Characterization of rpoB mutations in rifampin-resistant clinical Mycobacterium tuberculosis isolates from Kuwait and Dubai

Mutations conferring resistance to rifampin in rifampin-resistant clinical Mycobacterium tuberculosis isolates occur mostly in the 81 bp rifampin-resistance-determining region (RRDR) of the rpoB gene. In this study, 29 rifampin-resistant and 12 -susceptible clinical M. tuberculosis isolates were tested for characterization of mutations in the rpoB gene by line probe (INNO-LiPA Rif. TB) assay and the results were confirmed and extended by DNA sequencing of the PCR amplified target DNA. The line probe assay identified all 12 susceptible strains as rifampin-sensitive and the DNA sequence of RRDR in the amplified rpoB gene from two isolates matched perfectly with the wild-type sequence. The line probe assay identified 28 resistant isolates as rifampin-resistant with specific detection of mutation in 22 isolates including one isolate that exhibited hetro-resistance containing both the wild-type pattern as well as a specific mutation within RRDR while one of the rifampin-resistant strain was identified as rifampin-susceptible. DNA sequencing confirmed these results and, in addition, led to the specific detection of mutations in 5 rifampin-resistant isolates in which specific base changes within RRDR could not be determined by the line probe assay. These analyses identified 8 different mutations within RRDR of the rpoB gene including one novel mutation (S522W) that has not been reported so far. The genotyping performed on the isolates carrying similar mutations showed that majority of these isolates were unique as they exhibited varying DNA banding patterns. Correlating the ethnic origin of the infected TB patients with the occurrence of specific mutations at three main codon positions (516, 526 and 531) in the rpoB gene showed that most patients (11 of 15) from South Asian region contained mutations at codon 526 while majority of isolates from patients (6 of 11) of Middle Eastern origin contained mutations at codon 531.     

Characterization of rpoB mutations in rifampin-resistant Mycobacterium tuberculosis isolates from the Middle East

The nature and frequency of mutations in the rpoB gene of rifampin-resistant clinical Mycobacterium tuberculosis isolates vary considerably according to geographical locations. There is no information on the prevalence of specific mutations in clinical M. tuberculosis strains isolated from patients in Middle-Eastern countries. In this study, 13 rifampin-resistant and 6 susceptible clinical M. tuberculosis isolates were tested for identification and characterization of mutations in the rpoB gene by INNO-LiPA Rif. TB kit and DNA sequencing of the PCR amplified target DNA. The kit identified all six susceptible strains as rifampin-sensitive and the DNA sequence of the amplified rpoB gene in the target region matched perfectly with the wild-type sequence. The kit identified 12 resistant isolates as rifampin-resistant with specific detection of mutations in 8 isolates while one of the rifampin-resistant strain was identified as rifampin-susceptible. DNA sequencing confirmed these results and, in addition, led to the specific detection of mutations in 4 rifampin-resistant isolates in which specific base changes within the target region could not be determined by the INNO-LiPA Rif. TB kit. The majority (8 of 13) of resistant isolates involved base changes at codon 531 of the rpoB gene. Mutations at codon position 531 within the rpoB gene have also been reported in majority of rifampin-resistant strains from Greece and St. Petersburg, Russia but not from other geographical locations.     

Rifampin resistance in Mycobacterium kansasii is associated with rpoB mutations

Rifampin is the most potent drug used in the treatment of disease due to Mycobacterium kansasii. A 69-bp fragment of rpoB, the gene that encodes the beta subunit of the bacterial RNA polymerase, was sequenced and found to be identical in five rifampin-susceptible clinical isolates of M. kansasii. This sequence showed 87% homology with the Mycobacterium tuberculosis gene, with an identical deduced amino acid sequence. In contrast, missense mutations were detected in the same fragment amplified from five rifampin-resistant isolates. A rifampin-resistant strain generated in vitro also harbored an rpoB gene missense mutation that was not present in the parent isolate. All mutations detected (in codons 513, 526, and 531) have previously been described in rifampin-resistant M. tuberculosis isolates. Rifampin MICs determined by E-test were <1 mg/liter for all rifampin-susceptible isolates and >256 mg/liter for all rifampin-resistant ones. In addition, four of the five rifampin-resistant isolates were also resistant to rifabutin. We have thus shown a strong association between rpoB gene missense mutations and rifampin resistance in M. kansasii. Although our results are derived from a small number of isolates and confirmation with larger numbers would be useful, they strongly suggest that mutations within rpoB form the molecular basis of rifampin resistance in this species.     

