Cloning of a gene from Mycobacterium tuberculosis coding for a hypothetical 27 kDa protein and its use for the specific PCR identification of these mycobacteria

PCR targeting the IS 6110 has been considered specific for identification of Mycobacterium tuberculosis and is frequently applied to confirm the presence of this organism directly in biological specimens. However, several authors found that some M. tuberculosis strains failed to hybridize with the IS 6110 probe and other authors found that false-positive results may be obtained for clinical samples when some methods based on IS 6110 are used. In the present study, the p27 gene isolated from a cosmid library was found to be highly specific for M. tuberculosis complex strains and allowed us to develop a PCR-based assay for rapid detection and identification of this mycobacterium. One pair of primers and two oligonucleotide probes were successfully used to amplify and to detect the DNA of strains belonging to the M. tuberculosis complex. These primers and probes did not hybridize with DNA from any of the 21 other mycobacterial species tested. It is worth noting that the chosen primers and probes hybridize with DNA from the M. tuberculosis strain with no IS 6110, furthermore no strain without p27 was found among the 410 strains tested in the present study.     

Evaluation of multiple genetic markers for typing drug-resistant Mycobacterium tuberculosis strains from Poland

In the present study, 77 drug-resistant Mycobacterium tuberculosis strains isolated in Poland in 2000 were characterized by the mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing and our novel method based on PCR amplification of DNA regions between IS6110 and 16-bp GC-rich frequent repeats (designated IS6110-Mtb1/Mtb2 PCR). The results were compared with previous data of the more commonly used methods, IS6110 restriction fragment length polymorphism (RFLP) and spoligotyping. The discriminatory power of IS6110-Mtb1/Mtb2 method was only slightly lower than that of IS6110 RFLP, whereas MIRU-VNTR typing was the least discriminative among the 4 methods used. Clustering of strains by using results of IS6110-Mtb1/Mtb2 PCR correlated well with RFLP-defined clusters, further confirming epidemiologic relationships among patients. These results indicate that the novel genotyping method could be an attractive alternative for other PCR-based typing procedures, such as spoligotyping and MIRU-VNTR typing. Also, it seems to be a valuable adjunct to the reference IS6110 RFLP method for studying the genetic diversity of drug-resistant M. tuberculosis strains in Poland.     

Characterization of New Mutations in Pyrazinamide-Resistant Strains of Mycobacterium tuberculosis and Identification of Conserved Regions Important for the Catalytic Activity of the Pyrazinamidase PncA

A new set of mutations, including transposition of the insertion sequence IS6110, was identified in the pncA gene from 19 pyrazinamide-resistant Mycobacterium tuberculosis strains. Alignment of the PncA protein from M. tuberculosis with homologous proteins from different bacterial species revealed three highly conserved regions in PncA which may play an important role in the processing of pyrazinamide.     

Differentiation of Mycobacterium tuberculosis and Mycobacterium bovis BCG by a polymerase chain reaction assay

Mycobacterium tuberculosis and Mycobacterium bovis are closely related species which carry different numbers of the repetitive DNA element IS6110. A polymerase chain reaction assay was developed to assess the copy number of IS6110 in a strain and thereby differentiate these two important human pathogens.     

Mycobacterium tuberculosis detection by polymerase chain reaction and identification of M. tuberculosis strain

A rapid multiprimer PCR method for detection of Mycobacterium tuberculosis complex (MTC) and simultaneous identification of M. tuberculosis in clinical samples has been developed. The method is based on simultaneous amplification of two targets: a 401 bp region from the mtp40 species-specific gene sequence of M. tuberculosis and a 544 bp fragment from the RD1 genome region which is specific for MTC but absent in BCG strains. Polymerase inhibitors in this study were detected by internal control in each test. Detection sensitivity was 25 copies of M. tuberculosis genomic DNA. Seven methods for isolation of mycobacterial DNA were compared and the technique with chloroform extraction was selected as the most efficient. The proposed method was used for analysis of 37 clinical samples and the results were compared with the results of culturing, acid-fast bacilli staining, and clinical diagnosis. The method proved to be sufficiently sensitive and specific for detection of mycobacterial DNA. Moreover, in countries with only two main pathogenic species of MTC circulating (M. tuberculosis and M. bovis) this method can be used for differentiation of these two species.     

