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Sample records for mycobacterium tuberculosis suggest

  1. A functional role of Rv1738 in Mycobacterium tuberculosis persistence suggested by racemic protein crystallography.

    Science.gov (United States)

    Bunker, Richard D; Mandal, Kalyaneswar; Bashiri, Ghader; Chaston, Jessica J; Pentelute, Bradley L; Lott, J Shaun; Kent, Stephen B H; Baker, Edward N

    2015-04-07

    Protein 3D structure can be a powerful predictor of function, but it often faces a critical roadblock at the crystallization step. Rv1738, a protein from Mycobacterium tuberculosis that is strongly implicated in the onset of nonreplicating persistence, and thereby latent tuberculosis, resisted extensive attempts at crystallization. Chemical synthesis of the L- and D-enantiomeric forms of Rv1738 enabled facile crystallization of the D/L-racemic mixture. The structure was solved by an ab initio approach that took advantage of the quantized phases characteristic of diffraction by centrosymmetric crystals. The structure, containing L- and D-dimers in a centrosymmetric space group, revealed unexpected homology with bacterial hibernation-promoting factors that bind to ribosomes and suppress translation. This suggests that the functional role of Rv1738 is to contribute to the shutdown of ribosomal protein synthesis during the onset of nonreplicating persistence of M. tuberculosis.

  2. The Mycobacterium tuberculosis homologue of the Mycobacterium ...

    African Journals Online (AJOL)

    With the completion of genome sequencing of Mycobacterium tuberculosis and upsurge in the incidence of M. tuberculosis infection worldwide partly as a result of HIV pandemic, there is need for rationale approach to vaccine and chemotherapy discoveries for M. tuberculosis. The homologue of mig gene of. Mycobacterium ...

  3. Mycobacterium tuberculosis monoarthritis in a child

    Directory of Open Access Journals (Sweden)

    Rosenberg Alan M

    2008-09-01

    Full Text Available Abstract A child with isolated Mycobacterium tuberculosis monoarthritis, with features initially suggesting oligoarthritis subtype of juvenile idiopathic arthritis, is presented. This patient illustrates the need to consider the possibility of tuberculosis as the cause of oligoarthritis in high-risk pediatric populations even in the absence of a tuberculosis contact history and without evidence of overt pulmonary disease.

  4. Mycobacterium tuberculosis Metabolism

    Science.gov (United States)

    Warner, Digby F.

    2015-01-01

    Metabolism underpins the physiology and pathogenesis of Mycobacterium tuberculosis. However, although experimental mycobacteriology has provided key insights into the metabolic pathways that are essential for survival and pathogenesis, determining the metabolic status of bacilli during different stages of infection and in different cellular compartments remains challenging. Recent advances—in particular, the development of systems biology tools such as metabolomics—have enabled key insights into the biochemical state of M. tuberculosis in experimental models of infection. In addition, their use to elucidate mechanisms of action of new and existing antituberculosis drugs is critical for the development of improved interventions to counter tuberculosis. This review provides a broad summary of mycobacterial metabolism, highlighting the adaptation of M. tuberculosis as specialist human pathogen, and discusses recent insights into the strategies used by the host and infecting bacillus to influence the outcomes of the host–pathogen interaction through modulation of metabolic functions. PMID:25502746

  5. Drug Resistance of Mycobacterium tuberculosis Complex among ...

    African Journals Online (AJOL)

    BACKGROUND: In Burkina Faso, there is no recent data about the level of drug resistance in Mycobacterium tuberculosis strains among newly diagnosed tuberculosis cases. OBJECTIVE: To provide an update of the primary drug resistance of mycobacterium tuberculosis among patients in Burkina faso. METHODS: ...

  6. Targeting phenotypically tolerant Mycobacterium tuberculosis

    Science.gov (United States)

    Gold, Ben; Nathan, Carl

    2016-01-01

    While the immune system is credited with averting tuberculosis in billions of individuals exposed to Mycobacterium tuberculosis, the immune system is also culpable for tempering the ability of antibiotics to deliver swift and durable cure of disease. In individuals afflicted with tuberculosis, host immunity produces diverse microenvironmental niches that support suboptimal growth, or complete growth arrest, of M. tuberculosis. The physiological state of nonreplication in bacteria is associated with phenotypic drug tolerance. Many of these host microenvironments, when modeled in vitro by carbon starvation, complete nutrient starvation, stationary phase, acidic pH, reactive nitrogen intermediates, hypoxia, biofilms, and withholding streptomycin from the streptomycin-addicted strain SS18b, render M. tuberculosis profoundly tolerant to many of the antibiotics that are given to tuberculosis patients in a clinical setting. Targeting nonreplicating persisters is anticipated to reduce the duration of antibiotic treatment and rate of post-treatment relapse. Some promising drugs to treat tuberculosis, such as rifampicin and bedaquiline, only kill nonreplicating M. tuberculosis in vitro at concentrations far greater than their minimal inhibitory concentrations against replicating bacilli. There is an urgent demand to identify which of the currently used antibiotics, and which of the molecules in academic and corporate screening collections, have potent bactericidal action on nonreplicating M. tuberculosis. With this goal, we review methods of high throughput screening to target nonreplicating M. tuberculosis and methods to progress candidate molecules. A classification based on structures and putative targets of molecules that have been reported to kill nonreplicating M. tuberculosis revealed a rich diversity in pharmacophores. However, few of these compounds were tested under conditions that would exclude the impact of adsorbed compound acting during the recovery phase of

  7. In vitro susceptibility of Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium avium, Mycobacterium fortuitum, and Mycobacterium chelonae to ticarcillin in combination with clavulanic acid.

    OpenAIRE

    Casal, M J; Rodriguez, F C; Luna, M D; Benavente, M C

    1987-01-01

    The in vitro susceptibility of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium avium, Mycobacterium fortuitum, and Mycobacterium chelonae (M. chelonei) to ticarcillin in combination with calvulanic acid (CA) was studied by the agar dilution method. All the M. tuberculosis, M. bovis, and M. africanum strains were inhibited at a ticarcillin concentration of 32 micrograms/ml or lower in combination with 5 micrograms of CA. M. chelonae and M. avium strains ...

  8. Associations between Mycobacterium tuberculosis Strains and Phenotypes

    Science.gov (United States)

    Brown, Timothy; Nikolayevskyy, Vladyslav; Velji, Preya

    2010-01-01

    To inform development of tuberculosis (TB) control strategies, we characterized a total of 2,261 Mycobacterium tuberculosis complex isolates by using multiple phenotypic and molecular markers, including polymorphisms in repetitive sequences (spoligotyping and variable-number tandem repeats [VNTRs]) and large sequence and single-nucleotide polymorphisms. The Beijing family was strongly associated with multidrug resistance (p = 0.0001), and VNTR allelic variants showed strong associations with spoligotyping families: >5 copies at exact tandem repeat (ETR) A, >2 at mycobacterial interspersed repetitive unit 24, and >3 at ETR-B associated with the East African–Indian and M. bovis strains. All M. tuberculosis isolates were differentiated into 4 major lineages, and a maximum parsimony tree was constructed suggesting a more complex phylogeny for M. africanum. These findings can be used as a model of pathogen global diversity. PMID:20113558

  9. Comparative Mycobacteriology of the Mycobacterium tuberculosis complex

    OpenAIRE

    Gordon, Stephen V.; Behr, Marcel A.

    2015-01-01

    The Mycobacterium tuberculosis complex (MTBC) is a group of highly genetically related pathogens that cause tuberculosis (TB) in mammalian species. However, the very name of the complex underlines the fact that our knowledge of these pathogens is dominated by studies on the human pathogen, M. tuberculosis. Of course this is entirely justified; M. tuberculosis is a major global pathogen that exacts a horrendous burden in terms of mortality and morbidity so it is appropriate that it is...

  10. Tracing Mycobacterium tuberculosis transmission by whole genome sequencing in a high incidence setting

    DEFF Research Database (Denmark)

    Bjorn-Mortensen, K; Soborg, B; Koch, A

    2016-01-01

    In East Greenland, a dramatic increase of tuberculosis (TB) incidence has been observed in recent years. Classical genotyping suggests a genetically similar Mycobacterium tuberculosis (Mtb) strain population as cause, however, precise transmission patterns are unclear. We performed whole genome...

  11. Radiographic differentiation of atypical tuberculosis from mycobacterium tuberculosis

    International Nuclear Information System (INIS)

    Tarver, R.D.; Pearcy, E.A.; Conces, D.J. Jr.; Mathur, P.N.

    1987-01-01

    The chest radiographs of 95 patients with the new diagnosis of atypical turberculosis were reviewed to determine if any significant differences between atypical tuberculosis and that caused by Mycobacterium tuberculosis could be discerned. Findings included upper lobe involvement in B4 of the 95 patients and cavities in 76, with nearly equal groups having no, moderate, or extensive surrounding alveolar disease. Nodules were common; in six patients a nodule was the sole manifestation of disease. Adenopathy was seen in 12 of the 95 patients, atlectasis in 45, pleural thickening in 90, and effusions in three. These radiographic findings did not allow the radiographic differentiation of atypical tuberculosis from Mycobacterium tuberculosis infection

  12. Polymorphisms of twenty regulatory proteins between Mycobacterium tuberculosis and Mycobacterium bovis

    Science.gov (United States)

    Mycobacterium tuberculosis and Mycobacterium bovis are responsible for tuberculosis in humans or animals, respectively. Both species are closely related and belong to the Mycobacterium tuberculosis complex (MTC). M. tuberculosis is the most ancient species from which M. bovis and the other members o...

  13. Mycobacterium tuberculosis, Beijing genotype strains not associated with radiological presentation of pulmonary tuberculosis

    NARCIS (Netherlands)

    Borgdorff, Martien W.; van Deutekom, Henk; de Haas, Petra E. W.; Kremer, Kristin; van Soolingen, Dick

    2004-01-01

    Mycobacterium tuberculosis strains of the Beijing genotype have been involved in various outbreaks of multidrug-resistant tuberculosis. Some studies suggest that the infection with the Beijing genotype is associated with a different host immune response. Since this might also lead to a different

  14. Detection of Mycobacterium Tuberculosis by using PCR

    International Nuclear Information System (INIS)

    Suhadi, F; Dadang-Sudrajat; Maria-Lina, R.

    1996-01-01

    Polymerase Chain Reaction (PCR) procedure using three primary set derived from repetitive DNA sequence specific to mycobacteria was used to diagnose pathogenic Mycobacterium tuberculosis. The assay was specific for M. tuberculosis and could be used to detect the amount DNA less than 10 -9 g

  15. Safety assessment in primary Mycobacterium tuberculosis smear ...

    African Journals Online (AJOL)

    Introduction Tuberculosis (TB) is caused by Mycobacterium tuberculosis and is transmitted mainly through aerosolization of infected sputum which puts laboratory workers at risk in spite of the laboratory workersf risk of infection being at 3 to 9 times higher than the general public. Laboratory safety should therefore be ...

  16. Peritoneal tuberculosis due to Mycobacterium caprae

    Directory of Open Access Journals (Sweden)

    T. Nebreda

    2016-01-01

    Full Text Available The incidence of tuberculosis in humans due to Mycobacterium caprae is very low and is almost confined to Europe. We report a case of a previously healthy 41-year-old Moroccan with a 6 month history of abdominal pain, weight loss, fatigue and diarrhea. A diagnosis of peritoneal tuberculosis due to M. caprae was made.

  17. Modelling the Transitional Dynamics of Mycobacterium Tuberculosis ...

    African Journals Online (AJOL)

    The World Health Organization's targets of eliminating Tuberculosis (TB) by 2050 is challenged by the emergence and spread of drug resistance TB. However, the traditional mechanism of resistance is that of acquired resistance, whereby the mycobacterium Tuberculosis (MTB) strain develops mutations under selective ...

  18. Characterization of Mycobacterium tuberculosis nicotinamidase/pyrazinamidase.

    Science.gov (United States)

    Zhang, Hua; Deng, Jiao-Yu; Bi, Li-Jun; Zhou, Ya-Feng; Zhang, Zhi-Ping; Zhang, Cheng-Gang; Zhang, Ying; Zhang, Xian-En

    2008-02-01

    The nicotinamidase/pyrazinamidase (PncA) of Mycobacterium tuberculosis is involved in the activation of the important front-line antituberculosis drug pyrazinamide by converting it into the active form, pyrazinoic acid. Mutations in the pncA gene cause pyrazinamide resistance in M. tuberculosis. The properties of M. tuberculosis PncA were characterized in this study. The enzyme was found to be a 20.89 kDa monomeric protein. The optimal pH and temperature of enzymatic activity were pH 7.0 and 40 degrees C, respectively. Inductively coupled plasma-optical emission spectrometry revealed that the enzyme was an Mn(2+)/Fe(2+)-containing protein with a molar ratio of [Mn(2+)] to [Fe(2+)] of 1 : 1; furthermore, the external addition of either type of metal ion had no apparent effect on the wild-type enzymatic activity. The activity of the purified enzyme was determined by HPLC, and it was shown that it possessed similar pyrazinamidase and nicotinamidase activity, by contrast with previous reports. Nine PncA mutants were generated by site-directed mutagenesis. Determination of the enzymatic activity and metal ion content suggested that Asp8, Lys96 and Cys138 were key residues for catalysis, and Asp49, His51, His57 and His71 were essential for metal ion binding. Our data show that M. tuberculosis PncA may bind metal ions in a manner different from that observed in the case of Pyrococcus horikoshii PncA.

  19. Host immunity to Mycobacterium tuberculosis and risk of tuberculosis

    DEFF Research Database (Denmark)

    Michelsen, Sascha Wilk; Soborg, Bolette; Agger, Else-Marie

    2016-01-01

    BACKGROUND: Human immune responses to latent Mycobacterium tuberculosis (Mtb) infection (LTBI) may enable individuals to control Mtb infection and halt progression to tuberculosis (TB), a hypothesis applied in several novel TB vaccines. We aimed to evaluate whether immune responses to selected LTBI...

  20. A Case of False-Positive Mycobacterium tuberculosis Caused by Mycobacterium celatum

    Directory of Open Access Journals (Sweden)

    Edward Gildeh

    2016-01-01

    Full Text Available Mycobacterium celatum is a nontuberculous mycobacterium shown to cause symptoms similar to pulmonary M. tuberculosis. Certain strains have been shown to cross-react with the probes used to detect M. tuberculosis, making this a diagnostic challenge. We present a 56-year-old gentleman who developed signs and symptoms of lung infection with computed tomography scan of the chest showing right lung apex cavitation. Serial sputum samples were positive for acid-fast bacilli and nucleic acid amplification testing identified M. tuberculosis ribosomal RNA, resulting in treatment initiation. Further testing with high performance liquid chromatography showed a pattern consistent with M. celatum. This case illustrates the potential for M. celatum to mimic M. tuberculosis in both its clinical history and laboratory testing due to the identical oligonucleotide sequence contained in both. An increasing number of case reports suggest that early reliable differentiation could reduce unnecessary treatment and public health intervention associated with misdiagnosed tuberculosis.

  1. Infection caused by Mycobacterium tuberculosis.

    Science.gov (United States)

    Peloquin, C A; Berning, S E

    1994-01-01

    To update readers on the clinical management of infections caused by Mycobacterium tuberculosis, to provide a general description of the organism, culture and susceptibility testing, and clinical manifestations of the disease, and to provide several aspects of the treatment of the disease, including historical perspective, current approaches, and research opportunities for the future. The current medical literature, including abstracts presented at recent international meetings, is reviewed. References were identified through MEDLINE, MEDLARS II, Current Contents, and published meeting abstracts. Data regarding the epidemiology, clinical manifestations, culture and susceptibility testing, and treatment of tuberculosis are cited. Specific attention has been focused on the clinical management of patients with noncontagious infection and potentially contagious active disease (TB) caused by M. tuberculosis. Information contributing to the discussion of the topics selected by the authors is reviewed. Data supporting and disputing specific conclusions are presented. The incidence of TB is increasing in the US, despite the fact that available technologies are capable of controlling the vast majority of existing cases. Fueling the fire is the problem of coinfection with HIV and M. tuberculosis. Very few drugs are available for the treatment of TB, and few of these approach the potency of isoniazid and rifampin. Preventive therapy of patients exposed to multiple-drug-resistant M. tuberculosis (MDR-TB) is controversial and of unknown efficacy. Treatment of active disease caused by MDR-TB requires up to four times longer, is associated with increased toxicity, and is far less successful than the treatment of drug-susceptible TB. Strategies for the management of such cases are presented. The rising incidence of TB in the US reflects a breakdown in the healthcare systems responsible for controlling the disease, which reflects the past budgetary reductions. Although TB control

  2. Molecular Epidemiology of Mycobacterium Tuberculosis Strains in ...

    African Journals Online (AJOL)

    Doroudchi M, Kremer K, Basiri EA, Kadivar MR,. Van Soolingen D, Ghaderi AA. IS6110‑RFLP and spoligotyping of Mycobacterium tuberculosis isolates in Iran. Scand J Infect. Dis 2000;32:663‑8. 13. Farnia P, Masjedi MR, Mirsaeidi M, Mohammadi F,. Jallaledin‑Ghanavi, Vincent V, et al. Prevalence of Haarlem I and Beijing ...

  3. Signal peptide prediction suggests Mycobacterium tuberculosis curli pilin subunit secretion via the Sec pathway may hinder MTP overexpression in Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Naidoo, N.; Pillay, B.; Bubb, M.; Kumar, A.; Chiliza, T.; Pillay, M.

    2017-07-01

    Introduction Mycobacterium tuberculosis curli pili (MTP) are novel, potential TB diagnostic biomarkers as they are important virulence attributes, unique to the M. tuberculosis complex (MTBC). The production of high quality recombinant transmembrane and secretory proteins that can serve as biomarkers may be challenging due to their secretion attributes. For example, the signal peptide of MTP governed by the classical secretion pathway may hinder the purification of the protein in E. coli systems. In this study, the secretion characteristics of MTP were determined and the cloning, expression and purification of MTP was attempted in E.coli. Materials and methods A fragment of MTP unique to MTBC was cloned into pet101 and pGEX-6P-1 vectors. The clones were confirmed by nucleotide sequencing and expression of the protein was attempted at IPTG concentrations ranging from 0.1mM to 1mM and at temperatures between 25 °C to 37 °C. The pGEX-6P-1/mtp clone expressed protein was purified, yielding a MTP-GST fusion protein and a free GST band that were analysed by LC/MS mass spectrometry. Inclusion body preparation attempted from the pet101/mtp clone yielded two bands at 10 kDa and below 10 kDa, both of which were analysed by LC/MS mass spectrometry. Transcription activity of both the clones was interrogated by real time PCR on the cDNA derived from the induced clones at various time points after induction with IPTG. The signal peptide and protein secretion characteristics of the MTP protein were determined by bioinformatics analysis of the amino acid sequence using publically available software. Results The truncated MTP fragments were successfully cloned in both the vectors as confirmed by nucleotide sequencing. Expression of the pGEX-6P-1/mtp clone using 0.5 mM IPTG at 27 °C demonstrated the presence of the expected fragment at approximately 35 kDa. This was confirmed by Western Blotting using anti-GST antibodies. However, purification of MTP in adequate quantities as a

  4. Signal peptide prediction suggests Mycobacterium tuberculosis curli pilin subunit secretion via the Sec pathway may hinder MTP overexpression in Escherichia coli

    International Nuclear Information System (INIS)

    Naidoo, N.; Pillay, B.; Bubb, M.; Kumar, A.; Chiliza, T.; Pillay, M.

    2017-01-01

    Introduction Mycobacterium tuberculosis curli pili (MTP) are novel, potential TB diagnostic biomarkers as they are important virulence attributes, unique to the M. tuberculosis complex (MTBC). The production of high quality recombinant transmembrane and secretory proteins that can serve as biomarkers may be challenging due to their secretion attributes. For example, the signal peptide of MTP governed by the classical secretion pathway may hinder the purification of the protein in E. coli systems. In this study, the secretion characteristics of MTP were determined and the cloning, expression and purification of MTP was attempted in E.coli. Materials and methods A fragment of MTP unique to MTBC was cloned into pet101 and pGEX-6P-1 vectors. The clones were confirmed by nucleotide sequencing and expression of the protein was attempted at IPTG concentrations ranging from 0.1mM to 1mM and at temperatures between 25 °C to 37 °C. The pGEX-6P-1/mtp clone expressed protein was purified, yielding a MTP-GST fusion protein and a free GST band that were analysed by LC/MS mass spectrometry. Inclusion body preparation attempted from the pet101/mtp clone yielded two bands at 10 kDa and below 10 kDa, both of which were analysed by LC/MS mass spectrometry. Transcription activity of both the clones was interrogated by real time PCR on the cDNA derived from the induced clones at various time points after induction with IPTG. The signal peptide and protein secretion characteristics of the MTP protein were determined by bioinformatics analysis of the amino acid sequence using publically available software. Results The truncated MTP fragments were successfully cloned in both the vectors as confirmed by nucleotide sequencing. Expression of the pGEX-6P-1/mtp clone using 0.5 mM IPTG at 27 °C demonstrated the presence of the expected fragment at approximately 35 kDa. This was confirmed by Western Blotting using anti-GST antibodies. However, purification of MTP in adequate quantities as a

  5. Siderocalin inhibits the intracellular replication of Mycobacterium tuberculosis in macrophages

    DEFF Research Database (Denmark)

    Johnson, Erin E; Srikanth, Chittur V; Sandgren, Andreas

    2010-01-01

    Siderocalin is a secreted protein that binds to siderophores to prevent bacterial iron acquisition. While it has been shown to inhibit the growth of Mycobacterium tuberculosis (M.tb) in extracellular cultures, its effect on this pathogen within macrophages is not clear. Here, we show that sideroc......Siderocalin is a secreted protein that binds to siderophores to prevent bacterial iron acquisition. While it has been shown to inhibit the growth of Mycobacterium tuberculosis (M.tb) in extracellular cultures, its effect on this pathogen within macrophages is not clear. Here, we show...... findings are consistent with an important role for siderocalin in protection against M.tb infection and suggest that exogenously administered siderocalin may have therapeutic applications in tuberculosis....

  6. Microevolution of Mycobacterium tuberculosis in a tuberculosis patient.

    NARCIS (Netherlands)

    Al-Hajoj, S.A.; Akkerman, O.; Parwati, I.; Al-Gamdi, S.; Rahim, Z.; Soolingen, D. van; Ingen, J. van; Supply, P.; Zanden, A.G. van der

    2010-01-01

    Five Mycobacterium tuberculosis isolates were obtained from three body sites from a Dutch patient. The isolates displayed a single genotype by 24-locus MIRU-VNTR typing (except for a single locus not amplified from one isolate) but were differentiated by small variations in IS6110 fingerprints,

  7. Progression to active tuberculosis, but not transmission, varies by Mycobacterium tuberculosis lineage in The Gambia

    NARCIS (Netherlands)

    de Jong, Bouke C.; Hill, Philip C.; Aiken, Alex; Awine, Timothy; Antonio, Martin; Adetifa, Ifedayo M.; Jackson-Sillah, Dolly J.; Fox, Annette; Deriemer, Kathryn; Gagneux, Sebastien; Borgdorff, Martien W.; McAdam, Keith P. W. J.; Corrah, Tumani; Small, Peter M.; Adegbola, Richard A.

    2008-01-01

    BACKGROUND: There is considerable variability in the outcome of Mycobacterium tuberculosis infection. We hypothesized that Mycobacterium africanum was less likely than M. tuberculosis to transmit and progress to tuberculosis disease. METHODS: In a cohort study of patients with tuberculosis and their

  8. A non-sense mutation in the putative anti-mutator gene ada/alkA of Mycobacterium tuberculosis and M. bovis isolates suggests convergent evolution

    Directory of Open Access Journals (Sweden)

    Gicquel Brigitte

    2007-05-01

    Full Text Available Abstract Background Previous studies have suggested that variations in DNA repair genes of W-Beijing strains may have led to transient mutator phenotypes which in turn may have contributed to host adaptation of this strain family. Single nucleotide polymorphism (SNP in the DNA repair gene mutT1 was identified in MDR-prone strains from the Central African Republic. A Mycobacteriumtuberculosis H37Rv mutant inactivated in two DNA repair genes, namely ada/alkA and ogt, was shown to display a hypermutator phenotype. We then looked for polymorphisms in these genes in Central African Republic strains (CAR. Results In this study, 55 MDR and 194 non-MDR strains were analyzed. Variations in DNA repair genes ada/alkA and ogt were identified. Among them, by comparison to M. tuberculosis published sequences, we found a non-sense variation in ada/alkA gene which was also observed in M. bovis AF2122 strain. SNPs that are present in the adjacent regions to the amber variation are different in M. bovis and in M. tuberculosis strain. Conclusion An Amber codon was found in the ada/alkA locus of clustered M. tuberculosis isolates and in M. bovis strain AF2122. This is likely due to convergent evolution because SNP differences between strains are incompatible with horizontal transfer of an entire gene. This suggests that such a variation may confer a selective advantage and be implicated in hypermutator phenotype expression, which in turn contributes to adaptation to environmental changes.

  9. Three-dimensional models of Mycobacterium tuberculosis proteins Rv1555, Rv1554 and their docking analyses with sildenafil, tadalafil, vardenafil drugs, suggest interference with quinol binding likely to affect protein's function.

    Science.gov (United States)

    Dash, Pallabini; Bala Divya, M; Guruprasad, Lalitha; Guruprasad, Kunchur

    2018-04-18

    Earlier based on bioinformatics analyses, we had predicted the Mycobacterium tuberculosis (M.tb) proteins; Rv1555 and Rv1554, among the potential new tuberculosis drug targets. According to the 'TB-drugome' the Rv1555 protein is 'druggable' with sildenafil (Viagra), tadalafil (Cialis) and vardenafil (Levitra) drugs. In the present work, we intended to understand via computer modeling studies, how the above drugs are likely to inhibit the M.tb protein's function. The three-dimensional computer models for M.tb proteins; Rv1555 and Rv1554 constructed on the template of equivalent membrane anchor subunits of the homologous E.coli quinol fumarate reductase respiratory protein complex, followed by drug docking analyses, suggested that the binding of above drugs interferes with quinol binding sites. Also, we experimentally observed the in-vitro growth inhibition of E.coli bacteria containing the homologous M.tb protein sequences with sildenafil and tadalafil drugs. The predicted binding sites of the drugs is likely to affect the above M.tb proteins function as quinol binding is known to be essential for electron transfer function during anaerobic respiration in the homologous E.coli protein complex. Therefore, sildenafil and related drugs currently used in the treatment of male erectile dysfunction targeting the human phosphodiesterase 5 enzyme may be evaluated for their plausible role as repurposed drugs to treat human tuberculosis.

  10. [Increased IL-4 production in response to virulent Mycobacterium tuberculosis in tuberculosis patients with advanced disease].

    Science.gov (United States)

    Ordway, Diane J; Martins, Marta S; Costa, Leonor M; Freire, Mónica S; Arroz, Maria J; Dockrell, Hazel M; Ventura, Fernando A

    2005-01-01

    The study was designed to compare immune responses to Mycobacterium tuberculosis bacilli and antigens in healthy Portuguese subjects and pulmonary tuberculosis patients (TB), and to correlate immune status with clinical severity of tuberculosis disease. PBMC were cultured and stimulated with live and killed M. tuberculosis H37Rv and purified protein derivative (PPD) and lymphoproliferation and production of IFN-gamma and IL-5/IL-4 by these cultures were evaluated by the use of ELISA and multi-parameter flow cytometry. PBMC from 30 tuberculosis patients demonstrated significantly reduced amounts of proliferation and IFN-gamma when stimulated with live M. tuberculosis compared the control group. Of 15 tuberculosis patients tested for intracellular IL-4 following stimulation with M. tuberculosis, 7 showed greatly increased IL-4 production in CD8+ and gammadelta+ T cells. Tuberculosis patients demonstrated an increase of intracellular IL-4 after PBMC were stimulated with live M. tuberculosis in the CD4+ phenotype, but more notably in CD8+ and gammadelta TCR+ subsets. Increased production of IL-4 in tuberculosis patients was primarily in individuals with advanced involvement of lung parenchymal with high bacterial loads in sputum. These results suggest that an alteration in type 1 and type 2 cytokine balance can occur in patients with tuberculosis at an advanced clinical stage of disease.

  11. 21 CFR 866.3370 - Mycobacterium tuberculosis immunofluorescent reagents.

    Science.gov (United States)

    2010-04-01

    ... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents... used to identify Mycobacterium tuberculosis directly from clinical specimens. The identification aids...

  12. Virulence factors of the Mycobacterium tuberculosis complex

    Science.gov (United States)

    Forrellad, Marina A.; Klepp, Laura I.; Gioffré, Andrea; Sabio y García, Julia; Morbidoni, Hector R.; Santangelo, María de la Paz; Cataldi, Angel A.; Bigi, Fabiana

    2013-01-01

    The Mycobacterium tuberculosis complex (MTBC) consists of closely related species that cause tuberculosis in both humans and animals. This illness, still today, remains to be one of the leading causes of morbidity and mortality throughout the world. The mycobacteria enter the host by air, and, once in the lungs, are phagocytated by macrophages. This may lead to the rapid elimination of the bacillus or to the triggering of an active tuberculosis infection. A large number of different virulence factors have evolved in MTBC members as a response to the host immune reaction. The aim of this review is to describe the bacterial genes/proteins that are essential for the virulence of MTBC species, and that have been demonstrated in an in vivo model of infection. Knowledge of MTBC virulence factors is essential for the development of new vaccines and drugs to help manage the disease toward an increasingly more tuberculosis-free world. PMID:23076359

  13. Buoyant density of Mycobacterium tuberculosis: implications for sputum processing

    NARCIS (Netherlands)

    den Hertog, A. L.; Klatser, P. R.; Anthony, R. M.

    2009-01-01

    A tuberculosis (TB) research laboratory in the Netherlands. The concentration of Mycobacterium tuberculosis cells from sputum is almost universally performed by centrifugation after chemical liquefaction. These methods are thus dependent on the effective sedimentation of mycobacterial cells, and the

  14. Characterization of a Mycobacterium leprae antigen related to the secreted Mycobacterium tuberculosis protein MPT32

    NARCIS (Netherlands)

    Wieles, B.; van Agterveld, M.; Janson, A.; Clark-Curtiss, J.; Rinke de Wit, T.; Harboe, M.; Thole, J.

    1994-01-01

    Secreted proteins may serve as major targets in the immune response to mycobacteria. To identify potentially secreted Mycobacterium leprae antigens, antisera specific for culture filtrate proteins of Mycobacterium tuberculosis were used to screen a panel of recombinant antigens selected previously

  15. Mycobacterium tuberculosis effectors interfering host apoptosis signaling.

    Science.gov (United States)

    Liu, Minqiang; Li, Wu; Xiang, Xiaohong; Xie, Jianping

    2015-07-01

    Tuberculosis remains a serious human public health concern. The coevolution between its pathogen Mycobacterium tuberculosis and human host complicated the way to prevent and cure TB. Apoptosis plays subtle role in this interaction. The pathogen endeavors to manipulate the apoptosis via diverse effectors targeting key signaling nodes. In this paper, we summarized the effectors pathogen used to subvert the apoptosis, such as LpqH, ESAT-6/CFP-10, LAMs. The interplay between different forms of cell deaths, such as apoptosis, autophagy, necrosis, is also discussed with a focus on the modes of action of effectors, and implications for better TB control.

  16. Tuberculosis patients co-infected with Mycobacterium bovis and Mycobacterium tuberculosis in an urban area of Brazil.

