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.     

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。     

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.     

Population genetics study of isoniazid resistance mutations and evolution of multidrug-resistant Mycobacterium tuberculosis

The molecular basis for isoniazid resistance in Mycobacterium tuberculosis is complex. Putative isoniazid resistance mutations have been identified in katG, ahpC, inhA, kasA, and ndh. However, small sample sizes and related potential biases in sample selection have precluded the development of statistically valid and significant population genetic analyses of clinical isoniazid resistance. We present the first large-scale analysis of 240 alleles previously associated with isoniazid resistance in a diverse set of 608 isoniazid-susceptible and 403 isoniazid-resistant clinical M. tuberculosis isolates. We detected 12 mutant alleles in isoniazid-susceptible isolates, suggesting that these alleles are not involved in isoniazid resistance. However, mutations in katG, ahpC, and inhA were strongly associated with isoniazid resistance, while kasA mutations were associated with isoniazid susceptibility. Remarkably, the distribution of isoniazid resistance-associated mutations was different in isoniazid-monoresistant isolates from that in multidrug-resistant isolates, with significantly fewer isoniazid resistance mutations in the isoniazid-monoresistant group. Mutations in katG315 were significantly more common in the multidrug-resistant isolates. Conversely, mutations in the inhA promoter were significantly more common in isoniazid-monoresistant isolates. We tested for interactions among mutations and resistance to different drugs. Mutations in katG, ahpC, and inhA were associated with rifampin resistance, but only katG315 mutations were associated with ethambutol resistance. There was also a significant inverse association between katG315 mutations and mutations in ahpC or inhA and between mutations in kasA and mutations in ahpC. Our results suggest that isoniazid resistance and the evolution of multidrug-resistant strains are complex dynamic processes that may be influenced by interactions between genes and drug-resistant phenotypes.     

应用多重PCR-单链构象多态性分析同时检测耐异烟肼和利福平的结核分枝杆菌

目的 探讨应用多重PCR-单链构象多态性分析(multiplexpulymerase chain reaction-single strand conformation polymorphism,multi-PCR-SSCP)方法快速、特异地同时快速检测结核分枝杆菌对异烟肼和利福平耐药性的效能.方法 根据结核分枝杆菌的inhA序列、katG序列、rpoB序列,分别设计出3对特异性寡聚核苷酸引物.采用multi-PCR-SSCP技术,一次性检出耐异烟肼和利福平的结核分枝杆菌.新方法的有效性通过116株临床分离株(70株耐异烟肼,66株耐利福平)的验证.结果 名 Multi-PCR-SSCP方法检测临床分离株基因突变的有效性,以细菌培养和药敏试验结果为金标准.116株临床分离株和H37Rv标准株中除了4株katG缺失突变,其余菌株3个基因katG、inhA和rpoB在单基因PCR中都扩增成功.与H37Rv标准株相比,46株katG基因突变,14株inhA基因突变,58株rpoB基因突变.38株katG和rpoB,4株inhA和rpoB,4株inhA和katG同时突变,还有2株3个基因都有突变.multi-PCR-SSCP对于耐异烟肼和利福平的结核分枝杆菌检出的敏感度分别为80%、82%,特异度分别为100%和92%.结论 multi-PCR-SSCP方法敏感、特异,能同时快速有效地检测耐多药结核分枝杆菌,有望成为临床指导用药的好方法,为深入研究耐药基凶检测奠定了良好的基础.     

广西壮族自治区结核分枝杆菌异烟肼与利福平耐药基因联合突变特征分析

  目的   了解广西壮族自治区（广西）结核分枝杆菌异烟肼与利福平耐药基因联合突变特征，为耐多药结核病的分子诊断和治疗提供依据。  方法   2017—2018年从广西30个结核病防治定点机构收集的结核分枝杆菌中选取49株耐多药菌株和459株全敏感菌株进行全基因组测序。  结果   耐多药表型与基因联合突变的符合率为71.43%。 单基因突变率和基因联合突变率在耐多药菌株中均高于全敏感菌株（χ2=5.753，P=0.016; χ2=284.034，P<0.001）。 katG和rpoB的单基因突变率在耐多药菌株中高于全敏感菌株（χ2=7.524，P=0.006; χ2=4.353，P=0.037）。 katG＋rpoB基因联合突变在耐多药菌株中高于全敏感菌株（χ2=279.956，P<0.001）。 在基因联合突变的位点分布中，以katG315＋rpoB450和katG315＋rpoB445位点突变为主，占40.82%（20/49），2种形式的基因位点联合突变率在耐多药菌株中均高于全敏感菌株（χ2=144.232，P<0.001; χ2=19.014，P<0.001）。  结论   对异烟肼和利福平耐药基因联合突变的检测可作为广西耐多药筛查的重要指标。 广西结核分枝杆菌异烟肼和利福平耐药基因突变以katG315、rpoB450和rpoB445位点突变为主，基因联合突变以katG＋rpoB形式为主。 katG315＋rpoB450和katG315＋rpoB445位点突变是广西地区耐多药产生的主要分子机制。     