A multiplex polymerase chain reaction assay for genus-, group- and species-specific detection of mycobacteria

We developed and evaluated a single-step, multiplex polymerase chain reaction (PCR) assay for distinguishing (1) between the Mycobacterium tuberculosis complex (MTBC) and mycobacteria other than tuberculosis (MOTT) and (2) between M. tuberculosis and M. bovis species. The assay targeted the 16S and the 23S rDNA to distinguish between MTBC and MOTT species, and the oxyR gene to distinguish between M. tuberculosis and M. bovis strains. Clinical samples and reference strains (N = 156) comprised 93 strains of M. tuberculosis, 44 of M. bovis, 1 M. africanum strain, and 18 strains representing 9 different species of MOTTs. MOTTs generated only a single PCR product of about 2.5 kilobase; however, all of the MTBC strains produced a 118 base pair (bp) fragment and an additional 270 bp fragment was obtained for M. tuberculosis and M. africanum when the primer pair oxyRTB-2.1/oxyRMT-1 was used. When oxyRTB-2.1/oxyRMB-1 primers were used, the 270 bp fragment was obtained for only M. bovis. The assay needed as little as 1 pg of purified genomic DNA to make a positive identification.     

IS6110限制性片段长度多态性和间隔区寡核苷酸分型技术在结核分枝杆菌菌株鉴定中的应用

目的 评价IB6110-限制性片段长度多态性（IS6110-RFLP）和间隔区寡核苷酸分型技术（spoligotyping）两种方法在结核病流行病学上的应用，探讨我国各地区的结核分枝杆菌菌株特点。方法 收集158株结核分枝杆菌临床分离株，分别应用IS6110-RFLP和Spoligotyping两种方法进行鉴定。结果 （1）IS6110-RFLP的分辨力大于spoligotyping分型。（2）将本次试验结果与国际spoligotype数据库进行比较。结果 有14个类型属于共有类型，其中类型1为流行的类型，分布广泛，即所说的北京基因型。（3）广东地区与其他地区成簇率和北京基因型所占比例差异有统计学意义（P〈0．05）。广东地区成簇率和北京基因型所占比例均显著低于其他地区。结论 同时应用IS6110-RFLP分型和Spoligotyping两种方法进行结核病流行病学调查研究非常有效，在中国不同地区的菌株具有不同的特点。     

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.     

Genetic and phenotypic characterization of pyrazinamide-resistant mycobacterium tuberculosis complex isolates in Japan

The pncA gene mutations associated with pyrazinamide (PZA) resistance in Mycobacterium tuberculosis complex were determined in 26 PZA-resistant isolates in Japan. Of the 26 PZA-resistant isolates included, 21 were negative for pyrazinamidase (PZase). Of these, 20 isolates had various pncA mutations, resulting in alteration of primary amino acid sequence. However, 1 PZase-negative isolate did not have any mutation on pncA gene. The remaining 5 PZA-resistant isolates were positive for PZase and had identical pncA alleles with PZA-susceptible isolates. IS6110 RFLP analysis demonstrated various distinct IS6110 types and 5 pairs of isolates were very close to each other (>90% identical pattern). This study demonstrates that most of the PZA resistance is a result of various mutations on pncA resulting in loss of PZase activity. Further investigation, particularly on PZase-positive but PZA-resistant isolates and a PZase-negative isolate with no mutation on pncA, should be urgently done.     

