Mycobacterium tuberculosis mammalian cell entry operon (mce) homologs in Mycobacterium other than tuberculosis (MOTT)

The cloned mammalian cell entry gene mce1a from Mycobacterium tuberculosis confers to non-pathogenic Escherichia coli the ability to invade and survive inside macrophages and HeLa cells. The aim of this work was to search for and characterize homologs of the four M. tuberculosis mammalian cell entry operons (mce1, mce2, mce3 and mce4) in mycobacteria other than tuberculosis (MOTT). The dot-blot and polymerase chain reaction (PCR) experiments performed on 24 clinical isolates representing 20 different mycobacterial species indicated that the mce operons were widely distributed throughout the genus Mycobacterium. BLAST search results showed the presence of mce1, mce2 and mce4 homologs in Mycobacterium bovis, Mycobacterium avium and Mycobacterium smegmatis. A homologous region for the mce3 operon was also found in M. avium and M. smegmatis. DNA and protein alignments were done to compare the M. tuberculosis mce operons and the deduced M. bovis, M. avium, and M. smegmatis homologs. The deduced proteins of M. bovis mce1, mce2 and mce4 operons had 99.6-100% homology with the respective M. tuberculosis mce proteins (MTmce). The similarity between M. avium mce proteins and the individual M. tuberculosis homologs ranged from 56.2 to 85.5%. The alignment results between M. smegmatis mce proteins and the respective MTmce proteins ranged from 58.5% to 68.5%. Primer sets were designed from the M. tuberculosis mce4a gene for amplification of 379-bp fragments. Amplification was successful in 14 strains representing 11 different mycobacterial species. The PCR fragments were sequenced from 10 strains representing eight species. Alignment of the sequenced PCR products showed that mce4a homologs are highly conserved in the genus Mycobacterium. In conclusions, the four mce operons in different mycobacterial species are generally organized in the same manner. The phylogenetic tree comparing the different mce operons showed that the mce1 operon was closely related to the mce2 operon and mce3 diverged from the other operons. The wide distribution of the mce operons in pathogenic and non-pathogenic mycobacteria implicates that the presence of these putative virulence genes is not an indicator for the pathogenicity of the bacilli. Instead, the pathogenicity of these factors might be determined by their expression.     

Occurrence of RD9 region and 500 bp fragment among clinical isolates of Mycobacterium tuberculosis and Mycobacterium bovis

The present investigation dealt with the identification of Mycobacterium tuberculosis and M. bovis by RD9 region and 500 bp fragment PCR assays. Eight M. tuberculosis and 5 M. bovis characterized and identified from 40 human sputum and 41 bovine lung specimens and 20 M. tuberculosis and 9 M. bovis strains maintained at Mycobacteria Laboratory, Indian Veterinary Research Institute were included in this study. In this way, 28 M. tuberculosis and 14 M. bovis strains and, for comparison and control purpose, M. tuberculosis H37Rv, M. bovis BCG, M. canetti, M. smegmatis, M. phlei, M. chelonae, M. kansasii, M. xenopi and M. avium were subjected to RD9 and 500 bp amplification by PCR. All M. tuberculosis strains, M. tuberculosis H37 Rv and M. canetti yielded a product of 333 bp which showed presence of RD9 region in these strains, whereas all M. bovis yielded a product of 206 bp with RD9 PCR assay. There was no ampli-fication product found in M. bovis BCG, M. xenopi, M. smegmatis, M. phlei, M. chelonae, M. kansasii, and M. avium. PCR based on 500 bp fragment showed a product of 500 bp in all M. bovis strains and M. bovis BCG. There was no amplification product of 500 bp found in M. canetti, M. smegmatis, M. phlei, M. chelonae, M. avium, M. kansasii, M. xenopi and was absent in all M. tuberculosis strains. The PCR assay results correlated 100% with the culture and biochemical results of the isolates. Our study suggested that PCR based on RD9 and 500 bp may effectively identify two closely related species of M. tuberculosis and M. bovis.     

