结核分枝杆菌对利福平和异烟肼耐药基因突变快速检测方法的建立

目的建立结核分枝杆菌对利福平和异烟肼耐药基因突变的快速检测方法。方法根据结核分枝杆菌标准株H37Rv序列，自行设计覆盖rpoB、katG、inhA基因突变区的系列寡核苷酸探针，并检测临床样品中结核分枝杆菌的基因突变情况，以此来判断耐药结果。结果在56个利福平耐药菌株中，有50个菌株都在rpoB基因上榆出有突变，利福平耐药突变检出率为89．3％（50／56）；有30个利福平敏感菌株rpoB基因上都未检出突变。有58个异烟肼培养的耐药菌株中有47个在katG或inhA基因上检出有突变，异烟肼耐药突变检出率为81．0％（47／58）；有30个异烟肼敏感菌株katG或inhA基因上未检出突变。结论用膜芯片检测结核分枝杆菌对利福平和异烟肼的耐药性，具有较高的特异性和敏感性，可用于临床结核分枝杆菌耐药性的检测。     

耐药结核分枝杆菌基因突变分析

目的 探讨结核分枝杆菌耐药表型与基因突变位点之间的相互关系.方法 采用序列特异性引物分别扩增92株结核分枝杆菌利福平耐药基因rpoB,异烟肼耐药基因katG、inhA、ahpC,链霉素耐药基因rrs、rpsL,乙胺丁醇耐药基因embB及喹诺酮耐药基因gyrA,SSCP筛选出突变序列,DNA测序分析突变性质.结果 59株利福平耐药株rpoB基因突变检出率94.9%(56/59),以Ser450Trp突变最多;90株异烟肼耐药株中,katG基因突变检出率38.9%(35/90),以Ser315Thr最多,3株检出inhA基因突变,ahpC基因无突变检出;34株喹诺酮耐药株中gyrA基因突变检出率82.4%(28/34),主要为Asp94Gly,其次为Ala90Val;31株链霉素耐药株中,15株检出rrs突变,最常见为A514C和A1041G,10株发生rpsL Lys88Arg突变,总的链霉素基因突变检出率为77.4%(24/31);31株乙胺丁醇耐药株中embB 基因突变检出率19.4%(6/31),主要为Met306Val.结论 耐药结核分枝杆菌耐药情况较为严重,以DNA测序为基础的基因突变分析能快速有效地检测结核分枝杆菌的rpoB、katG、gyrA、rrs、rpsL、embB 等耐药分子标识,显示了西安地区耐药性结核分枝杆菌的突变特点,为结核病的临床诊断和合理用药提供了实验依据.     

结核分枝杆菌katG、inhA、ahpC等突变与异烟肼耐药关系的研究

目的：了解结核分枝杆菌katG、inhA、ahpC、fabG1、sodA及sodC基因突变的特征及其与耐异烟肼的关系。方法对127例活动性肺结核患者痰标本进行菌型鉴定及结核分枝杆菌药敏试验,提取结核分枝杆菌菌株DNA,应用PCR扩增katG、inhA及ahpC、fabG1、sodA及sodC基因片段,并进行DNA序列分析。结果结核分枝杆菌药物敏感试验显示127株结核分枝杆菌中,其中47株耐异烟肼,80株对异烟肼敏感,耐异烟肼率为37.01%。47株耐异烟肼中,29株存在katG和（或）inhA基因突变,其中22株（46.81%,22/47）存在katG基因单位点突变,3株（6.38%,3/47）存在inhA基因单位点突变,4株（8.51%,4/47）存在katG及inhA基因联合位点突变。22株katG基因单位点突变中,20株为AGC315ACC、AGC315AAC （42.55%,20/47）突变,2株（2.13%,1/47）分别为CTG378CCG（Leu378Pro）、ACG394ATG（Thr394Met）突变,该突变位点及突变形式尚未见文献报道。18株katG及inhA未突变结核分枝杆菌均未检测到ahpC、fabG1、sodA及sodC基因突变。结论结核分枝杆菌对异烟肼耐药主要与katG和inhA基因突变有关。耐异烟肼结核分枝杆菌临床分离株378和394新突变位点的发现为进一步研究耐药机制以及耐药结核病的快速检测提供了依据。     