武汉地区耐利福平结核分枝杆菌rpoB基因突变分析

摘要：目的：探讨武汉地区结核分枝杆菌（MTB）利福平耐药株rpoB基因的突变特征。 方法：对76例MTB临床分离株包括rpoB核心区域81 bp碱基在内的428 bp碱基进行PCR测定,并进行DNA序列分析。 结果：76例临床分离MTB中利福平耐药株56例,敏感株20例。耐药株中92.9%（52/56）存在突变,共涉及10个密码子的18种突变类型。 531、526为常见突变位点,其突变率分别为57.7%(30/52)、19.2%(10/52);联合突变率为13.5%（7/52）;同时发现了509位（AGC→AGA）新的突变类型和国内少见的517位CAG缺失类型。 结论：rpoB基因突变在武汉地区利福平耐药MTB中广泛存在,并存在新的突变位点。     

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.     

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。     

Relationship between rifampin MICs for and rpoB mutations of Mycobacterium tuberculosis strains isolated in Japan.

We analyzed the relationship between rifampin MICs and rpoB mutations of 40 clinical isolates of Mycobacterium tuberculosis. A point mutation in either codon 516, 526, or 531 was found in 13 strains requiring MICs of > or = 64 micrograms/ml, while 21 strains requiring MICs of < or = 1 microgram/ml showed no alteration in these codons. However, 3 of these 21 strains contained a point mutation in either codon 515 or 533. Of the other six strains requiring MICs between 2 and 32 micrograms/ml, three contained a point mutation in codon 516 or 526, while no alteration was detected in the other three. Our results indicate that the sequencing analysis of a 69-bp fragment in the rpoB gene is useful in predicting rifampin-resistant phenotypes.     

DNA芯片快速检测耐利福平结核分枝杆菌rpoB基因突变

目的 开发快速检测耐利福平结核分枝杆菌(结核菌)rpoB基因突变的DNA芯片。方法 根据结核菌rpoB基因序列设计探针并制作基因芯片，从临床样品中分离出结核菌的基因组DNA，PCR扩增含有rpoB基因突变位点的特异DNA片段，荧光标记后与芯片上含有的检测特异突变位点的寡核苷酸探针进行杂交，同时与DNA直接测序法测定序列比较。结果 35株耐利福平结核菌中有91．4％(32／35)用直接测序法检测出存在rpoB基因突变，DNA芯片的检测效率为71．4％(25／35)。结论 用DNA芯片检测结核菌对利福平的耐药性具有较高的特异性和敏感性，可用于临床结核菌耐药性检测。     

rpoB基因在结核分枝杆菌利福平/利福布丁交叉耐药株中突变情况的差异分析

目的 了解结核分枝杆菌利福平/利福布丁交叉耐药株与利福平耐药/利福布丁敏感株rpoB全基因突变特征的差异.方法 测定rpoB全基因序列,比较278株利福平/利福布丁交叉耐药(rifampicin/rifabutin cross-resistant,RIF/Rfb-R)株和40株利福平耐药/利福布丁敏感(rifampicinresistant/rifabutin-susceptible,RIF-R/Rfb-S)株,30株利福平/利福布丁敏感(rifampicin-susceptible/rifabutin-susceptible,RIF-S/Rfb-S)株及标准株H37 Rv之间的rpoB全基因序列突变差异.结果 利福平/利福布丁敏感株和H37Rv rpoB全基因未发现突变;利福平/利福布丁交叉耐药株的常见突变位点是531(70.5％)和526( 20.9％).223( 80.2％)株单点突变株分别含有S531L、S531W、H526D、H526Y、H526R、Q513K、Q513P、Q510H、V176F、P287R、Y395C及H404Y单点突变,55( 19.8％)株多重突变株分别以S531L、H526R、H526Y、H526D、D516G和Q513K位点与其他位点联合突变为主.利福平耐药/利福布丁敏感株的常见突变位点分别是516(65.0％)、526( 17.5％)和533( 10.0％).21(52.5％)株单点突变株分别含有L533P、H526L、H526S、S522L、D516V、D516Y和D516F单点突变,19(47.5％)株多重突变株分别以D516V和L533P位点与其他位点联合突变为主.结论 本批标本中,利福霉素耐药株rpoB基因突变率为100％,利福平/利福布丁交叉耐药株与利福平耐药/利福布丁敏感株rpoB全基因单点突变株的突变位置或氨基酸替换类型、多重突变株的突变位置或组合类型以及常见突变位点或其氨基酸替换类型完全不同,rpoB全基因DNA序列分析对临床科学应用利福平和利福布丁有指导意义.     