聚合酶链式反应检测结核杆菌的研究

以人型结核杆菌基因组DNA为模板,合成二段引物各20个碱基进行聚合酶链式反应(PCR)。经琼脂糖凝胶电泳证实,获得一条245bp扩增带。PCR检测的敏感性染色体基因组DNA为1pg,菌悬液为13个活菌／ml。在特异性试验中,人型结核杆趋,牛型结核杆菌、BCG可见此扩增带。被试的其它14种扰酸菌以及变铅青链霉菌、大肠杆菌质粒Puc19、星状诺卡氏菌、红球菌均未见该扩增带。54例肺结核痰标本3种方法检查的阳性率分别为：萋尼氏抗酸染色16．7％,培养法14．8％,PCR 37．0％。前2种检查方法分别与PCR比较,经统计学处理均有显著性差异(P<0．01)。12例非结核性肺部疾患痰标本抗酸染色和PCR均为阴性。结果表明,PCR技术是快速、敏感、特异诊断结核病的方法。     

Differentiation between Mycobacterium tuberculosis and Mycobacterium bovis by a multiplex-polymerase chain reaction

A multiplex-polymerase chain reaction (PCR) assay, based on one-step amplification and detection of three different mycobacterial genomic fragments, was designed for differentiation between Mycobacterium bovis and Mycobacterium tuberculosis. The oligonucleotide primers were chosen from the gro EL gene, present in the genus Mycobacterium sp., from the IS 6110 insertion sequence, present in Myco. tuberculosis complex and from the mtp 40 gene, identified as a specificspecies Myco. tuberculosis genomic fragment. This amplification method allowed the detection of two fragments of 576 and 317 base pairs in Myco. bovis and three fragments of 576, 396 and 317 base pairs in Myco. tuberculosis strains, including atypical strains of Myco. tuberculosis where the copy number of the IS 6110 element is low. The multiplex-PCR assay described may be a very useful tool for the rapid and specific differentiation of these related mycobacteria and easy to use in medical and veterinary microbiology laboratories.     

Identification of Mycobacterium tuberculosis complex based on amplification and sequencing of the oxyR pseudogene from stored Ziehl-Neelsen-stained sputum smears in Brazil

A cross-sectional analysis of stored Ziehl-Neelsen (ZN)-stained sputum smear slides (SSS) obtained from two public tuberculosis referral laboratories located in Juiz de Fora, Minas Gerais, was carried out to distinguish Mycobacterium bovis from other members of the Mycobacterium tuberculosis complex (MTC). A two-step approach was used to distinguish M. bovis from other members of MTC: (i) oxyR pseudogene amplification to detect MTC and, subsequently, (ii) allele-specific sequencing based on the polymorphism at position 285 of this gene. The oxyR pseudogene was successfully amplified in 100 of 177 (56.5%) SSS available from 99 individuals. No molecular profile of M. bovis was found. Multivariate analysis indicated that acid-fast bacilli (AFB) results and the source laboratory were associated (p < 0.05) with oxyR pseudogene amplification. SSS that were AFB++ SSS showed more oxyR pseudogene amplification than those with AFB0, possibly due to the amount of DNA. One of the two source laboratories presented a greater chance of oxyR pseudogene amplification, suggesting that differences in sputum conservation between laboratories could have influenced the preservation of DNA. This study provides evidence that stored ZN-SSS can be used for the molecular detection of MTC.     

人结核分枝杆菌特异性核酸探针制备及结核病PCR检测技术的初步临床应用

采用人结核分枝杆菌(Mycobacterium tuberculosis TB)染色体DNA为模板,选择位于插入片段IS6110中884～865和568～588碱基对处的两个片段为引物,扩增出317bp的特异性片段．将其克隆进pUCl9载体。酶切图谱分析和DNA序列测定证实为目的片段。该片段经DIG标记,分别与11种分枝杆菌DNA进行Southern杂交,结果证明只与人型复合分枝杆菌发生杂交反应。利用该对引物建立的PcR检测拄术对74份结核病痰液标本进行检测,并与临床细菌快速培养结果相比较,发现48份临床阳性均为PcR阳性,在26份临床阴性标本中亦发现11份PCR检测阳性。将标本PCR产物与克隆探针进行杂交,显示两者结果完全一致。说明PCR检测体系结果可靠,其灵敏度明显高于目前临床所采用的方法,可作为一种常规技术用于结核病的临床检测。     