    Science.gov (United States)

    Silva, Marcio Roberto; Rocha, Adalgiza da Silva; da Costa, Ronaldo Rodrigues; de Alencar, Andrea Padilha; de Oliveira, Vania Maria; Fonseca Júnior, Antônio Augusto; Sales, Mariana Lázaro; Issa, Marina de Azevedo; Filho, Paulo Martins Soares; Pereira, Omara Tereza Vianello; dos Santos, Eduardo Calazans; Mendes, Rejane Silva; Ferreira, Angela Maria de Jesus; Mota, Pedro Moacyr Pinto Coelho; Suffys, Philip Noel; Guimarães, Mark Drew Crosland

    2013-05-01

    In this cross-sectional study, mycobacteria specimens from 189 tuberculosis (TB) patients living in an urban area in Brazil were characterised from 2008-2010 using phenotypic and molecular speciation methods (pncA gene and oxyR pseudogene analysis). Of these samples, 174 isolates simultaneously grew on Löwenstein-Jensen (LJ) and Stonebrink (SB)-containing media and presented phenotypic and molecular profiles of Mycobacterium tuberculosis, whereas 12 had molecular profiles of M. tuberculosis based on the DNA analysis of formalin-fixed paraffin wax-embedded tissue samples (paraffin blocks). One patient produced two sputum isolates, the first of which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, and the second of which only grew on SB media and presented phenotypic profiles of Mycobacterium bovis. One patient provided a bronchial lavage isolate, which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, but had molecular profiles of M. bovis from paraffin block DNA analysis, and one sample had molecular profiles of M. tuberculosis and M. bovis identified from two distinct paraffin blocks. Moreover, we found a low prevalence (1.6%) of M. bovis among these isolates, which suggests that local health service procedures likely underestimate its real frequency and that it deserves more attention from public health officials.

  17. Tuberculosis patients co-infected with Mycobacterium bovis and Mycobacterium tuberculosis in an urban area of Brazil

    Directory of Open Access Journals (Sweden)

    Marcio Roberto Silva

    2013-05-01

    Full Text Available In this cross-sectional study, mycobacteria specimens from 189 tuberculosis (TB patients living in an urban area in Brazil were characterised from 2008-2010 using phenotypic and molecular speciation methods (pncA gene and oxyR pseudogene analysis. Of these samples, 174 isolates simultaneously grew on Löwenstein-Jensen (LJ and Stonebrink (SB-containing media and presented phenotypic and molecular profiles of Mycobacterium tuberculosis, whereas 12 had molecular profiles of M. tuberculosis based on the DNA analysis of formalin-fixed paraffin wax-embedded tissue samples (paraffin blocks. One patient produced two sputum isolates, the first of which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, and the second of which only grew on SB media and presented phenotypic profiles of Mycobacterium bovis. One patient provided a bronchial lavage isolate, which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, but had molecular profiles of M. bovis from paraffin block DNA analysis, and one sample had molecular profiles of M. tuberculosis and M. bovis identified from two distinct paraffin blocks. Moreover, we found a low prevalence (1.6% of M. bovis among these isolates, which suggests that local health service procedures likely underestimate its real frequency and that it deserves more attention from public health officials.

  18. Beta-lactamases of Mycobacterium tuberculosis and Mycobacterium kansasii.

    Science.gov (United States)

    Segura, C; Salvadó, M

    1997-09-01

    Re-emergence of infectious diseases caused by mycobacteria as well as the emergence of multiresistant strains of Mycobacterium has promoted the research on the use of beta-lactames in the treatment of such diseases. Mycobacteria produce beta-lactamases: M. tuberculosis produces a wide-spectrum beta-lactamase whose behaviour mimicks those of Gram-negative bacteria. M. kansasii produces also beta-lactamase which can be inhibited by clavulanic acid. An overview on beta-lactamases from both species is reported.

  19. Risk factors for Mycobacterium tuberculosis infection among children in Greenland

    DEFF Research Database (Denmark)

    Søborg, Bolette; Andersen, Aase Bengaard; Melbye, Mads

    2011-01-01

    To examine the risk factors for Mycobacterium tuberculosis infection (MTI) among Greenlandic children for the purpose of identifying those at highest risk of infection.......To examine the risk factors for Mycobacterium tuberculosis infection (MTI) among Greenlandic children for the purpose of identifying those at highest risk of infection....

  20. DNA repair in Mycobacterium tuberculosis revisited.

    Science.gov (United States)

    Dos Vultos, Tiago; Mestre, Olga; Tonjum, Tone; Gicquel, Brigitte

    2009-05-01

    Our understanding of Mycobacterium tuberculosis DNA repair mechanisms is still poor compared with that of other bacterial organisms. However, the publication of the first complete M. tuberculosis genome sequence 10 years ago boosted the study of DNA repair systems in this organism. A first step in the elucidation of M. tuberculosis DNA repair mechanisms was taken by Mizrahi and Andersen, who identified homologs of genes involved in the reversal or repair of DNA damage in Escherichia coli and related organisms. Genes required for nucleotide excision repair, base excision repair, recombination, and SOS repair and mutagenesis were identified. Notably, no homologs of genes involved in mismatch repair were identified. Novel characteristics of the M. tuberculosis DNA repair machinery have been found over the last decade, such as nonhomologous end joining, the presence of Mpg, ERCC3 and Hlr - proteins previously presumed to be produced exclusively in mammalian cells - and the recently discovered bifunctional dCTP deaminase:dUTPase. The study of these systems is important to develop therapeutic agents that can counteract M. tuberculosis evolutionary changes and to prevent adaptive events resulting in antibiotic resistance. This review summarizes our current understanding of the M. tuberculosis DNA repair system.

  1. Phenotypic assays for Mycobacterium tuberculosis infection.

    Science.gov (United States)

    Song, Ok-Ryul; Deboosere, Nathalie; Delorme, Vincent; Queval, Christophe J; Deloison, Gaspard; Werkmeister, Elisabeth; Lafont, Frank; Baulard, Alain; Iantomasi, Raffaella; Brodin, Priscille

    2017-10-01

    Tuberculosis (TB) is still a major global threat, killing more than one million persons each year. With the constant increase of Mycobacterium tuberculosis strains resistant to first- and second-line drugs, there is an urgent need for the development of new drugs to control the propagation of TB. Although screenings of small molecules on axenic M. tuberculosis cultures were successful for the identification of novel putative anti-TB drugs, new drugs in the development pipeline remains scarce. Host-directed therapy may represent an alternative for drug development against TB. Indeed, M. tuberculosis has multiple specific interactions within host phagocytes, which may be targeted by small molecules. In order to enable drug discovery strategies against microbes residing within host macrophages, we developed multiple fluorescence-based HT/CS phenotypic assays monitoring the intracellular replication of M. tuberculosis as well as its intracellular trafficking. What we propose here is a population-based, multi-parametric analysis pipeline that can be used to monitor the intracellular fate of M. tuberculosis and the dynamics of cellular events such as phagosomal maturation (acidification and permeabilization), zinc poisoning system or lipid body accumulation. Such analysis allows the quantification of biological events considering the host-pathogen interplay and may thus be derived to other intracellular pathogens. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

  2. Lymphatic endothelial cells are a replicative niche for Mycobacterium tuberculosis

    Science.gov (United States)

    Lerner, Thomas R.; de Souza Carvalho-Wodarz, Cristiane; Repnik, Urska; Russell, Matthew R.G.; Borel, Sophie; Diedrich, Collin R.; Rohde, Manfred; Wainwright, Helen; Collinson, Lucy M.; Wilkinson, Robert J.; Griffiths, Gareth; Gutierrez, Maximiliano G.

    2016-01-01

    In extrapulmonary tuberculosis, the most common site of infection is within the lymphatic system, and there is growing recognition that lymphatic endothelial cells (LECs) are involved in immune function. Here, we identified LECs, which line the lymphatic vessels, as a niche for Mycobacterium tuberculosis in the lymph nodes of patients with tuberculosis. In cultured primary human LECs (hLECs), we determined that M. tuberculosis replicates both in the cytosol and within autophagosomes, but the bacteria failed to replicate when the virulence locus RD1 was deleted. Activation by IFN-γ induced a cell-autonomous response in hLECs via autophagy and NO production that restricted M. tuberculosis growth. Thus, depending on the activation status of LECs, autophagy can both promote and restrict replication. Together, these findings reveal a previously unrecognized role for hLECs and autophagy in tuberculosis pathogenesis and suggest that hLECs are a potential niche for M. tuberculosis that allows establishment of persistent infection in lymph nodes. PMID:26901813

  3. Radiometric diagnosis of Mycobacterium tuberculosis

    International Nuclear Information System (INIS)

    Laszlo, A.

    1986-01-01

    The results of this study confirm that rapid radiometric diagnostic tests such as the NAP selective inhibition test for the M. tuberculosis complex followed by the radiometric drug susceptibility tests are extremely reliable and compare favourably with conventional methodologies. This study also shows that referred cultures growing on solid medium can be processed by radiometric procedures without prior subculture. This circumstance by itself shortens the time needed for reporting. (Auth.)

  4. Distinct Spatiotemporal Dynamics of Peptidoglycan Synthesis between Mycobacterium smegmatis and Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Helene Botella

    2017-09-01

    Full Text Available Peptidoglycan (PG, a polymer cross-linked by d-amino acid-containing peptides, is an essential component of the bacterial cell wall. We found that a fluorescent d-alanine analog (FDAA incorporates chiefly at one of the two poles in Mycobacterium smegmatis but that polar dominance varies as a function of the cell cycle in Mycobacterium tuberculosis: immediately after cytokinesis, FDAAs are incorporated chiefly at one of the two poles, but just before cytokinesis, FDAAs are incorporated comparably at both. These observations suggest that mycobacterial PG-synthesizing enzymes are localized in functional compartments at the poles and septum and that the capacity for PG synthesis matures at the new pole in M. tuberculosis. Deeper knowledge of the biology of mycobacterial PG synthesis may help in discovering drugs that disable previously unappreciated steps in the process.

  5. Immune Responses Involved in Mycobacterium Tuberculosis Infection

    Directory of Open Access Journals (Sweden)

    Roghayeh Teimourpour

    2016-09-01

    Full Text Available Background and Objectives: Mycobacterium tuberculosis is the causative agent of tuberculosis (TB. Approximately one-third of the world's population is infected with M. tuberculosis. Despite the availability of drug and vaccine, it remains one of the leading causes of death in humans especially in developing countries. Epidemiological studies have indicated that only 10-30% of people exposed to tubercle bacillus are infected with M. tuberculosis, and at least 90% of the infected people finally do not acquire TB. The studies have indicated that the host efficient immune system has essential roles in the control of TB infection such that the highest rate of mortality and morbidity is seen in immunocompromised patients such as people infected with HIV. M. tuberculosis is an obligatory intracellular bacterium. It enters the body mainly through the respiratory tract and alveolar macrophages combat this pathogen most commonly. In addition to alveolar macrophages, various T-cell subpopulations need to be activated to overcome this bacterium's resistance to the host defense systems. CD4+ T cells, through production of several cytokines such as IFN-γ and TNF-α, and CD8+ T cells, through cytotoxic activities and induction of apoptosis in infected cells, play critical roles in inducing appropriate immune responses against M. tuberculosis. Although cell-mediated immunity is the cornerstone of host responses against TB and the recent studies have provided evidence for the importance of humoral and innate immune system in the control of TB, a profound understanding of the immune responses would provide a basis for development of new generations of vaccines and drugs. The present study addresses immune responses involved in M. tuberculosis infection.

  6. Uracil excision repair in Mycobacterium tuberculosis cell-free extracts.

    Science.gov (United States)

    Kumar, Pradeep; Bharti, Sanjay Kumar; Varshney, Umesh

    2011-05-01

    Uracil excision repair is ubiquitous in all domains of life and initiated by uracil DNA glycosylases (UDGs) which excise the promutagenic base, uracil, from DNA to leave behind an abasic site (AP-site). Repair of the resulting AP-sites requires an AP-endonuclease, a DNA polymerase, and a DNA ligase whose combined activities result in either short-patch or long-patch repair. Mycobacterium tuberculosis, the causative agent of tuberculosis, has an increased risk of accumulating uracils because of its G + C-rich genome, and its niche inside host macrophages where it is exposed to reactive nitrogen and oxygen species, two major causes of cytosine deamination (to uracil) in DNA. In vitro assays to study DNA repair in this important human pathogen are limited. To study uracil excision repair in mycobacteria, we have established assay conditions using cell-free extracts of M. tuberculosis and M. smegmatis (a fast-growing mycobacterium) and oligomer or plasmid DNA substrates. We show that in mycobacteria, uracil excision repair is completed primarily via long-patch repair. In addition, we show that M. tuberculosis UdgB, a newly characterized family 5 UDG, substitutes for the highly conserved family 1 UDG, Ung, thereby suggesting that UdgB might function as backup enzyme for uracil excision repair in mycobacteria. Copyright © 2011 Elsevier Ltd. All rights reserved.

  7. Mycobacterium Tuberculosis Pyomyositis in an Infant

    Science.gov (United States)

    Malik, ZA; Shehab, M

    2013-01-01

    Mycobacterium tuberculosis is endemic to many parts of the world. It may have variable clinical presentations, especially in the pediatric age group. Presented here is the case of a 9-month old infant who was referred for infectious disease opinion when his thigh induration failed to improve after surgical drainage and a course of oral antibiotic therapy. Mycobacterial PCR on the operative sample fluid was found to be positive; and mycobacterial culture grew M. tuberculosis. He received 9 months of treatment with anti-TB medications, with excellent results and complete recovery. This is the first report of TB pyomyositis in an infant; and highlights the need to have a high index of suspicion for unusual organisms when conventional therapy fails to demonstrate expected results. PMID:23919207

  8. Consequences of genomic diversity in Mycobacterium tuberculosis

    Science.gov (United States)

    Coscolla, Mireia; Gagneux, Sebastien

    2014-01-01

    The causative agent of human tuberculosis, Mycobacterium tuberculosis complex (MTBC), comprises seven phylogenetically distinct lineages associated with different geographical regions. Here we review the latest findings on the nature and amount of genomic diversity within and between MTBC lineages. We then review recent evidence for the effect of this genomic diversity on mycobacterial phenotypes measured experimentally and in clinical settings. We conclude that overall, the most geographically widespread Lineage 2 (includes Beijing) and Lineage 4 (also known as Euro-American) are more virulent than other lineages that are more geographically restricted. This increased virulence is associated with delayed or reduced pro-inflammatory host immune responses, greater severity of disease, and enhanced transmission. Future work should focus on the interaction between MTBC and human genetic diversity, as well as on the environmental factors that modulate these interactions. PMID:25453224

  9. Comparison of the UDP-N-Acetylmuramate:l-Alanine Ligase Enzymes from Mycobacterium tuberculosis and Mycobacterium leprae

    Science.gov (United States)

    Mahapatra, Sebabrata; Crick, Dean C.; Brennan, Patrick J.

    2000-01-01

    In the peptidoglycan of Mycobacterium leprae, l-alanine of the side chain is replaced by glycine. When expressed in Escherichia coli, MurC (UDP-N-acetyl-muramate:l-alanine ligase) of M. leprae showed Km and Vmax for l-alanine and glycine similar to those of Mycobacterium tuberculosis MurC, suggesting that another explanation should be sought for the presence of glycine. PMID:11073931

  10. Comparison of the UDP-N-Acetylmuramate:l-Alanine Ligase Enzymes from Mycobacterium tuberculosis and Mycobacterium leprae

    OpenAIRE

    Mahapatra, Sebabrata; Crick, Dean C.; Brennan, Patrick J.

    2000-01-01

    In the peptidoglycan of Mycobacterium leprae, l-alanine of the side chain is replaced by glycine. When expressed in Escherichia coli, MurC (UDP-N-acetyl-muramate:l-alanine ligase) of M. leprae showed Km and Vmax for l-alanine and glycine similar to those of Mycobacterium tuberculosis MurC, suggesting that another explanation should be sought for the presence of glycine.

  11. Automatic identification of tuberculosis mycobacterium

    Directory of Open Access Journals (Sweden)

    Cicero Ferreira Fernandes Costa Filho

    Full Text Available Introduction According to the Global TB control report of 2013, “Tuberculosis (TB remains a major global health problem. In 2012, an estimated 8.6 million people developed TB and 1.3 million died from the disease. Two main sputum smear microscopy techniques are used for TB diagnosis: Fluorescence microscopy and conventional microscopy. Fluorescence microscopy is a more expensive diagnostic method because of the high costs of the microscopy unit and its maintenance. Therefore, conventional microscopy is more appropriate for use in developing countries. Methods This paper presents a new method for detecting tuberculosis bacillus in conventional sputum smear microscopy. The method consists of two main steps, bacillus segmentation and post-processing. In the first step, the scalar selection technique was used to select input variables for the segmentation classifiers from four color spaces. Thirty features were used, including the subtractions of the color components of different color spaces. In the post-processing step, three filters were used to separate bacilli from artifact: a size filter, a geometric filter and a Rule-based filter that uses the components of the RGB color space. Results In bacillus identification, an overall sensitivity of 96.80% and an error rate of 3.38% were obtained. An image database with 120-sputum-smear microscopy slices of 12 patients with objects marked as bacillus, agglomerated bacillus and artifact was generated and is now available online. Conclusions The best results were obtained with a support vector machine in bacillus segmentation associated with the application of the three post-processing filters.

  12. Immunological crossreactivity of the Mycobacterium leprae CFP-10 with its homologue in Mycobacterium tuberculosis

    NARCIS (Netherlands)

    Geluk, A.; van Meijgaarden, K. E.; Franken, K. L. M. C.; Wieles, B.; Arend, S. M.; Faber, W. R.; Naafs, B.; Ottenhoff, T. H. M.

    2004-01-01

    Mycobacterium tuberculosis culture filtrate protein-10 (CFP-10) (Rv3874) is considered a promising antigen for the immunodiagnosis of tuberculosis (TB) together with early secreted antigens of M. tuberculosis (ESAT-6). Both ESAT-6 and CFP-10 are encoded by the RD1 region that is deleted from all

  13. Rapid Detection of Cell-Free Mycobacterium tuberculosis DNA in Tuberculous Pleural Effusion.

    Science.gov (United States)

    Che, Nanying; Yang, Xinting; Liu, Zichen; Li, Kun; Chen, Xiaoyou

    2017-05-01

    Tuberculous pleurisy is one of the most common types of extrapulmonary tuberculosis, but its diagnosis remains difficult. In this study, we report for the first time on the detection of cell-free Mycobacterium tuberculosis DNA in pleural effusion and an evaluation of a newly developed molecular assay for the detection of cell-free Mycobacterium tuberculosis DNA. A total of 78 patients with pleural effusion, 60 patients with tuberculous pleurisy, and 18 patients with alternative diseases were included in this study. Mycobacterial culture, the Xpert MTB/RIF assay, the adenosine deaminase assay, the T-SPOT.TB assay, and the cell-free Mycobacterium tuberculosis DNA assay were performed on all the pleural effusion samples. The cell-free Mycobacterium tuberculosis DNA assay and adenosine deaminase assay showed significantly higher sensitivities of 75.0% and 68.3%, respectively, than mycobacterial culture and the Xpert MTB/RIF assay, which had sensitivities of 26.7% and 20.0%, respectively ( P pleural effusion showed the highest sensitivity of 95.0% but the lowest specificity of 38.9%. The cell-free Mycobacterium tuberculosis DNA assay detected as few as 1.25 copies of IS 6110 per ml of pleural effusion and showed good accordance of the results between repeated tests ( r = 0.978, P = 2.84 × 10 -10 ). These data suggest that the cell-free Mycobacterium tuberculosis DNA assay is a rapid and accurate molecular test which provides direct evidence of Mycobacterium tuberculosis etiology. Copyright © 2017 American Society for Microbiology.

  14. Variable host-pathogen compatibility in Mycobacterium tuberculosis.

    NARCIS (Netherlands)

    Gagneux, Sebastien; DeRiemer, Kathryn; Van, Tran; Kato-Maeda, Midori; Jong, Bouke C de; Narayanan, Sujatha; Nicol, Mark; Niemann, Stefan; Kremer, Kristin; Gutierrez, M Cristina; Hilty, Markus; Hopewell, Philip C; Small, Peter M

    2006-01-01

    Mycobacterium tuberculosis remains a major cause of morbidity and mortality worldwide. Studies have reported human pathogens to have geographically structured population genetics, some of which have been linked to ancient human migrations. However, no study has addressed the potential evolutionary

  15. A robust SNP barcode for typing Mycobacterium tuberculosis complex strains

    KAUST Repository

    Coll, Francesc; McNerney, Ruth; Guerra-Assunç ã o, José Afonso; Glynn, Judith R.; Perdigã o, Joã o; Viveiros, Miguel; Portugal, Isabel; Pain, Arnab; Martin, Nigel; Clark, Taane G.

    2014-01-01

    Strain-specific genomic diversity in the Mycobacterium tuberculosis complex (MTBC) is an important factor in pathogenesis that may affect virulence, transmissibility, host response and emergence of drug resistance. Several systems have been proposed

  16. Drug susceptibility testing of Mycobacterium tuberculosis to fluoroquinolones

    DEFF Research Database (Denmark)

    Johansen, I S; Larsen, A R; Sandven, P

    2003-01-01

    In the first attempt to establish a quality assurance programme for susceptibility testing of Mycobacterium tuberculosis to fluoroquinolones, 20 strains with different fluoroquinolone susceptibility patterns were distributed by the Supranational Reference Laboratory in Stockholm to the other...

  17. Bloodstream Infections with Mycobacterium tuberculosis among HIV patients

    Centers for Disease Control (CDC) Podcasts

    This podcast looks at bloodstream infections with Mycobacterium tuberculosis and other pathogens among outpatients infected with HIV in Southeast Asia. CDC health scientist Kimberly McCarthy discusses the study and why bloodstream infections occur in HIV-infected populations.

  18. Insights on the Emergence of Mycobacterium tuberculosis from the Analysis of Mycobacterium kansasii

    Science.gov (United States)

    Wang, Joyce; McIntosh, Fiona; Radomski, Nicolas; Dewar, Ken; Simeone, Roxane; Enninga, Jost; Brosch, Roland; Rocha, Eduardo P.; Veyrier, Frédéric J.; Behr, Marcel A.

    2015-01-01

    By phylogenetic analysis, Mycobacterium kansasii is closely related to Mycobacterium tuberculosis. Yet, although both organisms cause pulmonary disease, M. tuberculosis is a global health menace, whereas M. kansasii is an opportunistic pathogen. To illuminate the differences between these organisms, we have sequenced the genome of M. kansasii ATCC 12478 and its plasmid (pMK12478) and conducted side-by-side in vitro and in vivo investigations of these two organisms. The M. kansasii genome is 6,432,277 bp, more than 2 Mb longer than that of M. tuberculosis H37Rv, and the plasmid contains 144,951 bp. Pairwise comparisons reveal conserved and discordant genes and genomic regions. A notable example of genomic conservation is the virulence locus ESX-1, which is intact and functional in the low-virulence M. kansasii, potentially mediating phagosomal disruption. Differences between these organisms include a decreased predicted metabolic capacity, an increased proportion of toxin–antitoxin genes, and the acquisition of M. tuberculosis-specific genes in the pathogen since their common ancestor. Consistent with their distinct epidemiologic profiles, following infection of C57BL/6 mice, M. kansasii counts increased by less than 10-fold over 6 weeks, whereas M. tuberculosis counts increased by over 10,000-fold in just 3 weeks. Together, these data suggest that M. kansasii can serve as an image of the environmental ancestor of M. tuberculosis before its emergence as a professional pathogen, and can be used as a model organism to study the switch from an environmental opportunistic pathogen to a professional host-restricted pathogen. PMID:25716827

  19. Origins and properties of Mycobacterium tuberculosis isolates in London.

    Science.gov (United States)

    Dale, Jeremy W; Bothamley, Graham H; Drobniewski, Francis; Gillespie, Stephen H; McHugh, Timothy D; Pitman, Richard

    2005-06-01

    Using similarities of IS6110 banding patterns, isolates of Mycobacterium tuberculosis from a population-based study in London were assigned to 12 large groups termed 'superfamilies' (sfams). Analysis of patient data showed a marked geographical association in the distribution of these sfams. In particular, isolates from patients born in Europe were from different sfams than those born elsewhere, indicating that there had been relatively little transmission of tuberculosis in London from immigrant communities into the endogenous population. Multivariate analysis showed that certain sfams were significantly associated with pulmonary rather than extrapulmonary disease, or with sputum smear negativity, independently of country of birth or ethnicity, suggesting that the properties of the infecting organism play a role in the nature of the disease process.

  20. Mycobacterium bovis and Other Uncommon Members of the Mycobacterium tuberculosis Complex.

    Science.gov (United States)

    Esteban, Jaime; Muñoz-Egea, Maria-Carmen

    2016-12-01

    Since its discovery by Theobald Smith, Mycobacterium bovis has been a human pathogen closely related to animal disease. At present, M. bovis tuberculosis is still a problem of importance in many countries and is considered the main cause of zoonotic tuberculosis throughout the world. Recent development of molecular epidemiological tools has helped us to improve our knowledge about transmission patterns of this organism, which causes a disease indistinguishable from that caused by Mycobacterium tuberculosis. Diagnosis and treatment of this mycobacterium are similar to those for conventional tuberculosis, with the important exceptions of constitutive resistance to pyrazinamide and the fact that multidrug-resistant and extremely drug-resistant M. bovis strains have been described. Among other members of this complex, Mycobacterium africanum is the cause of many cases of tuberculosis in West Africa and can be found in other areas mainly in association with immigration. M. bovis BCG is the currently available vaccine for tuberculosis, but it can cause disease in some patients. Other members of the M. tuberculosis complex are mainly animal pathogens with only exceptional cases of human disease, and there are even some strains, like "Mycobacterium canettii," which is a rare human pathogen that could have an important role in the knowledge of the evolution of tuberculosis in the history.

  1. The Use Of Rap-PCR In Studying Mycobacterium tuberculosis ...

    African Journals Online (AJOL)

    Mycobacterium tuberculosis is the second leading cause of death from infectious agent. This study sought to detect M. tuberculosis genes, which were specifically expressed, or upregulated during intracellular infection of. J774 murine macrophages; as such genes may be potential targets for novel drug action. J774 murine ...

  2. Advances in the Laboratory Diagnosis of Mycobacterium Tuberculosis

    African Journals Online (AJOL)

    Mycobacterium tuberculosis (MTB), the agent of human tuberculosis remains a leading cause of mortality globally. Its resurgence during the last two decades is a reflection of its opportunistic relationship with HIV. The challenges associated with the disease are enormous and often debilitating. The role of clinical and ...

  3. Adaptation and evolution of drug-resistant Mycobacterium tuberculosis

    NARCIS (Netherlands)

    Bergval, I.L.

    2013-01-01

    Many studies have been conducted on drug resistance and the evolution of Mycobacterium tuberculosis. Notwithstanding, many molecular mechanisms facilitating the emergence, adaptation and spread of drug-resistant tuberculosis have yet to be discovered. This thesis reports studies of the adaptive

  4. Benzothiazinones kill Mycobacterium tuberculosis by blocking arabinan synthesis

    DEFF Research Database (Denmark)

    Makarov, Vadim; Manina, Giulia; Mikusova, Katarina

    2009-01-01

    New drugs are required to counter the tuberculosis (TB) pandemic. Here, we describe the synthesis and characterization of 1,3-benzothiazin-4-ones (BTZs), a new class of antimycobacterial agents that kill Mycobacterium tuberculosis in vitro, ex vivo, and in mouse models of TB. Using genetics...

  5. Management of latent Mycobacterium tuberculosis infection: WHO guidelines for low tuberculosis burden countries

    NARCIS (Netherlands)

    Getahun, Haileyesus; Matteelli, Alberto; Abubakar, Ibrahim; Aziz, Mohamed Abdel; Baddeley, Annabel; Barreira, Draurio; den Boon, Saskia; Borroto Gutierrez, Susana Marta; Bruchfeld, Judith; Burhan, Erlina; Cavalcante, Solange; Cedillos, Rolando; Chaisson, Richard; Chee, Cynthia Bin-Eng; Chesire, Lucy; Corbett, Elizabeth; Dara, Masoud; Denholm, Justin; de Vries, Gerard; Falzon, Dennis; Ford, Nathan; Gale-Rowe, Margaret; Gilpin, Chris; Girardi, Enrico; Go, Un-Yeong; Govindasamy, Darshini; D Grant, Alison; Grzemska, Malgorzata; Harris, Ross; Horsburgh, C. Robert; Ismayilov, Asker; Jaramillo, Ernesto; Kik, Sandra; Kranzer, Katharina; Lienhardt, Christian; LoBue, Philip; Lönnroth, Knut; Marks, Guy; Menzies, Dick; Migliori, Giovanni Battista; Mosca, Davide; Mukadi, Ya Diul; Mwinga, Alwyn; Nelson, Lisa; Nishikiori, Nobuyuki; Oordt-Speets, Anouk; Rangaka, Molebogeng Xheedha; Reis, Andreas; Rotz, Lisa; Sandgren, Andreas; Sañé Schepisi, Monica; Schünemann, Holger J.; Sharma, Surender Kumar; Sotgiu, Giovanni; Stagg, Helen R.; Sterling, Timothy R.; Tayeb, Tamara; Uplekar, Mukund; van der Werf, Marieke J.; Vandevelde, Wim; van Kessel, Femke; van't Hoog, Anna; Varma, Jay K.; Vezhnina, Natalia; Voniatis, Constantia; Vonk Noordegraaf-Schouten, Marije; Weil, Diana; Weyer, Karin; Wilkinson, Robert John; Yoshiyama, Takashi; Zellweger, Jean Pierre; Raviglione, Mario

    2015-01-01

    Latent tuberculosis infection (LTBI) is characterised by the presence of immune responses to previously acquired Mycobacterium tuberculosis infection without clinical evidence of active tuberculosis (TB). Here we report evidence-based guidelines from the World Health Organization for a public health

  6. The cell envelope glycoconjugates of Mycobacterium tuberculosis

    Science.gov (United States)

    Angala, Shiva Kumar; Belardinelli, Juan Manuel; Huc-Claustre, Emilie; Wheat, William H.; Jackson, Mary

    2015-01-01

    Tuberculosis (TB) remains the second most common cause of death due to a single infectious agent. The cell envelope of Mycobacterium tuberculosis (Mtb), the causative agent of the disease in humans, is a source of unique glycoconjugates and the most distinctive feature of the biology of this organism. It is the basis of much of Mtb pathogenesis and one of the major causes of its intrinsic resistance to chemotherapeutic agents. At the same time, the unique structures of Mtb cell envelope glycoconjugates, their antigenicity and essentiality for mycobacterial growth provide opportunities for drug, vaccine, diagnostic and biomarker development, as clearly illustrated by recent advances in all of these translational aspects. This review focuses on our current understanding of the structure and biogenesis of Mtb glycoconjugates with particular emphasis on one of most intriguing and least understood aspect of the physiology of mycobacteria: the translocation of these complex macromolecules across the different layers of the cell envelope. It further reviews the rather impressive progress made in the last ten years in the discovery and development of novel inhibitors targeting their biogenesis. PMID:24915502

  7. Gamma Interferon Release Assays for Detection of Mycobacterium tuberculosis Infection

    Science.gov (United States)

    Denkinger, Claudia M.; Kik, Sandra V.; Rangaka, Molebogeng X.; Zwerling, Alice; Oxlade, Olivia; Metcalfe, John Z.; Cattamanchi, Adithya; Dowdy, David W.; Dheda, Keertan; Banaei, Niaz

    2014-01-01

    SUMMARY Identification and treatment of latent tuberculosis infection (LTBI) can substantially reduce the risk of developing active disease. However, there is no diagnostic gold standard for LTBI. Two tests are available for identification of LTBI: the tuberculin skin test (TST) and the gamma interferon (IFN-γ) release assay (IGRA). Evidence suggests that both TST and IGRA are acceptable but imperfect tests. They represent indirect markers of Mycobacterium tuberculosis exposure and indicate a cellular immune response to M. tuberculosis. Neither test can accurately differentiate between LTBI and active TB, distinguish reactivation from reinfection, or resolve the various stages within the spectrum of M. tuberculosis infection. Both TST and IGRA have reduced sensitivity in immunocompromised patients and have low predictive value for progression to active TB. To maximize the positive predictive value of existing tests, LTBI screening should be reserved for those who are at sufficiently high risk of progressing to disease. Such high-risk individuals may be identifiable by using multivariable risk prediction models that incorporate test results with risk factors and using serial testing to resolve underlying phenotypes. In the longer term, basic research is necessary to identify highly predictive biomarkers. PMID:24396134

  8. Human B cells produce chemokine CXCL10 in the presence of Mycobacterium tuberculosis specific T cells

    DEFF Research Database (Denmark)

    Hoff, Soren T; Salman, Ahmed M; Ruhwald, Morten

    2015-01-01

    BACKGROUND: The role of B cells in human host response to Mycobacterium tuberculosis (Mtb) infection is still controversial, but recent evidence suggest that B cell follicle like structures within the lung may influence host responses through regulation of the local cytokine environment. A candid......BACKGROUND: The role of B cells in human host response to Mycobacterium tuberculosis (Mtb) infection is still controversial, but recent evidence suggest that B cell follicle like structures within the lung may influence host responses through regulation of the local cytokine environment...