焦磷酸测序技术检测结核分枝杆菌四种药物耐药性

目的 利用焦磷酸测序技术检测结核分枝杆菌对利福平、异烟肼、氧氟沙星、阿米卡星的耐药性,并对其临床应用进行评价.方法 收集上海市肺科医院2008-2009年临床确诊的肺结核患者的痰标本培养阳性结核分枝杆菌89株.所有菌株按<结核病诊断实验室检验规程>进行分枝杆菌培养、菌种鉴定.另外10株已知药敏结果的结核分枝杆菌来自上海市肺科医院菌株库.利用焦磷酸测序技术,以10株已知药敏结果和经直接测序已知耐药基因突变情况的结核分枝杆菌为试验菌株,探索焦磷酸测序检测结核分枝杆菌rpoB、katG、gyrA、rrs耐药基因的最佳模式,并用该条件检测89株结核分枝杆菌临床分离株,检测结果与Bactec 960药敏法进行比较.结果 rpoB、gyrA基因检测宜采用焦磷酸测序序列分析模式,katG、rrs基因检测宜采用焦磷酸测序单核苷酸多态性模式.以Bactec 960药敏法测定结果为判断标准,则焦磷酸测序法检测89株结核分枝杆菌临床分离株对利福平、异烟肼、氧氟沙星、阿米卡星耐药性的敏感度分别为98.0%、64.1%、79.5%、78.3%,特异度分别为97.5%、100.0%、90.0%、100.0%,准确性分别为97.8%、77.5%、85.4%、94.4%,该法检测利福平、氧氟沙星、阿米卡星的检测结果与Bactec 960药敏检测结果一致性较好,Kappa值均≥0.7.结论 焦磷酸测序技术检测结核分枝杆菌对利福平、异烟肼、氧氟沙星、阿米卡星耐药性具有快速、准确、高通量的优点,是一种可对结核分枝杆菌多种药物耐药性进行快速检测的方法.

Abstract：

Objective To develop an assay to determine Mycobacterium tuberculosis resistance to rifampin, isoniazid, ofloxacin and amikacin by pyrosequencing and evaluate the value of this method in clinical application. Methods Eighty-nine clinical isolates of Mycobacterium tuberculosis from tuberculosis patients were collected from Shanghai Pulmonary Hospital during 2008 to 2009. All strains were isolated from decontaminated sputum, cultured on Lowenstein-Jensen media and identified by traditional biochemical tests with standard methods. Ten Mycobacterium tuberculosis were selected from the strain bank of Shanghai Pulmonary Hospital. The optimal conditions of detection of rpoB, katG, gyrA and rrs gene by pyroseuencing were determined, using the 10 Mycobacterium tuberculosis strains whose drug susceptibility of Bactec 960 and sequence of rpoB, katG, gyrA, rrs gene were known. Then the drug susceptibility of 89 Mycobacterium tuberculosis clinical isolate strains were detected by pyrosequencing using this conditions and the results were compared with that of the Bactec 960 methods. Results The pyrosequencing program of sequence analysis was suitable for the detection of the mutations of rpoB and gyrA genes, and the program of single nucleotide polymorphism was suitable for katG and rrs genes. Among the 89 Mycobacterium tuberculosis clinical isolates,when using the drug susceptibility of Bactec 960 as the standard, the sensitivity of rifampin, isoniazid,ofloxacin and amikacin is 98.0%, 64. 1%, 79.5%, 78. 3% respectively, the specificity is 97.5%,100. 0%, 90. 0%, 100. 0% respectively, the accuracy is 97.8%, 77. 5%, 85.4%, 94. 4% respectively,tested by pyrosequencing. The results of the detection of resistance to rifampin, isoniazid, ofloxacin and amikacin in Mycobacterium tuberculosis using pyrosequencing technique were almost the same with that of Bactec 960, and Kappa ≥0. 7 in each detection. Conclusion Pyrosequencing is thus a rapid, accurate and high throughput method for detecting Mycobacterium tuberculosis resistance to these four drugs.     