Mycobacterium bovis identification by a molecular method from post-mortem inspected cattle obtained in abattoirs of Mato Grosso do Sul, Brazil

The presence of Mycobacterium bovis in bovine carcasses with lesions suggestive of tuberculosis was evaluated. Seventy-two carcass samples were selected during slaughter inspection procedures in abattoirs in the state of Mato Grosso do Sul, Brazil. Seventeen (23.6%) of samples showed colonies suggestive of mycobacteria that were confirmed to be acid-fast bacilli by Ziehl-Neelsen staining. Polymerase chain reaction (PCR) using primers specific for M. bovis identified M. bovis in 13 (76.5%) isolates. The PCR-restriction enzyme pattern analysis using gene encoding for the 65-kDa protein and two restriction enzymes identified the remaining four isolates that were represented by two M. tuberculosis complex and two nontuberculous mycobacteria. The results are indicative of infection of slaughter cattle by M. bovis and other mycobacteria in the state of Mato Grosso do Sul.     

Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis.

The live attenuated bacillus Calmette-Guérin (BCG) vaccine for the prevention of disease associated with Mycobacterium tuberculosis was derived from the closely related virulent tubercle bacillus, Mycobacterium bovis. Although the BCG vaccine has been one of the most widely used vaccines in the world for over 40 years, the genetic basis of BCG's attenuation has never been elucidated. We employed subtractive genomic hybridization to identify genetic differences between virulent M. bovis and M. tuberculosis and avirulent BCG. Three distinct genomic regions of difference (designated RD1 to RD3) were found to be deleted from BCG, and the precise junctions and DNA sequence of each deletion were determined. RD3, a 9.3-kb genomic segment present in virulent laboratory strains of M. bovis and M. tuberculosis, was absent from BCG and 84% of virulent clinical isolates. RD2, a 10.7-kb DNA segment containing a novel repetitive element and the previously identified mpt-64 gene, was conserved in all virulent laboratory and clinical tubercle bacilli tested and was deleted only from substrains derived from the original BCG Pasteur strain after 1925. Thus, the RD2 deletion occurred after the original derivation of BCG. RD1, a 9.5-kb DNA segment found to be deleted from all BCG substrains, was conserved in all virulent laboratory and clinical isolates of M. bovis and M. tuberculosis tested. The reintroduction of RD1 into BCG repressed the expression of at least 10 proteins and resulted in a protein expression profile almost identical to that of virulent M. bovis and M. tuberculosis, as determined by two-dimensional gel electrophoresis. These data indicate a role for RD1 in the regulation of multiple genetic loci, suggesting that the loss of virulence by BCG is due to a regulatory mutation. These findings may be applicable to the rational design of a new attenuated tuberculosis vaccine and the development of new diagnostic tests to distinguish BCG vaccination from tuberculosis infection.     

Assessment of the N-PCR Assay in Diagnosis of Pleural Tuberculosis: Detection of M.tuberculosis in Pleural Fluid and Sputum Collected in Tandem

Background

The nonspecific clinical presentation and paucibacillary nature of tuberculous pleuritis remains a challenge for diagnosis. Diagnosis of tuberculous pleural effusion depends on the demonstration of the presence of tubercle bacilli in the sputum, pleural fluid, or pleural biopsy specimen, or demonstration of granuloma in pleura by histological examination. We examined the clinical utility of the diagnosis of pleural tuberculosis using the in house N-PCR assay, AFB smear microscopy and culture. Besides pleural fluid the inclusion of sputum in the efficacy of diagnosis of pleural tuberculosis was scrutinized.