  9. Evaluation of the Mycobacterium tuberculosis SO2 vaccine using a natural tuberculosis infection model in goats.

    Science.gov (United States)

    Bezos, J; Casal, C; Álvarez, J; Roy, A; Romero, B; Rodríguez-Bertos, A; Bárcena, C; Díez, A; Juste, R; Gortázar, C; Puentes, E; Aguiló, N; Martín, C; de Juan, L; Domínguez, L

    2017-05-01

    The development of new vaccines against animal tuberculosis (TB) is a priority for improving the control and eradication of this disease, particularly in those species not subjected to compulsory eradication programmes. In this study, the protection conferred by the Mycobacterium tuberculosis SO 2 experimental vaccine was evaluated using a natural infection model in goats. Twenty-six goats were distributed in three groups: (1) 10 goats served as a control group; (2) six goats were subcutaneously vaccinated with BCG; and (3) 10 goats were subcutaneously vaccinated with SO 2 . Four months after vaccination, all groups were merged with goats infected with Mycobacterium bovis or Mycobacterium caprae, and tested over a 40 week period using a tuberculin intradermal test and an interferon-γ assay for mycobacterial reactivity. The severity of lesions was determined at post-mortem examination and the bacterial load in tissues were evaluated by culture. The two vaccinated groups had significantly lower lesion and bacterial culture scores than the control group (P<0.05); at the end of the study, the SO 2 vaccinated goats had the lowest lesion and culture scores. These results suggest that the SO 2 vaccine provides some protection against TB infection acquired from natural exposure. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. T-cell recognition of Mycobacterium tuberculosis culture filtrate fractions in tuberculosis patients and their household contacts

    DEFF Research Database (Denmark)

    Demissie, A; Ravn, P; Olobo, J

    1999-01-01

    We examined the immune responses of patients with active pulmonary tuberculosis (TB) and their healthy household contacts to short-term culture filtrate (ST-CF) of Mycobacterium tuberculosis or molecular mass fractions derived from it. Our goal was to identify fractions strongly recognized......, to secreted mycobacterial antigens is suggestive of an early stage of infection by M. tuberculosis, which could in time result in overt disease or containment of the infection. This possibility is currently being investigated by follow-up studies of the household contacts....

  11. First molecular epidemiology study of Mycobacterium tuberculosis in Kiribati.

    Directory of Open Access Journals (Sweden)

    Eman Aleksic

    Full Text Available Tuberculosis incidence rates in Kiribati are among the highest in the Western Pacific Region, however the genetic diversity of circulating Mycobacterium tuberculosis complex strains (MTBC and transmission dynamics are unknown. Here, we analysed MTBC strains isolated from culture positive pulmonary tuberculosis (TB cases from the main TB referral centre between November 2007 and October 2009. Strain genotyping (IS6110 typing, spoligotyping, 24-loci MIRU-VNTR and SNP typing was performed and demographic information collected. Among 73 MTBC strains analysed, we identified seven phylogenetic lineages, dominated by Beijing strains (49%. Beijing strains were further differentiated in two main branches, Beijing-A (n = 8 and -B (n = 28, that show distinct genotyping patterns and are characterized by specific deletion profiles (Beijing A: only RD105, RD207 deleted; Beijing B: RD150 and RD181 additionally deleted. Many Kiribati strains (59% based on IS6110 typing of all strains occurred in clusters, suggesting ongoing local transmission. Beijing-B strains and over-crowded living conditions were associated with strain clustering (likely recent transmission, however little evidence of anti-tuberculous drug resistance was observed. We suggest enhanced case finding amongst close contacts and continued supervised treatment of all identified cases using standard first-line drugs to reduce TB burden in Kiribati. Beijing strains can be subdivided in different principle branches that might be associated with differential spreading patterns in the population.

  12. Microbe Profile: Mycobacterium tuberculosis: Humanity's deadly microbial foe.

    Science.gov (United States)

    Gordon, Stephen V; Parish, Tanya

    2018-04-01

    Mycobacterium tuberculosis is an expert and deadly pathogen, causing the disease tuberculosis (TB) in humans. It has several notable features: the ability to enter non-replicating states for long periods and cause latent infection; metabolic remodelling during chronic infection; a thick, waxy cell wall; slow growth rate in culture; and intrinsic drug resistance and antibiotic tolerance. As a pathogen, M. tuberculosis has a complex relationship with its host, is able to replicate inside macrophages, and expresses diverse immunomodulatory molecules. M. tuberculosis currently causes over 1.8 million deaths a year, making it the world's most deadly human pathogen.

  13. Is adipose tissue a place for Mycobacterium tuberculosis persistence?

    Directory of Open Access Journals (Sweden)

    Olivier Neyrolles

    Full Text Available BACKGROUND: Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB, has the ability to persist in its human host for exceptionally long periods of time. However, little is known about the location of the bacilli in latently infected individuals. Long-term mycobacterial persistence in the lungs has been reported, but this may not sufficiently account for strictly extra-pulmonary TB, which represents 10-15% of the reactivation cases. METHODOLOGY/PRINCIPAL FINDINGS: We applied in situ and conventional PCR to sections of adipose tissue samples of various anatomical origins from 19 individuals from Mexico and 20 from France who had died from causes other than TB. M. tuberculosis DNA could be detected by either or both techniques in fat tissue surrounding the kidneys, the stomach, the lymph nodes, the heart and the skin in 9/57 Mexican samples (6/19 individuals, and in 8/26 French samples (6/20 individuals. In addition, mycobacteria could be immuno-detected in perinodal adipose tissue of 1 out of 3 biopsy samples from individuals with active TB. In vitro, using a combination of adipose cell models, including the widely used murine adipose cell line 3T3-L1, as well as primary human adipocytes, we show that after binding to scavenger receptors, M. tuberculosis can enter within adipocytes, where it accumulates intracytoplasmic lipid inclusions and survives in a non-replicating state that is insensitive to the major anti-mycobacterial drug isoniazid. CONCLUSIONS/SIGNIFICANCE: Given the abundance and the wide distribution of the adipose tissue throughout the body, our results suggest that this tissue, among others, might constitute a vast reservoir where the tubercle bacillus could persist for long periods of time, and avoid both killing by antimicrobials and recognition by the host immune system. In addition, M. tuberculosis-infected adipocytes might provide a new model to investigate dormancy and to evaluate new drugs for the treatment of

  14. The transcriptional regulatory network of Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Joaquín Sanz

    Full Text Available Under the perspectives of network science and systems biology, the characterization of transcriptional regulatory (TR networks beyond the context of model organisms offers a versatile tool whose potential remains yet mainly unexplored. In this work, we present an updated version of the TR network of Mycobacterium tuberculosis (M.tb, which incorporates newly characterized transcriptional regulations coming from 31 recent, different experimental works available in the literature. As a result of the incorporation of these data, the new network doubles the size of previous data collections, incorporating more than a third of the entire genome of the bacterium. We also present an exhaustive topological analysis of the new assembled network, focusing on the statistical characterization of motifs significances and the comparison with other model organisms. The expanded M.tb transcriptional regulatory network, considering its volume and completeness, constitutes an important resource for diverse tasks such as dynamic modeling of gene expression and signaling processes, computational reliability determination or protein function prediction, being the latter of particular relevance, given that the function of only a small percent of the proteins of M.tb is known.

  15. Sputum is a surrogate for bronchoalveolar lavage for monitoring Mycobacterium tuberculosis transcriptional profiles in TB patients.

    Science.gov (United States)

    Garcia, Benjamin J; Loxton, Andre G; Dolganov, Gregory M; Van, Tran T; Davis, J Lucian; de Jong, Bouke C; Voskuil, Martin I; Leach, Sonia M; Schoolnik, Gary K; Walzl, Gerhard; Strong, Michael; Walter, Nicholas D

    2016-09-01

    Pathogen-targeted transcriptional profiling in human sputum may elucidate the physiologic state of Mycobacterium tuberculosis (M. tuberculosis) during infection and treatment. However, whether M. tuberculosis transcription in sputum recapitulates transcription in the lung is uncertain. We therefore compared M. tuberculosis transcription in human sputum and bronchoalveolar lavage (BAL) samples from 11 HIV-negative South African patients with pulmonary tuberculosis. We additionally compared these clinical samples with in vitro log phase aerobic growth and hypoxic non-replicating persistence (NRP-2). Of 2179 M. tuberculosis transcripts assayed in sputum and BAL via multiplex RT-PCR, 194 (8.9%) had a p-value <0.05, but none were significant after correction for multiple testing. Categorical enrichment analysis indicated that expression of the hypoxia-responsive DosR regulon was higher in BAL than in sputum. M. tuberculosis transcription in BAL and sputum was distinct from both aerobic growth and NRP-2, with a range of 396-1020 transcripts significantly differentially expressed after multiple testing correction. Collectively, our results indicate that M. tuberculosis transcription in sputum approximates M. tuberculosis transcription in the lung. Minor differences between M. tuberculosis transcription in BAL and sputum suggested lower oxygen concentrations or higher nitric oxide concentrations in BAL. M. tuberculosis-targeted transcriptional profiling of sputa may be a powerful tool for understanding M. tuberculosis pathogenesis and monitoring treatment responses in vivo. Published by Elsevier Ltd.

  16. Characterization of extensively drug-resistant Mycobacterium tuberculosis in Nepal.

    Science.gov (United States)

    Poudel, Ajay; Maharjan, Bhagwan; Nakajima, Chie; Fukushima, Yukari; Pandey, Basu D; Beneke, Antje; Suzuki, Yasuhiko

    2013-01-01

    The emergence of extensively drug-resistant tuberculosis (XDR-TB) has raised public health concern for global control of TB. Although molecular characterization of drug resistance-associated mutations in multidrug-resistant isolates in Nepal has been made, mutations in XDR isolates and their genotypes have not been reported previously. In this study, we identified and characterized 13 XDR Mycobacterium tuberculosis isolates from clinical isolates in Nepal. The most prevalent mutations involved in rifampicin, isoniazid, ofloxacin, and kanamycin/capreomycin resistance were Ser531Leu in rpoB gene (92.3%), Ser315Thr in katG gene (92.3%), Asp94Gly in gyrA gene (53.9%) and A1400G in rrs gene (61.5%), respectively. Spoligotyping and multilocus sequence typing revealed that 69% belonged to Beijing family, especially modern types. Further typing with 26-loci variable number of tandem repeats suggested the current spread of XDR M. tuberculosis. Our result highlights the need to reinforce the TB policy in Nepal with regard to control and detection strategies. Copyright © 2012 Elsevier Ltd. All rights reserved.

  17. Immune responses to the Mycobacterium tuberculosis-specific antigen ESAT-6 signal subclinical infection among contacts of tuberculosis patients

    DEFF Research Database (Denmark)

    Doherty, T Mark; Demissie, Abebech; Olobo, Joseph

    2002-01-01

    Diagnosis of latent Mycobacterium tuberculosis infection is considered essential for tuberculosis control but is hampered by the lack of specific reagents. We report that strong recognition of tuberculosis complex-specific antigen ESAT-6 by healthy household contacts of tuberculosis patients...... correlates with the subsequent development of active tuberculosis during a 2-year follow-up period....

  18. Spoligotyping of Mycobacterium tuberculosis isolates from patients with pulmonary tuberculosis in Mumbai, India.

    Science.gov (United States)

    Kulkarni, Savita; Sola, Christophe; Filliol, Ingrid; Rastogi, Nalin; Kadival, Gururaj

    2005-05-01

    Tuberculosis remains a major health problem in India, with 2 million new cases and 421,000 deaths each year. In this paper, we describe the spoligotyping results of 216 Mycobacterium tuberculosis culture isolates from patients with pulmonary tuberculosis in Mumbai, India. As spoligotyping data from India have rarely been described until now, and as there is limited information on the major circulating clades of M. tuberculosis, the data obtained were also compared to an international spoligotype database (SpolDB4) that contained patterns from 22,546 isolates from more than 100 countries. Eighty-four (39%) of the isolates were definitively marked as orphan strains, indicating the paucity of such data from India. The remaining 132 isolates clustered among 59 shared types; among these, 42 shared types were already present in the database, 17 were newly created, and 5 of them were specifically reported from Mumbai. A total of 9 major types in this study clustered 32% of the isolates. At the phylogenetic level, 30% of the isolates belonged to the Central Asian families CAS1 and CAS2, of the major genetic group (MGG) 1, 29% to MGG 2 and 3 families (spacers 33-36 missing) and 17% to the ancestral East African Indian (EAI) family. Finally, nearly 10% of the isolates belonged to the W-Beijing family in a broad sense, also in the MGG 1 group. In conclusion, historic clones of the MGG 1 group of M. tuberculosis are responsible for roughly 60% of all tuberculosis cases in Mumbai. Together with the fact that organisms presumably of European descent (such as the Haarlem family) were only rarely found, our observations suggest that tuberculosis in Mumbai, India is essentially caused by historical clones of tubercle bacilli undergoing active circulation due to uncontrolled demography, high prevalence of the disease, and a paucity of resources.

  19. Mycobacterium tuberculosis Isolates from Single Outpatient Clinic in Panama City Exhibit Wide Genetic Diversity

    Science.gov (United States)

    Sambrano, Dilcia; Correa, Ricardo; Almengor, Pedro; Domínguez, Amada; Vega, Silvio; Goodridge, Amador

    2014-01-01

    Understanding Mycobacterium tuberculosis biodiversity and transmission is significant for tuberculosis control. This short report aimed to determine the genetic diversity of M. tuberculosis isolates from an outpatient clinic in Panama City. A total of 62 M. tuberculosis isolates were genotyped by 12 loci mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) and Spoligotyping. Forty-five (72.6%) of the isolates showed unique MIRU-VNTR genotypes, and 13 (21%) of the isolates were grouped into four clusters. Four isolates showed polyclonal MIRU-VNTR genotypes. The MIRU-VNTR Hunter-Gaston discriminatory index reached 0.988. The Spoligotyping analysis revealed 16 M. tuberculosis families, including Latin American-Mediterranean, Harlem, and Beijing. These findings suggest a wide genetic diversity of M. tuberculosis isolates at one outpatient clinic. A detailed molecular epidemiology survey is now warranted, especially following second massive immigration for local Panama Canal expansion activities. PMID:24865686

  20. Intraocular manifestations of mycobacterium tuberculosis: A review of the literature

    Directory of Open Access Journals (Sweden)

    Lauren A. Dalvin

    2017-05-01

    Full Text Available Mycobacterium tuberculosis: is most commonly associated with pulmonary infection. However, tuberculosis (TB can also affect the eye. TB can affect nearly any tissue in the eye, and a high index of suspicion is required for accurate diagnosis, as many of the intraocular manifestations of TB can mimic other, more common diseases. Correct diagnosis is critical because systemic anti-tuberculosis treatment may be required, and vision loss or even loss of the affected eye can occur without proper treatment. Thus, it is important for ophthalmologists and infectious disease specialists to work together to accurately diagnose and treat intraocular TB. This article reports the various known presentations of intraocular TB and reviews important elements of diagnosis and treatment. Keywords: Mycobacterium, Tuberculosis, Choroidal granuloma, Retinal vasculitis

  1. Diversity and evolution of drug resistance mechanisms in Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Al-Saeedi M

    2017-10-01

    Full Text Available Mashael Al-Saeedi, Sahal Al-Hajoj Department of Infection and Immunity, Mycobacteriology Research Section, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia Abstract: Despite the efficacy of antibiotics to protect humankind against many deadly pathogens, such as Mycobacterium tuberculosis, nothing can prevent the emergence of drug-resistant strains. Several mechanisms facilitate drug resistance in M. tuberculosis including compensatory evolution, epistasis, clonal interference, cell wall integrity, efflux pumps, and target mimicry. In this study, we present recent findings relevant to these mechanisms, which can enable the discovery of new drug targets and subsequent development of novel drugs for treatment of drug-resistant M. tuberculosis. Keywords: Mycobacterium tuberculosis, antibiotic resistance, compensatory evolution, epistasis, efflux pumps, fitness cost

  2. A Mycobacterium tuberculosis cluster demonstrating the use of genotyping in urban tuberculosis control

    NARCIS (Netherlands)

    G. de Vries (Gerard); R.M. van Hest (Reinier); C.C.A. Burdo (Conny); D. van Soolingen (Dick); J.H. Richardus (Jan Hendrik)

    2009-01-01

    textabstractBackground: DNA fingerprinting of Mycobacterium tuberculosis isolates offers better opportunities to study links between tuberculosis (TB) cases and can highlight relevant issues in urban TB control in low-endemic countries. Methods: A medium-sized molecular cluster of TB cases with

  3. Tuberculosis relapse in Vietnam is significantly associated with Mycobacterium tuberculosis Beijing genotype infections

    NARCIS (Netherlands)

    Huyen, Mai N. T.; Buu, Tran N.; Tiemersma, Edine; Lan, Nguyen T. N.; Dung, Nguyen H.; Kremer, Kristin; Soolingen, Dick V.; Cobelens, Frank G. J.

    2013-01-01

    In Vietnam, the Mycobacterium tuberculosis Beijing genotype is associated with multi-drug resistance and is emerging. A possible explanation for this genotype's success is an increased rate of relapse. In a prospective cohort study, isolates from patients with smear-positive tuberculosis were

  4. Real-Time Measurement of Host Bioenergetics During Mycobacterium Tuberculosis Infection

    Science.gov (United States)

    2015-05-01

    AWARD NUMBER: W81XWH-13-1-0149 TITLE: “Real-Time Measurement of Host Bioenergetics During Mycobacterium Tuberculosis Infection...successfully adapted metabolic flux analysis using a Seahorse XF96 metabolic flux analyzer to study Mycobacterium tuberculosis energy metabolism in an...Mycobacterium tuberculosis function. In: Systems Biology of Tuberculosis . Editors: J McFadden, D Beste and A Kierzek. 2013. Springer, New York, NY. 2

  5. Expression of Mycobacterium smegmatis pyrazinamidase in Mycobacterium tuberculosis confers hypersensitivity to pyrazinamide and related amides.

    Science.gov (United States)

    Boshoff, H I; Mizrahi, V

    2000-10-01

    A pyrazinamidase (PZase)-deficient pncA mutant of Mycobacterium tuberculosis, constructed by allelic exchange, was used to investigate the effects of heterologous amidase gene expression on the susceptibility of this organism to pyrazinamide (PZA) and related amides. The mutant was highly resistant to PZA (MIC, >2,000 microg/ml), in accordance with the well-established role of pncA in the PZA susceptibility of M. tuberculosis (A. Scorpio and Y. Zhang, Nat. Med. 2:662-667, 1996). Integration of the pzaA gene encoding the major PZase/nicotinamidase from Mycobacterium smegmatis (H. I. M. Boshoff and V. Mizrahi, J. Bacteriol. 180:5809-5814, 1998) or the M. tuberculosis pncA gene into the pncA mutant complemented its PZase/nicotinamidase defect. In both pzaA- and pncA-complemented mutant strains, the PZase activity was detected exclusively in the cytoplasm, suggesting an intracellular localization for PzaA and PncA. The pzaA-complemented strain was hypersensitive to PZA (MIC, /=20 microg/ml) and was also sensitive to benzamide (MIC, 20 microg/ml), unlike the wild-type and pncA-complemented mutant strains, which were highly resistant to this amide (MIC, >500 microg/ml). This finding was consistent with the observation that benzamide is hydrolyzed by PzaA but not by PncA. Overexpression of PzaA also conferred sensitivity to PZA, nicotinamide, and benzamide on M. smegmatis (MIC, 150 microg/ml in all cases) and rendered Escherichia coli hypersensitive for growth at low pH.

  6. Nicotine Impairs Macrophage Control of Mycobacterium tuberculosis.

    Science.gov (United States)

    Bai, Xiyuan; Stitzel, Jerry A; Bai, An; Zambrano, Cristian A; Phillips, Matthew; Marrack, Philippa; Chan, Edward D

    2017-09-01

    Pure nicotine impairs macrophage killing of Mycobacterium tuberculosis (MTB), but it is not known whether the nicotine component in cigarette smoke (CS) plays a role. Moreover, the mechanisms by which nicotine impairs macrophage immunity against MTB have not been explored. To neutralize the effects of nicotine in CS extract, we used a competitive inhibitor to the nicotinic acetylcholine receptor (nAChR)-mecamylamine-as well as macrophages derived from mice with genetic disruption of specific subunits of nAChR. We also determined whether nicotine impaired macrophage autophagy and whether nicotine-exposed T regulatory cells (Tregs) could subvert macrophage anti-MTB immunity. Mecamylamine reduced the CS extract increase in MTB burden by 43%. CS extract increase in MTB was also significantly attenuated in macrophages from mice with genetic disruption of either the α7, β2, or β4 subunit of nAChR. Nicotine inhibited autophagosome formation in MTB-infected THP-1 cells and primary murine alveolar macrophages, as well as increased the intracellular MTB burden. Nicotine increased migration of THP-1 cells, consistent with the increased number of macrophages found in the lungs of smokers. Nicotine induced Tregs to produce transforming growth factor-β. Naive mouse macrophages co-cultured with nicotine-exposed Tregs had significantly greater numbers of viable MTB recovered with increased IL-10 production and urea production, but no difference in secreted nitric oxide as compared with macrophages cocultured with unexposed Tregs. We conclude that nicotine in CS plays an important role in subverting macrophage control of MTB infection.

  7. The draft genome of Mycobacterium aurum , a potential model organism for investigating drugs against Mycobacterium tuberculosis and Mycobacterium leprae

    Directory of Open Access Journals (Sweden)

    Jody Phelan

    2015-01-01

    Full Text Available Mycobacterium aurum (M. aurum is an environmental mycobacteria that has previously been used in studies of anti-mycobacterial drugs due to its fast growth rate and low pathogenicity. The M. aurum genome has been sequenced and assembled into 46 contigs, with a total length of 6.02 Mb containing 5684 annotated protein-coding genes. A phylogenetic analysis using whole genome alignments positioned M. aurum close to Mycobacterium vaccae and Mycobacterium vanbaalenii, within a clade related to fast-growing mycobacteria. Large-scale genomic rearrangements were identified by comparing the M. aurum genome to those of Mycobacterium tuberculosis and Mycobacterium leprae. M. aurum orthologous genes implicated in resistance to anti-tuberculosis drugs in M. tuberculosis were observed. The sequence identity at the DNA level varied from 68.6% for pncA (pyrazinamide drug-related to 96.2% for rrs (streptomycin, capreomycin. We observed two homologous genes encoding the catalase-peroxidase enzyme (katG that is associated with resistance to isoniazid. Similarly, two emb B homologues were identified in the M. aurum genome. In addition to describing for the first time the genome of M. aurum , this work provides a resource to aid the use of M. aurum in studies to develop improved drugs for the pathogenic mycobacteria M. tuberculosis and M. leprae.

  8. The draft genome of Mycobacterium aurum, a potential model organism for investigating drugs against Mycobacterium tuberculosis and Mycobacterium leprae

    KAUST Repository

    Phelan, Jody

    2015-06-04

    Mycobacterium aurum (M. aurum) is an environmental mycobacteria that has previously been used in studies of anti-mycobacterial drugs due to its fast growth rate and low pathogenicity. The M. aurum genome has been sequenced and assembled into 46 contigs, with a total length of 6.02 Mb containing 5684 annotated protein-coding genes. A phylogenetic analysis using whole genome alignments positioned M. aurum close to Mycobacterium vaccae and Mycobacterium vanbaalenii, within a clade related to fast-growing mycobacteria. Large-scale genomic rearrangements were identified by comparing the M. aurum genome to those of Mycobacterium tuberculosis and Mycobacterium leprae. M. aurum orthologous genes implicated in resistance to anti-tuberculosis drugs in M. tuberculosis were observed. The sequence identity at the DNA level varied from 68.6% for pncA (pyrazinamide drug-related) to 96.2% for rrs (streptomycin, capreomycin). We observed two homologous genes encoding the catalase-peroxidase enzyme (katG) that is associated with resistance to isoniazid. Similarly, two embB homologues were identified in the M. aurum genome. In addition to describing for the first time the genome of M. aurum, this work provides a resource to aid the use of M. aurum in studies to develop improved drugs for the pathogenic mycobacteria M. tuberculosis and M. leprae.

  9. The draft genome of Mycobacterium aurum, a potential model organism for investigating drugs against Mycobacterium tuberculosis and Mycobacterium leprae

    KAUST Repository

    Phelan, Jody; Maitra, Arundhati; McNerney, Ruth; Nair, Mridul; Gupta, Antima; Coll, Francesc; Pain, Arnab; Bhakta, Sanjib; Clark, Taane G.

    2015-01-01

    Mycobacterium aurum (M. aurum) is an environmental mycobacteria that has previously been used in studies of anti-mycobacterial drugs due to its fast growth rate and low pathogenicity. The M. aurum genome has been sequenced and assembled into 46 contigs, with a total length of 6.02 Mb containing 5684 annotated protein-coding genes. A phylogenetic analysis using whole genome alignments positioned M. aurum close to Mycobacterium vaccae and Mycobacterium vanbaalenii, within a clade related to fast-growing mycobacteria. Large-scale genomic rearrangements were identified by comparing the M. aurum genome to those of Mycobacterium tuberculosis and Mycobacterium leprae. M. aurum orthologous genes implicated in resistance to anti-tuberculosis drugs in M. tuberculosis were observed. The sequence identity at the DNA level varied from 68.6% for pncA (pyrazinamide drug-related) to 96.2% for rrs (streptomycin, capreomycin). We observed two homologous genes encoding the catalase-peroxidase enzyme (katG) that is associated with resistance to isoniazid. Similarly, two embB homologues were identified in the M. aurum genome. In addition to describing for the first time the genome of M. aurum, this work provides a resource to aid the use of M. aurum in studies to develop improved drugs for the pathogenic mycobacteria M. tuberculosis and M. leprae.

  10. The draft genome of Mycobacterium aurum, a potential model organism for investigating drugs against Mycobacterium tuberculosis and Mycobacterium leprae.

    Science.gov (United States)

    Phelan, Jody; Maitra, Arundhati; McNerney, Ruth; Nair, Mridul; Gupta, Antima; Coll, Francesc; Pain, Arnab; Bhakta, Sanjib; Clark, Taane G

    2015-09-01

    Mycobacterium aurum (M. aurum) is an environmental mycobacteria that has previously been used in studies of anti-mycobacterial drugs due to its fast growth rate and low pathogenicity. The M. aurum genome has been sequenced and assembled into 46 contigs, with a total length of 6.02Mb containing 5684 annotated protein-coding genes. A phylogenetic analysis using whole genome alignments positioned M. aurum close to Mycobacterium vaccae and Mycobacterium vanbaalenii, within a clade related to fast-growing mycobacteria. Large-scale genomic rearrangements were identified by comparing the M. aurum genome to those of Mycobacterium tuberculosis and Mycobacterium leprae. M. aurum orthologous genes implicated in resistance to anti-tuberculosis drugs in M. tuberculosis were observed. The sequence identity at the DNA level varied from 68.6% for pncA (pyrazinamide drug-related) to 96.2% for rrs (streptomycin, capreomycin). We observed two homologous genes encoding the catalase-peroxidase enzyme (katG) that is associated with resistance to isoniazid. Similarly, two embB homologues were identified in the M. aurum genome. In addition to describing for the first time the genome of M. aurum, this work provides a resource to aid the use of M. aurum in studies to develop improved drugs for the pathogenic mycobacteria M. tuberculosis and M. leprae. Copyright © 2015 Asian-African Society for Mycobacteriology. Published by Elsevier Ltd. All rights reserved.

  11. Spoligotyping of Mycobacterium tuberculosis isolates from tuberculosis diagnosed patients at Dilla University Referral Hospital and other private clinics, Southern Ethiopia

    Directory of Open Access Journals (Sweden)

    Gebremedhin Gebrezgabiher

    2015-04-01

    Full Text Available Objective: To assess Mycobacterium tuberculosis (M. tuberculosis strains exsisting in Gedeo zone and the surrounding areas of the Southern Ethiopia using spoligotyping. Methods: A cross sectional study was carried out from February, 2012 to June, 2013 and 97 (76 sputum and 21 fine needle aspirate samples were taken from tuberculosis diagnosed patients at Dilla University Referral Hospital and other private clinics. Culturing, region of difference (RD9 deletion typing and spoligotyping techniques were employed to isolate M. tuberculosis strains. Results: Growth of mycobacteria was observed in 35.1% (34/97. Speciation of isolates showed that 91.2% (31/34 of the isolates were M. tuberculosis. Further characterization led to the identification of 23 different spoligotype patterns of M. tuberculosis of which 61% and 39% displayed unique and cluster patterns, respectively. The most dominant shared type was spoligotype international type 53. Of the 23 strains, 12 have not been registered in the international spoligotyping database (SpolDB4. Seventy one percent of the strains belonged to the Euro-American lineage. Conclusions: This study revealed the existence of both genetically diverse and clustered M. tuberculosis strains from tuberculosis patients in the area, suggesting reactivation of infection and recent transmission, respectively. Molecular epidemiology of M. tuberculosis should be done nationwide in order to set appropriate control measures.

  12. Mycobacterium tuberculosis infection of domesticated Asian elephants, Thailand.

    OpenAIRE

    2011-01-01

    Four Asian elephants were confirmed to be infected with Mycobacterium tuberculosis by bacterial culture, other diagnostic procedures, and sequencing of 16S–23S rDNA internal transcribed spacer region, 16S rRNA, and gyrase B gene sequences. Genotyping showed that the infectious agents originated from 4 sources in Thailand. To identify infections, a combination of diagnostic assays is essential.

  13. Siderocalin inhibits the intracellular replication of Mycobacterium tuberculosis in macrophages

    DEFF Research Database (Denmark)

    Johnson, Erin E; Srikanth, Chittur V; Sandgren, Andreas

    2010-01-01

    Siderocalin is a secreted protein that binds to siderophores to prevent bacterial iron acquisition. While it has been shown to inhibit the growth of Mycobacterium tuberculosis (M.tb) in extracellular cultures, its effect on this pathogen within macrophages is not clear. Here, we show that sideroc...

