resistant tuberculosis in Russia may be due, at least partly, to the prevalence of the Beijing genotype in a local population of M. tuberculosis.     

耐吡嗪酰胺结核分枝杆菌基因突变研究

目的 研究吡嗪酰胺酶编码基因pncA突变与结核分枝杆菌吡嗪酰胺(pyrazinamide,PZA)耐药的关系.方法 将临床分离的36株结核分枝杆菌进行常规PZA药敏试验和pncA基因序列分析.采用绝对浓度法进行PZA药敏试验;PCR扩增pncA基因及其上游、下游碱基序列,纯化回收PCR产物克隆到T载体并进行全自动测序.结果 36株临床分离株中25株PZA耐药,11株PZA敏感.5株高耐PZA(PZA浓度为250 μg/ml)临床分离株4株pncA基因突变;20株低耐PZA(PZA浓度为50 μg/ml)6株pncA基因突变;25株耐PZA结核分枝杆菌pncA基因突变率为40.0%.3株PZA敏感临床分离株pncA基因出现突变、其中2株为同义突变.11株耐PZA结核分枝杆菌pncA基因上游调控序列突变,其中5株同时有pncA基因突变;2株PZA敏感结核分枝杆菌pncA基因上游序列突变.结论 pncA基因突变可引起结核分枝杆菌对PZA耐药,但pncA基因突变只是结核分枝杆菌耐PZA的一种机制,可能还存在PZA的其他耐药机制.