耐多药结核分枝杆菌耐药相关基因突变特征分析

目的了解中国耐多药结核分枝杆菌耐药相关基因的分子特征。方法对138株耐多药结核分枝杆菌和45株敏感菌的耐药相关基因inhA、katG和oxyR-ahpC间隔区(异烟肼)、rpob(利福平)、gyrA(氧氟沙星)和rrs(卡那霉素)进行序列测定,分析其基因突变特点。结果 138株耐多药结核分枝杆菌中,14.4%的菌株inhA基因发生突变,72.5%菌株的katG基因发生突变,15.9%菌株的oxyR-ahpC基因发生突变,同时考虑这3种基因,异烟肼耐药相关基因突变检出率可达90.6%;94.2%菌株的rpoB基因发生突变,74.5%菌株的gyrA基因发生突变,61.1%菌株的rrs基因发生突变,主要的突变位点为katG315(66.7%),inhA-15(9.4%),oxyR-ahpC-10(5.1%),rpoB516(13.8%),526(26.1%)和531(49.3%),gyrA90(21.6%)和94(51%),rrs1401(61.1%)。结论我国耐多药结核菌异烟肼、利福平、氧氟沙星和卡那霉素耐药相关基因最常见突变为katG315、inhA-15,rpoB531、526和516,gyrA94和90,rrs1401。     

艾滋病合并结核病患者结核分枝杆菌耐药基因突变特征分析

目的了解云南省艾滋病合并结核病患者中结核分枝杆菌(MTB)耐利福平RNA聚合酶β亚单位编码基因(rpoB)和耐异烟肼过氧化氢酶-过氧化物酶编码基因(katG)、烯酰基还原酶编码基因(inhA)突变特点。方法采用基因芯片技术,对该省208例艾滋病合并结核病患者标本进行MTB利福平耐药相关基因rpoB和异烟肼耐药相关基因katG、inhA分析。结果 208例标本中共34例(16.3%)耐药,其中单耐利福平6例(2.9%),单耐异烟肼10例(4.8%),二者同时耐药18例(8.7%)。24例利福平耐药MTB的rpoB基因突变位点主要为531(TCG→TTG),占66.7%(16/24);28例异烟肼耐药MTB的基因突变位点主要为katG基因315(AGC→ACC),占78.6%(22/28)。结论云南省艾滋病合并结核病患者中MTB利福平和异烟肼耐药基因突变具有多态性,其中耐利福平rpoB基因的主要突变位点为531(TCG→TTG),耐异烟肼katG、inhA基因的主要突变位点为katG基因315(AGC→ACC)。     

实时荧光PCR分子信标检测耐利福平结核分枝杆菌印rpoB基因

目的 应用实时荧光PCR分子信标技术,建立快速检测临床标本中结核分枝杆菌利福平rpoB相关耐药突变点方法,探讨其缩短耐药实验报告时间的临床应用价值.方法 以分枝杆菌药物敏感性实验绝对浓度法为标准,12株非结核分枝杆菌、4株非分枝杆菌作对照,对174例结核患者临床分离株应用实时荧光PCR分子信标方法,检测利福平rpoB核心区域的耐药突变点并将结果与直接测序进行比较.结果 (1)实时荧光PCR分子信标方法:82例结核分枝杆菌利福平敏感菌株中,3例发生rpoB基因突变,特异度为96.3%;92例结核分枝杆菌利福平耐药菌株中,82例检出耐药突变,敏感度为89.1%;准确性为92.5%.(2)DNA直接测序分析:82例结核分枝杆菌利福平敏感株中,1例发生rpoB基因突变,特异度为98.8%;92例结核分枝杆菌利福平耐药菌株中,83例发生:rpoB基因突变,敏感度为90.2%;准确性为94.2%.检测174株结核分枝杆菌临床分离菌株,与实时荧光PCR分子信标方法检测一致性为98.3%(171/174).结论 实时荧光PCR分子信标方法检测耐利福平结核分枝杆菌rpoB基因突变点可作为结核患者快速耐药检测的初筛方法之一.     