耐利福平结核分枝杆菌rpoB基因序列研究

目的 研究杭州地区结核分枝杆菌（Mycobacterium tuberculosis）对利福平（RFP）的耐受与rpoB基因突变的关系。方法 随机挑选了90例结核杆菌感染的病例。选择了利福平作为主要的药物进行药敏实验，PCR扩增39株耐RFP结核分支杆菌的rpoB基因片段（580bp）；测定扩增片段的序列。并与美国国立生物信息中心（www．ncbi．nlm．nih．gov／nucleotide）检索获得结核杆菌野生株（利福平敏感株）序列（登陆号：AJ749948）作对比分析。结果 结果显示PCR结核测序方法得到了39例结核杆菌耐药株。51例敏感株，与药敏实验的方法检测的结果完全一致。测定的11株临床分离的耐药株中，526位或531位有突变，其中526位有突变的有7株，占耐药测定株63．6%，531位突变的有4株。占耐药测定株36．4％。未见两位点同时突变。检测的11株敏感株，未见526位或531位有突变。结论 杭州地区结核分支杆菌耐利福平的发生与rpoB基因的526位或531位突变密切相关，与国外的报告基本相似。但526位突变占耐药测定株高达63．6％。如此高比例目前国内外未见相似报道。PCR扩增和产物测序将是临床检测结核分支杆菌耐利福平和耐多药的一种迅速、准确的方法。     

Mutation analysis of mycobacterial rpoB genes and rifampin resistance using recombinant Mycobacterium smegmatis

Rifampin is a major drug used to treat leprosy and tuberculosis. The rifampin resistance of Mycobacterium leprae and Mycobacterium tuberculosis results from a mutation in the rpoB gene, encoding the β subunit of RNA polymerase. A method for the molecular determination of rifampin resistance in these two mycobacteria would be clinically valuable, but the relationship between the mutations and susceptibility to rifampin must be clarified before its use. Analyses of mutations responsible for rifampin resistance using clinical isolates present some limitations. Each clinical isolate has its own genetic variations in some loci other than rpoB, which might affect rifampin susceptibility. For this study, we constructed recombinant strains of Mycobacterium smegmatis carrying the M. leprae or M. tuberculosis rpoB gene with or without mutation and disrupted their own rpoB genes on the chromosome. The rifampin and rifabutin susceptibilities of the recombinant bacteria were measured to examine the influence of the mutations. The results confirmed that several mutations detected in clinical isolates of these two pathogenic mycobacteria can confer rifampin resistance, but they also suggested that some mutations detected in M. leprae isolates or rifampin-resistant M. tuberculosis isolates are not involved in rifampin resistance.     

结核分枝杆菌耐利福平基因变异株的体外最小抑菌浓度研究

目的：研究结核分枝杆菌（MTB）耐利福平临床分离株基因突变对其体外最小抑菌浓度（MIC）测定结果的影响。方法用基因芯片对临床分离的对利福平耐药的175株 MTB及对利福平敏感的48株 MTB进行耐药基因检测;并对所有标本进行利福平的 MIC测定,比较不同变异株的 MIC测定结果。结果对利福平耐药的 MTB 临床分离株基因突变以531、526、516、533位点单突变为主;531位点单突变的利福平耐药变异株 MIC 高于526位点单突变的耐药变异株,差异有统计学意义（t′＝2．2237,t′α＝2．0449,P＜0．05）;526位点单突变的利福平耐药变异株 MIC高于516位点单突变的耐药变异株,差异有统计学意义（t＝2．2056,P＝0．0329,P＜0．05）。双突变者均有较高的 MIC测定值。结论合理的基因芯片设计能检出95．0％以上的对利福平耐药 MTB变异株;对利福平耐药的 MTB变异株有不同的基因突变模式,且不同突变模式的 MTB耐药程度不同,可以为临床用药提供参考。     