Methodology/Principal Findings

Pleural fluid and sputum samples of 58 tuberculous and 42 non-tuberculous pleural effusion patients were processed for AFB smear microscopy, culture and the N-PCR assay. Mycobacteria were detected exclusively in tuberculous pleural effusion samples. None of the non-tuberculous pleural effusion samples were positive for mycobacteria. Comparative analysis showed that the N-PCR assay had the highest sensitivity. Inclusion of sputum along with pleural fluid increased N-PCR sensitivity from 51.7 to 70.6% (p<0.0001).This improved sensitivity was reflected in AFB smear microscopy and isolation by culture. The sensitivity enhanced on inclusion of sputum from 3.4 (p = 0.50) to 10.3% (p = 0.038) for AFB smear microscopy and for isolation of mycobacteria from 10.3(p = 0.03) to 22.4% (p = 0.0005). Thirteen isolates were obtained from 58 pleural tuberculosis patients. Eleven mycobacterial isolates were identified as M.tuberculosis and two as M.fortuitum and M.chelonae. Complete concordance was seen between the biochemical identification of isolates and the N-PCR identification of mycobacterial species prior to isolation.

Conclusions/Significance

To the best of our knowledge this is the first PCR based report on utility of sputum for diagnosis of pleural tuberculosis. The present study demonstrates that a combination of pleural fluid with sputum sample and N-PCR improved the diagnosis of pleural tuberculosis.     

PCR amplification of shorter fragments from the devR (Rv3133c) gene significantly increases the sensitivity of tuberculosis diagnosis

This study was designed to assess the vital issue of gene target length and PCR assay performance in relation to the detection of Mycobacterium tuberculosis in clinical specimens. Two PCR assays that amplify fragments of varying lengths from the devR gene of M. tuberculosis were evaluated. Using M. tuberculosis DNA the 'short-length' PCR assay detected 250-500 genome equivalents vs. 500-1,000 genome equivalents by the 'long-length' assay. In comparison to a highly sensitive smear microscopy test (universal sample processing smear), the sensitivity of the 'short-length' assay was 97.8% vs. 69.9% of the 'long-length' assay in sputum specimens (n=506) from patients being evaluated for a possible diagnosis of tuberculosis. The 27.9% absolute increase in sensitivity was statistically significant (P<0.001). Our results indicate that in a clinical setting when all other conditions are equal, the amplification of a shorter gene fragment of devR increases the sensitivity and efficiency of the PCR assay in spite of using a single copy gene as target.     

Prevalence of mutations at codon 463 of katG gene in MDR and XDR clinical isolates of Mycobacterium tuberculosis in Belarus and application of the method in rapid diagnosis

Isoniazid (INH) is a central component of drug regimens used worldwide to treat tuberculosis. In respect to high GC content of Mycobacterium tuberculosis, nonsynonymous mutations are dominant in this group. In this study a collection of 145 M. tuberculosis isolates was used to evaluate the conferring mutations in nucleotide 1388 of katG gene (KatG463) in resistance to isoniazid. A PCR-RFLP method was applied in comparison with DNA sequencing and anti-mycobacterial susceptibility testing. From all studied patients, 98 (67.6%) were men, 47 (32.4%) were women, 3% were <15 and 9% were >65 years old; male to female ratio was 1:2.4. PCR result of katG for a 620-bp amplicon was successful for all purified M. tuberculosis isolates and there was no positive M. tuberculosis culture with PCR negative results (100% specificity). Subsequent PCR RFLP of the katG identified mutation at KatG463 in 33.3%, 57.8% and 59.2% of our clinically susceptible, multidrug resistant TB (MDR) and extensively drug resistant (XDR) isolates, respectively. Strains of H37Rv and Academic had no any mutations in this codon. M. bovis was used as a positive control for mutation in KatG463. Automated DNA sequencing of the katG amplicon from randomly selected INH-susceptible and resistant isolates verified 100% sequence accuracy of the point mutations detected by PCR-RFLP. We concluded that codon 463 was a polymorphic site that is associated to INH resistance (a missense or "quiet" mutation). RFLP results of katG amplicons were identical to those of sequence method. Our PCR-RFLP method has a potential application for rapid diagnosis of M. tuberculosis with a high specificity.     

Can 15-locus mycobacterial interspersed repetitive unit-variable-number tandem repeat analysis provide insight into the evolution of Mycobacterium tuberculosis?