  14. Transmission of Mycobacterium tuberculosis Undetected by Tuberculin Skin Testing

    Czech Academy of Sciences Publication Activity Database

    Anderson, S. T.; Williams, A. J.; Brown, J. R.; Newton, S. M.; Šimšová, Marcela; Nicol, M. P.; Šebo, Peter; Levin, M.; Wilkinson, R. J.; Wilkinson, K. A.

    2006-01-01

    Roč. 173, - (2006), s. 1038-1042 ISSN 1073-449X R&D Projects: GA AV ČR IAA5020406 Institutional research plan: CEZ:AV0Z50200510 Keywords : adenylate cyclase * diagnostic tests and procedures * mycobacterium tuberculosis Subject RIV: EE - Microbiology, Virology Impact factor: 9.091, year: 2006

  15. Structural studies on Mycobacterium tuberculosis RecA

    Indian Academy of Sciences (India)

    Structures of crystals of Mycobacterium tuberculosis RecA, grown and analysed under different conditions, provide insights into hitherto underappreciated details of molecular structure and plasticity. In particular, they yield information on the invariant and variable features of the geometry of the P-loop, whose binding to ATP ...

  16. Modern lineages of Mycobacterium tuberculosis in Addis Ababa ...

    African Journals Online (AJOL)

    Background: The genotyping of Mycobacterium tuberculosis strains is important to have unique insights into the dissemination dynamics and evolutionary genetics of this pathogen and for TB control as it allows the detection of suspected outbreaks and the tracing of transmission chains. Objective: To characterize M.

  17. Beijing/W genotype Mycobacterium tuberculosis and drug resistance.

    NARCIS (Netherlands)

    Glynn, Judith R; Kremer, Kristin; Borgdorff, Martien W; Rodriguez, Mar Pujades; Soolingen, Dick van

    2006-01-01

    Beijing/W genotype Mycobacterium tuberculosis is widespread, may be increasing, and may have a predilection for drug resistance. Individual-level data on >29,000 patients from 49 studies in 35 countries were combined to assess the Beijing genotype's prevalence worldwide, trends over time and with

  18. The transmission of Mycobacterium tuberculosis in high burden settings

    NARCIS (Netherlands)

    Yates, Tom A.; Khan, Palwasha Y.; Knight, Gwenan M.; Taylor, Jonathon G.; McHugh, Timothy D.; Lipman, Marc; White, Richard G.; Cohen, Ted; Cobelens, Frank G.; Wood, Robin; Moore, David A. J.; Abubakar, Ibrahim

    2016-01-01

    Unacceptable levels of Mycobacterium tuberculosis transmission are noted in high burden settings and a renewed focus on reducing person-to-person transmission in these communities is needed. We review recent developments in the understanding of airborne transmission. We outline approaches to measure

  19. A case of Manila type Mycobacterium tuberculosis infection in Japan

    Science.gov (United States)

    Usami, Osamu; Nakajima, Chie; Endo, Shiro; Inomata, Shinya; Kanamori, Hajime; Hirakata, Yoichi; Uchiyama, Bine; Kaku, Mitsuo; Suzuki, Yasuhiko; Hattori, Toshio

    2015-01-01

    Key Clinical Message A 76-year-old Japanese woman contracted a Mycobacterium tuberculosis (TB, Manila type) infection in Japan, despite never having traveled. However, her son was treated for TB in the Philippines 3 years before he stayed at her house. Spoligotyping allows us to identify the TB genotype and identify the route of infection. PMID:26273455

  20. Deciphering the biology of Mycobacterium tuberculosis from thecomplete genome sequence

    DEFF Research Database (Denmark)

    Cole, S.T.; Krogh, Anders Stærmose

    1998-01-01

    Countless millions of people have died from tuberculosis, a chronic infectious disease caused by the tubercle bacillus. The complete genome sequence of the best-characterized strain of Mycobacterium tuberculosis, H37Rv, has been determined and analysed in order to improve our understanding....... tuberculosis differs radically from other bacteria in that a very large portion of its coding capacity is devoted to the production of enzymes involved in lipogenesis and lipolysis, and to two new families of glycine-rich proteins with a repetitive structure that may represent a source of antigenic variation....

  1. Antibacterial Activity of Medicinal Aqueous Plant Extracts against Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Muna Mohammed Buzayan

    2012-09-01

    Full Text Available Tuberculosis (TB remains a serious health problem in many regions of the world, and the development of resistance to antibiotics by this microbe created the need for new drugs to replace those which have lost effectiveness. This study assesses the medicinal anti-Mycobacterium tuberculosis properties of natural products obtained from plants collected from Eastern Libya. In this study aqueous extracts of nine different plants were assayed for their Mycobacterium tuberculosis inhibitory activity using the BACTEC MGIT960 susceptibility test method. The aqueous extracts of Ceratonia siliqua L, Helichrysum stoechas (L. Moench and Thymus algeriensis did not show any activity against M. tuberculosis in different concentrations. The aqueous extract of Marrubium vulgare L. from Syria showed high activity against M. tuberculosis. Marrubium alysson L., Marrubium vulgare L., Pistacia lentiscus L, Quercus coccifera L, Thymus capitatus (L. Hoffm. & Link, showed varying degrees of activity against M. tuberculosis. The results of this study show that aqueous extracts from six different medicinal plants have different effects against M. tuberculosis in vitro.

  2. Characterization of drug susceptibility of Mycobacterium tuberculosis isolated from new cases of tuberculosis concurrent with HIV infection

    Directory of Open Access Journals (Sweden)

    G. V. Panov

    2015-01-01

    Full Text Available The paper characterizes drug susceptibility in Mycobacterium tuberculosis isolated from new cases of tuberculosis concurrent with HIV infection. The investigators have studied the spectrum of drug resistance in Mycobacterium tuberculosis isolated from new cases of tuberculosis concurrent with and without HIV infection (172 and 309 clinical isolates, respectively. There are differences in the rate of primary drug resistance to antituberculosis drugs in patients with and without HIV infection (59 and 43.5% of the cases, respectively. The HIV-infected have also shown high rifampicin resistance rates in Mycobacterium tuberculosis (41.7%. The reasons for these differences are as yet unknown and call for further investigation.

  3. Comparative genomics of archived pyrazinamide resistant Mycobacterium tuberculosis complex isolates from Uganda

    Science.gov (United States)

    Bovine tuberculosis is a ‘neglected zoonosis’ and its contribution to the proportion of Mycobacterium tuberculosis complex infections in humans is unknown. A retrospective study on archived Mycobacterium tuberculosis complex (MTC) isolates from a reference laboratory in Uganda was undertaken to iden...

  4. Study on drug resistance of mycobacterium tuberculosis in patients with pulmonary tuberculosis by drug resistance gene detecting

    International Nuclear Information System (INIS)

    Wang Wei; Li Hongmin; Wu Xueqiong; Wang Ansheng; Ye Yixiu; Wang Zhongyuan; Liu Jinwei; Chen Hongbing; Lin Minggui; Wang Jinhe; Li Sumei; Jiang Ping; Feng Bai; Chen Dongjing

    2004-01-01

    To investigate drug resistance of mycobacterium tuberculosis in different age group, compare detecting effect of two methods and evaluate their the clinical application value, all of the strains of mycobacterium tuberculosis were tested for resistance to RFP, INH SM PZA and EMB by the absolute concentration method on Lowenstein-Jensen medium and the mutation of the rpoB, katG, rpsL, pncA and embB resistance genes in M. tuberculosis was tested by PCR-SSCP. In youth, middle and old age group, the rate of acquired drug resistance was 89.2%, 85.3% and 67.6% respectively, the gene mutation rate was 76.2%, 81.3% and 63.2% respectively. The rate of acquired drug resistance and multiple drug resistance in youth group was much higher than those in other groups. The gene mutation was correlated with drug resistance level of mycobacterium tuberculosis. The gene mutation rate was higher in strains isolated from high concentration resistance than those in strains isolated from low concentration resistance. The more irregular treatment was longer, the rate of drug resistance was higher. Acquired drug resistance varies in different age group. It suggested that surveillance of drug resistence in different age group should be taken seriously, especially in youth group. PCR - SSCP is a sensitive and specific method for rapid detecting rpoB, katG, rpsL, pncA and embB genes mutations of MTB. (authors)

  5. Phylogenetic analysis of vitamin B12-related metabolism in Mycobacterium tuberculosis

    OpenAIRE

    Young, Douglas B.; Comas, I?aki; de Carvalho, Luiz P. S.

    2015-01-01

    Comparison of genome sequences from clinical isolates of Mycobacterium tuberculosis with phylogenetically-related pathogens Mycobacterium marinum, Mycobacterium kansasii, and Mycobacterium leprae reveals diversity amongst genes associated with vitamin B12-related metabolism. Diversity is generated by gene deletion events, differential acquisition of genes by horizontal transfer, and single nucleotide polymorphisms (SNPs) with predicted impact on protein function and transcriptional regulation...

  6. Biochemical characterization of uracil phosphoribosyltransferase from Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Anne Drumond Villela

    Full Text Available Uracil phosphoribosyltransferase (UPRT catalyzes the conversion of uracil and 5-phosphoribosyl-α-1-pyrophosphate (PRPP to uridine 5'-monophosphate (UMP and pyrophosphate (PP(i. UPRT plays an important role in the pyrimidine salvage pathway since UMP is a common precursor of all pyrimidine nucleotides. Here we describe cloning, expression and purification to homogeneity of upp-encoded UPRT from Mycobacterium tuberculosis (MtUPRT. Mass spectrometry and N-terminal amino acid sequencing unambiguously identified the homogeneous protein as MtUPRT. Analytical ultracentrifugation showed that native MtUPRT follows a monomer-tetramer association model. MtUPRT is specific for uracil. GTP is not a modulator of MtUPRT ativity. MtUPRT was not significantly activated or inhibited by ATP, UTP, and CTP. Initial velocity and isothermal titration calorimetry studies suggest that catalysis follows a sequential ordered mechanism, in which PRPP binding is followed by uracil, and PP(i product is released first followed by UMP. The pH-rate profiles indicated that groups with pK values of 5.7 and 8.1 are important for catalysis, and a group with a pK value of 9.5 is involved in PRPP binding. The results here described provide a solid foundation on which to base upp gene knockout aiming at the development of strategies to prevent tuberculosis.

  7. Immune responses to the Mycobacterium tuberculosis-specific antigen ESAT-6 signal subclinical infection among contacts of tuberculosis patients

    DEFF Research Database (Denmark)

    Doherty, T Mark; Demissie, Abebech; Olobo, Joseph

    2002-01-01

    Diagnosis of latent Mycobacterium tuberculosis infection is considered essential for tuberculosis control but is hampered by the lack of specific reagents. We report that strong recognition of tuberculosis complex-specific antigen ESAT-6 by healthy household contacts of tuberculosis patients...

  8. Networked T cell death following macrophage infection by Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Stephen H-F Macdonald

    Full Text Available BACKGROUND: Depletion of T cells following infection by Mycobacterium tuberculosis (Mtb impairs disease resolution, and interferes with clinical test performance that relies on cell-mediated immunity. A number of mechanisms contribute to this T cell suppression, such as activation-induced death and trafficking of T cells out of the peripheral circulation and into the diseased lungs. The extent to which Mtb infection of human macrophages affects T cell viability however, is not well characterised. METHODOLOGY/PRINCIPAL FINDINGS: We found that lymphopenia (<1.5 × 10(9 cells/l was prevalent among culture-positive tuberculosis patients, and lymphocyte counts significantly improved post-therapy. We previously reported that Mtb-infected human macrophages resulted in death of infected and uninfected bystander macrophages. In the current study, we sought to examine the influence of infected human alveolar macrophages on T cells. We infected primary human alveolar macrophages (the primary host cell for Mtb or PMA-differentiated THP-1 cells with Mtb H37Ra, then prepared cell-free supernatants. The supernatants of Mtb-infected macrophages caused dose-dependent, caspase-dependent, T cell apoptosis. This toxic effect of infected macrophage secreted factors did not require TNF-α or Fas. The supernatant cytotoxic signal(s were heat-labile and greater than 50 kDa in molecular size. Although ESAT-6 was toxic to T cells, other Mtb-secreted factors tested did not influence T cell viability; nor did macrophage-free Mtb bacilli or broth from Mtb cultures. Furthermore, supernatants from Mycobacterium bovis Bacille de Calmette et Guerin (BCG- infected macrophages also elicited T cell death suggesting that ESAT-6 itself, although cytotoxic, was not the principal mediator of T cell death in our system. CONCLUSIONS: Mtb-Infected macrophages secrete heat-labile factors that are toxic to T cells, and may contribute to the immunosuppression seen in tuberculosis as well as

  9. Triple valve endocarditis by mycobacterium tuberculosis. A case report

    Directory of Open Access Journals (Sweden)

    Shaikh Quratulain

    2012-09-01

    Full Text Available Abstract Background Granulomas caused by Mycobacterium Tuberculosis have been observed at autopsy in the heart, pre-dominantly in the myocardium and endocardium, but rarely involving the coronary vessels and valvular structures. Mycobacterium tuberculosis valvular endocarditis is extremely rare, with most reports coming from autopsy series. Case presentation We report the case of a 17 year old immunocompetent girl who presented with history of fever, malaise, foot gangrene and a left sided hemiparesis. On investigation she was found to have infective endocarditis involving the aortic, mitral and tricuspid valves. She had developed a right middle cerebral artery stroke. She underwent dual valve replacement and tricuspid repair. The vegetations showed granulomatous inflammation but blood cultures and other biological specimen cultures were negative for any organisms. She was started on antituberculous treatment and anticoagulation. Conclusion This is the first reported case of triple valve endocarditis by Mycobacterium Tuberculosis in an immunocompetent host. Especially important is the fact that the right heart is involved which has been historically described in the setting of intravenous drug abuse. This implies that Tuberculosis should be considered in cases of culture negative endocarditis in endemic areas like Pakistan even in immunocompetent hosts.

  10. Proteogenomic Investigation of Strain Variation in Clinical Mycobacterium tuberculosis Isolates

    KAUST Repository

    Heunis, Tiaan; Dippenaar, Anzaan; Warren, Robin M.; van Helden, Paul D.; van der Merwe, Ruben G.; Gey van Pittius, Nicolaas C.; Pain, Arnab; Sampson, Samantha L.; Tabb, David L.

    2017-01-01

    Mycobacterium tuberculosis consists of a large number of different strains that display unique virulence characteristics. Whole-genome sequencing has revealed substantial genetic diversity among clinical M. tuberculosis isolates, and elucidating the phenotypic variation encoded by this genetic diversity will be of utmost importance to fully understand M. tuberculosis biology and pathogenicity. In this study we integrated whole-genome sequencing and mass spectrometry (GeLC-MS/MS) to reveal strain-specific characteristics in the proteomes of two clinical M. tuberculosis Latin American-Mediterranean isolates. Using this approach we identified 59 peptides containing single amino acid variants, which covered ~9% of all total coding nonsynonymous single nucleotide variants detected by whole-genome sequencing. Furthermore, we identified 29 distinct peptides that mapped to a hypothetical protein not present in the M. tuberculosis H37Rv reference proteome. Here we provide evidence for the expression of this protein in the clinical M. tuberculosis SAWC3651 isolate. The strain-specific databases enabled confirmation of genomic differences (i.e. large genomic regions of difference and nonsynonymous single nucleotide variants) in these two clinical M. tuberculosis isolates and allowed strain differentiation at the proteome level. Our results contribute to the growing field of clinical microbial proteogenomics and can improve our understanding of phenotypic variation in clinical M. tuberculosis isolates.

  11. Proteogenomic Investigation of Strain Variation in Clinical Mycobacterium tuberculosis Isolates

    KAUST Repository

    Heunis, Tiaan

    2017-08-18

    Mycobacterium tuberculosis consists of a large number of different strains that display unique virulence characteristics. Whole-genome sequencing has revealed substantial genetic diversity among clinical M. tuberculosis isolates, and elucidating the phenotypic variation encoded by this genetic diversity will be of utmost importance to fully understand M. tuberculosis biology and pathogenicity. In this study we integrated whole-genome sequencing and mass spectrometry (GeLC-MS/MS) to reveal strain-specific characteristics in the proteomes of two clinical M. tuberculosis Latin American-Mediterranean isolates. Using this approach we identified 59 peptides containing single amino acid variants, which covered ~9% of all total coding nonsynonymous single nucleotide variants detected by whole-genome sequencing. Furthermore, we identified 29 distinct peptides that mapped to a hypothetical protein not present in the M. tuberculosis H37Rv reference proteome. Here we provide evidence for the expression of this protein in the clinical M. tuberculosis SAWC3651 isolate. The strain-specific databases enabled confirmation of genomic differences (i.e. large genomic regions of difference and nonsynonymous single nucleotide variants) in these two clinical M. tuberculosis isolates and allowed strain differentiation at the proteome level. Our results contribute to the growing field of clinical microbial proteogenomics and can improve our understanding of phenotypic variation in clinical M. tuberculosis isolates.

  12. Proteogenomic Investigation of Strain Variation in Clinical Mycobacterium tuberculosis Isolates.

    Science.gov (United States)

    Heunis, Tiaan; Dippenaar, Anzaan; Warren, Robin M; van Helden, Paul D; van der Merwe, Ruben G; Gey van Pittius, Nicolaas C; Pain, Arnab; Sampson, Samantha L; Tabb, David L

    2017-10-06

    Mycobacterium tuberculosis consists of a large number of different strains that display unique virulence characteristics. Whole-genome sequencing has revealed substantial genetic diversity among clinical M. tuberculosis isolates, and elucidating the phenotypic variation encoded by this genetic diversity will be of the utmost importance to fully understand M. tuberculosis biology and pathogenicity. In this study, we integrated whole-genome sequencing and mass spectrometry (GeLC-MS/MS) to reveal strain-specific characteristics in the proteomes of two clinical M. tuberculosis Latin American-Mediterranean isolates. Using this approach, we identified 59 peptides containing single amino acid variants, which covered ∼9% of all coding nonsynonymous single nucleotide variants detected by whole-genome sequencing. Furthermore, we identified 29 distinct peptides that mapped to a hypothetical protein not present in the M. tuberculosis H37Rv reference proteome. Here, we provide evidence for the expression of this protein in the clinical M. tuberculosis SAWC3651 isolate. The strain-specific databases enabled confirmation of genomic differences (i.e., large genomic regions of difference and nonsynonymous single nucleotide variants) in these two clinical M. tuberculosis isolates and allowed strain differentiation at the proteome level. Our results contribute to the growing field of clinical microbial proteogenomics and can improve our understanding of phenotypic variation in clinical M. tuberculosis isolates.

  13. In vitro and in vivo activities of the nitroimidazole TBA-354 against Mycobacterium tuberculosis.

    Science.gov (United States)

    Upton, A M; Cho, S; Yang, T J; Kim, Y; Wang, Y; Lu, Y; Wang, B; Xu, J; Mdluli, K; Ma, Z; Franzblau, S G

    2015-01-01

    Nitroimidazoles are a promising new class of antitubercular agents. The nitroimidazo-oxazole delamanid (OPC-67683, Deltyba) is in phase III trials for the treatment of multidrug-resistant tuberculosis, while the nitroimidazo-oxazine PA-824 is entering phase III for drug-sensitive and drug-resistant tuberculosis. TBA-354 (SN31354[(S)-2-nitro-6-((6-(4-trifluoromethoxy)phenyl)pyridine-3-yl)methoxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine]) is a pyridine-containing biaryl compound with exceptional efficacy against chronic murine tuberculosis and favorable bioavailability in preliminary rodent studies. It was selected as a potential next-generation antituberculosis nitroimidazole following an extensive medicinal chemistry effort. Here, we further evaluate the pharmacokinetic properties and activity of TBA-354 against Mycobacterium tuberculosis. TBA-354 is narrow spectrum and bactericidal in vitro against replicating and nonreplicating Mycobacterium tuberculosis, with potency similar to that of delamanid and greater than that of PA-824. The addition of serum protein or albumin does not significantly alter this activity. TBA-354 maintains activity against Mycobacterium tuberculosis H37Rv isogenic monoresistant strains and clinical drug-sensitive and drug-resistant isolates. Spontaneous resistant mutants appear at a frequency of 3 × 10(-7). In vitro studies and in vivo studies in mice confirm that TBA-354 has high bioavailability and a long elimination half-life. In vitro studies suggest a low risk of drug-drug interactions. Low-dose aerosol infection models of acute and chronic murine tuberculosis reveal time- and dose-dependent in vivo bactericidal activity that is at least as potent as that of delamanid and more potent than that of PA-824. Its superior potency and pharmacokinetic profile that predicts suitability for once-daily oral dosing suggest that TBA-354 be studied further for its potential as a next-generation nitroimidazole. Copyright © 2015, American

  14. Metabolic principles of persistence and pathogenicity in Mycobacterium tuberculosis.

    Science.gov (United States)

    Ehrt, Sabine; Schnappinger, Dirk; Rhee, Kyu Y

    2018-04-24

    Metabolism was once relegated to the supply of energy and biosynthetic precursors, but it has now become clear that it is a specific mediator of nearly all physiological processes. In the context of microbial pathogenesis, metabolism has expanded outside its canonical role in bacterial replication. Among human pathogens, this expansion has emerged perhaps nowhere more visibly than for Mycobacterium tuberculosis, the causative agent of tuberculosis. Unlike most pathogens, M. tuberculosis has evolved within humans, which are both host and reservoir. This makes unrestrained replication and perpetual quiescence equally incompatible strategies for survival as a species. In this Review, we summarize recent work that illustrates the diversity of metabolic functions that not only enable M. tuberculosis to establish and maintain a state of chronic infection within the host but also facilitate its survival in the face of drug pressure and, ultimately, completion of its life cycle.

  15. Rifampicin resistance in Mycobacterium tuberculosis - rapid ...

    African Journals Online (AJOL)

    PH.D. T. C. VJCtor. PH.O. National Tuberculosis Research Programme, Pretoria ... together with. MDR profiles of clinical isolates of M. tuberculosis to assess ..... values in a population." This analysis ..... increased after the mid-1980s but the contribution of social science has ... Schall argues that this is a worst- case scenario ...

  16. Mycobacterium tuberculosis bacteremia detected by the Isolator lysis-centrifugation blood culture system.

    OpenAIRE

    Kiehn, T E; Gold, J W; Brannon, P; Timberger, R J; Armstrong, D

    1985-01-01

    Mycobacterium tuberculosis was detected by the Isolator lysis-centrifugation blood culture system from the blood of a patient with tuberculosis of the breast. The organism also grew on conventional laboratory media inoculated with pleural fluid from the patient.

  17. Total Hip Arthroplasty Loosening Due to Mycobacterium Tuberculosis: A Case Report and Review of the Literature

    Directory of Open Access Journals (Sweden)

    Anis Tebourbi

    2017-04-01

    Full Text Available Context: Prosthetic joint infection due to Mycobacterium tuberculosis with no previous history of pulmonary or extra pulmonary tuberculosis is an extremely rare complication. Aims To report the case of a patient with tuberculous mycobacterial prosthetic hip infection, 14 years after surgery for post traumatic osteoarthritis, with no previous history of tuberculosis. Methods A 46-year-old male presented an acetabular loosening of a cemented total hip arthroplasty with subnormal biologic parameters. A one stage revision surgery was planned. Intraoperative findings suggested mycobacterial tuberculous infection with presence of periacetabular yellowish rice-shaped granules. Results A one-stage prosthesis exchange was performed; Culture on Löwenstein-Jensen medium grew MTB days after inoculation and histological examination confirmed tuberculous infection. Patient was treated by antituberculous agents for 12 months with optimal clinical and biological response and no prosthetic loosening signs at eighteen months follow up. Conclusions Total hip arthroplasty loosening due to mycobacterium tuberculosis is a rare entity, which should be evoked even when no inflammatory signs are shown. Discovery of yellowish rice-shaped granules is an indicator to investigate for tuberculosis. Management of prosthetic joint infection due to M.tuberculosis must involve both medical and surgical approach.

  18. The endothelin system has a significant role in the pathogenesis and progression of Mycobacterium tuberculosis infection.

    Science.gov (United States)

    Correa, Andre F; Bailão, Alexandre M; Bastos, Izabela M D; Orme, Ian M; Soares, Célia M A; Kipnis, Andre; Santana, Jaime M; Junqueira-Kipnis, Ana Paula

    2014-12-01

    Tuberculosis (TB) remains a major global health problem, and although multiple studies have addressed the relationship between Mycobacterium tuberculosis and the host on an immunological level, few studies have addressed the impact of host physiological responses. Proteases produced by bacteria have been associated with important alterations in the host tissues, and a limited number of these enzymes have been characterized in mycobacterial species. M. tuberculosis produces a protease called Zmp1, which appears to be associated with virulence and has a putative action as an endothelin-converting enzyme. Endothelins are a family of vasoactive peptides, of which 3 distinct isoforms exist, and endothelin 1 (ET-1) is the most abundant and the best-characterized isoform. The aim of this work was to characterize the Zmp1 protease and evaluate its role in pathogenicity. Here, we have shown that M. tuberculosis produces and secretes an enzyme with ET-1 cleavage activity. These data demonstrate a possible role of Zmp1 for mycobacterium-host interactions and highlights its potential as a drug target. Moreover, the results suggest that endothelin pathways have a role in the pathogenesis of M. tuberculosis infections, and ETA or ETB receptor signaling can modulate the host response to the infection. We hypothesize that a balance between Zmp1 control of ET-1 levels and ETA/ETB signaling can allow M. tuberculosis adaptation and survival in the lung tissues. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  19. MicroRNA-365 in macrophages regulates Mycobacterium tuberculosis-induced active pulmonary tuberculosis via interleukin-6.

    Science.gov (United States)

    Song, Qingzhang; Li, Hui; Shao, Hua; Li, Chunling; Lu, Xiao

    2015-01-01

    The present study is to investigate the relationship between microRNA (miR)-365 expression and the levels of interleukin (IL)-6 mRNA and protein in patients with active tuberculosis. From June 2011 to June 2014, 48 patients with active pulmonary tuberculosis induced by Mycobacterium tuberculosis were included in the study. In addition, 23 healthy subjects were enrolled as control. Macrophages were collected by pulmonary alveolus lavage. In addition, serum and mononuclear cells were isolated from peripheral blood. The levels of miR-365 and IL-6 in macrophages, mononuclear cells and serum were determined using quantitative real-time polymerase chain reaction. The protein expression of IL-6 in macrophages and mononuclear cells was measured using Western blotting, while that in serum was detected by enzyme-linked immunoabsorbent assay. Expression of IL-6 mRNA and protein was significantly enhanced in patients with active pulmonary tuberculosis. Increase of IL-6 protein concentration in serum was probably due to the release of IL-6 protein from mononuclear cells in the blood. In addition, miR-365 levels were significantly lowered in patients with active pulmonary tuberculosis. Up-regulated IL-6 expression in macrophages, mononuclear cells and serum in patients with active pulmonary tuberculosis is related to the down-regulation of miR-365, suggesting that miR-365 may regulate the occurrence and immune responses of active pulmonary tuberculosis via IL-6.

  20. Bloodstream Infections with Mycobacterium tuberculosis among HIV patients

    Centers for Disease Control (CDC) Podcasts

    2010-09-23

    This podcast looks at bloodstream infections with Mycobacterium tuberculosis and other pathogens among outpatients infected with HIV in Southeast Asia. CDC health scientist Kimberly McCarthy discusses the study and why bloodstream infections occur in HIV-infected populations.  Created: 9/23/2010 by National Center for Emerging and Zoonotic Infectious Diseases (NCEZID).   Date Released: 9/23/2010.

  1. Structural and Functional Studies of Phosphoenolpyruvate Carboxykinase from Mycobacterium tuberculosis

    Czech Academy of Sciences Publication Activity Database

    Machová, Iva; Snášel, Jan; Dostál, Jiří; Brynda, Jiří; Fanfrlík, Jindřich; Singh, M.; Tarábek, Ján; Vaněk, O.; Bednárová, Lucie; Pichová, Iva

    2015-01-01

    Roč. 10, č. 3 (2015), e0120682/1-e0120682/21 E-ISSN 1932-6203 R&D Projects: GA MŠk LO1302 EU Projects: European Commission(XE) 241587 - SYSTEMTB Institutional support: RVO:61388963 Keywords : crystal structure * noncovalent complexes * Mycobacterium tuberculosis * mechanism Subject RIV: CE - Biochemistry Impact factor: 3.057, year: 2015 http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0120682

  2. Studium role fosfofruktokinasy A a B v metabolismu Mycobacterium tuberculosis

    Czech Academy of Sciences Publication Activity Database

    Machová, Iva; Snášel, Jan; Pichová, Iva

    2014-01-01

    Roč. 108, č. 5 (2014), s. 542 ISSN 0009-2770. [Mezioborové setkání mladých biologů, biochemiků a chemiků /14./. 13.05.2014-16.05.2014, Milovy] Grant - others:European Research Council(XE) FP7-245187 Institutional support: RVO:61388963 Keywords : Mycobacterium tuberculosis * phosphofructokinase Subject RIV: CE - Biochemistry

  3. Tumor necrosis factor blockers influence macrophage responses to Mycobacterium tuberculosis

    OpenAIRE

    HARRIS, JAMES; HARRIS, JAMES

    2008-01-01

    PUBLISHED umor necrosis factor (TNF)?? is a proinflammatory cytokine that mediates inflammation in response to various pathogens, including Mycobacterium tuberculosis, but is also a key factor in the pathogenesis of rheumatoid arthritis and other autoimmune diseases. Three TNF???suppressing drugs have been approved to treat selected autoimmune diseases; 2 are monoclonal antibodies against TNF?? (adalimumab and infliximab), and the other is a soluble TNF receptor/Fc fusion protein (etanerce...

  4. Highly structured genetic diversity of the Mycobacterium tuberculosis population in Djibouti.

    Science.gov (United States)

    Godreuil, S; Renaud, F; Choisy, M; Depina, J J; Garnotel, E; Morillon, M; Van de Perre, P; Bañuls, A L

    2010-07-01

    Djibouti is an East African country with a high tuberculosis incidence. This study was conducted over a 2-month period in Djibouti, during which 62 consecutive patients with pulmonary tuberculosis (TB) were included. Genetic characterization of Mycobacterium tuberculosis, using mycobacterial interspersed repetitive-unit variable-number tandem-repeat typing and spoligotyping, was performed. The genetic and phylogenetic analysis revealed only three major families (Central Asian, East African Indian and T). The high diversity and linkage disequilibrium within each family suggest a long period of clonal evolution. A Bayesian approach shows that the phylogenetic structure observed in our sample of 62 isolates is very likely to be representative of the phylogenetic structure of the M. tuberculosis population in the total number of TB cases.