Rapid detection of specific gene mutations associated with isoniazid or rifampicin resistance in Mycobacterium tuberculosis clinical isolates using non-fluorescent low-density DNA microarrays

BACKGROUND: A new, fast 'low cost and density' DNA microarray (LCD array), designed for the detection of mutations that confer isoniazid or rifampicin resistance in Mycobacterium tuberculosis isolates, has been developed and was evaluated using 46 resistant clinical isolates from Barcelona. METHODS: LCD chips are pre-structured polymer supports using a non-fluorescent detection principle based on the precipitation of a clearly visible dark substrate. One LCD chip consists of eight identical microarrays, designed to detect mutations within the 90 bp rpoB region, codon 315 in the katG gene and the mabA-inhA regulatory region. A total of 22 strains with a katG 315 mutation, 19 strains with alterations in the mabA-inhA regulatory region and 16 strains with mutations in the rpoB region, characterized previously, were studied. RESULTS: The identification of S315T and S315N mutations using the LCD was 100% concordant with the sequencing data. A strain with the S315R mutation, which is not tiled on the LCD array, was detected by the absence of hybridization using the wild-type probe. Of 19 strains with low-level isoniazid resistance related to the mabA-inhA regulatory region, 18 were identified correctly. The detection of mutations in the rpoB region was 93.8% concordant with the sequencing data. One mabA-inhA and rpoB mutated strain showed a cross-hybridization. CONCLUSIONS: The LCD array protocol takes 45 min (15 min 'hands-on' time) after prior PCR amplification. Only minimal laboratory equipment is required. LCD arrays provide a rapid and economical method to characterize mutations in codon 315 of the katG gene, in the mabA-inhA regulatory region and in the rpoB gene.     

Molecular Characterization of Multidrug-Resistant Isolates of Mycobacterium tuberculosis from Patients in North India

The World Health Organization has identified India as a major hot-spot region for Mycobacterium tuberculosis infection. We have characterized the sequences of the loci associated with multidrug resistance in 126 clinical isolates of M. tuberculosis from India to identify the respective mutations. The loci selected were rpoB (rifampin), katG and the ribosomal binding site of inhA (isoniazid), gyrA and gyrB (ofloxacin), and rpsL and rrs (streptomycin). We found known as well as novel mutations at these loci. Few of the mutations at the rpoB locus could be correlated with the drug resistance levels exhibited by the M. tuberculosis isolates and occurred with frequencies different from those reported earlier. Missense mutations at codons 526 to 531 seemed to be crucial in conferring a high degree of resistance to rifampin. We identified a common Arg463Leu substitution in the katG locus and certain novel insertions and deletions. Mutations were also mapped in the ribosomal binding site of the inhA gene. A Ser95Thr substitution in the gyrA locus was the most common mutation observed in ofloxacin-resistant isolates. A few isolates showed other mutations in this locus. Seven streptomycin-resistant isolates had a silent mutation at the lysine residue at position 121. While certain mutations are widely present, pointing to the magnitude of the polymorphisms at these loci, others are not common, suggesting diversity in the multidrug-resistant M. tuberculosis strains prevalent in this region. Our results additionally have implications for the development of methods for multidrug resistance detection and are also relevant in the shaping of future clinical treatment regimens and drug design strategies.     

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.     

浙江省丽水市结核分枝杆菌临床分离株耐药性及基因突变情况

目的 调查浙江省丽水市结核病定点医院患者结核分枝杆菌（MTB）临床分离株耐药特征及耐药基因突变情况，为该地区耐药结核病防控提供依据。方法 采用荧光PCR熔解曲线法对培养自丽水市结核病定点医院结核病患者MTB临床分离株进行抗结核药物耐药性及耐药基因突变位点检测及分析。结果 242株MTB总耐药突变占16.53%(40/242)，耐多药率为4.96%(12/242)，耐药顺位分别为异烟肼>链霉素>利福平>氟喹诺酮类>乙胺丁醇。链霉素耐药株基因突变位点主要发生在rpsL43位密码子，占比为65.00%(13/20)；异烟肼耐药基因突变位点主要发生在katG315密码子，占比为76.19%(14/21)；利福平耐药基因突变位点主要发生在rpoB基因529-533位密码子，占比为61.11%(11/18)；乙胺丁醇耐药基因突变位点主要发生在embB306位密码子，占77.78%(7/9)。结论 丽水市结核患者总体耐药突变低于全国耐药水平，但耐药模式和耐药基因突变情况仍较为复杂，需进一步加强耐药结核病监测及防控工作。     