PCR-DNA测序技术检测结核分支杆菌耐多药基因突变的研究

目的应用PCR—DNA测序技术快速检测同时耐利福平和异烟肼结核分支杆菌分离株rpoB、KatG基因突变，评价其在检测结核分支杆菌耐多药性方面的价值。方法47株耐利福平和异烟肼结核分支杆菌临床分离株及30株结核分支杆菌敏感分离株用PCR—DNA测序技术分别检测rpoB、KatG基因突变。结果47株耐多药结核分支杆菌分离株中，检出43株rpoB基因突变，突变检出率为91．5％（43／47）；31株KatG基因突变，突变检出率为66．0％（31／47）；rpoB和KatG基因同时突变者31株，突变检出率为66．0％（31／47）。30株结核分支杆菌敏感株检出1株KatG基因突变。结论PCR—DNA测序技术方法敏感、准确、特异，可快速检测结核分支杆菌rpoB、KatG耐药基因突变，有利于耐多药结核分支杆菌耐药性的快速检测。     

焦磷酸测序技术快速检测结核分枝杆菌利福平耐药性研究

目的利用焦磷酸测序技术，建立一种高通量结核分枝杆菌利福平耐药性快速基因检测方法。方法应用生物素标记的引物扩增rpoB基因片段，经链亲和素标记的磁珠分离制备单链模板，设计2个测序引物在焦磷酸测序仪上检测rpoB基因耐药突变区的81bp序列突变情况，与H。Rv序列比对后判断耐药结果。结果通过建立的基于焦磷酸测序技术的结核分枝杆菌rpoB基因突变检测平台，32株利福平耐药株均在rpoB基因片段上检测到突变，22株利福平敏感株未检测到突变，检测结果与直接测序符合率达100％。结论本方法可快速、准确、高通量检测结核分枝杆菌耐利福平rpoB基因突变。     

吉林省耐多药结核分枝杆菌rpoB及katG基因突变特征分析

目的分析吉林省耐多药结核分枝杆菌临床分离株rpoB、katG基因突变特点,为建立本省快速耐多药检测方法提供参考。方法对吉林省耐多药结核分枝杆菌菌株扩增rpoB、katG基因测序后比对分析。结果耐多药结核分枝杆菌rpoB、katG基因的突变率分别为83.53%和70.6%,突变类型包括点突变、联合突变和缺失。在rpoB基因中,82.30%突变发生在RRDR区域,最常见突变位点为rpoB 531、rpoB 526、rpoB 516。KatG基因突变率为70.6%,最常见突变位点为katG315,占所有突变的68.55%。结论吉林省耐多药结核分枝杆菌最常见突变位点为rpoB531、rpoB 526、rpoB 516和katG 315。     

结核分枝杆菌耐利福平和异烟肼相关基因快速检测的应用研究

目的 利用DNA芯片检测技术直接检测痰标本中结核分枝杆菌耐利福平(RFP)、异烟肼(INH)相关的耐药基因(rpoB、katG/inhA),评价DNA芯片检测技术临床应用的可行性.方法 对586份涂阳痰标本使用L-J培养并用终点法确定其耐药性,同时利用DNA芯片检测技术检测痰标觋本中结核分枝杆菌的rpoB、katG/inhA常见基因突变位点的突变情况,比对两种方法的检测结果,对不符合的菌株测定其相应DNA序列,评估上述试验的准确性.结果 (1)586份涂阳痰标本,其中3(+)163份、2(+)204份、1(+)217份,培养阳性584份.耐药结果显示,对INH、RFP敏感的菌株分别为361株和327株,耐药菌株分别为223株和247株,其中低浓度耐药、高浓度敏感菌株分别为93株和59株,低浓度、高浓度均耐药菌株分别为130株和188株.(2)耐药基因特异性片段扩增阳性标本367份(62.8%)、阴性217份(37.2%).对INH耐药相关基因(katG/inhA)突变检出率是28.4%,突发发生位点集中在katG315位密码子(89.8%);对RFP耐药相关基因(rpoB)突变检出率是55.9%(137/247),突变发生位点主要在rpoB 531和rpoB 526位密码子,发生率分别是68.6%和16.1%.(3)对L-J药敏结果与DNA芯片检测结果不符的菌株进行DNA序列分析,发现有漏检现象.结论 DNA芯片技术直接检测样本中结核分枝杆菌的相关耐药基因存在可行性,如直接应用于临床样本检测,关键要解决样本中DNA的提取效率、PCR的扩增效率和试验的质量控制.     