  5. High genetic diversity among Mycobacterium tuberculosis strains in Tehran, Iran

    Directory of Open Access Journals (Sweden)

    Taher Azimi

    2018-05-01

    Full Text Available Introduction: Tuberculosis (TB still remains an important public health problem in Iran. The genotyping of Mycobacterium tuberculosis isolates is expected to lead to a better understanding of M. tuberculosis transmission in Tehran, the most populated city of Iran. Materials and Methods: A total of 2300 clinical specimens were obtained from TB suspected patients who were referred to a TB center in Tehran from Jan 2014 to Dec 2016. Identification was performed using both conventional and molecular methods. The presence of resistance to rifampicin was examined by the GeneXpert MTB/RIF. The standard 15-locus mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR typing method was applied to genotype of clinical isolates. Results: Of 2300 specimens, 80 isolates were identified as M. tuberculosis by using biochemical and molecular tests. Of 80 M. tuberculosis isolates, 76 (95% had unique genotypic profiles and 4 (5% shared a profile with one or more other strains. Based on single loci variation (SLV 4 clonal complexes were observed. NEW-1 was found to be the most predominant lineage (22.5% followed by West African (1.25%, Central Asian (CAS/Delhi (1.25%, Bovis (1.25%, H37Rv (1.25% and multiple matches (1.25%. Loci MIRU10, MIRU26, MTUB21 and QUB26 were found as highly discriminative. No mutation was detected in the hotspot region of rifampicin by using GeneXpert MTB/RIF. Conclusions: Our study findings show that there was considerable genotypic diversity among M. tuberculosis isolates in Tehran. The 15-locus MIRU-VNTR showed high HGDI and could be used as a first-line genotyping method for epidemiological studies. Keywords: Mycobacterium tuberculosis, Genotyping, MIRU-VNTR, Tehran, Iran

  6. Drug Resistance and Population Structure of Mycobacterium tuberculosis Beijing Strains Isolated in Poland.

    Science.gov (United States)

    Kozińska, Monika; Augustynowicz-Kopeć, Ewa

    2015-01-01

    In total, 1095 Mycobacterium tuberculosis clinical isolates from 282 patients with drug-resistant and 813 with drug-sensitive tuberculosis (TB) in Poland during 2007-2011 were analysed. Seventy-one (6.5%) patients were found to have strains of Beijing genotype as defined by spoligotyping. The majority of patients were Polish-born; among foreign-born a large proportion came from Chechnya and Vietnam. Analysis showed strong associations between Beijing genotype infection and MDR, pre-XDR and XDR resistance, with a considerable relative risk among new patients, suggesting that this is due to increased spread of drug-resistant strains rather than acquisition of resistance during treatment.

  7. Diversity of DNA fingerprints of Mycobacterium tuberculosis isolates in the United States.

    Science.gov (United States)

    Yang, Z; Barnes, P F; Chaves, F; Eisenach, K D; Weis, S E; Bates, J H; Cave, M D

    1998-04-01

    To investigate the diversity of IS6110 fingerprints of Mycobacterium tuberculosis isolates in the United States and to determine if matching IS6110 fingerprints represent recent interstate tuberculosis transmission, we performed restriction fragment length polymorphism analysis of M. tuberculosis isolates from 1,326 patients in three geographically separated states. Seven hundred ninety-five different IS6110 fingerprint patterns were generated, and pattern diversity was similar in each state. Ninety-six percent of the fingerprint patterns were observed in only one state, demonstrating that most IS6110 fingerprint patterns are confined to a single geographic location. Of the IS6110 fingerprint patterns that were shared by isolates from more than one state, most isolates with 1 to 5 IS6110 copies were separable by pTBN12 fingerprinting whereas those with > 15 copies were not. One high-copy-number M. tuberculosis strain had identical IS6110 and pTBN12 fingerprints and included 57 isolates from three states. Epidemiological data demonstrated significant recent transmission of tuberculosis within each city but not among the states. This suggests that identical fingerprints of isolates from geographically separate locations most likely reflect interstate tuberculosis transmission in the past, with subsequent intrastate spread of disease. Further evaluation of M. tuberculosis strains that cause outbreaks in different geographic locations will provide insight into the epidemiological and bacteriological factors that facilitate the spread of tuberculosis.

  8. DNA alkylation damage as a sensor of nitrosative stress in Mycobacterium tuberculosis

    OpenAIRE

    Durbach, S I; Springer, B; Machowski, E E; North, R J; Papavinasasundaram, K G; Colston, M J; Böttger, E C; Mizrahi, V

    2003-01-01

    One of the cellular consequences of nitrosative stress is alkylation damage to DNA. To assess whether nitrosative stress is registered on the genome of Mycobacterium tuberculosis, mutants lacking an alkylation damage repair and reversal operon were constructed. Although hypersensitive to the genotoxic effects of N-methyl-N′-nitro-N-nitrosoguanidine in vitro, the mutants displayed no phenotype in vivo, suggesting that permeation of nitrosative stress to the level of cytotoxic DNA damage is res...

  9. Lipoprotein Processing Is Essential for Resistance of Mycobacterium tuberculosis to Malachite Green▿

    OpenAIRE

    Banaei, Niaz; Kincaid, Eleanor Z.; Lin, S.-Y. Grace; Desmond, Edward; Jacobs, William R.; Ernst, Joel D.

    2009-01-01

    Malachite green, a synthetic antimicrobial dye, has been used for over 50 years in mycobacterial culture medium to inhibit the growth of contaminants. The molecular basis of mycobacterial resistance to malachite green is unknown, although the presence of malachite green-reducing enzymes in the cell envelope has been suggested. The objective of this study was to investigate the role of lipoproteins in resistance of Mycobacterium tuberculosis to malachite green. The replication of an M. tubercu...

  10. The Evolution of Strain Typing in the Mycobacterium tuberculosis Complex.

    Science.gov (United States)

    Merker, Matthias; Kohl, Thomas A; Niemann, Stefan; Supply, Philip

    2017-01-01

    Tuberculosis (TB) is a contagious disease with a complex epidemiology. Therefore, molecular typing (genotyping) of Mycobacterium tuberculosis complex (MTBC) strains is of primary importance to effectively guide outbreak investigations, define transmission dynamics and assist global epidemiological surveillance of the disease. Large-scale genotyping is also needed to get better insights into the biological diversity and the evolution of the pathogen. Thanks to its shorter turnaround and simple numerical nomenclature system, mycobacterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR) typing, based on 24 standardized plus 4 hypervariable loci, optionally combined with spoligotyping, has replaced IS6110 DNA fingerprinting over the last decade as a gold standard among classical strain typing methods for many applications. With the continuous progress and decreasing costs of next-generation sequencing (NGS) technologies, typing based on whole genome sequencing (WGS) is now increasingly performed for near complete exploitation of the available genetic information. However, some important challenges remain such as the lack of standardization of WGS analysis pipelines, the need of databases for sharing WGS data at a global level, and a better understanding of the relevant genomic distances for defining clusters of recent TB transmission in different epidemiological contexts. This chapter provides an overview of the evolution of genotyping methods over the last three decades, which culminated with the development of WGS-based methods. It addresses the relative advantages and limitations of these techniques, indicates current challenges and potential directions for facilitating standardization of WGS-based typing, and provides suggestions on what method to use depending on the specific research question.

  11. Rapid drug susceptibility test of mycobacterium tuberculosis by bioluminescence sensor

    Science.gov (United States)

    Lu, Bin; Xu, Shunqing; Chen, Zifei; Zhou, Yikai

    2001-09-01

    With the persisting increase of drug-resistant stains of M. Tuberculosis around the world, rapid and sensitive detection of antibiotic of M. Tuberculosis is becoming more and more important. In the present study, drug susceptibility of M. tuberculosis were detected by recombination mycobacteriophage combined with bioluminescence sensor. It is based on the use of recombination mycobacteriophage which can express firefly luciferase when it infects viable mycobacteria, and can effectively produce quantifiable photon. Meanwhile, in mycobacterium cells treated with active antibiotic, no light is observed. The emitted light is recorded by a bioluminscence sensor, so the result of drug-resistant test can be determined by the naked eye. 159 stains of M. tuberculosis were applied to this test on their resistant to rifampin, streptomycin and isoniazid. It is found that the agreement of this assay with Liewenstein- Jensen slat is: rifampin 95.60 percent, isoniazid 91.82 percent, streptomycin 88.68 percent, which showed that it is a fast and practical method to scene and detect drug resistant of mycobacterium stains.

  12. Phenotypic and genomic comparison of Mycobacterium aurum and surrogate model species to Mycobacterium tuberculosis: implications for drug discovery.

    Science.gov (United States)

    Namouchi, Amine; Cimino, Mena; Favre-Rochex, Sandrine; Charles, Patricia; Gicquel, Brigitte

    2017-07-13

    Tuberculosis (TB) is caused by Mycobacterium tuberculosis and represents one of the major challenges facing drug discovery initiatives worldwide. The considerable rise in bacterial drug resistance in recent years has led to the need of new drugs and drug regimens. Model systems are regularly used to speed-up the drug discovery process and circumvent biosafety issues associated with manipulating M. tuberculosis. These include the use of strains such as Mycobacterium smegmatis and Mycobacterium marinum that can be handled in biosafety level 2 facilities, making high-throughput screening feasible. However, each of these model species have their own limitations. We report and describe the first complete genome sequence of Mycobacterium aurum ATCC23366, an environmental mycobacterium that can also grow in the gut of humans and animals as part of the microbiota. This species shows a comparable resistance profile to that of M. tuberculosis for several anti-TB drugs. The aims of this study were to (i) determine the drug resistance profile of a recently proposed model species, Mycobacterium aurum, strain ATCC23366, for anti-TB drug discovery as well as Mycobacterium smegmatis and Mycobacterium marinum (ii) sequence and annotate the complete genome sequence of this species obtained using Pacific Bioscience technology (iii) perform comparative genomics analyses of the various surrogate strains with M. tuberculosis (iv) discuss how the choice of the surrogate model used for drug screening can affect the drug discovery process. We describe the complete genome sequence of M. aurum, a surrogate model for anti-tuberculosis drug discovery. Most of the genes already reported to be associated with drug resistance are shared between all the surrogate strains and M. tuberculosis. We consider that M. aurum might be used in high-throughput screening for tuberculosis drug discovery. We also highly recommend the use of different model species during the drug discovery screening process.

  13. Collectin CL-LK Is a Novel Soluble Pattern Recognition Receptor for Mycobacterium tuberculosis

    DEFF Research Database (Denmark)

    Troegeler, Anthony; Lugo-Villarino, Geanncarlo; Hansen, Søren

    2015-01-01

    Understanding the molecular components of immune recognition of the tuberculosis (TB) bacillus, Mycobacterium tuberculosis, can help designing novel strategies to combat TB. Here, we identify collectin CL-LK as a novel soluble C-type lectin able to bind M. tuberculosis, and characterize mycobacte......Understanding the molecular components of immune recognition of the tuberculosis (TB) bacillus, Mycobacterium tuberculosis, can help designing novel strategies to combat TB. Here, we identify collectin CL-LK as a novel soluble C-type lectin able to bind M. tuberculosis, and characterize...

  14. Molecular epidemiology and evolutionary genetics of Mycobacterium tuberculosis in Taipei.

    Science.gov (United States)

    Dou, Horng-Yunn; Tseng, Fan-Chen; Lin, Chih-Wei; Chang, Jia-Ru; Sun, Jun-Ren; Tsai, Wen-Shing; Lee, Shi-Yi; Su, Ih-Jen; Lu, Jang-Jih

    2008-12-22

    The control of tuberculosis in densely populated cities is complicated by close human-to-human contacts and potential transmission of pathogens from multiple sources. We conducted a molecular epidemiologic analysis of 356 Mycobacterium tuberculosis (MTB) isolates from patients presenting pulmonary tuberculosis in metropolitan Taipei. Classical antibiogram studies and genetic characterization, using mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing and spoligotyping, were applied after culture. A total of 356 isolates were genotyped by standard spoligotyping and the strains were compared with in the international spoligotyping database (SpolDB4). All isolates were also categorized using the 15 loci MIRU-VNTR typing method and combin with NTF locus and RD deletion analyses. Of 356 isolates spoligotyped, 290 (81.4%) displayed known spoligotypes and 66 were not identified in the database. Major spoligotypes found were Beijing lineages (52.5%), followed by Haarlem lineages (13.5%) and EAI plus EAI-like lineages (11%). When MIRU-VNTR was employed, 140 patterns were identified, including 36 clusters by 252 isolates and 104 unique patterns, and the largest cluster comprised 95 isolates from the Beijing family. The combination of spoligotyping and MIRU-VNTR revealed that 236 (67%) of the 356 isolates were clustered in 43 genotypes. Strains of the Beijing family was more likely to be of modern strain and a higher percentage of multiple drug resistance than other families combined (P = 0.08). Patients infected with Beijing strains were younger than those with other strains (mean 58.7 vs. 64.2, p = 0.02). Moreover, 85.3% of infected persons younger than 25 years had Beijing modern strain, suggesting a possible recent spread in the young population by this family of TB strain in Taipei. Our data on MTB genotype in Taipei suggest that MTB infection has not been optimally controlled. Control efforts should be reinforced in view of the

  15. The Influence of Host and Bacterial Genotype on the Development of Disseminated Disease with Mycobacterium tuberculosis

    Science.gov (United States)

    Caws, Maxine; Thwaites, Guy; Dunstan, Sarah; Hawn, Thomas R.; Thi Ngoc Lan, Nguyen; Thuong, Nguyen Thuy Thuong; Stepniewska, Kasia; Huyen, Mai Nguyet Thu; Bang, Nguyen Duc; Huu Loc, Tran; Gagneux, Sebastien; van Soolingen, Dick; Kremer, Kristin; van der Sande, Marianne; Small, Peter; Thi Hoang Anh, Phan; Chinh, Nguyen Tran; Thi Quy, Hoang; Thi Hong Duyen, Nguyen; Quang Tho, Dau; Hieu, Nguyen T.; Torok, Estee; Hien, Tran Tinh; Dung, Nguyen Huy; Thi Quynh Nhu, Nguyen; Duy, Phan Minh; van Vinh Chau, Nguyen; Farrar, Jeremy

    2008-01-01

    The factors that govern the development of tuberculosis disease are incompletely understood. We hypothesized that some strains of Mycobacterium tuberculosis (M. tuberculosis) are more capable of causing disseminated disease than others and may be associated with polymorphisms in host genes responsible for the innate immune response to infection. We compared the host and bacterial genotype in 187 Vietnamese adults with tuberculous meningitis (TBM) and 237 Vietnamese adults with uncomplicated pulmonary tuberculosis. The host genotype of tuberculosis cases was also compared with the genotype of 392 cord blood controls from the same population. Isolates of M. tuberculosis were genotyped by large sequence polymorphisms. The hosts were defined by polymorphisms in genes encoding Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) and Toll-like receptor-2 (TLR-2). We found a significant protective association between the Euro-American lineage of M. tuberculosis and pulmonary rather than meningeal tuberculosis (Odds ratio (OR) for causing TBM 0.395, 95% confidence intervals (C.I.) 0.193–0.806, P = 0.009), suggesting these strains are less capable of extra-pulmonary dissemination than others in the study population. We also found that individuals with the C allele of TLR-2 T597C allele were more likely to have tuberculosis caused by the East-Asian/Beijing genotype (OR = 1.57 [95% C.I. 1.15–2.15]) than other individuals. The study provides evidence that M. tuberculosis genotype influences clinical disease phenotype and demonstrates, for the first time, a significant interaction between host and bacterial genotypes and the development of tuberculosis. PMID:18369480

  16. The influence of host and bacterial genotype on the development of disseminated disease with Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Maxine Caws

    2008-03-01

    Full Text Available The factors that govern the development of tuberculosis disease are incompletely understood. We hypothesized that some strains of Mycobacterium tuberculosis (M. tuberculosis are more capable of causing disseminated disease than others and may be associated with polymorphisms in host genes responsible for the innate immune response to infection. We compared the host and bacterial genotype in 187 Vietnamese adults with tuberculous meningitis (TBM and 237 Vietnamese adults with uncomplicated pulmonary tuberculosis. The host genotype of tuberculosis cases was also compared with the genotype of 392 cord blood controls from the same population. Isolates of M. tuberculosis were genotyped by large sequence polymorphisms. The hosts were defined by polymorphisms in genes encoding Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP and Toll-like receptor-2 (TLR-2. We found a significant protective association between the Euro-American lineage of M. tuberculosis and pulmonary rather than meningeal tuberculosis (Odds ratio (OR for causing TBM 0.395, 95% confidence intervals (C.I. 0.193-0.806, P = 0.009, suggesting these strains are less capable of extra-pulmonary dissemination than others in the study population. We also found that individuals with the C allele of TLR-2 T597C allele were more likely to have tuberculosis caused by the East-Asian/Beijing genotype (OR = 1.57 [95% C.I. 1.15-2.15] than other individuals. The study provides evidence that M. tuberculosis genotype influences clinical disease phenotype and demonstrates, for the first time, a significant interaction between host and bacterial genotypes and the development of tuberculosis.

  17. LIGAND-BINDING SITES ON THE MYCOBACTERIUM TUBERCULOSIS UREASE

    Directory of Open Access Journals (Sweden)

    Lisnyak Yu. V.

    2017-10-01

    Full Text Available Introduction. Mycobacterium tuberculosis is the causative agent of tuberculosis that remains a serious medical and social health problem. Despite intensive efforts have been made in the past decade, there are no new efficient anti-tuberculosis drugs today, and that need is growing due to the spread of drug-resistant strains of M.tuberculosis. M. tuberculosis urease (MTU, being an important factor of the bacterium viability and virulence, is an attractive target for anti-tuberculosis drugs acting by inhibition of urease activity. However, the commercially available urease inhibitors are toxic and unstable, that prevent their clinical use. Therefore, new more potent anti-tuberculosis drugs inhibiting new targets are urgently needed. A useful tool for the search of novel inhibitors is a computational drug design. The inhibitor design is significantly easier if binding sites on the enzyme are identified in advance. This paper aimed to determine the probable ligand binding sites on the surface of M. tuberculosis urease. Methods. To identify ligand binding sites on MTU surface, сomputational solvent mapping method FTSite was applied by the use of MTU homology model we have built earlier. The method places molecular probes (small organic molecules containing various functional groups on a dense grid defined around the enzyme, and for each probe finds favorable positions. The selected poses are refined by free energy minimization, the low energy conformations are clustered, and the clusters are ranked on the basis of the average free energy. FTSite server outputs the protein residues delineating a binding sites and the probe molecules representing each cluster. To predict allosteric pockets on MTU, AlloPred and AlloSite servers were applied. AlloPred uses the normal mode analysis (NMA and models how the dynamics of a protein would be altered in the presence of a modulator at a specific pocket. Pockets on the enzyme are predicted using the Fpocket

  18. Diminished Adherence and/or Ingestion of Virulent Mycobacterium tuberculosis by Monocyte-Derived Macrophages from Patients with Tuberculosis

    Science.gov (United States)

    Zabaleta, J.; Arias, M.; Maya, J. R.; García, L. F.

    1998-01-01

    The interaction between the macrophage and Mycobacterium tuberculosis is mediated by a variety of macrophage membrane-associated proteins. Complement receptors have been implicated in the adherence of M. tuberculosis to macrophages. In the present work, the adherence and/or ingestion of M. tuberculosis H37Rv to human monocyte-derived macrophages (MDM) from patients with tuberculosis (TB) and healthy controls was measured by microscopical examination, [3H]uracil incorporation, and CFU. The adherence and/or ingestion was enhanced by fresh serum and inhibited by heat inactivation, EDTA treatment, and anti-CR1 and anti-CR3 antibodies. Comparison of MDM from TB patients and healthy controls showed that the former exhibited a significantly decreased capacity to adhere and/or ingest M. tuberculosis, as determined by the number of CFU and 3H incorporation. The expression of CR1 (CD35) and CR3 (CD11b/CD18) on MDM from TB patients and healthy controls, as determined by flow cytometry, did not show significant differences. These results suggest that the lower ingestion of M. tuberculosis by MDM from TB patients is not due to defects in complement receptors, and therefore, there might be other molecules involved in the adherence and/or ingestion process that render MDM from TB patients ingest less mycobacteria than those from healthy controls. PMID:9729537

  19. Inactivation of Mycobacterium paratuberculosis and Mycobacterium tuberculosis in fresh soft cheese by gamma radiation

    International Nuclear Information System (INIS)

    Badr, Hesham M.

    2011-01-01

    The effectiveness of gamma irradiation on the inactivation of Mycobacterium paratuberculosis, Mycobacterium bovis and Mycobacterium tuberculosis in fresh soft cheese that prepared from artificially inoculated milk samples was studied. Irradiation at dose of 2 kGy was sufficient for the complete inactivation of these mycobacteria as they were not detected in the treated samples during storage at 4±1 o C for 15 days. Moreover, irradiation of cheese samples, that were prepared from un-inoculated milk, at this effective dose had no significant effects on their gross composition and contents from riboflavin, niacin and pantothenic acid, while significant decreases in vitamin A and thiamin were observed. In addition, irradiation of cheese samples had no significant effects on their pH and nitrogen fractions contents, except for the contents of ammonia, which showed a slight, but significant, increases due to irradiation. The analysis of cheese fats indicated that irradiation treatment induced significant increase in their oxidation parameters and contents from free fatty acids; however, the observed increases were relatively low. On the other hand, irradiation of cheese samples induced no significant alterations on their sensory properties. Thus, irradiation dose of 2 kGy can be effectively applied to ensure the safety of soft cheese with regards to these harmful mycobacteria. - Highlights: → We examined the effectiveness of gamma irradiation on inactivation of Mycobacterium paratuberculosis, Mycobacterium bovis and Mycobacterium tuberculosis in fresh soft cheese. → Irradiation at dose of 2 kGy was sufficient for complete inactivation of these mycobacteria. → Irradiation of cheese samples induced no significant alterations on their sensory properties.

  20. Inactivation of Mycobacterium paratuberculosis and Mycobacterium tuberculosis in fresh soft cheese by gamma radiation

    Energy Technology Data Exchange (ETDEWEB)

    Badr, Hesham M., E-mail: heshambadr_aea@yahoo.co.uk [Atomic Energy Authority, Nuclear Research Center, Abou Zaabal, P.O. Box 13759 Cairo (Egypt)

    2011-11-15

    The effectiveness of gamma irradiation on the inactivation of Mycobacterium paratuberculosis, Mycobacterium bovis and Mycobacterium tuberculosis in fresh soft cheese that prepared from artificially inoculated milk samples was studied. Irradiation at dose of 2 kGy was sufficient for the complete inactivation of these mycobacteria as they were not detected in the treated samples during storage at 4{+-}1 {sup o}C for 15 days. Moreover, irradiation of cheese samples, that were prepared from un-inoculated milk, at this effective dose had no significant effects on their gross composition and contents from riboflavin, niacin and pantothenic acid, while significant decreases in vitamin A and thiamin were observed. In addition, irradiation of cheese samples had no significant effects on their pH and nitrogen fractions contents, except for the contents of ammonia, which showed a slight, but significant, increases due to irradiation. The analysis of cheese fats indicated that irradiation treatment induced significant increase in their oxidation parameters and contents from free fatty acids; however, the observed increases were relatively low. On the other hand, irradiation of cheese samples induced no significant alterations on their sensory properties. Thus, irradiation dose of 2 kGy can be effectively applied to ensure the safety of soft cheese with regards to these harmful mycobacteria. - Highlights: > We examined the effectiveness of gamma irradiation on inactivation of Mycobacterium paratuberculosis, Mycobacterium bovis and Mycobacterium tuberculosis in fresh soft cheese. > Irradiation at dose of 2 kGy was sufficient for complete inactivation of these mycobacteria. > Irradiation of cheese samples induced no significant alterations on their sensory properties.

  1. Comparative analysis of mycobacterium and related actinomycetes yields insight into the evolution of mycobacterium tuberculosis pathogenesis

    Directory of Open Access Journals (Sweden)

    McGuire Abigail

    2012-03-01

    Full Text Available Abstract Background The sequence of the pathogen Mycobacterium tuberculosis (Mtb strain H37Rv has been available for over a decade, but the biology of the pathogen remains poorly understood. Genome sequences from other Mtb strains and closely related bacteria present an opportunity to apply the power of comparative genomics to understand the evolution of Mtb pathogenesis. We conducted a comparative analysis using 31 genomes from the Tuberculosis Database (TBDB.org, including 8 strains of Mtb and M. bovis, 11 additional Mycobacteria, 4 Corynebacteria, 2 Streptomyces, Rhodococcus jostii RHA1, Nocardia farcinia, Acidothermus cellulolyticus, Rhodobacter sphaeroides, Propionibacterium acnes, and Bifidobacterium longum. Results Our results highlight the functional importance of lipid metabolism and its regulation, and reveal variation between the evolutionary profiles of genes implicated in saturated and unsaturated fatty acid metabolism. It also suggests that DNA repair and molybdopterin cofactors are important in pathogenic Mycobacteria. By analyzing sequence conservation and gene expression data, we identify nearly 400 conserved noncoding regions. These include 37 predicted promoter regulatory motifs, of which 14 correspond to previously validated motifs, as well as 50 potential noncoding RNAs, of which we experimentally confirm the expression of four. Conclusions Our analysis of protein evolution highlights gene families that are associated with the adaptation of environmental Mycobacteria to obligate pathogenesis. These families include fatty acid metabolism, DNA repair, and molybdopterin biosynthesis. Our analysis reinforces recent findings suggesting that small noncoding RNAs are more common in Mycobacteria than previously expected. Our data provide a foundation for understanding the genome and biology of Mtb in a comparative context, and are available online and through TBDB.org.

  2. Comparative analysis of Mycobacterium and related Actinomycetes yields insight into the evolution of Mycobacterium tuberculosis pathogenesis.

    Science.gov (United States)

    McGuire, Abigail Manson; Weiner, Brian; Park, Sang Tae; Wapinski, Ilan; Raman, Sahadevan; Dolganov, Gregory; Peterson, Matthew; Riley, Robert; Zucker, Jeremy; Abeel, Thomas; White, Jared; Sisk, Peter; Stolte, Christian; Koehrsen, Mike; Yamamoto, Robert T; Iacobelli-Martinez, Milena; Kidd, Matthew J; Maer, Andreia M; Schoolnik, Gary K; Regev, Aviv; Galagan, James

    2012-03-28

    The sequence of the pathogen Mycobacterium tuberculosis (Mtb) strain H37Rv has been available for over a decade, but the biology of the pathogen remains poorly understood. Genome sequences from other Mtb strains and closely related bacteria present an opportunity to apply the power of comparative genomics to understand the evolution of Mtb pathogenesis. We conducted a comparative analysis using 31 genomes from the Tuberculosis Database (TBDB.org), including 8 strains of Mtb and M. bovis, 11 additional Mycobacteria, 4 Corynebacteria, 2 Streptomyces, Rhodococcus jostii RHA1, Nocardia farcinia, Acidothermus cellulolyticus, Rhodobacter sphaeroides, Propionibacterium acnes, and Bifidobacterium longum. Our results highlight the functional importance of lipid metabolism and its regulation, and reveal variation between the evolutionary profiles of genes implicated in saturated and unsaturated fatty acid metabolism. It also suggests that DNA repair and molybdopterin cofactors are important in pathogenic Mycobacteria. By analyzing sequence conservation and gene expression data, we identify nearly 400 conserved noncoding regions. These include 37 predicted promoter regulatory motifs, of which 14 correspond to previously validated motifs, as well as 50 potential noncoding RNAs, of which we experimentally confirm the expression of four. Our analysis of protein evolution highlights gene families that are associated with the adaptation of environmental Mycobacteria to obligate pathogenesis. These families include fatty acid metabolism, DNA repair, and molybdopterin biosynthesis. Our analysis reinforces recent findings suggesting that small noncoding RNAs are more common in Mycobacteria than previously expected. Our data provide a foundation for understanding the genome and biology of Mtb in a comparative context, and are available online and through TBDB.org.

  3. Rapid detection of Mycobacterium tuberculosis by recombinase polymerase amplification.

    Directory of Open Access Journals (Sweden)

    David S Boyle

    Full Text Available Improved access to effective tests for diagnosing tuberculosis (TB has been designated a public health priority by the World Health Organisation. In high burden TB countries nucleic acid based TB tests have been restricted to centralised laboratories and specialised research settings. Requirements such as a constant electrical supply, air conditioning and skilled, computer literate operators prevent implementation of such tests in many settings. Isothermal DNA amplification technologies permit the use of simpler, less energy intensive detection platforms more suited to low resource settings that allow the accurate diagnosis of a disease within a short timeframe. Recombinase Polymerase Amplification (RPA is a rapid, low temperature isothermal DNA amplification reaction. We report here RPA-based detection of Mycobacterium tuberculosis complex (MTC DNA in <20 minutes at 39 °C. Assays for two MTC specific targets were investigated, IS6110 and IS1081. When testing purified MTC genomic DNA, limits of detection of 6.25 fg (IS6110 and 20 fg (IS1081were consistently achieved. When testing a convenience sample of pulmonary specimens from suspected TB patients, RPA demonstrated superior accuracy to indirect fluorescence microscopy. Compared to culture, sensitivities for the IS1081 RPA and microscopy were 91.4% (95%CI: 85, 97.9 and 86.1% (95%CI: 78.1, 94.1 respectively (n = 71. Specificities were 100% and 88.6% (95% CI: 80.8, 96.1 respectively. For the IS6110 RPA and microscopy sensitivities of 87.5% (95%CI: 81.7, 93.2 and 70.8% (95%CI: 62.9, 78.7 were obtained (n = 90. Specificities were 95.4 (95% CI: 92.3,98.1 and 88% (95% CI: 83.6, 92.4 respectively. The superior specificity of RPA for detecting tuberculosis was due to the reduced ability of fluorescence microscopy to distinguish Mtb complex from other acid fast bacteria. The rapid nature of the RPA assay and its low energy requirement compared to other amplification technologies suggest RPA-based TB

  4. Human ULK1 Variation and Susceptibility to Mycobacterium tuberculosis Infection.

    Science.gov (United States)

    Horne, David J; Graustein, Andrew D; Shah, Javeed A; Peterson, Glenna; Savlov, Meg; Steele, Sergio; Narita, Masahiro; Hawn, Thomas R

    2016-10-15

    Unlike tuberculosis, few studies have evaluated a host genetic basis for variability in susceptibility to latent Mycobacterium tuberculosis infection (LTBI). We performed a candidate gene association study of autophagy-related genes and LTBI. We enrolled close contacts of individuals with pulmonary tuberculosis, assessed LTBI status, and determined clinical and sociodemographic risk factors for LTBI. In participants who self-identified as Asian or black, we compared haplotype-tagging single-nucleotide polymorphisms (SNPs) in ULK1 and GABARAP between cases (n = 143) and controls (n = 106). Using CRISPR/Cas9 in U937 monocytes, we investigated the effect of ULK1 deficiency on cytokine expression, autophagy, and M. tuberculosis replication. In Asian participants, we identified 2 ULK1 SNPs (rs12297124 and rs7300908) associated with LTBI. After adjustment for population admixture and clinical risk for LTBI, each rs12297124 minor allele conferred 80% reduction in LTBI risk (odds ratio, 0.18; 95% confidence interval, .07-.46). Compared with controls, ULK1-deficient cells exhibited decreased tumor necrosis factor secretion after stimulation with Toll-like receptor ligands and M. tuberculosis whole-cell lysate, increased M. tuberculosis replication, and decreased selective autophagy. These results demonstrate a strong association of rs12297124, a noncoding ULK1 SNP, with LTBI and a role for ULK1 regulation of TNF secretion, nonspecific and M. tuberculosis-induced autophagy, and M. tuberculosis replication in monocytes. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  5. Dendritic Cells Activate and Mature after Infection with Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Mamo Gezahagne

    2011-07-01

    Full Text Available Abstract Background Dendritic cells (DCs can take up an array of different antigens, including microorganisms which they can process and present more effectively than any other antigen presenting cell. However, whether the interaction between the human DC and Mycobacterium tuberculosis represents a defense mechanism by the invaded host, or helping the invader to evade the defense mechanism of the host is still not clearly understood. Findings To analyze the interactions between M. tuberculosis and immune cells, human peripheral blood monocyte-derived immature DCs were infected with M. tuberculosis H37Rv wild type strain and flow cytometry was used to analyse cell surface expression markers. The ability of the M. tuberculosis infected DC to induce T cell proliferation using 5 and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE dilution technique was also investigated. DCs were found to internalize the mycobacteria and show dose dependent infection and necrosis with different multiplicity of infection. Flow cytometry analysis of cell surface expression markers CD40, CD54, CD80, CD83, CD86 and HLA DR in infected DC revealed significant (p M. tuberculosis in comparison to immature DC with no stimulation. Lipopolysaccharide (LPS from Salmonella abortus equi, a known DC maturation agent, was used as a positive control and showed a comparable up regulation of cell surface markers as observed with M. tuberculosis infected DC. It was revealed that the M. tuberculosis infected DC induced T cell proliferation. Conclusion These data clearly demonstrate that M. tuberculosis induces activation and maturation of human monocyte-derived immature DC as well as induces T cell proliferation in vitro.