The phylogeny and evolution of the bacterium Mycobacterium tuberculosis is still poorly understood despite the application of a variety of molecular techniques. We analyzed 469 M. tuberculosis and 49 Mycobacterium bovis isolates to evaluate if the mycobacterial interspersed repetitive units-variable-number tandem repeats (MIRU-VNTR) commonly used for epidemiological studies can define the phylogeny of the M. tuberculosis complex. This population was characterized by previously identified silent single-nucleotide polymorphisms (sSNPs) or by a macroarray based on these sSNPs that was developed in this study. MIRU-VNTR phylogenetic codes capable of differentiating between phylogenetic lineages were identified. Overall, there was 90.9% concordance between the lineages of isolates as defined by the MIRU-VNTR and sSNP analyses. The MIRU-VNTR phylogenetic code was unique to M. bovis and was not observed in any M. tuberculosis isolates. The codes were able to differentiate between different M. tuberculosis strain families such as Beijing, Delhi, and East African-Indian. Discrepant isolates with similar but not identical MIRU-VNTR codes often displayed a stepwise trend suggestive of bidirectional evolution. A lineage-specific panel of MIRU-VNTR can be used to subdivide each lineage for epidemiological purposes. MIRU-VNTR is a valuable tool for phylogenetic studies and could define an evolutionarily uncharacterized population of M. tuberculosis complex organisms.     

Zoonotic transmission of tuberculosis between pastoralists and their livestock in South-East Ethiopia

Despite huge global efforts in tuberculosis (TB) control, pastoral areas remain under-investigated. During two years sputum and fine needle aspirate (FNA) specimens were collected from 260 Ethiopian pastoralists of Oromia and Somali Regional States with suspected pulmonary TB and from 32 cases with suspected TB lymphadenitis. In parallel, 207 suspected tuberculous lesions were collected from cattle, camels and goats at abattoirs. All specimens were processed and cultured for mycobacteria; samples with acid-fast stained bacilli (AFB) were further characterized by molecular methods including genus and deletion typing as well as spoligotyping. Non-tuberculous mycobacteria (NTM) were sequenced at the 16S rDNA locus. Culturing of AFB from human sputum and FNA samples gave a yield of 174 (67%) and 9 (28%) isolates, respectively. Molecular typing was performed on 173 of these isolates and 160 were confirmed as Mycobacterium tuberculosis, three as M. bovis, and the remaining 10 were typed as NTMs. Similarly, 48 AFB isolates (23%) yielded from tuberculous lesions of livestock, of which 39 were molecular typed, including 24 M. bovis and 4 NTMs from cattle, 1 M. tuberculosis and 1 NTM from camels and 9 NTMs from goats. Isolation of M. bovis from humans and M. tuberculosis from livestock suggests transmission between livestock and humans in the pastoral areas of South-East Ethiopia.     

痰涂片标本用于结核分枝杆菌耐药性检测的研究

目的 研究抗酸染色结核分枝杆菌(简称结核杆菌)阳性痰涂片标本直接用于耐药性检测的方法。方法 对18株临床分离培养的结核杆菌用利福平进行药敏试验。分别提取菌株DNA和与之对应的痰涂片标本的菌体DNA,用聚合酶链反应(PcR)扩增ropB基因后进行固相杂交和核酸测序检测结核杆菌的耐药性。结果 18株结核杆菌中有12株对利福平耐药。经PCR扩增的ropB片段与探针杂交后,敏感菌株未发现rpoB基因的突变,自耐药菌株提取的DNA中rpoB突变体的检出率为100％(12／12),痰涂片提取DNA的检出率为91．7％(11／12)。所有耐药菌株DNA与痰涂片DNA核酸测序结果相吻合,都有rpoB基因核心区域碱基突变。结论 抗酸染色痰涂片阳性标本可直接用于检测结核杆菌利福平耐药基因rpoB突变体,是一种值得临床实验室推广使用的耐药菌诊断方法。     

A promoter mutation causes differential nitrate reductase activity of Mycobacterium tuberculosis and Mycobacterium bovis

The recent publication of the genome sequence of Mycobacterium bovis showed >99.95% identity to M. tuberculosis. No genes unique to M. bovis were found. Instead numerous single-nucleotide polymorphisms (SNPs) were identified. This has led to the hypothesis that differential gene expression due to SNPs might explain the differences between the human and bovine tubercle bacilli. One phenotypic distinction between M. tuberculosis and M. bovis is nitrate reduction, which not only is an essential diagnostic tool but also contributes to mycobacterial pathogenesis. We previously showed that narGHJI encodes a nitrate reductase in both M. tuberculosis and M. bovis and that NarGHJI-mediated nitrate reductase activity was substantially higher in the human tubercle bacillus. In the present study we used a genetic approach to demonstrate that an SNP within the promoter of the nitrate reductase gene cluster narGHJI is responsible for the different nitrate reductase activity of M. tuberculosis and M. bovis. This is the first example of an SNP that leads to differential gene expression between the human and bovine tubercle bacilli.     