Molecular characterization of multidrug-resistant Mycobacterium tuberculosis isolated in Nepal

Despite the fact that Nepal is one of the first countries globally to introduce multidrug-resistant tuberculosis (MDR-TB) case management, the number of MDR-TB cases is continuing to rise in Nepal. Rapid molecular tests applicable in this setting to identify resistant organisms would be an effective tool in reversing this trend. To develop such tools, information about the frequency and distribution of mutations that are associated with phenotypic drug resistance in Mycobacterium tuberculosis is required. In the present study, we investigated the prevalence of mutations in rpoB and katG genes and the inhA promoter region in 158 M. tuberculosis isolates (109 phenotypically MDR and 49 non-MDR isolates collected in Nepal) by DNA sequencing. Mutations affecting the 81-bp rifampin (RIF) resistance-determining region (RRDR) of rpoB were identified in 106 of 109 (97.3%) RIF-resistant isolates. Codons 531, 526, and 516 were the most commonly affected, at percentages of 58.7, 15.6, and 15.6%, respectively. Of 113 isoniazid (INH)-resistant isolates, 99 (87.6%) had mutations in the katG gene, with Ser315Thr being the most prevalent (81.4%) substitution. Mutations in the inhA promoter region were detected in 14 (12.4%) INH-resistant isolates. The results from this study provide an overview of the current situation of RIF and INH resistance in M. tuberculosis in Nepal and can serve as a basis for developing or improving rapid molecular tests to monitor drug-resistant strains in this country.     

Evaluation of the CombiChip Mycobacteria Drug-Resistance detection DNA chip for identifying mutations associated with resistance to isoniazid and rifampin in Mycobacterium tuberculosis

The CombiChip Mycobacteriatrade mark Drug-Resistance Detection DNA chip, recently developed by GeneIn (Pusan, South Korea), is an oligonucleotide microchip coupled with polymerase chain reaction for the detection of mutations associated with resistance to isoniazid (INH) and rifampin (RIF). This oligonucleotide chip was compared with DNA sequencing and phenotypic drug susceptibility testing with 69 INH- and/or RIF-resistant and 27 all tested drug-susceptible Mycobacterium tuberculosis isolates. Two selected codons (the katG codon 315 and inhA15) allowed identification of 84.1% of INH-resistant isolates and 100% of RIF resistance were detected by screening for 7 codons: rpoB511, rpoB513, rpoB516, rpoB522, rpoB526, rpoB531, and rpoB533. The overall specificity of this oligonucleotide chip for detecting INH and RIF resistance were 100 and 95.3%, respectively. This level of sensitivity and specificity is concordant with that from the determination of M. tuberculosis drug resistance by DNA sequencing. This oligonucleotide chip is a rapid and reliable genotypic method capable of detecting multiple mutations associated with INH and RIF resistance simultaneously in a single microchip slide.     

Single nucleotide polymorphisms in genes associated with isoniazid resistance in Mycobacterium tuberculosis