  6. Microbial sensor for drug susceptibility testing of Mycobacterium tuberculosis.

    Science.gov (United States)

    Zhang, Z-T; Wang, D-B; Li, C-Y; Deng, J-Y; Zhang, J-B; Bi, L-J; Zhang, X-E

    2018-01-01

    Drug susceptibility testing (DST) of clinical isolates of Mycobacterium tuberculosis is critical in treating tuberculosis. We demonstrate the possibility of using a microbial sensor to perform DST of M. tuberculosis and shorten the time required for DST. The sensor is made of an oxygen electrode with M. tuberculosis cells attached to its surface. This sensor monitors the residual oxygen consumption of M. tuberculosis cells after treatment with anti-TB drugs with glycerine as a carbon source. In principle, after drug pretreatment for 4-5 days, the response differences between the sensors made of drug-sensitive isolates are distinguishable from the sensors made of drug-resistant isolates. The susceptibility of the M. tuberculosis H37Ra strain, its mutants and 35 clinical isolates to six common anti-TB drugs: rifampicin, isoniazid, streptomycin, ethambutol, levofloxacin and para-aminosalicylic acid were tested using the proposed method. The results agreed well with the gold standard method (LJ) and were determined in significantly less time. The whole procedure takes approximately 11 days and therefore has the potential to inform clinical decisions. To our knowledge, this is the first study that demonstrates the possible application of a dissolved oxygen electrode-based microbial sensor in M. tuberculosis drug resistance testing. This study used the microbial sensor to perform DST of M. tuberculosis and shorten the time required for DST. The overall detection result of the microbial sensor agreed well with that of the conventional LJ proportion method and takes less time than the existing phenotypic methods. In future studies, we will build an O 2 electrode array microbial sensor reactor to enable a high-throughput drug resistance analysis. © 2017 The Authors. Journal of Applied Microbiology published by John Wiley & Sons Ltd on behalf of The Society for Applied Microbiology.

  7. Bim is a crucial regulator of apoptosis induced by Mycobacterium tuberculosis

    Science.gov (United States)

    Aguiló, N; Uranga, S; Marinova, D; Martín, C; Pardo, J

    2014-01-01

    Mycobacterium tuberculosis, the causative agent of tuberculosis, induces apoptosis in infected macrophages in vitro and in vivo. However, the molecular mechanism controlling this process is not known. In order to study the involvement of the mitochondrial apoptotic pathway in M. tuberculosis-induced apoptosis, we analysed cell death in M. tuberculosis-infected embryonic fibroblasts (MEFs) derived from different knockout mice for genes involved in this route. We found that apoptosis induced by M. tuberculosis is abrogated in the absence of Bak and Bax, caspase 9 or the executioner caspases 3 and 7. Notably, we show that MEF deficient in the BH3-only BCL-2-interacting mediator of cell death (Bim) protein were also resistant to this process. The relevance of these results has been confirmed in the mouse macrophage cell line J774, where cell transfection with siRNA targeting Bim impaired apoptosis induced by virulent mycobacteria. Notably, only infection with a virulent strain, but not with attenuated ESX-1-defective strains, such as Bacillus Calmette-Guerin and live-attenuated M. tuberculosis vaccine strain MTBVAC, induced Bim upregulation and apoptosis, probably implicating virulence factor early secreted antigenic target 6-kDa protein in this process. Our results suggest that Bim upregulation and apoptosis is mediated by the p38MAPK-dependent pathway. Our findings show that Bim is a master regulator of apoptosis induced by M. tuberculosis. PMID:25032866

  8. Conserved hypothetical protein Rv1977 in Mycobacterium tuberculosis strains contains sequence polymorphisms and might be involved in ongoing immune evasion.

    Science.gov (United States)

    Jiang, Yi; Liu, Haican; Wang, Xuezhi; Li, Guilian; Qiu, Yan; Dou, Xiangfeng; Wan, Kanglin

    2015-01-01

    Host immune pressure and associated parasite immune evasion are key features of host-pathogen co-evolution. A previous study showed that human T cell epitopes of Mycobacterium tuberculosis are evolutionarily hyperconserved and thus it was deduced that M. tuberculosis lacks antigenic variation and immune evasion. Here, we selected 151 clinical Mycobacterium tuberculosis isolates from China, amplified gene encoding Rv1977 and compared the sequences. The results showed that Rv1977, a conserved hypothetical protein, is not conserved in M. tuberculosis strains and there are polymorphisms existed in the protein. Some mutations, especially one frameshift mutation, occurred in the antigen Rv1977, which is uncommon in M.tb strains and may lead to the protein function altering. Mutations and deletion in the gene all affect one of three T cell epitopes and the changed T cell epitope contained more than one variable position, which may suggest ongoing immune evasion.

  9. Dehalogenation of Haloalkanes by Mycobacterium tuberculosis H37Rv and Other Mycobacteria

    Science.gov (United States)

    Jesenská, Andrea; Sedlác̆ek, Ivo; Damborský, Jir̆í

    2000-01-01

    Haloalkane dehalogenases convert haloalkanes to their corresponding alcohols by a hydrolytic mechanism. To date, various haloalkane dehalogenases have been isolated from bacteria colonizing environments that are contaminated with halogenated compounds. A search of current databases with the sequences of these known haloalkane dehalogenases revealed the presence of three different genes encoding putative haloalkane dehalogenases in the genome of the human parasite Mycobacterium tuberculosis H37Rv. The ability of M. tuberculosis and several other mycobacterial strains to dehalogenate haloaliphatic compounds was therefore studied. Intact cells of M. tuberculosis H37Rv were found to dehalogenate 1-chlorobutane, 1-chlorodecane, 1-bromobutane, and 1,2-dibromoethane. Nine isolates of mycobacteria from clinical material and four strains from a collection of microorganisms were found to be capable of dehalogenating 1,2-dibromoethane. Crude extracts prepared from two of these strains, Mycobacterium avium MU1 and Mycobacterium smegmatis CCM 4622, showed broad substrate specificity toward a number of halogenated substrates. Dehalogenase activity in the absence of oxygen and the identification of primary alcohols as the products of the reaction suggest a hydrolytic dehalogenation mechanism. The presence of dehalogenases in bacterial isolates from clinical material, including the species colonizing both animal tissues and free environment, indicates a possible role of parasitic microorganisms in the distribution of degradation genes in the environment. PMID:10618227

  10. Interaction of Mycobacterium tuberculosis with human respiratory mucosa.

    Science.gov (United States)

    Middleton, A M; Chadwick, M V; Nicholson, A G; Dewar, A; Groger, R K; Brown, E J; Ratliff, T L; Wilson, R

    2002-01-01

    Endobronchial infection is associated with pulmonary tuberculosis in the majority of cases. We have investigated the adherence of Mycobacterium tuberculosis to the human respiratory mucosa. Organ cultures constructed with human tissue were infected with M. tuberculosis in the presence or absence of mycobacterial fibronectin attachment cell surface proteins and examined by scanning electron microscopy. M. tuberculosis adhered mainly to extracellular matrix (ECM) in areas of mucosal damage, but not to ciliated mucosa, intact extruded cells, basement membrane or collagen fibres. Bacteria also adhered to fibrous but not globular mucus and occasionally to healthy unciliated mucosa, open tight junctions and to extruded cells that had degenerated, exposing their contents. There was a significant reduction (pprotein (FAP) and M. bovis antigen 85B protein, in a concentration dependent manner. The combined effect of FAP and antigen 85B protein was significantly greater than either protein alone. Bacterial adherence to fibrous mucus was not influenced by fibronectin. We conclude that M. tuberculosis adheres to ECM in areas of mucosal damage at least in part via FAP and antigen 85B protein.

  11. Combating highly resistant emerging pathogen Mycobacterium abscessus and Mycobacterium tuberculosis with novel salicylanilide esters and carbamates.

    Science.gov (United States)

    Baranyai, Zsuzsa; Krátký, Martin; Vinšová, Jarmila; Szabó, Nóra; Senoner, Zsuzsanna; Horváti, Kata; Stolaříková, Jiřina; Dávid, Sándor; Bősze, Szilvia

    2015-08-28

    In the Mycobacterium genus over one hundred species are already described and new ones are periodically reported. Species that form colonies in a week are classified as rapid growers, those requiring longer periods (up to three months) are the mostly pathogenic slow growers. More recently, new emerging species have been identified to lengthen the list, all rapid growers. Of these, Mycobacterium abscessus is also an intracellular pathogen and it is the most chemotherapy-resistant rapid-growing mycobacterium. In addition, the cases of multidrug-resistant Mycobacterium tuberculosis infection are also increasing. Therefore there is an urgent need to find new active molecules against these threatening strains. Based on previous results, a series of salicylanilides, salicylanilide 5-chloropyrazinoates and carbamates was designed, synthesized and characterised. The compounds were evaluated for their in vitro activity on M. abscessus, susceptible M. tuberculosis H37Rv, multidrug-resistant (MDR) M. tuberculosis MDR A8, M. tuberculosis MDR 9449/2006 and on the extremely-resistant Praha 131 (XDR) strains. All derivatives exhibited a significant activity with minimum inhibitory concentrations (MICs) in the low micromolar range. Eight salicylanilide carbamates and two salicylanilide esters exhibited an excellent in vitro activity on M. abscessus with MICs from 0.2 to 2.1 μM, thus being more effective than ciprofloxacin and gentamicin. This finding is potentially promising, particularly, as M. abscessus is a threateningly chemotherapy-resistant species. M. tuberculosis H37Rv was inhibited with MICs from 0.2 μM, and eleven compounds have lower MICs than isoniazid. Salicylanilide esters and carbamates were found that they were effective also on MDR and XDR M. tuberculosis strains with MICs ≥1.0 μM. The in vitro cytotoxicity (IC50) was also determined on human MonoMac-6 cells, and selectivity index (SI) of the compounds was established. In general, salicylanilide

  12. Mycobacterium tuberculosis Complex Members Adapted to Wild and Domestic Animals.

    Science.gov (United States)

    Malone, Kerri M; Gordon, Stephen V

    2017-01-01

    The Mycobacterium tuberculosis complex (MTBC) is composed of several highly genetically related species that can be broadly classified into those that are human-host adapted and those that possess the ability to propagate and transmit in a variety of wild and domesticated animals. Since the initial description of the bovine tubercle bacillus, now known as Mycobacterium bovis, by Theobald Smith in the late 1800's, isolates originating from a wide range of animal hosts have been identified and characterized as M. microti, M. pinnipedii, the Dassie bacillus, M. mungi, M. caprae, M. orygis and M. suricattae. This chapter outlines the events resulting in the identification of each of these animal-adapted species, their close genetic relationships, and how genome-based phylogenetic analyses of species-specific variation amongst MTBC members is beginning to unravel the events that resulted in the evolution of the MTBC and the observed host tropism between the human- and animal-adapted member species.

  13. Multiplexed Quantitation of Intraphagocyte Mycobacterium tuberculosis Secreted Protein Effectors

    Directory of Open Access Journals (Sweden)

    Fadel Sayes

    2018-04-01

    Full Text Available Summary: The pathogenic potential of Mycobacterium tuberculosis largely depends on ESX secretion systems exporting members of the multigenic Esx, Esp, and PE/PPE protein families. To study the secretion and regulation patterns of these proteins while circumventing immune cross-reactions due to their extensive sequence homologies, we developed an approach that relies on the recognition of their MHC class II epitopes by highly discriminative T cell receptors (TCRs of a panel of T cell hybridomas. The latter were engineered so that each expresses a unique fluorescent reporter linked to specific antigen recognition. The resulting polychromatic and multiplexed imaging assay enabled us to measure the secretion of mycobacterial effectors inside infected host cells. We applied this novel technology to a large panel of mutants, clinical isolates, and host-cell types to explore the host-mycobacteria interplay and its impact on the intracellular bacterial secretome, which also revealed the unexpected capacity of phagocytes from lung granuloma to present mycobacterial antigens via MHC class II. : Sayes et al. develop an approach to express distinct fluorescent reporters that is based on the recognition of specific Mycobacterium tuberculosis MHC class II epitopes by highly discriminative T cell hybridomas. This multiplexed technology allows the study of secretion, subcellular location, and regulation patterns of these instrumental protein members. Keywords: mycobacterium tuberculosis, type VII secretion systems, intracellular bacteria, T-cell hybridomas, mycobacterial virulence factors, bacterial antigen presentation, lentiviral vectors, reporter T cells, in vivo antigen presentation, protein localization

  14. Mycobacterium tuberculosis whole genome sequencing and protein structure modelling provides insights into anti-tuberculosis drug resistance

    KAUST Repository

    Phelan, Jody; Coll, Francesc; McNerney, Ruth; Ascher, David B.; Pires, Douglas E. V.; Furnham, Nick; Coeck, Nele; Hill-Cawthorne, Grant A.; Nair, Mridul; Mallard, Kim; Ramsay, Andrew; Campino, Susana; Hibberd, Martin L.; Pain, Arnab; Rigouts, Leen; Clark, Taane G.

    2016-01-01

    of 144 Mycobacterium tuberculosis clinical isolates from The Special Programme for Research and Training in Tropical Diseases (TDR) collection sourced from 20 countries in four continents. A genome-wide approach was applied to 127 isolates to identify

  15. Diffuse Type Primary Mycobacterium Tuberculosis of the Breast: A Case Report

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Hyun A; Kang, Bong Joo; Kim, Sung Hun; Kim, Hnana; Lee, Ah Won [Seoul St. Mary' s Hospital, The Catholic University of Korea, Seoul (Korea, Republic of)

    2011-12-15

    Tuberculous mastitis is a rare manifestation of mycobacterium tuberculosis infection. It mimics inflammatory breast cancer or other pyogenic inflammations. In most of the tuberculous mastitis reports, coexisting or prior tuberculosis infection and secondary infection of the breast by direct spread via axillary or cervical lymphadenopathy, or hematogenous spread have been noted. We describe the mammographic and ultrasonographic findings of a case of diffuse type mycobacterium tuberculosis of the breast showing diffuse edema which was confirmed as tuberculosis through biopsy and had no evidence of old or concurrent pulmonary tuberculosis on chest computed tomography

  16. Structure of Rv1848 (UreA), the Mycobacterium tuberculosis urease γ subunit

    International Nuclear Information System (INIS)

    Habel, Jeff E.; Bursey, Evan H.; Rho, Beom-Seop; Kim, Chang-Yub; Segelke, Brent W.; Rupp, Bernhard; Park, Min S.; Terwilliger, Thomas C.; Hung, Li-Wei

    2010-01-01

    Crystal and solution structures of Rv1848 protein and their implications in the biological assembly of Mtb urease is presented. The crystal structure of the urease γ subunit (UreA) from Mycobacterium tuberculosis, Rv1848, has been determined at 1.8 Å resolution. The asymmetric unit contains three copies of Rv1848 arranged into a homotrimer that is similar to the UreA trimer in the structure of urease from Klebsiella aerogenes. Small-angle X-ray scattering experiments indicate that the Rv1848 protein also forms trimers in solution. The observed homotrimer and the organization of urease genes within the M. tuberculosis genome suggest that M. tuberculosis urease has the (αβγ) 3 composition observed for other bacterial ureases. The γ subunit may be of primary importance for the formation of the urease quaternary structure

  17. Improved method for testing susceptibility of Mycobacterium tuberculosis to pyrazinamide.

    OpenAIRE

    Butler, W R; Kilburn, J O

    1982-01-01

    The acid medium required to test susceptibility of Mycobacterium tuberculosis to pyrazinamide (PZA) is a major problem in obtaining reliable test results. Satisfactory growth is usually obtained on Middlebrook and Cohn 7H10 medium at pH 5.5 if albumin-dextrose-catalase (ADC) supplement rather than oleic acid-albumin-dextrose-catalase is used; however, some lots of ADC supplement still fail to support growth at this low pH. A rapid turbidimetric test was developed to determine the growth-suppo...

  18. Anti-Mycobacterium tuberculosis activity of fungus Phomopsis stipata

    Directory of Open Access Journals (Sweden)

    Karina Andrade de Prince

    2012-03-01

    Full Text Available Our purpose was to determine the anti-Mycobacterium tuberculosis activity of the metabolites produced by the endophitic fungus Phomopsis stipata (Lib. B. Sutton, (Diaporthaceae, cultivated in different media. The antimycobacterial activity was assessed through the Resazurin Microtiter Assay (REMA and the cytotoxicity test performed on macrophage cell line. The extracts derived from fungi grown on Corn Medium and Potato Dextrose Broth presented the smallest values of Minimum Inhibitory Concentration (MIC and low cytotoxicity, which implies a high selectivity index. This is the first report on the chemical composition and antitubercular activity of metabolites of P. stipata, as well as the influence of culture medium on these properties.

  19. Drug Susceptibility of Mycobacterium tuberculosis Beijing Genotype and Association with MDR TB

    Science.gov (United States)

    ten Kate, Marian T.; de Knegt, Gerjo J.; Kremer, Kristin; Aarnoutse, Rob E.; Boeree, Martin J.; Verbrugh, Henri A.; van Soolingen, Dick; Bakker-Woudenberg, Irma A.J.M.

    2012-01-01

    To determine differences in the ability of Mycobacterium tuberculosis strains to withstand antituberculosis drug treatment, we compared the activity of antituberculosis drugs against susceptible Beijing and East-African/Indian genotype M. tuberculosis strains. Beijing genotype strains showed high rates of mutation within a wide range of drug concentrations, possibly explaining this genotype’s association with multidrug-resistant tuberculosis. PMID:22469099

  20. Mycobacterium tuberculosis Genotype and Case Notification Rates, Rural Vietnam, 2003-2006

    NARCIS (Netherlands)

    Buu, T.N.; Huyen, M.N.T.; Lan, N.N.T.; Quy, H.T.; Hen, N.V.; Zignol, M.; Borgdorff, M.W.; van Soolingen, D.; Cobelens, F.G.J.

    2009-01-01

    Tuberculosis case notification rates (CNRs) for young adults in Vietnam are increasing. To determine whether this finding could reflect emergence of Mycobacterium tuberculosis Beijing genotype, we studied all new sputum smear-positive pulmonary tuberculosis patients registered for treatment in 3

  1. Revaccination of Guinea Pigs With the Live Attenuated Mycobacterium tuberculosis Vaccine MTBVAC Improves BCG's Protection Against Tuberculosis.

    Science.gov (United States)

    Clark, Simon; Lanni, Faye; Marinova, Dessislava; Rayner, Emma; Martin, Carlos; Williams, Ann

    2017-09-01

    The need for an effective vaccine against human tuberculosis has driven the development of different candidates and vaccination strategies. Novel live attenuated vaccines are being developed that promise greater safety and efficacy than BCG against tuberculosis. We combined BCG with the vaccine MTBVAC to evaluate whether the efficacy of either vaccine would be affected upon revaccination. In a well-established guinea pig model of aerosol infection with Mycobacterium tuberculosis, BCG and MTBVAC delivered via various prime-boost combinations or alone were compared. Efficacy was determined by a reduction in bacterial load 4 weeks after challenge. Efficacy data suggests MTBVAC-associated immunity is longer lasting than that of BCG when given as a single dose. Long and short intervals between BCG prime and MTBVAC boost resulted in improved efficacy in lungs, compared with BCG given alone. A shorter interval between MTBVAC prime and BCG boost resulted in improved efficacy in lungs, compared with BCG given alone. A longer interval resulted in protection equivalent to that of BCG given alone. These data indicate that, rather than boosting the waning efficacy of BCG, a vaccination schedule involving a combination of the 2 vaccines yielded stronger immunity to M. tuberculosis infection. This work supports development of MTBVAC use as a revaccination strategy to improve on the effects of BCG in vaccinated people living in tuberculosis-endemic countries. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

  2. Identification of a novel 27-kDa protein from Mycobacterium tuberculosis culture fluid by a monoclonal antibody specific for the Mycobacterium tuberculosis complex

    NARCIS (Netherlands)

    Rambukkana, A.; Das, P. K.; Kolk, A. H.; Burggraaf, J. D.; Kuijper, S.; Harboe, M.

    1993-01-01

    Mycobacterium tuberculosis antigens inducing species-specific immune responses are likely to be particularly important for serodiagnosis or for skin testing of tuberculosis. In the present study, we describe the characterization of two novel monoclonal antibodies (MoAbs) A3h4 (IgG2a) and B5g1 (IgM)

  3. Mycobacterium tuberculosis Infection following Kidney Transplantation

    Directory of Open Access Journals (Sweden)

    Karima Boubaker

    2013-01-01

    Full Text Available Introduction and Aims. Post-transplant tuberculosis (TB is a problem in successful long-term outcome of renal transplantation recipients. Our objective was to describe the pattern and risk factors of TB infection and the prognosis in our transplant recipients. Patients and Methods. This study was a retrospective review of the records of 491 renal transplant recipients in our hospital during the period from January 1986 to December 2009. The demographic data, transplant characteristics, clinical manifestations, diagnostic criteria, treatment protocol, and long-term outcome of this cohort of patients were analyzed. Results. 16 patients (3,2% developed post-transplant TB with a mean age of 32,5 ± 12,7 (range: 13–60 years and a mean post-transplant period of 36,6months (range: 12,3 months–15,9 years. The forms of the diseases were pulmonary in 10/16 (62,6%, disseminated in 3/16 (18,7%, and extrapulmonary in 3/16 (18,7%. Graft dysfunction was observed in 7 cases (43,7% with tissue-proof acute rejection in 3 cases and loss of the graft in 4 cases. Hepatotoxicity developed in 3 patients (18,7% during treatment. Recurrences were observed in 4 cases after early stop of treatment. Two patients (12.5% died. Conclusion. Extra pulmonary and disseminated tuberculosis were observed in third of our patients. More than 9months of treatment may be necessary to prevent recurrence.

  4. Attenuated Mycobacterium tuberculosis SO2 vaccine candidate is unable to induce cell death.

    Directory of Open Access Journals (Sweden)

    Adriana Aporta

    Full Text Available It has been proposed that Mycobacterium tuberculosis virulent strains inhibit apoptosis and trigger cell death by necrosis of host macrophages to evade innate immunity, while non-virulent strains induce typical apoptosis activating a protective host response. As part of the characterization of a novel tuberculosis vaccine candidate, the M. tuberculosis phoP mutant SO2, we sought to evaluate its potential to induce host cell death. The parental M. tuberculosis MT103 strain and the current vaccine against tuberculosis Bacillus Calmette-Guérin (BCG were used as comparators in mouse models in vitro and in vivo. Our data reveal that attenuated SO2 was unable to induce apoptotic events neither in mouse macrophages in vitro nor during lung infection in vivo. In contrast, virulent MT103 triggers typical apoptotic events with phosphatidylserine exposure, caspase-3 activation and nuclear condensation and fragmentation. BCG strain behaved like SO2 and did not induce apoptosis. A clonogenic survival assay confirmed that viability of BCG- or SO2-infected macrophages was unaffected. Our results discard apoptosis as the protective mechanism induced by SO2 vaccine and provide evidence for positive correlation between classical apoptosis induction and virulent strains, suggesting apoptosis as a possible virulence determinant during M. tuberculosis infection.

  5. Profiling the Proteome of Mycobacterium tuberculosis during Dormancy and Reactivation*

    Science.gov (United States)

    Gopinath, Vipin; Raghunandanan, Sajith; Gomez, Roshna Lawrence; Jose, Leny; Surendran, Arun; Ramachandran, Ranjit; Pushparajan, Akhil Raj; Mundayoor, Sathish; Jaleel, Abdul; Kumar, Ramakrishnan Ajay

    2015-01-01

    Tuberculosis, caused by Mycobacterium tuberculosis, still remains a major global health problem. The main obstacle in eradicating this disease is the ability of this pathogen to remain dormant in macrophages, and then reactivate later under immuno-compromised conditions. The physiology of hypoxic nonreplicating M. tuberculosis is well-studied using many in vitro dormancy models. However, the physiological changes that take place during the shift from dormancy to aerobic growth (reactivation) have rarely been subjected to a detailed investigation. In this study, we developed an in vitro reactivation system by re-aerating the virulent laboratory strain of M. tuberculosis that was made dormant employing Wayne's dormancy model, and compared the proteome profiles of dormant and reactivated bacteria using label-free one-dimensional LC/MS/MS analysis. The proteome of dormant bacteria was analyzed at nonreplicating persistent stage 1 (NRP1) and stage 2 (NRP2), whereas that of reactivated bacteria was analyzed at 6 and 24 h post re-aeration. Proteome of normoxially grown bacteria served as the reference. In total, 1871 proteins comprising 47% of the M. tuberculosis proteome were identified, and many of them were observed to be expressed differentially or uniquely during dormancy and reactivation. The number of proteins detected at different stages of dormancy (764 at NRP1, 691 at NRP2) and reactivation (768 at R6 and 983 at R24) was very low compared with that of the control (1663). The number of unique proteins identified during normoxia, NRP1, NRP2, R6, and R24 were 597, 66, 56, 73, and 94, respectively. We analyzed various biological functions during these conditions. Fluctuation in the relative quantities of proteins involved in energy metabolism during dormancy and reactivation was the most significant observation we made in this study. Proteins that are up-regulated or uniquely expressed during reactivation from dormancy offer to be attractive targets for therapeutic

  6. Strain specific transcriptional response in Mycobacterium tuberculosis infected macrophages

    Directory of Open Access Journals (Sweden)

    Koo Mi-Sun

    2012-01-01

    Full Text Available Abstract Background Tuberculosis (TB, a bacterial infection caused by Mycobacterium tuberculosis (Mtb remains a significant health problem worldwide with a third of the world population infected and nearly nine million new cases claiming 1.1 million deaths every year. The outcome following infection by Mtb is determined by a complex and dynamic host-pathogen interaction in which the phenotype of the pathogen and the immune status of the host play a role. However, the molecular mechanism by which Mtb strains induce different responses during intracellular infection of the host macrophage is not fully understood. To explore the early molecular events triggered upon Mtb infection of macrophages, we studied the transcriptional responses of murine bone marrow-derived macrophages (BMM to infection with two clinical Mtb strains, CDC1551 and HN878. These strains have previously been shown to differ in their virulence/immunogenicity in the mouse and rabbit models of pulmonary TB. Results In spite of similar intracellular growth rates, we observed that compared to HN878, infection by CDC1551 of BMM was associated with an increased global transcriptome, up-regulation of a specific early (6 hours immune response network and significantly elevated nitric oxide production. In contrast, at 24 hours post-infection of BMM by HN878, more host genes involved in lipid metabolism, including cholesterol metabolism and prostaglandin synthesis were up-regulated, compared to infection with CDC1551. In association with the differences in the macrophage responses to infection with the 2 Mtb strains, intracellular CDC1551 expressed higher levels of stress response genes than did HN878. Conclusions In association with the early and more robust macrophage activation, intracellular CDC1551 cells were exposed to a higher level of stress leading to increased up-regulation of the bacterial stress response genes. In contrast, sub-optimal activation of macrophages and induction of

  7. Design of Thymidine Analogues Targeting Thymidilate Kinase of Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Luc Calvin Owono Owono

    2013-01-01

    Full Text Available We design here new nanomolar antituberculotics, inhibitors of Mycobacterium tuberculosis thymidine monophosphate kinase (TMPKmt, by means of structure-based molecular design. 3D models of TMPKmt-inhibitor complexes have been prepared from the crystal structure of TMPKmt cocrystallized with the natural substrate deoxythymidine monophosphate (dTMP (1GSI for a training set of 15 thymidine analogues (TMDs with known activity to prepare a QSAR model of interaction establishing a correlation between the free energy of complexation and the biological activity. Subsequent validation of the predictability of the model has been performed with a 3D QSAR pharmacophore generation. The structural information derived from the model served to design new subnanomolar thymidine analogues. From molecular modeling investigations, the agreement between free energy of complexation (ΔΔGcom and Ki values explains 94% of the TMPKmt inhibition (pKi=-0.2924ΔΔGcom+3.234;R2=0.94 by variation of the computed ΔΔGcom and 92% for the pharmacophore (PH4 model (pKi=1.0206×pKipred-0.0832,  R2=0.92. The analysis of contributions from active site residues suggested substitution at the 5-position of pyrimidine ring and various groups at the 5′-position of the ribose. The best inhibitor reached a predicted Ki of 0.155 nM. The computational approach through the combined use of molecular modeling and PH4 pharmacophore is helpful in targeted drug design, providing valuable information for the synthesis and prediction of activity of novel antituberculotic agents.

  8. A multiplex PCR method for detection of Aspergillus spp. and Mycobacterium tuberculosis in BAL specimens.

    Science.gov (United States)

    Amini, F; Kachuei, R; Noorbakhsh, F; Imani Fooladi, A A

    2015-06-01

    The aim of this study was the detection of Aspergillus species and Mycobacterium tuberculosis together in bronchoalveolar lavage (BAL) using of multiplex PCR. In this study, from September 2012 until June 2013, 100 bronchoalveolar lavage (BAL) specimens were collected from patients suspected of tuberculosis (TB). After the direct and culture test, multiplex PCR were utilized in order to diagnose Aspergillus species and M. tuberculosis. Phenol-chloroform manual method was used in order to extract DNA from these microorganisms. Aspergillus specific primers, M. tuberculosis designed primers and beta actin primers were used for multiplex PCR. In this study, by multiplex PCR method, Aspergillus species were identified in 12 samples (12%), positive samples in direct and culture test were respectively 11% and 10%. Sensitivity and specificity of this method in comparison to direct test were respectively 100% and 98.8%, also sensitivity and specificity of this method in comparison to culture test were respectively 100% and 97.7%. In this assay, M. tuberculosis was identified in 8 samples (8%). Mycobacterium-positive samples in molecular method, direct and culture test were respectively 6%, 5% and 7%. Sensitivity and specificity of PCR method in comparison to direct test were 80% and 97.8% also sensitivity and specificity of this method in comparison to culture test was 71.4% and 98.9%. In the present study, multiplex PCR method had higher sensitivity than direct and culture test in order to identify and detect Aspergillus, also this method had lower sensitivity for identification of M. tuberculosis, suggesting that the method of DNA extraction was not suitable. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  9. Mycobacterium tuberculosis Infection in a Domesticated Korean Wild Boar ( Sus scrofa coreanus).