多位点数目可变串联重复序列分析在北京基因型结核分枝杆菌基因分型中的应用

目的 评价多位点MLVA的15位点组合在北京基因型结核分枝杆菌(MTB)基因分型研究中的应用价值.方法 采用MLVA(15位点)对来源于北京胸科医院的72株北京基因型MTB菌株进行基因分型,并将结果与金标准IS6110-RFLP的分型结果进行比较.结果 MLVA(15位点)分型后共得到59个类型,其中53个为独特类型,其总体分辨力为0.990,多态性较好的位点有Qub-11b、Mtub 21和QUB-26;IS6110-RFLP分型后共得到69个类型,其中66个为独特类型,分辨力高达0.999,并可对MLVA(15位点)中成簇的菌株进一步区分.结论 MLVA(15位点)在北京基因型菌株中具有较好的分辨能力,但成簇菌株仍需采用金标准IS6110-RFLP进一步细分.     

结核分枝杆菌DNA指纹技术及其应用研究

目的　从分子流行病学角度探讨北京及其他地区结核分枝杆菌(结核菌)的分布特征。方法　构建以IS6110为基础的结核菌DNA指纹图谱,应用MINTS软件进行处理,并用χ2检验比较不同组别结核病人临床分离菌株成簇率的差别。结果　H37Rv、BCG两个标准菌株和62例结核病人临床分离菌株IS6110DNA指纹结果与国外同类报道一致;其中70%（44/62）的临床分离菌株IS6110DNA指纹相似值在1.00～0.65之间;分组统计结果显示,男性组与女性组成簇率之间差异有显著性（P<0.05）,其他各组之间差异无显著性（P>0.05）。结论　DNA指纹技术对结核菌在株水平的鉴定具有特异、灵敏等优点,可应用于结核流行病学研究。研究表明,北京及其他地区结核菌临床分离菌株多数遗传关系较近,且在基因水平上相关程度较强;结核菌在男性人群中的传播频率可能较女性更高。     

Study of the gyrB gene polymorphism as a tool to differentiate among Mycobacterium tuberculosis complex subspecies further underlines the older evolutionary age of 'Mycobacterium canettii'

The present investigation evaluated the PCR-restriction fragment length polymorphism (RFLP) analysis of hsp65 and gyrB targets for differentiation of the species within the Mycobacterium tuberculosis complex (MTC) both by including new restriction enzymes and previously unstudied species. The hsp65 restriction analysis using HhaI resulted in a characteristic 'Mycobacterium canettii' pattern. A study of the gyrB gene polymorphism using TaqIalpha and HinfI allowed the initial division of MTC into two major groups, one consisting of M. tuberculosis and 'M. canettii' as opposed to another single group with other species. Three different patterns were observed with RsaI, the first characteristic of Mycobacterium microti, the second with Mycobacterium bovis, M. bovis BCG and Mycobacterium caprae (M. caprae was easily separated from M. bovis, and M. bovis BCG by SacII digestion), and the third with M. tuberculosis, 'M. canettii', Mycobacterium africanum, Mycobacterium pinnipedii, and the dassie bacillus. Although further discrimination within the last group was not obtained using additional restriction enzymes, the HaeIII and RsaI digestions highlighted an important gyrB polymorphism among 'M. canettii' strains. A study of the single nucleotide polymorphisms (SNP) within the gyrB by sequence analysis not only confirmed the results of the restriction analysis, but showed further differences among 'M. canettii' isolates that were not picked up using the existing battery of restriction enzymes. As many as 11 different SNPs were identified in the collection of eight 'M. canettii' isolates studied. Considering that gyrB variability among MTC member species other than 'M. canettii' is as restricted as hsp65 variability among MTC, our data corroborate a recent proposition that the 'M. canettii' group is evolutionary much older than the other MTC members. In conclusion, gyrB PCR-RFLP is a simple and rapid low-cost method that combined with phenotypic characteristics, may be helpful to differentiate most of the subspecies within the MTC.     