Isoniazid (INH) is a central component of drug regimens used worldwide to treat tuberculosis. Previous studies have identified resistance-associated mutations in katG, inhA, kasA, ndh, and the oxyR-ahpC intergenic region. DNA microarray-based experiments have shown that INH induces several genes in Mycobacterium tuberculosis that encode proteins physiologically relevant to the drug's mode of action. To gain further insight into the molecular genetic basis of INH resistance, 20 genes implicated in INH resistance were sequenced for INH resistance-associated mutations. Thirty-eight INH-monoresistant clinical isolates and 86 INH-susceptible isolates of M. tuberculosis were obtained from the Texas Department of Health and the Houston Tuberculosis Initiative. Epidemiologic independence was established for all isolates by IS6110 restriction fragment length polymorphism analysis. Susceptible isolates were matched with resistant isolates by molecular genetic group and IS6110 profiles. Spoligotyping was done with isolates with five or fewer IS6110 copies. A major genetic group was established on the basis of the polymorphisms in katG codon 463 and gyrA codon 95. MICs were determined by the E-test. Semiquantitative catalase assays were performed with isolates with mutations in the katG gene. When the 20 genes were sequenced, it was found that 17 (44.7%) INH-resistant isolates had a single-locus, resistance-associated mutation in the katG, mabA, or Rv1772 gene. Seventeen (44.7%) INH-resistant isolates had resistance-associated mutations in two or more genes, and 76% of all INH-resistant isolates had a mutation in the katG gene. Mutations were also identified in the fadE24, Rv1592c, Rv1772, Rv0340, and iniBAC genes, recently shown by DNA-based microarray experiments to be upregulated in response to INH. In general, the MICs were higher for isolates with mutations in katG and the isolates had reduced catalase activities. The results show that a variety of single nucleotide polymorphisms in multiple genes are found exclusively in INH-resistant clinical isolates. These genes either are involved in mycolic acid biosynthesis or are overexpressed as a response to the buildup or cellular toxicity of INH.     

基因芯片技术在结核分支杆菌耐药突变基因检测中的应用

目的通过基因芯片检测系统，快速检测临床样品中结核分支杆菌耐药突变情况。方法根据结核分支杆菌标准株H37Rv序列，设计了覆盖rpoB、katG，inhA基因突变区的系列寡核苷酸探针，制作膜芯片，检测临床样品中结核分支杆菌基因突变情况，以此判断耐药结果。结果在305例临床病例中，共检出阳性病例125例，其中阳性敏感病例64例，阳性突变病例61例，阳性率为40．98％，在125例阳性样品中，共发现有8种突变类型，其中10例531L，占7．94％，19例315M，占阳性样品中总数的15．08％。结论PCR与膜芯片杂交技术可临床检测结核分支杆菌对利福平和异烟肼的耐药性，并具有快速、简便、敏感的特点。     

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.     

Prevalence of and molecular basis for tuberculosis drug resistance in the Republic of Georgia: validation of a QIAplex system for detection of drug resistance-related mutations

We developed a QIAplex system for the simultaneous detection of 24 Mycobacterium tuberculosis gene mutations responsible for resistance to isoniazid (INH), rifampin (RIF), streptomycin (STM), and ethambutol (EMB) in 196 M. tuberculosis isolates recovered in the Republic of Georgia. In comparison to phenotypic susceptibility tests, the QIAplex showed sensitivity and specificity of 85.4% and 96.1% for INH, 94.4% and 99.4% for RIF, 69.6% and 99.2% for STM, 50.0% and 98.8% for EBM, and 86.7% and 100.0% for multidrug resistance, respectively. The dominant resistance mutations revealed were a mutation in katG resulting in S315T (katG S315T), rpsL K43R, and rpoB S531L. Mutations katG S315G and S315T and rpoB S531L were detected with higher frequencies in pretreated patients than in naive patients (P < 0.05). Simultaneous detection of 24 common drug resistance-related mutations provides a molecular tool for studying and monitoring M. tuberculosis resistance mechanism and epidemiology.     

Molecular detection of mutations associated with first- and second-line drug resistance compared with conventional drug susceptibility testing of Mycobacterium tuberculosis

The emergence of multi- and extensively drug-resistant tuberculosis is a significant impediment to the control of this disease because treatment becomes more complex and costly. Reliable and timely drug susceptibility testing is critical to ensure that patients receive effective treatment and become noninfectious. Molecular methods can provide accurate and rapid drug susceptibility results. We used DNA sequencing to detect resistance to the first-line antituberculosis drugs isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and ethambutol (EMB) and the second-line drugs amikacin (AMK), capreomycin (CAP), kanamycin (KAN), ciprofloxacin (CIP), and ofloxacin (OFX). Nine loci were sequenced: rpoB (for resistance to RIF), katG and inhA (INH), pncA (PZA), embB (EMB), gyrA (CIP and OFX), and rrs, eis, and tlyA (KAN, AMK, and CAP). A total of 314 clinical Mycobacterium tuberculosis complex isolates representing a variety of antibiotic resistance patterns, genotypes, and geographical origins were analyzed. The molecular data were compared to the phenotypic data and the accuracy values were calculated. Sensitivity and specificity values for the first-line drug loci were 97.1% and 93.6% for rpoB, 85.4% and 100% for katG, 16.5% and 100% for inhA, 90.6% and 100% for katG and inhA together, 84.6% and 85.8% for pncA, and 78.6% and 93.1% for embB. The values for the second-line drugs were also calculated. The size and scope of this study, in numbers of loci and isolates examined, and the phenotypic diversity of those isolates support the use of DNA sequencing to detect drug resistance in the M. tuberculosis complex. Further, the results can be used to design diagnostic tests utilizing other mutation detection technologies.     