    Science.gov (United States)

    Seo, Min-Goo; Ouh, In-Ohk; Kim, Munki; Lee, Jienny; Kim, Young-Hoan; Do, Jae-Cheul; Kwak, Dongmi

    2017-06-01

    Tuberculosis, a chronic progressive disease, has been reported in bovine, swine, and primate species. Here, we report the first case of Mycobacterium tuberculosis infection in a Korean wild boar ( Sus scrofa coreanus). The owners this domesticated boar brought it to the Gyeongbuk Veterinary Service Laboratory in Korea after it was found dead and severely emaciated. Demarcated yellowish white nodules were found around the larynx and retropharyngeal lymph node during necropsy. The lungs had diffuse fibrinous pleuritis, severe congestion, and scattered nodules. More nodules were found in the spleen. Tuberculosis is characterized by massive macrophage infiltration and central caseous necrosis; both characteristics were found in the lungs. Histopathologic examination revealed that the alveolar lumen had marked fibrosis and exudates. Examination of the fluid revealed extensive macrophage permeation. To confirm a Mycobacterium infection, PCR was performed using two primer sets specific to the rpoB gene of Mycobacterium; Mycobacterium was detected in the lungs and spleen. To identify the species of Mycobacterium, immunohistochemical evaluation was performed using antibodies against Mycobacterium tuberculosis and Mycobacterium bovis . The results revealed immunoreactivity against M. tuberculosis but not against M. bovis . The consumption of undercooked or raw meat from game animals may expose humans and other animals to sylvatic infection. Consequently, Koreans who ingest wild boar may be at risk of a tuberculosis infection. To reduce the risk of foodborne infection and maintain public health, continuous monitoring and control strategies are required.

  10. Laboratory Diagnosis and Susceptibility Testing for Mycobacterium tuberculosis.

    Science.gov (United States)

    Procop, Gary W

    2016-12-01

    The laboratory, which utilizes some of the most sophisticated and rapidly changing technologies, plays a critical role in the diagnosis of tuberculosis. Some of these tools are being employed in resource-challenged countries for the rapid detection and characterization of Mycobacterium tuberculosis. Foremost, the laboratory defines appropriate specimen criteria for optimal test performance. The direct detection of mycobacteria in the clinical specimen, predominantly done by acid-fast staining, may eventually be replaced by rapid-cycle PCR. The widespread use of the Xpert MTB/RIF (Cepheid) assay, which detects both M. tuberculosis and key genetic determinants of rifampin resistance, is important for the early detection of multidrug-resistant strains. Culture, using both broth and solid media, remains the standard for establishing the laboratory-based diagnosis of tuberculosis. Cultured isolates are identified far less commonly by traditional biochemical profiling and more commonly by molecular methods, such as DNA probes and broad-range PCR with DNA sequencing. Non-nucleic acid-based methods of identification, such as high-performance liquid chromatography and, more recently, matrix-assisted laser desorption/ionization-time of flight mass spectrometry, may also be used for identification. Cultured isolates of M. tuberculosis should be submitted for susceptibility testing according to standard guidelines. The use of broth-based susceptibility testing is recommended to significantly decrease the time to result. Cultured isolates may also be submitted for strain typing for epidemiologic purposes. The use of massive parallel sequencing, also known as next-generation sequencing, promises to continue to this molecular revolution in mycobacteriology, as whole-genome sequencing provides identification, susceptibility, and typing information simultaneously.

  11. Activity of Scottish plant, lichen and fungal endophyte extracts against Mycobacterium aurum and Mycobacterium tuberculosis.

    Science.gov (United States)

    Gordien, Andréa Y; Gray, Alexander I; Ingleby, Kevin; Franzblau, Scott G; Seidel, Véronique

    2010-05-01

    With tuberculosis the leading bacterial killer worldwide and other mycobacterial diseases on the increase, the search for new antimycobacterial agents is timely. In this study, extracts from plants, lichens and fungal endophytes of Scottish provenance were screened for activity against Mycobacterium aurum and M. tuberculosis H(37)Rv. The best activity against M. aurum was observed for extracts of Juniperus communis roots and Cladonia arbuscula (MIC = 4 microg/mL), and a fungal endophyte isolated from Vaccinium myrtillus (MIC = 8 microg/mL). The best activity against M. tuberculosis was observed for extracts of C. arbuscula, Empetrum nigrum, J. communis roots, Calluna vulgaris aerial parts, Myrica gale roots and stems (93 to 99% inhibition at 100 microg/mL). Potent antitubercular activity (90 to 96% inhibition at 100 microg/mL) was also observed for the ethanol extracts of Xerocomus badius, Chalciporus piperatus, Suillus luteus and of endophytes isolated from C. vulgaris, E. nigrum, Vaccinium vitis-idaea and V. myrtillus. The results obtained this study provide, in part, some scientific basis for the traditional use of some of the selected plants in the treatment of tuberculosis. They also indicate that fungal endophytes recovered from Scottish plants are a source of antimycobacterial agents worthy of further investigation. Copyright (c) 2009 John Wiley & Sons, Ltd.

  12. Increased level of acute phase reactants in patients infected with modern Mycobacterium tuberculosis genotypes in Mwanza, Tanzania

    DEFF Research Database (Denmark)

    Stavrum, Ruth; PrayGod, George; Range, Nyagosya

    2014-01-01

    a distinct genetic ancestry. This study describes the genetic biodiversity of M. tuberculosis genotypes in Mwanza city, Tanzania and the clinical presentation of the disease caused by isolates of different lineages. METHODS: Two-hundred-fifty-two isolates from pulmonary TB patients in Mwanza, Tanzania were......BACKGROUND: There is increasing evidence to suggest that different Mycobacterium tuberculosis lineages cause variations in the clinical presentation of tuberculosis (TB). Certain M. tuberculosis genotypes/lineages have been shown to be more likely to cause active TB in human populations from....... tuberculosis lineage of the infectious agent for each patient. RESULTS: The most frequent genotype was ST59 (48 out of 248 [19.4%]), belonging to the Euro-American lineage LAM11_ZWE, followed by ST21 (CAS_KILI lineage [44 out of 248 [17.7%]). A low degree of diversity (15.7% [39 different ST's out of 248...

  13. Genetic Determinants of Drug Resistance in Mycobacterium tuberculosis and Their Diagnostic Value

    NARCIS (Netherlands)

    Farhat, M.R.; Sultana, R.; Iartchouk, O.; Bozeman, S.; Galagan, J.; Sisk, P.; Stolte, C.; Nebenzahl-Guimaraes, H.; Jacobson, K.; Sloutsky, A.; Kaur, D.; Posey, J.; Kreiswirth, B.N.; Kurepina, N.; Rigouts, L.; Streicher, E.M.; Victor, T.C.; Warren, R.M.; Soolingen, D. van; Murray, M.

    2016-01-01

    RATIONALE: The development of molecular diagnostics that detect both the presence of Mycobacterium tuberculosis in clinical samples and drug resistance-conferring mutations promises to revolutionize patient care and interrupt transmission by ensuring early diagnosis. However, these tools require the

  14. Supplementary Material for: Whole genome sequencing reveals genomic heterogeneity and antibiotic purification in Mycobacterium tuberculosis isolates

    KAUST Repository

    Black, PA; Vos, M. de; Louw, GE; Merwe, RG van der; Dippenaar, A.; Streicher, EM; Abdallah, AM; Sampson, SL; Victor, TC; Dolby, T.; Simpson, JA; Helden, PD van; Warren, RM; Pain, Arnab

    2015-01-01

    Abstract Background Whole genome sequencing has revolutionised the interrogation of mycobacterial genomes. Recent studies have reported conflicting findings on the genomic stability of Mycobacterium tuberculosis during the evolution of drug

  15. DIGITAL DETECTION SYSTEM DESIGN OF MYCOBACTERIUM TUBERCULOSIS THROUGH EXTRACTION OF SPUTUM IMAGE USING NEURAL NETWORK METHOD

    Directory of Open Access Journals (Sweden)

    Franky Arisgraha

    2012-01-01

    Full Text Available Tuberculosis (TBC is an dangerous disease and many people has been infected. One of many important steps to control TBC effectively and efficiently is by increasing case finding using right method and accurate diagnostic. One of them is to detect Mycobacterium Tuberculosis inside sputum. Conventional detection of Mycobacterium Tuberculosis inside sputum can need a lot of time, so digitally detection method of Mycobacterium Tuberculosis was designed as an effort to get better result of detection. This method was designed by using combination between digital image processing method and Neural Network method. From testing report that was done, Mycobacterium can be detected with successful value reach 77.5% and training error less than 5%.

  16. Genome-wide analysis of multi- and extensively drug-resistant Mycobacterium tuberculosis

    KAUST Repository

    Coll, Francesc; Phelan, Jody; Hill-Cawthorne, Grant A.; Nair, Mridul; Mallard, Kim; Ali, Shahjahan; Abdallah, Abdallah; Alghamdi, Saad; Alsomali, Mona; Ahmed, Abdallah O.; Portelli, Stephanie; Oppong, Yaa; Alves, Adriana; Bessa, Theolis Barbosa; Campino, Susana; Caws, Maxine; Chatterjee, Anirvan; Crampin, Amelia C.; Dheda, Keertan; Furnham, Nicholas; Glynn, Judith R.; Grandjean, Louis; Minh Ha, Dang; Hasan, Rumina; Hasan, Zahra; Hibberd, Martin L.; Joloba, Moses; Jones-Ló pez, Edward C.; Matsumoto, Tomoshige; Miranda, Anabela; Moore, David J.; Mocillo, Nora; Panaiotov, Stefan; Parkhill, Julian; Penha, Carlos; Perdigã o, Joã o; Portugal, Isabel; Rchiad, ‍ Zineb; Robledo, Jaime; Sheen, Patricia; Shesha, Nashwa Talaat; Sirgel, Frik A.; Sola, Christophe; Oliveira Sousa, Erivelton; Streicher, Elizabeth M.; Helden, Paul Van; Viveiros, Miguel; Warren, Robert M.; McNerney, Ruth; Pain, Arnab; Clark, Taane G.

    2018-01-01

    To characterize the genetic determinants of resistance to antituberculosis drugs, we performed a genome-wide association study (GWAS) of 6,465 Mycobacterium tuberculosis clinical isolates from more than 30 countries. A GWAS approach within a mixed

  17. Genotyping of ancient Mycobacterium tuberculosis strains reveals historic genetic diversity.

    Science.gov (United States)

    Müller, Romy; Roberts, Charlotte A; Brown, Terence A

    2014-04-22

    The evolutionary history of the Mycobacterium tuberculosis complex (MTBC) has previously been studied by analysis of sequence diversity in extant strains, but not addressed by direct examination of strain genotypes in archaeological remains. Here, we use ancient DNA sequencing to type 11 single nucleotide polymorphisms and two large sequence polymorphisms in the MTBC strains present in 10 archaeological samples from skeletons from Britain and Europe dating to the second-nineteenth centuries AD. The results enable us to assign the strains to groupings and lineages recognized in the extant MTBC. We show that at least during the eighteenth-nineteenth centuries AD, strains of M. tuberculosis belonging to different genetic groups were present in Britain at the same time, possibly even at a single location, and we present evidence for a mixed infection in at least one individual. Our study shows that ancient DNA typing applied to multiple samples can provide sufficiently detailed information to contribute to both archaeological and evolutionary knowledge of the history of tuberculosis.

  18. PolyTB: A genomic variation map for Mycobacterium tuberculosis

    KAUST Repository

    Coll, Francesc

    2014-02-15

    Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is the second major cause of death from an infectious disease worldwide. Recent advances in DNA sequencing are leading to the ability to generate whole genome information in clinical isolates of M. tuberculosis complex (MTBC). The identification of informative genetic variants such as phylogenetic markers and those associated with drug resistance or virulence will help barcode Mtb in the context of epidemiological, diagnostic and clinical studies. Mtb genomic datasets are increasingly available as raw sequences, which are potentially difficult and computer intensive to process, and compare across studies. Here we have processed the raw sequence data (>1500 isolates, eight studies) to compile a catalogue of SNPs (n = 74,039, 63% non-synonymous, 51.1% in more than one isolate, i.e. non-private), small indels (n = 4810) and larger structural variants (n = 800). We have developed the PolyTB web-based tool (http://pathogenseq.lshtm.ac.uk/polytb) to visualise the resulting variation and important meta-data (e.g. in silico inferred strain-types, location) within geographical map and phylogenetic views. This resource will allow researchers to identify polymorphisms within candidate genes of interest, as well as examine the genomic diversity and distribution of strains. PolyTB source code is freely available to researchers wishing to develop similar tools for their pathogen of interest. 2014 Elsevier Ltd. All rights reserved.

  19. Mycobacterium tuberculosis Transcription Machinery: Ready To Respond to Host Attacks

    Science.gov (United States)

    Flentie, Kelly; Garner, Ashley L.

    2016-01-01

    Regulating responses to stress is critical for all bacteria, whether they are environmental, commensal, or pathogenic species. For pathogenic bacteria, successful colonization and survival in the host are dependent on adaptation to diverse conditions imposed by the host tissue architecture and the immune response. Once the bacterium senses a hostile environment, it must enact a change in physiology that contributes to the organism's survival strategy. Inappropriate responses have consequences; hence, the execution of the appropriate response is essential for survival of the bacterium in its niche. Stress responses are most often regulated at the level of gene expression and, more specifically, transcription. This minireview focuses on mechanisms of regulating transcription initiation that are required by Mycobacterium tuberculosis to respond to the arsenal of defenses imposed by the host during infection. In particular, we highlight how certain features of M. tuberculosis physiology allow this pathogen to respond swiftly and effectively to host defenses. By enacting highly integrated and coordinated gene expression changes in response to stress, M. tuberculosis is prepared for battle against the host defense and able to persist within the human population. PMID:26883824

  20. First insights into circulating Mycobacterium tuberculosis complex lineages and drug resistance in Guinea

    Science.gov (United States)

    Ejo, Mebrat; Gehre, Florian; Barry, Mamadou Dian; Sow, Oumou; Bah, Nene Mamata; Camara, Mory; Bah, Boubacar; Uwizeye, Cecile; Nduwamahoro, Elie; Fissette, Kristina; Rijk, Pim De; Merle, Corinne; Olliaro, Piero; Burgos, Marcos; Lienhardt, Christian; Rigouts, Leen; de Jong, Bouke C.

    2015-01-01

    In this study we assessed first-line anti-tuberculosis drug resistance and the genotypic distribution of Mycobacterium tuberculosis complex (MTBC) isolates that had been collected from consecutive new tuberculosis patients enrolled in two clinical trials conducted in Guinea between 2005 and 2010. Among the total 359 MTBC strains that were analyzed in this study, 22.8% were resistant to at least one of the first line anti-tuberculosis drugs, including 2.5% multidrug resistance and 17.5% isoniazid resistance, with or without other drugs. In addition, further characterization of isolates from a subset of the two trials (n = 184) revealed a total of 80 different spoligotype patterns, 29 “orphan” and 51 shared patterns. We identified the six major MTBC lineages of human relevance, with predominance of the Euro-American lineage. In total, 132 (71.7%) of the strains were genotypically clustered, and further analysis (using the DESTUS model) suggesting significantly faster spread of LAM10_CAM family (p = 0.00016). In conclusion, our findings provide a first insight into drug resistance and the population structure of the MTBC in Guinea, with relevance for public health scientists in tuberculosis control programs. PMID:26004194

  1. Bacterial immunostat: Mycobacterium tuberculosis lipids and their role in the host immune response

    Directory of Open Access Journals (Sweden)

    Adriano Queiroz

    Full Text Available Abstract: The lipid-rich cell wall of Mycobacterium tuberculosis is a dynamic structure that is involved in the regulation of the transport of nutrients, toxic host-cell effector molecules, and anti-tuberculosis drugs. It is therefore postulated to contribute to the long-term bacterial survival in an infected human host. Accumulating evidence suggests that M. tuberculosis remodels the lipid composition of the cell wall as an adaptive mechanism against host-imposed stress. Some of these lipid species (trehalose dimycolate, diacylated sulphoglycolipid, and mannan-based lipoglycans trigger an immunopathologic response, whereas others (phthiocerol dimycocerosate, mycolic acids, sulpholipid-1, and di-and polyacyltrehalose appear to dampen the immune responses. These lipids appear to be coordinately expressed in the cell wall of M. tuberculosis during different phases of infection, ultimately determining the clinical fate of the infection. This review summarizes the current state of knowledge on the metabolism, transport, and homeostatic or immunostatic regulation of the cell wall lipids, and their orchestrated interaction with host immune responses that results in bacterial clearance, persistence, or tuberculosis.

  2. Identification of Secretory Proteins in Mycobacterium tuberculosis Using Pseudo Amino Acid Composition

    Directory of Open Access Journals (Sweden)

    Huan Yang

    2016-01-01

    Full Text Available Tuberculosis is killing millions of lives every year and on the blacklist of the most appalling public health problems. Recent findings suggest that secretory protein of Mycobacterium tuberculosis may serve the purpose of developing specific vaccines and drugs due to their antigenicity. Responding to global infectious disease, we focused on the identification of secretory proteins in Mycobacterium tuberculosis. A novel method called MycoSec was designed by incorporating g-gap dipeptide compositions into pseudo amino acid composition. Analysis of variance-based technique was applied in the process of feature selection and a total of 374 optimal features were obtained and used for constructing the final predicting model. In the jackknife test, MycoSec yielded a good performance with the area under the receiver operating characteristic curve of 0.93, demonstrating that the proposed system is powerful and robust. For user’s convenience, the web server MycoSec was established and an obliging manual on how to use it was provided for getting around any trouble unnecessary.

  3. Phosphorylation of InhA inhibits mycolic acid biosynthesis and growth of Mycobacterium tuberculosis

    Energy Technology Data Exchange (ETDEWEB)

    Molle, Virginie; Gulten, Gulcin; Vilchèze, Catherine; Veyron-Churlet, Romain; Zanella-Cléon, Isabelle; Sacchettini, James C.; Jacobs, Jr, William R.; Kremer, Laurent (CNRS-UMR); (Einstein); (TAM)

    2011-08-24

    The remarkable survival ability of Mycobacterium tuberculosis in infected hosts is related to the presence of cell wall-associated mycolic acids. Despite their importance, the mechanisms that modulate expression of these lipids in response to environmental changes are unknown. Here we demonstrate that the enoyl-ACP reductase activity of InhA, an essential enzyme of the mycolic acid biosynthetic pathway and the primary target of the anti-tubercular drug isoniazid, is controlled via phosphorylation. Thr-266 is the unique kinase phosphoacceptor, both in vitro and in vivo. The physiological relevance of Thr-266 phosphorylation was demonstrated using inhA phosphoablative (T266A) or phosphomimetic (T266D/E) mutants. Enoyl reductase activity was severely impaired in the mimetic mutants in vitro, as a consequence of a reduced binding affinity to NADH. Importantly, introduction of inhA{_}T266D/E failed to complement growth and mycolic acid defects of an inhA-thermosensitive Mycobacterium smegmatis strain, in a similar manner to what is observed following isoniazid treatment. This study suggests that phosphorylation of InhA may represent an unusual mechanism that allows M. tuberculosis to regulate its mycolic acid content, thus offering a new approach to future anti-tuberculosis drug development.

  4. Phosphorylation of InhA inhibits mycolic acid biosynthesis and growth of Mycobacterium tuberculosis.

    Science.gov (United States)

    Molle, Virginie; Gulten, Gulcin; Vilchèze, Catherine; Veyron-Churlet, Romain; Zanella-Cléon, Isabelle; Sacchettini, James C; Jacobs, William R; Kremer, Laurent

    2010-12-01

    The remarkable survival ability of Mycobacterium tuberculosis in infected hosts is related to the presence of cell wall-associated mycolic acids. Despite their importance, the mechanisms that modulate expression of these lipids in response to environmental changes are unknown. Here we demonstrate that the enoyl-ACP reductase activity of InhA, an essential enzyme of the mycolic acid biosynthetic pathway and the primary target of the anti-tubercular drug isoniazid, is controlled via phosphorylation. Thr-266 is the unique kinase phosphoacceptor, both in vitro and in vivo. The physiological relevance of Thr-266 phosphorylation was demonstrated using inhA phosphoablative (T266A) or phosphomimetic (T266D/E) mutants. Enoyl reductase activity was severely impaired in the mimetic mutants in vitro, as a consequence of a reduced binding affinity to NADH. Importantly, introduction of inhA_T266D/E failed to complement growth and mycolic acid defects of an inhA-thermosensitive Mycobacterium smegmatis strain, in a similar manner to what is observed following isoniazid treatment. This study suggests that phosphorylation of InhA may represent an unusual mechanism that allows M. tuberculosis to regulate its mycolic acid content, thus offering a new approach to future anti-tuberculosis drug development. © 2010 Blackwell Publishing Ltd.

  5. Immunogenic membrane-associated proteins of Mycobacterium tuberculosis revealed by proteomics.

    Science.gov (United States)

    Sinha, Sudhir; Kosalai, K; Arora, Shalini; Namane, Abdelkader; Sharma, Pawan; Gaikwad, Anil N; Brodin, Priscille; Cole, Stewart T

    2005-07-01

    Membrane-associated proteins of Mycobacterium tuberculosis offer a challenge, as well as an opportunity, in the quest for better therapeutic and prophylactic interventions against tuberculosis. The authors have previously reported that extraction with the detergent Triton X-114 (TX-114) is a useful step in proteomic analysis of mycobacterial cell membranes, and detergent-soluble membrane proteins of mycobacteria are potent stimulators of human T cells. In this study 1-D and 2-D gel electrophoresis-based protocols were used for the analysis of proteins in the TX-114 extract of M. tuberculosis membranes. Peptide mass mapping (using MALDI-TOF-MS, matrix assisted laser desorption/ionization time of flight mass spectrometry) of 116 samples led to the identification of 105 proteins, 9 of which were new to the M. tuberculosis proteome. Functional orthologues of 73 of these proteins were also present in Mycobacterium leprae, suggesting their relative importance. Bioinformatics predicted that as many as 73% of the proteins had a hydrophobic disposition. 1-D gel electrophoresis revealed more hydrophobic/transmembrane and basic proteins than 2-D gel electrophoresis. Identified proteins fell into the following major categories: protein synthesis, cell wall biogenesis/architecture and conserved hypotheticals/unknowns. To identify immunodominant proteins of the detergent phase (DP), 14 low-molecular-mass fractions prepared by continuous-elution gel electrophoresis were subjected to T cell activation assays using blood samples from BCG-vaccinated healthy donors from a tuberculosis endemic area. Analysis of the responses (cell proliferation and IFN-gamma production) showed that the immunodominance of certain DP fractions was most probably due to ribosomal proteins, which is consistent with both their specificity for mycobacteria and their abundance. Other membrane-associated proteins, including transmembrane proteins/lipoproteins and ESAT-6, did not appear to contribute

  6. Role of P27 -P55 operon from Mycobacterium tuberculosis in the resistance to toxic compounds

    Directory of Open Access Journals (Sweden)

    Cataldi Angel A

    2011-07-01

    Full Text Available Abstract Background The P27-P55 (lprG-Rv1410c operon is crucial for the survival of Mycobacterium tuberculosis, the causative agent of human tuberculosis, during infection in mice. P55 encodes an efflux pump that has been shown to provide Mycobacterium smegmatis and Mycobacterium bovis BCG with resistance to several drugs, while P27 encodes a mannosylated glycoprotein previously described as an antigen that modulates the immune response against mycobacteria. The objective of this study was to determine the individual contribution of the proteins encoded in the P27-P55 operon to the resistance to toxic compounds and to the cell wall integrity of M. tuberculosis. Method In order to test the susceptibility of a mutant of M. tuberculosis H37Rv in the P27-P55 operon to malachite green, sodium dodecyl sulfate, ethidium bromide, and first-line antituberculosis drugs, this strain together with the wild type strain and a set of complemented strains were cultivated in the presence and in the absence of these drugs. In addition, the malachite green decolorization rate of each strain was obtained from decolorization curves of malachite green in PBS containing bacterial suspensions. Results The mutant strain decolorized malachite green faster than the wild type strain and was hypersensitive to both malachite green and ethidium bromide, and more susceptible to the first-line antituberculosis drugs: isoniazid and ethambutol. The pump inhibitor reserpine reversed M. tuberculosis resistance to ethidium bromide. These results suggest that P27-P55 functions through an efflux-pump like mechanism. In addition, deletion of the P27-P55 operon made M. tuberculosis susceptible to sodium dodecyl sulfate, suggesting that the lack of both proteins causes alterations in the cell wall permeability of the bacterium. Importantly, both P27 and P55 are required to restore the wild type phenotypes in the mutant. Conclusions The results clearly indicate that P27 and P55 are

  7. Comparative Genomics and Proteomic Analysis of Four Non-tuberculous Mycobacterium Species and Mycobacterium tuberculosis Complex : Occurrence of Shared Immunogenic Proteins

    NARCIS (Netherlands)

    Gcebe, Nomakorinte; Michel, Anita; Gey van Pittius, Nicolaas C; Rutten, Victor

    2016-01-01

    The Esx and PE/PPE families of proteins are among the most immunodominant mycobacterial antigens and have thus been the focus of research to develop vaccines and immunological tests for diagnosis of bovine and human tuberculosis, mainly caused by Mycobacterium bovis and Mycobacterium tuberculosis,

  8. Prevalence of latent Mycobacterium tuberculosis infection in prisoners.

    Science.gov (United States)

    Navarro, Pedro Daibert de; Almeida, Isabela Neves de; Kritski, Afrânio Lineu; Ceccato, Maria das Graças; Maciel, Mônica Maria Delgado; Carvalho, Wânia da Silva; Miranda, Silvana Spindola de

    2016-01-01

    To determine the prevalence of and the factors associated with latent Mycobacterium tuberculosis infection (LTBI) in prisoners in the state of Minas Gerais, Brazil. This was a cross-sectional cohort study conducted in two prisons in Minas Gerais. Tuberculin skin tests were performed in the individuals who agreed to participate in the study. A total of 1,120 individuals were selected for inclusion in this study. The prevalence of LTBI was 25.2%. In the multivariate analysis, LTBI was associated with self-reported contact with active tuberculosis patients within prisons (adjusted OR = 1.51; 95% CI: 1.05-2.18) and use of inhaled drugs (adjusted OR = 1.48; 95% CI: 1.03-2.13). Respiratory symptoms were identified in 131 (11.7%) of the participants. Serological testing for HIV was performed in 940 (83.9%) of the participants, and the result was positive in 5 (0.5%). Two cases of active tuberculosis were identified during the study period. Within the prisons under study, the prevalence of LTBI was high. In addition, LTBI was associated with self-reported contact with active tuberculosis patients and with the use of inhaled drugs. Our findings demonstrate that it is necessary to improve the conditions in prisons, as well as to introduce strategies, such as chest X-ray screening, in order to detect tuberculosis cases and, consequently, reduce M. tuberculosis infection within the prison system. Determinar a prevalência e os fatores associados à infecção latente por Mycobacterium tuberculosis (ILTB) em pessoas privadas de liberdade no Estado de Minas Gerais. Estudo de coorte transversal realizado em duas penitenciárias em Minas Gerais. Foi realizada a prova tuberculínica nos indivíduos que aceitaram participar do estudo. Foram selecionados 1.120 indivíduos para a pesquisa. A prevalência da ILTB foi de 25,2%. Na análise multivariada, a ILTB esteve associada com relato de contato com caso de tuberculose ativa dentro da penitenciária (OR ajustada = 1,51; IC95%: 1

  9. Extracellular Sphingomyelinase Rv0888 of Mycobacterium tuberculosis Contributes to Pathological Lung Injury of Mycobacterium smegmatis in Mice via Inducing Formation of Neutrophil Extracellular Traps.

    Science.gov (United States)

    Dang, Guanghui; Cui, Yingying; Wang, Lei; Li, Tiantian; Cui, Ziyin; Song, Ningning; Chen, Liping; Pang, Hai; Liu, Siguo

    2018-01-01

    Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), which mainly causes pulmonary injury and tubercles. Although macrophages are generally considered to harbor the main cells of M. tuberculosis , new evidence suggests that neutrophils are rapidly recruited to the infected lung. M. tuberculosis itself, or its early secreted antigenic target protein 6 (ESAT-6), can induce formation of neutrophil extracellular traps (NETs). However, NETs trap mycobacteria but are unable to kill them. The role of NETs' formation in the pathogenesis of mycobacteria remains unclear. Here, we report a new M. tuberculosis extracellular factor, bifunctional enzyme Rv0888, with both nuclease and sphingomyelinase activities. Rv0888 sphingomyelinase activity can induce NETs' formation in vitro and in the lung of the mice and enhance the colonization ability of Mycobacterium smegmatis in the lungs of mice. Mice infected by M. smegmatis harboring Rv0888 sphingomyelinase induced pathological injury and inflammation of the lung, which was mainly mediated by NETs, induced by Rv0888 sphingomyelinase, associated protein (myeloperoxidase) triggered caspase-3. In summary, the study sheds new light on the pathogenesis of mycobacteria and reveals a novel target for TB treatment.

  10. Evaluation of Genetic Pattern of Non-Tuberculosis Mycobacterium Using VNTR Method

    Directory of Open Access Journals (Sweden)

    Noorozi J

    2011-06-01

    Full Text Available Background and Objectives: Epidemiological studies of Non-tuberculosis Mycobacterium is important because of the drug resistance pattern and worldwide dissemination of these organisms. One of genetic fingerprinting methods for epidemiological studies is VNTR (Variable Number Tandem Repeat. In this study genetic pattern of atypical Mycobacterium was evaluated by VNTR method for epidemiologic studies. Methods: 48 pulmonary and non pulmonary specimens separated from patients with the symptoms of pulmonary tuberculosis (PTB and identified as Non-tuberculosis Mycobacteriumby phenotypic and PCR-RFLP methods were selected for this study. Clinical samples and their standard strains were evaluated according to VNTR pattern using the 7 genetic loci including ETR-B. ETR-F. ETR-C. MPTR-A. ETR-A. ETR-E. ETR-D.Results: The results of VNTR method showed that none of the 7 loci had any polymorphism in the standard strains of atypical mycobacterium. Some of these variable number tandem repeat in 42 clinical samples of non-tuberculosis Mycobacterium were polymorphic while the PCR product (for any loci was not found in the remaining 6 specimens. Conclusion: Although the used genetic loci of this study were suitable for epidemiological studies of Mycobacterium tuberculosis, these loci were not able to determine the diversity of genetics of non-tuberculosis Mycobacterium Therefore, it seems necessary that other loci be studied using VNTR method.

  11. DETECTION OF MYCOBACTERIUM TUBERCULOSIS IN BLOOD FOR DIAGNOSIS OF GENERALISED TUBERCULOSIS IN HIV-POSITIVE PATIENTS

    Directory of Open Access Journals (Sweden)

    V. N. Zimina

    2017-01-01

    Full Text Available Objective: To study the informative value of the detection of mycobacteria in blood with the cultural method in patients with suspected tuberculous sepsis and to determine the most significant clinical and laboratory criteria for testing. Materials and methods: The investigation to detect M.tuberculosis was fulfilled in 159 HIV-positive patients with suspected tuberculosis sepsis. Blood culture was completed with culture medium Myco/F Lytic Culture Vials and analyzer BACTEC 9050. Results: Mycobacteria were detected in blood of 19 patients (11,9% of all patients: in 18 patients the growth of М. tuberculosis complex was detected (25,3% of all patients with diagnosed tuberculosis and in 1 patient it was Mycobacterium avium complex (0,6% of all patients. It was shown, that the probability of M.tuberculosis detection was especially associated with the severity of the disease, immunosupression (less than 100 cells/mkl, hemoglobin quantity less than 90 g/l (levels were determined through the seeking for the most significant cutoffs. It was not proofed, that meningoencephalitis develops more often in patients with proven bacteremia. There were no evident differences in detection frequency of mycobacteria in sputum between patients with tuberculous sepsis and without it.