耐利福平结核分枝杆菌 rpoA、rpoB、rpoC和rpoZ突变的研究

目的：了解结核分枝杆菌rpoA、rpoB、rpoC和rpoZ突变特征及其与利福平耐药的关系。方法对140株临床分离结核分枝杆菌进行利福平敏感性试验,并对耐利福平株分别进行rpoB耐药决定区及rpoA、rpoB、rpoC及rpoZ全基因测序。结果140株结核分枝杆菌中57株对利福平耐药。57株耐利福平结核分枝杆菌中,52株（91.2%）存在突变,其中50株为rpoB基因突变,2株为rpoC突变。在rpoB 765、1001、1156位及rpoC 51位发现新的突变位点。结论结核分枝杆菌对利福平耐药主要与rpoB和rpoC基因突变有关。     

银染单链构象多态性技术检测结核杆菌rpoB基因突变

目的探讨分子生物学方法在临床标本结核分枝杆菌利福平（ＲＦＰ）耐药性评估中的应用价值。方法设计针对ｒｐｏＢ基因１８３ｂｐ扩增引物,运用聚合酶链反应单链构象多态性分析（ＰＣＲＳＳＣＰ）银染色方法,检测了４５株结核杆菌临床分离株,并对其中部分菌株进一步做了ＤＮＡ序列分析。结果以药敏试验结果为对照,有１６株ＲＦＰ敏感株及２６株ＲＦＰ耐药株经ＳＣＰ方法得到检测,阳性占９３．３％,特异性１００％。对部分菌株１８３ｂｐ片断测序结果显示,ＲＦＰ敏感株无ｒｐｏＢ基因突变,耐药株均发生碱基突变,分别导致编码５３１及５２６位点的氨基酸改变。结论银染ＰＣＲＳＣＰ方法具有简便,快速,准确的特点,提高敏感性,可以对临床标本结核杆菌利福平耐药突变进行鉴定。序列分析证明结核分枝杆菌利福平耐药菌株有ｒｐｏＢ基因突变。     

Characterization of rifampin-resistance in pathogenic mycobacteria.

The emergence of rifampin-resistant strains of pathogenic mycobacteria has threatened the usefulness of this drug in treating mycobacterial diseases. Critical to the treatment of individuals infected with resistant strains is the rapid identification of these strains directly from clinical specimens. It has been shown that resistance to rifampin in Mycobacterium tuberculosis and Mycobacterium leprae apparently involves mutations in the rpoB gene encoding the beta-subunit of the RNA polymerases of these species. DNA sequences were obtained from a 305-bp fragment of the rpoB gene from 110 rifampin-resistant and 10 rifampin-susceptible strains of M. tuberculosis from diverse geographical regions throughout the world. In 102 of 110 rifampin-resistant strains 16 mutations affecting 13 amino acids were observed. No mutations were observed in rifampin-susceptible strains. No association was found between particular mutations in the rpoB gene and drug susceptibility patterns of multidrug-resistant M. tuberculosis strains. Drug-resistant M. tuberculosis strains from the same outbreak and exhibiting the same IS6110 DNA fingerprint and drug susceptibility pattern contained the same mutation in the rpoB gene. However, mutations are not correlated with IS6110 profiling outside of epidemics. The evolution of rifampin resistance as a consequence of mutations in the rpoB gene was documented in a patient who developed rifampin resistance during the course of treatment. Rifampin-resistant strains of M. leprae, Mycobacterium avium, and Mycobacterium africanum contained mutations in the rpoB gene similar to that documented for M. tuberculosis. This information served as the basis for developing a rapid DNA diagnostic assay (PCR-heteroduplex formation) for the detection of rifampin susceptibility of M. tuberculosis.     

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.     

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.     

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.     

DNA芯片快速检测结核分枝杆菌利福平耐药rpoB基因突变

目的建立快速检测结核分枝杆菌利福平（RFP）耐药rpoB基因突变的DNA芯片。方法从34株临床痰样本中提取结核分枝杆菌的基因组DNA，采用聚合酶链反应（PCR）扩增含有rpoB基因突变位点的特异DNA片段，荧光标记后与芯片上含有特异突变位点的寡核苷酸探针进行杂交，同时与DNA测序法比较。结果用DNA芯片检测出19株耐药株和15株敏感株，且结果与DNA测序相同，准确率可达88．2％，敏感性为78．9％，特异性为100．0％。结论DNA芯片快速检测结核分枝杆菌对RFP的耐药rpoB基因突变是一种操作简便、准确性、敏感性和特异性较高，且易于开展的方法。     

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.