基因芯片诊断耐多药结核病的临床多中心研究

目的 评估基因芯片法检测耐多药结核临床分离株的临床意义。方法采取分层抽样的方法分别从北京胸科医院、同济大学附属上海市肺科医院和广州市胸科医院保存的临床菌株库的耐药组和敏感组中,随机抽取利福平耐药株800株,异烟肼耐药株797株,耐多药株791株,利福平/异烟肼双敏感380株。用基因芯片法检测包括rpoB基因的511(T→C)、513(A→C,C→A)、516( G→T,A→T,A→G)、526( C→T,C→G,A→T,A→G)、531( C→T,C→G)、533( T→C)位点、katG的315(G→C,G→A)位点和inhA的-15(C→T)位点的耐药突变。以绝对浓度法药敏结果为金标准,计算基因芯片法的符合率、敏感度和特异度。同时对基因芯片法的核酸扩增产物进行测序,以验证基因芯片对核酸序列检测的准确性。结果以绝对浓度法药敏结果作为标准,基因芯片法检测利福平、异烟肼耐药和耐多药的符合率分别是93.7％(1 108/1 183)、83.8％ (994/1 186)、82.4％ (975/1 183)。检测利福平耐药的敏感度为92.0％ (733/797),特异度为97.2％( 375/386);检测异烟肼的敏感度为77.4％ (617/797),特异度为96.9％ (377/389);检测耐多药的敏感度为74.6％( 588/788),特异度为98.0％(387/395)。在利福平基因芯片检测为突变的菌株中,突变频率最高的位点是531( TCG),突变率为64.5％(480/744);在katG/ inhA突变菌株中,基因芯片检测为katG 315( AGC)单突变的为77.4％(487/629);且与测序结果基本一致,仅有5例菌株中不完全相符。其中1株异烟肼耐药菌基因芯片法检测为katG 315(G→C)突变,而测序结果为野生型,其余4株为基因芯片法未包含的突变类型。结论基因芯片法可快速可靠地检测结核临床分离株利福平和异烟肼的耐药性,有望在临床诊断中广泛应用。     

Isoniazid induces its own resistance in nonreplicating Mycobacterium tuberculosis

Isoniazid (INH) resistance is most frequent among drug-resistant Mycobacterium tuberculosis clinical isolates. This study was conducted to investigate whether INH could induce its own resistance. During INH susceptibility testing in BACTEC 12B and MGIT 960 media, weekly subcultures were made from the drug-containing media into fresh medium without drug and susceptibility testing was performed. Rifampin (RIF) was used as a control drug. M. tuberculosis H37Rv and three clinical isolates were tested in this study. INH-resistant subcultures were analyzed for catalase activity, INH susceptibility, and mutations associated with INH resistance. With inoculum size (10(4) bacilli) smaller than a size that contains spontaneously INH-resistant mutants, INH was found to induce resistance to itself in INH-tolerant persisters but not to other drugs. The minimum time required for induction of INH resistance was 5 to 6 days. In contrast, RIF did not induce RIF resistance. Eight subcultures with INH-induced resistance were analyzed, and two had a MIC of 0.4 microg/ml INH and six had MICs of over 2 microg/ml INH. Four of the eight subcultures with INH-induced resistance had lost catalase activity, with three having katG mutations. Despite being a powerful frontline tuberculosis drug, INH has the potential drawback of inducing its own stable genetic resistance in INH-tolerant persisters. This finding helps to explain the higher frequency and prevalence of INH-resistant isolates than isolates with resistance to other drugs in patients.