Development and evaluation of triplex PCR for direct detection of mycobacteria in respiratory specimens

AIMS: To develop a method for the simultaneous detection of Mycobacterium tuberculosis, Mycobacterium avium-intracellularecomplex (MAC), and other mycobacteria in clinical specimens using triplex PCR. METHODS AND RESULTS: The target of amplification was the internal transcribed spacer region between the l6S and 23S rDNA genes. Twenty-two mycobacterial type strains, 118 M. tuberculosis, 87 other mycobacteria, 75 nonmycobacterial pathogens, 115 respiratory specimens from confirmed cases of tuberculous or other mycobacterial diseases, and sputa from 50 patients with nonmycobacterial respiratory diseases were tested. In M. tuberculosis, 322 bp pan-mycobacterial and 133 bp species-specific bands were observed. In MAC, the respective bands were 322 and 84 bp. The other mycobacteria showed single pan-mycobacterial bands of approx. 300-350 bp. Nonspecific amplicons were not found in any of the nonmycobacterial pathogens. In the tuberculosis specimens, 96.4% of smear-positive specimens and 70.2% of smear-negative specimens showed positive reactions. Specimens from two patients with MAC infection were MAC positive. Only 1 of 50 specimens from nonmycobacterial diseases was positive (2.0%). CONCLUSION, SIGNIFICANCE AND IMPACT OF THE STUDY: Triplex PCR enables accurate and rapid diagnosis of tuberculosis and probably is useful for the detection of MAC and other mycobacteria in respiratory specimens.     

Role of the pks15/1 gene in the biosynthesis of phenolglycolipids in the Mycobacterium tuberculosis complex. Evidence that all strains synthesize glycosylated p-hydroxybenzoic methyl esters and that strains devoid of phenolglycolipids harbor a frameshift mutation in the pks15/1 gene

Diesters of phthiocerol and phenolphthiocerol are important virulence factors of Mycobacterium tuberculosis and Mycobacterium leprae, the two main mycobacterial pathogens in humans. They are both long-chain beta-diols, and their biosynthetic pathway is beginning to be elucidated. Although the two classes of molecules share a common lipid core, phthiocerol diesters have been found in all the strains of the M. tuberculosis complex examined although phenolphthiocerol diesters are produced by only a few groups of strains. To address the question of the origin of this diversity 8 reference strains and 10 clinical isolates of M. tuberculosis were analyzed. We report the presence of glycosylated p-hydroxybenzoic acid methyl esters, structurally related to the type-specific phenolphthiocerol glycolipids, in the culture media of all reference strains of M. tuberculosis, suggesting that the strains devoid of phenolphthiocerol derivatives are unable to elongate the putative p-hydroxybenzoic acid precursor. We also show that all the strains of M. tuberculosis examined and deficient in the production of phenolphthiocerol derivatives are natural mutants with a frameshift mutation in pks15/1 whereas a single open reading frame for pks15/1 is found in Mycobacterium bovis BCG, M. leprae, and strains of M. tuberculosis that produce phenolphthiocerol derivatives. Complementation of the H37Rv strain of M. tuberculosis, which is devoid of phenolphthiocerol derivatives, with the fused pks15/1 gene from M. bovis BCG restored phenolphthiocerol glycolipids production. Conversely, disruption of the pks15/1 gene in M. bovis BCG led to the abolition of the synthesis of type-specific phenolphthiocerol glycolipid. These data indicate that Pks15/1 is involved in the elongation of p-hydroxybenzoic acid to give p-hydroxyphenylalkanoates, which in turn are converted, presumably by the PpsA-E synthase, to phenolphthiocerol derivatives.