环介导等温扩增技术快速检测结核分枝杆菌核酸

摘要:目的：建立一种快速准确的检测结核分枝杆菌（MTB）核酸的环介导等温扩增（LAMP）方法。 方法：以重复插入序列IS6110为目的基因,设计LAMP引物,特异检测MTB核酸。用本法与痰涂片抗酸染色镜检法、实时荧光PCR法对100例可疑患者痰标本进行对比检查。 结果：LAMP法特异性强,仅扩增MTB复合群核酸;灵敏度高,检测限达100 fg;而实时荧光PCR检测限为1 pg。对100例疑似结核病患者痰液标本检测,涂片抗酸染色法、LAMP法、实时荧光PCR法的阳性率分别为28%、39%和38%。 结论：本研究建立的LAMP方法检测MTB核酸特异性强、灵敏度高、时间短且操作简便,有望成为临床快速检测MTB的新方法。     

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.     

FQ-PCR检测肠黏膜结核杆菌DNA对肠结核的诊断价值

目的建立一种实时定量检测结核分枝杆菌（MTB）插入序列IS6110 DNA的方法,探讨其在肠结核（ITB）诊断中的价值。方法采用FQ-PCR技术对36例内镜活检ITB标本（30例石蜡、6例新鲜组织）、36例Crohn’s disease标本（16例石蜡手术标本,15例石蜡内镜活检标本,5例新鲜内镜活检组织）及34例阴性对照标本进行IS6110 DNA的实时定量检测,并比较上述标本抗酸染色结果。结果13例ITB抗酸染色阳性,CD无1例阳性,抗酸染色对ITB诊断的敏感性36.11%。MTBIS6110DNA检测结果：23例ITB（63.89%）阳性,6例CD（16.67%）阳性,另有1例阳性为升结肠腺癌癌旁正常组织。MTBIS6110 DNA在ITB与CD中的阳性率差异有统计学意义（P〈0.05）。FQ-PCR对ITB诊断的敏感性为63.89%,特异性83.33%。FQ-PCR敏感性显著高于抗酸染色（P〈0.05）。MTBIS6110 DNA阳性的ITB标本其定量结果范围为2.44×10^-4-2.26×10^1拷贝/细胞。结论FQ-PCR检测MTBIS6110 DNA是一种快速有效的ITB诊断方法,其定量范围广,检测灵敏度高。对活检组织少、病理改变不典型、抗酸染色阴性组织的诊断和鉴别诊断尤有意义。     

Occurrence of the IS900 gene in Mycobacterium avium complex derived from HIV patients.

The occurrence of the insertion sequence IS900 in Mycobacterium avium subsp. avium strains isolated from HIV infected patients has been investigated. In this study, genomic DNA from 62 mycobacterial isolates [31 strains of M. avium complex (MAC) consisting of 26 M. avium subsp. avium HIV-isolates and five non-HIV isolates and 31 additional Mycobacterium species] were analysed by an IS900 -based polymerase chain reaction (PCR) assay and Southern hybridization using a non-radioactive-labelled 251 bp DNA fragment located at the 5'-region of the IS900 sequence. As expected, none of the 28 Mycobacterium species contained the IS900 in their genomic DNA. Of the 26 M. avium subsp. avium HIV-isolates, 15 (57.6%) were strongly positive for the IS900 or IS900 related sequence. The five pulmonary non-HIV MAC isolates were negative for the IS900. As expected, the three strains of M. avium subsp. paratuberculosis were positive. This PCR guided study suggest that the IS900 gene is very common in clinical strains of M. avium subsp. avium especially those isolated from HIV patients. Ultimately the IS900 PCR-based assay may provide a useful tool for diagnostic and epidemiological studies related to MAC infections in HIV patients.     