Characterization of mutations conferring extensive drug resistance to Mycobacterium tuberculosis isolates in Pakistan

The increasing incidence of extensively drug-resistant (XDR) Mycobacterium tuberculosis in high-tuberculosis-burden countries further highlights the need for improved rapid diagnostic assays. An increasing incidence of XDR M. tuberculosis strains in Pakistan has been reported, but drug resistance-associated mutations in these strains have not been evaluated previously. We sequenced the "hot-spot" regions of rpoB, katG, inhA, ahpC, gyrA, gyrB, and rrs genes in 50 XDR M. tuberculosis strains. It was observed that 2% of rifampin, 6% of isoniazid, 24% of fluoroquinolone, and 32% of aminoglycoside/capreomycin resistance in XDR M. tuberculosis strains would be undetected if only these common hot-spot regions were tested. The frequencies of resistance-conferring mutations were found to be comparable among all XDR M. tuberculosis strain families present, including the Central Asian Strain, Beijing, and East African Indian genogroups and the Unique isolates. Additional genetic loci need to be tested for detection of mutations conferring fluoroquinolone, aminoglycoside, and capreomycin resistance in order to improve molecular diagnosis of regional XDR M. tuberculosis strains.     

Direct detection in clinical samples of multiple gene mutations causing resistance of Mycobacterium tuberculosis to isoniazid and rifampicin using fluorogenic probes

BACKGROUND: This study evaluates a method based on real-time PCR for direct detection in clinical samples of the common mutations responsible for isoniazid and rifampicin resistance of Mycobacterium tuberculosis. METHODS: Six pairs of fluorogenic 5' exonuclease probes (Taqman), mutated and wild-type, were designed for six targets: codon 315 of katG, substitution C209T in the regulatory region of inhA, and codons 513, 516, 526 and 531 of rpoB. RESULTS: A total of 98 clinical samples harbouring resistant bacilli from 55 patients and 126 samples harbouring susceptible bacilli from 126 patients were processed. The isolates from samples were tested for drug susceptibility with the radiometric method and sequenced for the same genetic targets. Among the samples, 93 harboured isoniazid-resistant bacilli. According to the sequencing results, 30 had mutations in katG, 30 in inhA and 33 (35.4%) had no mutations in these targets. All 27 clinical specimens harbouring rifampicin-resistant bacilli showed mutations in rpoB. The detection threshold of this method in detecting target genes in serial dilutions of artificial samples was 1.5 x 10(3) cfu/mL. In clinical samples, the sensitivity ranged from 30.4 to 35.3% for smear-negative samples and from 95.1 to 99.2% for smear-positive samples, with a specificity of 100%. In this study, the overall sensitivity in detecting patients having the target mutations was 74.3%. CONCLUSIONS: The main advantage of the described method is the possibility of detecting rifampicin and isoniazid resistance within 48-72 h after sample collection, with a sensitivity of nearly 100% in smear-positive samples if the chosen target is responsible for the resistance.     

基因检测及MODS技术快速诊断结核分枝杆菌对左氧氟沙星的敏感性研究

目的 探讨基因检测技术和显微镜观察药物敏感度检测技术(MODS)对结核分枝杆菌(MTB)耐左氧氟沙星(LFX)快速诊断价值.方法 选取MTB标准株(H37Rv)和32株耐LFX临床分离株,行gyrA基因喹诺酮类药物耐药决定区(QRDR)序列测定,同时,用24孔细胞培养板进行MTB液体药敏检测.结果 基因检测结果显示,H37Rv未发生gyrA基因突变;与H37Rv株序列的差异比较显示,29株耐LFX临床株发生了有义突变,突变率90.6%.MODS与罗氏绝对浓度法药敏(L-J)比较,H37Rv完全相符;32株耐LFX临床分离株,相符31株,不符1株,符合率为96.9%.结论 gyrA基因突变检测可作为快速诊断结核分枝杆菌对LFX敏感性的一种方法,但不能完全区别低耐药和高耐药而使用受到限制.MODS技术检测LFX耐药性具有快速、操作简便、灵敏度和特异性高等优点,可作为结核分枝杆菌对LFX敏感性的快速检测新方法.     

Analysis of drug resistance and mutation profiles in Mycobacterium tuberculosis isolates in a surveillance site in Beijing,China

ObjectiveThis study analyzed drug resistance and mutations profiles in Mycobacterium tuberculosis isolates in a surveillance site in Huairou District, Beijing, China.MethodsThe proportion method was used to assess drug resistance profiles for four first-line and seven second-line anti-tuberculosis (TB) drugs. Molecular line probe assays were used for the rapid detection of resistance to rifampicin (RIF) and isoniazid (INH).ResultsAmong 235 strains of M. tuberculosis, 79 (33.6%) isolates were resistant to one or more drugs. The isolates included 18 monoresistant (7.7%), 19 polyresistant (8.1%), 28 RIF-resistant (11.9%), 24 multidrug-resistant (MDR) (10.2%), 7 pre-extensively drug-resistant (XDR, 3.0%), and 2 XDR strains (0.9%). A higher rate of MDR-TB was detected among previously treated patients than among patients with newly diagnosed TB (34.5% vs. 6.8%). The majority (62.5%) of RIF-resistant isolates exhibited a mutation at S531L in the DNA-dependent RNA polymerase gene. Meanwhile, 62.9% of INH-resistant isolates carried a mutation at S315T1 in the katG gene.ConclusionOur results confirmed the high rate of drug-resistant TB, especially MDR-TB, in Huairou District, Beijing, China. Therefore, detailed drug testing is crucial in the evaluation of MDR-TB treatment.     

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.     

Characterization of rifampicin-resistant clinical Helicobacter pylori isolates from Germany

OBJECTIVES: The aim of this study was to assess the rate of rifampicin resistance in Helicobacter pylori isolated from patients in Germany, to detect rifampicin resistance-associated mutations and to identify non-resistance-associated genetic variants in the rpoB gene. METHODS: Susceptibility to rifampicin in a total of 1585 clinical isolates obtained between January 2003 and July 2006 was tested by disc diffusion and/or by the Etest method. The rpoB genes of a selection of both resistant (n=17) and susceptible (n=100) clinical isolates were sequenced in order to distinguish between resistance- and non-resistance-associated genetic alterations. In vitro mutagenesis experiments such as site-directed mutagenesis were carried out to demonstrate the pivotal role of rpoB mutations in rifampicin resistance. RESULTS: From 1585 clinical isolates examined, 22 (1.4%) showed phenotypic resistance to rifampicin (MIC>4 mg/L). The majority of the resistant strains harboured point mutations in their rpoB genes at codons 530, 540 and 545 and showed cross-resistance to rifabutin. Four clinical isolates with moderate rifampicin resistance (8 mg/L) showed a rifabutin-susceptible phenotype and did not harbour any mutation in the sequenced rpoB fragments. Sequence analysis of 100 rifampicin-susceptible isolates revealed numerous novel silent mutations in the rpoB genes resulting in amino acid exchanges, but not in resistance. CONCLUSIONS: Resistance to rifampicin/rifabutin in H. pylori strains isolated in Germany is still low and is associated with mutations in the rpoB gene. Further surveillance studies analysing the use of rifabutin in H. pylori eradication and its association with the occurrence of rifabutin-resistant strains are required.     

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。     

四川德阳地区结核分枝杆菌耐药基因突变位点的分布特点及基因型耐药的分析

目的探讨四川德阳地区结核分枝杆菌耐药基因突变位点的分布特点及基因型耐药的分析。方法收集在2010年2月-2013年3月检出的257例肺结核病患者结核分枝杆菌DNA阳性的痰液标本,采用聚合酶链式反应（PCR）和反向点杂交（RDB）相结合的基因芯片技术对结核分枝杆菌耐药基因突变位点进行检测和分析。结果 257例肺结核患者中检测出49例存在结核分枝杆菌耐药基因位点突变,其中发生katG 315、rpsL43、embB 306和rpoB 531丝氨酸（S531L）位点突变的分别为30例（11.67%）、18例（7.00%）、11例（4.28%）和10例（3.89%）。234例初治肺结核患者对异烟肼、利福平、乙胺丁醇及链霉素的基因型耐药率分别为9.83%、4.27%、3.42%和5.13%,耐多药率为2.99%;23例复治肺结核患者对上述药物的基因型耐药率分别为52.17%、26.09%、13.04%和43.48%,耐多药率为13.04%。初治患者的基因型耐药率及耐多药率均明显低于复治患者。结论四川德阳地区结核分枝杆菌常见耐药基因突变位点分别为katG 315、rpsL 43、embB 306和rpoB 531丝氨酸（S531L）突变位点;肺结核初治患者的基因型耐药率及耐多药率均明显低于复治患者;该地区肺结核的耐多药率相对较低。