Molecular basis of rifampin and isoniazid resistance in Mycobacterium bovis strains isolated in Sardinia, Italy

Fourteen of 22 (68%) Mycobacterium bovis strains isolated from cattle in Sardinia were found to be resistant to rifampin and isoniazid. Analysis of the rpoB and the katG, oxyR-ahpC, and inhA gene regions of these strains was performed in order to investigate the molecular basis of rifampin and isoniazid resistance, respectively. The most frequent mutation, encountered in 6 of 10 strains (60%), was in the rpoB gene; it occurred, at codon position 521 and resulted in leucine changed to proline. This suggests that codon 521 may be important for the development of rifampin resistance in M. bovis. Resistance to isoniazid is associated in Mycobacterium tuberculosis with a variety of mutations affecting one or more genes. Our results confirm the difficulty of interpreting the sequence variations observed in clinical strains of M. bovis. M. bovis strains isolated from the same geographic area showed similar mutations within the genes responsible for rifampin and isoniazid resistance. Our results represent the first study to elucidate the molecular genetic basis of drug resistance in M. bovis isolated from cattle.     

Mutations associated with pyrazinamide resistance in pncA of Mycobacterium tuberculosis complex organisms.

A gene (pncA) with mutations associated with pyrazinamide resistance in Mycobacterium tuberculosis complex members was characterized in 67 pyrazinamide-resistant and 51 pyrazinamide-susceptible isolates recovered from diverse geographic localities and anatomic sites and typed by IS6110 profiling. All pyrazinamide-susceptible organisms had identical pncA alleles. In striking contrast, 72% of the 67 resistant organisms had pncA mutations that altered the primary amino acid sequence of pyrazinamidase. A total of 17 previously undescribed mutations were found, including upstream mutations, missense changes, nucleotide insertions and deletions, and termination mutations. The mutations were arrayed along virtually the entire length of the gene. These data are further evidence that most drug resistance in M. tuberculosis is due to simple mutations occurring in chromosomally encoded genes rather than to acquisition of resistance genes by horizontal transfer events.     

杂交信号放大方法快速检测结核分枝杆菌的实验研究

目的确定杂交信号放大方法（rlSAM）检测结核分枝杆菌的灵敏度和特异性,进而探讨该方法用于检测临床标本的可行性。方法HSAM法检测合成的IS6110靶基因、结核分枝杆菌标准菌株、大肠埃希菌和表皮葡萄球菌。确定该方法的灵敏度和特异性,并与PCR法进行比较。结果HSAM最少能检测到10合成的靶基因和结核分枝杆菌,与PCR方法灵敏度相近,标准菌株检测结果阳性,大肠埃希菌和表皮葡萄球菌均为阴性,该方法具有较高的特异性。结论HSAM具有高灵敏度、高特异性及操作简便快速等特点,可作为结核分枝杆菌临床标本检测方法。     

Comparison of the in vitro activities of rifapentine and rifampicin against Mycobacterium tuberculosis complex

The in vitro activity of rifapentine for 44 clinical isolates of Mycobacterium tuberculosis complex was compared with that of rifampicin using the Bactec radiometric method and the absolute concentration method for susceptibility testing. Twenty-nine M. tuberculosis, 11 Mycobacterium bovis and four Mycobacterium africanum strains were studied. Control tests showed that rifapentine was stable for 14 days in 7H9 broth and for 3 weeks in 7H10 agar medium. The 44 M. tuberculosis complex strains were more susceptible to rifapentine than to rifampicin, irrespective of the testing method. In the radiometric system, the MIC50 and MIC90 of rifapentine for M. tuberculosis complex strains were one or two two-fold dilutions lower than those of rifampicin (0.06-0.125 mg/L versus 0.25 mg/L, respectively). By the absolute concentration method, the MIC50 and MIC90 of rifapentine for M. tuberculosis complex strains were two two-fold dilutions lower than those of rifampicin (0.125-0.25 mg/L versus 0.5-1 mg/L, respectively). The MIC90 of rifapentine for the 44 M. tuberculosis complex strains was always 0.25 mg/L, irrespective of the method used, but the radiometric method was more reliable and more reproducible than the agar 7H10 method.