  12. Transcriptional and physiological changes during Mycobacterium tuberculosis reactivation from non-replicating persistence

    Directory of Open Access Journals (Sweden)

    Peicheng Du

    2016-08-01

    Full Text Available Mycobacterium tuberculosis can persist for years in the hostile environment of the host in a non-replicating or slowly replicating state. While active disease predominantly results from reactivation of a latent infection, the molecular mechanisms of M. tuberculosis reactivation are still poorly understood. We characterized the physiology and global transcriptomic profiles of M. tuberculosis during reactivation from hypoxia-induced non-replicating persistence. We found that M. tuberculosis reactivation upon reaeration was associated with a lag phase, in which the recovery of cellular physiological and metabolic functions preceded the resumption of cell replication. Enrichment analysis of the transcriptomic dynamics revealed changes to many metabolic pathways and transcription regulons/subnetworks that orchestrated the metabolic and physiological transformation in preparation for cell division. In particular, we found that M. tuberculosis reaeration lag phase is associated with down-regulation of persistence-associated regulons/subnetworks, including DosR, MprA, SigH, SigE and ClgR, as well as metabolic pathways including those involved in the uptake of lipids and their catabolism. More importantly, we identified a number of up-regulated transcription regulons and metabolic pathways, including those involved in metal transport and remobilization, second messenger-mediated responses, DNA repair and recombination, and synthesis of major cell wall components. We also found that inactivation of the major alternative sigma factors SigE or SigH disrupted exit from persistence, underscoring the importance of the global transcriptional reprogramming during M. tuberculosis reactivation. Our observations suggest that M. tuberculosis lag phase is associated with a global gene expression reprogramming that defines the initiation of a reactivation process.

  13. Mycobacterium tuberculosis Lineage 4 comprises globally distributed and geographically restricted sublineages

    Science.gov (United States)

    Coscolla, Mireia; Liu, Qingyun; Trauner, Andrej; Fenner, Lukas; Rutaihwa, Liliana; Borrell, Sonia; Luo, Tao; Gao, Qian; Kato-Maeda, Midori; Ballif, Marie; Egger, Matthias; Macedo, Rita; Mardassi, Helmi; Moreno, Milagros; Tudo Vilanova, Griselda; Fyfe, Janet; Globan, Maria; Thomas, Jackson; Jamieson, Frances; Guthrie, Jennifer L.; Asante-Poku, Adwoa; Yeboah-Manu, Dorothy; Wampande, Eddie; Ssengooba, Willy; Joloba, Moses; Henry Boom, W.; Basu, Indira; Bower, James; Saraiva, Margarida; Vaconcellos, Sidra E. G.; Suffys, Philip; Koch, Anastasia; Wilkinson, Robert; Gail-Bekker, Linda; Malla, Bijaya; Ley, Serej D.; Beck, Hans-Peter; de Jong, Bouke C.; Toit, Kadri; Sanchez-Padilla, Elisabeth; Bonnet, Maryline; Gil-Brusola, Ana; Frank, Matthias; Penlap Beng, Veronique N.; Eisenach, Kathleen; Alani, Issam; Wangui Ndung’u, Perpetual; Revathi, Gunturu; Gehre, Florian; Akter, Suriya; Ntoumi, Francine; Stewart-Isherwood, Lynsey; Ntinginya, Nyanda E.; Rachow, Andrea; Hoelscher, Michael; Cirillo, Daniela Maria; Skenders, Girts; Hoffner, Sven; Bakonyte, Daiva; Stakenas, Petras; Diel, Roland; Crudu, Valeriu; Moldovan, Olga; Al-Hajoj, Sahal; Otero, Larissa; Barletta, Francesca; Jane Carter, E.; Diero, Lameck; Supply, Philip; Comas, Iñaki; Niemann, Stefan; Gagneux, Sebastien

    2016-01-01

    Generalist and specialist species differ in the breadth of their ecological niche. Little is known about the niche width of obligate human pathogens. Here we analyzed a global collection of Mycobacterium tuberculosis Lineage 4 clinical isolates, the most geographically widespread cause of human tuberculosis. We show that Lineage 4 comprises globally distributed and geographically restricted sublineages, suggesting a distinction between generalists and specialists. Population genomic analyses showed that while the majority of human T cell epitopes were conserved in all sublineages, the proportion of variable epitopes was higher in generalists. Our data further support a European origin for the most common generalist sublineage. Hence, the global success of Lineage 4 reflects distinct strategies adopted by different sublineages and the influence of human migration. PMID:27798628

  14. Ready Experimental Translocation of Mycobacterium canettii Yields Pulmonary Tuberculosis.

    Science.gov (United States)

    Bouzid, Fériel; Brégeon, Fabienne; Lepidi, Hubert; Donoghue, Helen D; Minnikin, David E; Drancourt, Michel

    2017-12-01

    Mycobacterium canettii , which has a smooth colony morphology, is the tuberculous organism retaining the most genetic traits from the putative last common ancestor of the rough-morphology Mycobacterium tuberculosis complex. To explore whether M. canettii can infect individuals by the oral route, mice were fed phosphate-buffered saline or 10 6 M. canettii mycobacteria and sacrificed over a 28-day experiment. While no M. canettii was detected in negative controls, M. canettii -infected mice yielded granuloma-like lesions for 4/4 lungs at days 14 and 28 postinoculation (p.i.) and positive PCR detection of M. canettii for 5/8 mesenteric lymph nodes at days 1 and 3 p.i. and 5/6 pooled stools collected from day 1 to day 28 p.i. Smooth M. canettii colonies grew from 68% of lungs and 36% of spleens and cervical lymph nodes but fewer than 20% of axillary lymph nodes, livers, brown fat samples, kidneys, or blood samples throughout the 28-day experiment. Ready translocation in mice after digestive tract challenge demonstrates the potential of ingested M. canettii organisms to relocate to distant organs and lungs. The demonstration of this relocation supports the possibility that populations may be infected by environmental M. canettii . Copyright © 2017 American Society for Microbiology.

  15. Molecular characterization of Mycobacterium tuberculosis isolates from elephants of Nepal.

    Science.gov (United States)

    Paudel, Sarad; Mikota, Susan K; Nakajima, Chie; Gairhe, Kamal P; Maharjan, Bhagwan; Thapa, Jeewan; Poudel, Ajay; Shimozuru, Michito; Suzuki, Yasuhiko; Tsubota, Toshio

    2014-05-01

    Mycobacterium tuberculosis was cultured from the lung tissues of 3 captive elephants in Nepal that died with extensive lung lesions. Spoligotyping, TbD1 detection and multi-locus variable number of tandem repeat analysis (MLVA) results suggested 3 isolates belonged to a specific lineage of Indo-Oceanic clade, EAI5 SIT 138. One of the elephant isolates had a new synonymous single nucleotide polymorphism (SNP) T231C in the gyrA sequence, and the same SNP was also found in human isolates in Nepal. MLVA results and transfer history of the elephants suggested that 2 of them might be infected with M. tuberculosis from the same source. These findings indicated the source of M. tuberculosis infection of those elephants were local residents, presumably their handlers. Further investigation including detailed genotyping of elephant and human isolates is needed to clarify the infection route and eventually prevent the transmission of tuberculosis to susceptible hosts. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Ancient origin and gene mosaicism of the progenitor of Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    M Cristina Gutierrez

    2005-09-01

    Full Text Available The highly successful human pathogen Mycobacterium tuberculosis has an extremely low level of genetic variation, which suggests that the entire population resulted from clonal expansion following an evolutionary bottleneck around 35,000 y ago. Here, we show that this population constitutes just the visible tip of a much broader progenitor species, whose extant representatives are human isolates of tubercle bacilli from East Africa. In these isolates, we detected incongruence among gene phylogenies as well as mosaic gene sequences, whose individual elements are retrieved in classical M. tuberculosis. Therefore, despite its apparent homogeneity, the M. tuberculosis genome appears to be a composite assembly resulting from horizontal gene transfer events predating clonal expansion. The amount of synonymous nucleotide variation in housekeeping genes suggests that tubercle bacilli were contemporaneous with early hominids in East Africa, and have thus been coevolving with their human host much longer than previously thought. These results open novel perspectives for unraveling the molecular bases of M. tuberculosis evolutionary success.

  17. Mycobacterium tuberculosis: nepřekonatelný nepřítel

    Czech Academy of Sciences Publication Activity Database

    Machová, Iva; Pichová, Iva

    2014-01-01

    Roč. 24, č. 4 (2014), s. 98-100 ISSN 1210-1737 R&D Projects: GA MŠk LO1302; GA MŠk(CZ) 7E11070 EU Projects: European Commission(XE) 241587 - SYSTEMTB Institutional support: RVO:61388963 Keywords : Mycobacterium tuberculosis * tuberculosis * latent infection * resistance * metabolism * drugs Subject RIV: CE - Biochemistry

  18. Beijing Lineage of MDR Mycobacterium tuberculosis in Bulgaria, 2007-2011

    NARCIS (Netherlands)

    Panaiotov, Stefan; Bachiyska, Elizabeta; Yordanova, Stanislava; Atanasova, Yuliana; Brankova, Nadia; Levterova, Viktoria; Sengstake, Sarah; Anthony, Richard; Bergval, Indra; Sola, Christophe; Kantardjiev, Todor

    2014-01-01

    To assess the spread of the Mycobacterium tuberculosis Beijing genotype among patients with multidrug-resistant and extensively resistant tuberculosis in Bulgaria, we genotyped 188 (72%) of 261 microbiologically confirmed resistant isolates obtained during 2007-2011. The estimated prevalence of the

  19. Revisiting host preference in the Mycobacterium tuberculosis complex: experimental infection shows M. tuberculosis H37Rv to be avirulent in cattle.

    Directory of Open Access Journals (Sweden)

    Adam O Whelan

    Full Text Available Experiments in the late 19th century sought to define the host specificity of the causative agents of tuberculosis in mammals. Mycobacterium tuberculosis, the human tubercle bacillus, was independently shown by Smith, Koch, and von Behring to be avirulent in cattle. This finding was erroneously used by Koch to argue the converse, namely that Mycobacterium bovis, the agent of bovine tuberculosis, was avirulent for man, a view that was subsequently discredited. However, reports in the literature of M. tuberculosis isolation from cattle with tuberculoid lesions suggests that the virulence of M. tuberculosis for cattle needs to be readdressed. We used an experimental bovine infection model to test the virulence of well-characterized strains of M. tuberculosis and M. bovis in cattle, choosing the genome-sequenced strains M. tuberculosis H37Rv and M. bovis 2122/97. Cattle were infected with approximately 10(6 CFU of M. tuberculosis H37Rv or M. bovis 2122/97, and sacrificed 17 weeks post-infection. IFN-gamma and tuberculin skin tests indicated that both M. bovis 2122 and M. tuberculosis H37Rv were equally infective and triggered strong cell-mediated immune responses, albeit with some indication of differential antigen-specific responses. Postmortem examination revealed that while M. bovis 2122/97-infected animals all showed clear pathology indicative of bovine tuberculosis, the M. tuberculosis-infected animals showed no pathology. Culturing of infected tissues revealed that M. tuberculosis was able to persist in the majority of animals, albeit at relatively low bacillary loads. In revisiting the early work on host preference across the M. tuberculosis complex, we have shown M. tuberculosis H37Rv is avirulent for cattle, and propose that the immune status of the animal, or genotype of the infecting bacillus, may have significant bearing on the virulence of a strain for cattle. This work will serve as a baseline for future studies into the genetic basis

  20. Nanoparticle-Fusion Protein Complexes Protect against Mycobacterium tuberculosis Infection.

    Science.gov (United States)

    Hart, Peter; Copland, Alastair; Diogo, Gil Reynolds; Harris, Shane; Spallek, Ralf; Oehlmann, Wulf; Singh, Mahavir; Basile, Juan; Rottenberg, Martin; Paul, Matthew John; Reljic, Rajko

    2018-03-07

    Tuberculosis (TB) is the leading cause of death from infectious disease, and the current vaccine, Bacillus Calmette-Guerin (BCG), is inadequate. Nanoparticles (NPs) are an emerging vaccine technology, with recent successes in oncology and infectious diseases. NPs have been exploited as antigen delivery systems and also for their adjuvantic properties. However, the mechanisms underlying their immunological activity remain obscure. Here, we developed a novel mucosal TB vaccine (Nano-FP1) based upon yellow carnauba wax NPs (YC-NPs), coated with a fusion protein consisting of three Mycobacterium tuberculosis (Mtb) antigens: Acr, Ag85B, and HBHA. Mucosal immunization of BCG-primed mice with Nano-FP1 significantly enhanced protection in animals challenged with low-dose, aerosolized Mtb. Bacterial control by Nano-FP1 was associated with dramatically enhanced cellular immunity compared to BCG, including superior CD4 + and CD8 + T cell proliferation, tissue-resident memory T cell (Trm) seeding in the lungs, and cytokine polyfunctionality. Alongside these effects, we also observed potent humoral responses, such as the generation of Ag85B-specific serum IgG and respiratory IgA. Finally, we found that YC-NPs were able to activate antigen-presenting cells via an unconventional IRF-3-associated activation signature, without the production of potentially harmful inflammatory mediators, providing a mechanistic framework for vaccine efficacy and future development. Copyright © 2017. Published by Elsevier Inc.

  1. Amperometric immunosensor for rapid detection of Mycobacterium tuberculosis

    International Nuclear Information System (INIS)

    Hiraiwa, Morgan; Lee, Hyun-Boo; Inoue, Shinnosuke; Chung, Jae-Hyun; Kim, Jong-Hoon; Becker, Annie L; Weigel, Kris M; Cangelosi, Gerard A; Lee, Kyong-Hoon

    2015-01-01

    Tuberculosis (TB) has been a major public health problem, which can be better controlled by using accurate and rapid diagnosis in low-resource settings. A simple, portable, and sensitive detection method is required for point-of-care (POC) settings. This paper studies an amperometric biosensor using a microtip immunoassay for a rapid and low-cost detection of Mycobacterium tuberculosis (MTB) in sputum. MTB in sputum is specifically captured on the functionalized microtip surface and detected by electric current. According to the numerical study, the current signal on the microtip surface is linearly changed with increasing immersion depth. Using a reference microtip, the immersion depth is compensated for a sensing microtip. On the microtip surface, target bacteria are concentrated and organized by a coffee-ring effect, which amplifies the electric current. To enhance the signal-to-noise ratio, both the sample processing and rinsing steps are presented with the use of deionized water as a medium for the amperometric measurement. When applied to cultured MTB cells spiked into human sputum, the detection limit was 100 CFU mL −1 , comparable to a more labor-intensive fluorescence detection method reported previously. (paper)

  2. Roles of Mucosal Immunity against Mycobacterium tuberculosis Infection

    Directory of Open Access Journals (Sweden)

    Wu Li

    2012-01-01

    Full Text Available Mycobacterium tuberculosis (Mtb, the causative agent of tuberculosis (TB, is one of the world's leading infectious causes of morbidity and mortality. As a mucosal-transmitted pathogen, Mtb infects humans and animals mainly through the mucosal tissue of the respiratory tract. Apart from providing a physical barrier against the invasion of pathogen, the major function of the respiratory mucosa may be to serve as the inductive sites to initiate mucosal immune responses and sequentially provide the first line of defense for the host to defend against this pathogen. A large body of studies in the animals and humans have demonstrated that the mucosal immune system, rather than the systemic immune system, plays fundamental roles in the host’s defense against Mtb infection. Therefore, the development of new vaccines and novel delivery routes capable of directly inducing respiratory mucosal immunity is emphasized for achieving enhanced protection from Mtb infection. In this paper, we outline the current state of knowledge regarding the mucosal immunity against Mtb infection, including the development of TB vaccines, and respiratory delivery routes to enhance mucosal immunity are discussed.

  3. Amperometric immunosensor for rapid detection of Mycobacterium tuberculosis

    Science.gov (United States)

    Hiraiwa, Morgan; Kim, Jong-Hoon; Lee, Hyun-Boo; Inoue, Shinnosuke; Becker, Annie L.; Weigel, Kris M.; Cangelosi, Gerard A.; Lee, Kyong-Hoon; Chung, Jae-Hyun

    2015-05-01

    Tuberculosis (TB) has been a major public health problem, which can be better controlled by using accurate and rapid diagnosis in low-resource settings. A simple, portable, and sensitive detection method is required for point-of-care (POC) settings. This paper studies an amperometric biosensor using a microtip immunoassay for a rapid and low-cost detection of Mycobacterium tuberculosis (MTB) in sputum. MTB in sputum is specifically captured on the functionalized microtip surface and detected by electric current. According to the numerical study, the current signal on the microtip surface is linearly changed with increasing immersion depth. Using a reference microtip, the immersion depth is compensated for a sensing microtip. On the microtip surface, target bacteria are concentrated and organized by a coffee-ring effect, which amplifies the electric current. To enhance the signal-to-noise ratio, both the sample processing and rinsing steps are presented with the use of deionized water as a medium for the amperometric measurement. When applied to cultured MTB cells spiked into human sputum, the detection limit was 100 CFU mL-1, comparable to a more labor-intensive fluorescence detection method reported previously.

  4. Curcumin enhances human macrophage control of Mycobacterium tuberculosis infection.

    Science.gov (United States)

    Bai, Xiyuan; Oberley-Deegan, Rebecca E; Bai, An; Ovrutsky, Alida R; Kinney, William H; Weaver, Michael; Zhang, Gong; Honda, Jennifer R; Chan, Edward D

    2016-07-01

    With the worldwide emergence of highly drug-resistant tuberculosis (TB), novel agents that have direct antimycobacterial effects or that enhance host immunity are urgently needed. Curcumin is a polyphenol responsible for the bright yellow-orange colour of turmeric, a spice derived from the root of the perennial herb Curcuma longa. Curcumin is a potent inducer of apoptosis-an effector mechanism used by macrophages to kill intracellular Mycobacterium tuberculosis (MTB). An in vitro human macrophage infection model was used to determine the effects of curcumin on MTB survival. We found that curcumin enhanced the clearance of MTB in differentiated THP-1 human monocytes and in primary human alveolar macrophages. We also found that curcumin was an inducer of caspase-3-dependent apoptosis and autophagy. Curcumin mediated these anti-MTB cellular functions, in part, via inhibition of nuclear factor-kappa B (NFκB) activation. Curcumin protects against MTB infection in human macrophages. The host-protective role of curcumin against MTB in macrophages needs confirmation in an animal model; if validated, the immunomodulatory anti-TB effects of curcumin would be less prone to drug resistance development. © 2016 Asian Pacific Society of Respirology.

  5. Molecular epidemiology of Mycobacterium tuberculosis clinical isolates in Southwest Ireland.

    LENUS (Irish Health Repository)

    Ojo, Olabisi O

    2010-10-01

    Tuberculosis has had significant effects on Ireland over the past two centuries, causing persistently higher morbidity and mortality than in neighbouring countries until the last decade. This study describes the results of genotyping and drug susceptibility testing of 171 strains of Mycobacterium tuberculosis complex isolated between January 2004 and December 2006 in a region of Ireland centred on the city of Cork. Spoligotype comparisons were made with the SpolDB4 database and clustered 130 strains in 23 groups, forty-one strains showed unique Spoligotyping patterns. The commonest spoligotypes detected were ST0137 (X2) (16.9%), and ST0351 (15.8%) (\\'U\\' clade). The major spoligotype clades were X (26.2%), U (19.3%), T (15.2%), Beijing (5.9%), Haarlem (4.7%), LAM (4.1%), BOVIS (1.75%), with 12.9% unassigned strains. A 24-locus VNTR genotyping produced 15 clusters containing 49 isolates, with high discrimination index (HGDI>0.99). A combination of Spoligotyping and VNTR reduced the number of clustered isolates to 47 in 15 clusters (27.5%). This study identified ST351 as common among Irish nationals, and found a low rate of drug resistance with little evidence of transmission of drug resistant strains. Strain clustering was significantly associated with age under 55 years and Irish nationality. Only strains of Euro-American lineage formed clusters. Molecular typing did not completely coincide with the results of contact investigations.

  6. CCL2 responses to Mycobacterium tuberculosis are associated with disease severity in tuberculosis.

    Directory of Open Access Journals (Sweden)

    Zahra Hasan

    Full Text Available BACKGROUND: Leucocyte activating chemokines such as CCL2, CCL3, and CXCL8 together with proinflammatory IFNgamma, TNFalpha and downmodulatory IL10 play a central role in the restriction of M. tuberculosis infections, but is unclear whether these markers are indicative of tuberculosis disease severity. METHODOLOGY: We investigated live M. tuberculosis- and M. bovis BCG-induced peripheral blood mononuclear cell responses in patients with tuberculosis (TB and healthy endemic controls (ECs, n = 36. TB patients comprised pulmonary (PTB, n = 34 and extrapulmonary groups, subdivided into those with less severe localized extrapulmonary TB (L-ETB, n = 16 or severe disseminated ETB (D-ETB, n = 16. Secretion of CCL2, IFNgamma, IL10 and CCL3, and mRNA expression of CCL2, TNFalpha, CCL3 and CXCL8 were determined. RESULTS: M. tuberculosis- and BCG-induced CCL2 secretion was significantly increased in both PTB and D-ETB (p<0.05, p<0.01 as compared with L-ETB patients. CCL2 secretion in response to M. tuberculosis was significantly greater than to BCG in the PTB and D-ETB groups. M. tuberculosis-induced CCL2 mRNA transcription was greater in PTB than L-ETB (p = 0.023, while CCL2 was reduced in L-ETB as compared with D-ETB (p = 0.005 patients. M. tuberculosis-induced IFNgamma was greater in L-ETB than PTB (p = 0.04, while BCG-induced IFNgamma was greater in L-ETB as compared with D-ETB patients (p = 0.036. TNFalpha mRNA expression was raised in PTB as compared with L-ETB group in response to M. tuberculosis (p = 0.02 and BCG (p = 0.03. Mycobacterium-induced CCL3 and CXCL8 was comparable between TB groups. CONCLUSIONS: The increased CCL2 and TNFalpha in PTB patients may support effective leucocyte recruitment and M. tuberculosis localization. CCL2 alone is associated with severity of TB, possibly due to increased systemic inflammation found in severe disseminated TB or due to increased monocyte infiltration to lung parenchyma in pulmonary disease.

  7. Line probe assay for differentiation within Mycobacterium tuberculosis complex. Evaluation on clinical specimens and isolates including Mycobacterium pinnipedii

    DEFF Research Database (Denmark)

    Kjeldsen, Marianne Kirstine; Bek, Dorte; Rasmussen, Erik Michael

    2009-01-01

    A line probe assay (GenoType MTBC) was evaluated for species differentiation within the Mycobacterium tuberculosis complex (MTBC). We included 387 MTBC isolates, 43 IS6110 low-copy MTBC isolates, 28 clinical specimens with varying microscopy grade, and 30 isolates of non-tuberculous mycobacteria...

  8. Characterization of Mycobacterium tuberculosis Complex DNAs from Egyptian Mummies by Spoligotyping

    Science.gov (United States)

    Zink, Albert R.; Sola, Christophe; Reischl, Udo; Grabner, Waltraud; Rastogi, Nalin; Wolf, Hans; Nerlich, Andreas G.

    2003-01-01

    Bone and soft tissue samples from 85 ancient Egyptian mummies were analyzed for the presence of ancient Mycobacterium tuberculosis complex DNA (aDNA) and further characterized by spoligotyping. The specimens were obtained from individuals from different tomb complexes in Thebes West, Upper Egypt, which were used for upper social class burials between the Middle Kingdom (since ca. 2050 BC) and the Late Period (until ca. 500 BC). A total of 25 samples provided a specific positive signal for the amplification of a 123-bp fragment of the repetitive element IS6110, indicating the presence of M. tuberculosis DNA. Further PCR-based tests for the identification of subspecies failed due to lack of specific amplification products in the historic tissue samples. Of these 25 positive specimens, 12 could be successfully characterized by spoligotyping. The spoligotyping signatures were compared to those in an international database. They all show either an M. tuberculosis or an M. africanum pattern, but none revealed an M. bovis-specific pattern. The results from a Middle Kingdom tomb (used exclusively between ca. 2050 and 1650 BC) suggest that these samples bear an M. africanum-type specific spoligotyping signature. The samples from later periods provided patterns typical for M. tuberculosis. This study clearly demonstrates that spoligotyping can be applied to historic tissue samples. In addition, our results do not support the theory that M. tuberculosis originated from the M. bovis type but, rather, suggest that human M. tuberculosis may have originated from a precursor complex probably related to M. africanum. PMID:12517873

  9. Pulmonary Mycobacterium tuberculosis (Beijing strain infection in a stray dog : clinical communication

    Directory of Open Access Journals (Sweden)

    S.D.C. Parsons

    2008-05-01

    Full Text Available Mycobacterium tuberculosis infection in dogs is rarely reported and has not previously been documented in South Africa. A case of a stray Maltese crossbreed dog with extensive multifocal pulmonary tuberculosis due to M. tuberculosis is described. Pulmonary granulomas in this case were poorly encapsulated and contained large numbers of acid-fast bacteria, highlighting the potential for infected companion animals to excrete the pathogen. Treatment of canine tuberculosis is generally not advised, and for this reason, euthanasia of diseased animals must be advocated in most instances. Physicians and veterinarians must be aware that companion animals with active disease caused by M. tuberculosis could act as a potential source of infection.

  10. Genome sequencing and annotation of multidrug resistant Mycobacterium tuberculosis (MDR-TB PR10 strain

    Directory of Open Access Journals (Sweden)

    Mohd Zakihalani A. Halim

    2016-03-01

    Full Text Available Here, we report the draft genome sequence and annotation of a multidrug resistant Mycobacterium tuberculosis strain PR10 (MDR-TB PR10 isolated from a patient diagnosed with tuberculosis. The size of the draft genome MDR-TB PR10 is 4.34 Mbp with 65.6% of G + C content and consists of 4637 predicted genes. The determinants were categorized by RAST into 400 subsystems with 4286 coding sequences and 50 RNAs. The whole genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number CP010968. Keywords: Mycobacterium tuberculosis, Genome, MDR, Extrapulmonary

  11. Assessment of trends of ofloxacin resistance in Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    J S Verma

    2011-01-01

    Full Text Available Purpose: Ofloxacin (OFX is one of the potent fluoroquinolone (FQ recommended to treat MDR-TB. Over a decade, the preexposure of this drug for the treatment of other bacterial infections has resulted in acquisition of FQ resistance among Mycobacterium tuberculosis strains. Considering this possibility, a study was undertaken in a tertiary care center in the capital city (India to assess the drug resistance trends of OFX among susceptible and multidrug resistant (MDR strains of M. tuberculosis. Materials and Methods: A total of 102 M. tuberculosis isolates (47 susceptible to first-line drugs and 55 MDR isolates were screened for susceptibility testing of OFX with a critical concentration of 2 μg/ml by Lowenstein Jensen (LJ proportion method. Results: The results showed 40 (85.1% isolates among 47 susceptible isolates and 34 (61.8% isolates among 55 MDR isolates, were found to be susceptible to OFX. Fisher′s exact test showed significant P-value (0.0136 demonstrating 1.377 fold (95% confidence interval increased risk to become resistant to OFX than susceptible isolates. These finding shows decreased OFX susceptibility is not only limited to MDR isolates but also increasingly seen in susceptible strains as a result of drug abuse. Conclusions: Our finding were not alarming, but highlights the general risk of acquiring resistance to OFX, jeopardizing the potential for these drugs to be used as second-line anti-TB agents in the management of drug-resistant TB and creating incurable TB strains .

  12. Optimising Mycobacterium tuberculosis detection in resource limited settings.

    Science.gov (United States)

    Alfred, Nwofor; Lovette, Lawson; Aliyu, Gambo; Olusegun, Obasanya; Meshak, Panwal; Jilang, Tunkat; Iwakun, Mosunmola; Nnamdi, Emenyonu; Olubunmi, Onuoha; Dakum, Patrick; Abimiku, Alash'le

    2014-03-03

    The light-emitting diode (LED) fluorescence microscopy has made acid-fast bacilli (AFB) detection faster and efficient although its optimal performance in resource-limited settings is still being studied. We assessed the optimal performances of light and fluorescence microscopy in routine conditions of a resource-limited setting and evaluated the digestion time for sputum samples for maximum yield of positive cultures. Cross-sectional study. Facility-based involving samples of routine patients receiving tuberculosis treatment and care from the main tuberculosis case referral centre in northern Nigeria. The study included 450 sputum samples from 150 new patients with clinical diagnosis of pulmonary tuberculosis. The 450 samples were pooled into 150 specimens, examined independently with mercury vapour lamp (FM), LED CysCope (CY) and Primo Star iLED (PiLED) fluorescence microscopies, and with the Ziehl-Neelsen (ZN) microscopy to assess the performance of each technique compared with liquid culture. The cultured specimens were decontaminated with BD Mycoprep (4% NaOH-1% NLAC and 2.9% sodium citrate) for 10, 15 and 20 min before incubation in Mycobacterium growth incubator tube (MGIT) system and growth examined for acid-fast bacilli (AFB). Of the 150 specimens examined by direct microscopy: 44 (29%), 60 (40%), 49 (33%) and 64 (43%) were AFB positive by ZN, FM, CY and iLED microscopy, respectively. Digestion of sputum samples for 10, 15 and 20 min yielded mycobacterial growth in 72 (48%), 81 (54%) and 68 (45%) of the digested samples, respectively, after incubation in the MGIT system. In routine laboratory conditions of a resource-limited setting, our study has demonstrated the superiority of fluorescence microscopy over the conventional ZN technique. Digestion of sputum samples for 15 min yielded more positive cultures.

  13. Molecular epidemiology and evolutionary genetics of Mycobacterium tuberculosis in Taipei

    OpenAIRE

    Su Ih-Jen; Lee Shi-Yi; Tsai Wen-Shing; Sun Jun-Ren; Chang Jia-Ru; Lin Chih-Wei; Tseng Fan-Chen; Dou Horng-Yunn; Lu Jang-Jih

    2008-01-01

    Abstract Background The control of tuberculosis in densely populated cities is complicated by close human-to-human contacts and potential transmission of pathogens from multiple sources. We conducted a molecular epidemiologic analysis of 356 Mycobacterium tuberculosis (MTB) isolates from patients presenting pulmonary tuberculosis in metropolitan Taipei. Classical antibiogram studies and genetic characterization, using mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (M...

  14. Tuberculosis transmission of predominant genotypes of Mycobacterium tuberculosis in northern suburbs of Buenos Aires city region.

    Science.gov (United States)

    Morcillo, N; Zumarraga, M; Imperiale, B; Di Giulio, B; Chirico, C; Kuriger, A; Alito, A; Kremer, K; Cataldi, A

    2007-01-01

    In 2003, the incidence of tuberculosis in Argentina showed an increase compared to 2002. The severe national crisis at the end of the 90s has probably strongly contributed to this situation. The goal of this work was to estimate the extent of the spread of the most predominant Mycobacterium tuberculosis strains and to assess the spread of predominant M. tuberculosis clusters as determined by spoligotyping and IS6110 RFLP. The study involved 590 pulmonary, smear-positive TB cases receiving medical attention at health centers and hospitals in Northern Buenos Aires (NBA) suburbs, from October 2001 to December 2002. From a total of 208 clinical isolates belonging to 6 major clusters, 63 (30.2%) isolates had identical spoligotyping and IS6110 RFLP pattern. Only 22.2% were shown to have epidemiological connections with another member of their respective cluster. In these major clusters, 30.2% of the 208 TB cases studied by both molecular techniques and contact tracing could be convincingly attributable to a recently acquired infection. This knowledge may be useful to assess the clonal distribution of predominant M. tuberculosis clusters in Argentina, which may make an impact on TB control strategies.

  15. Mycobacterium tuberculosis strains of the Beijing genotype are rarely observed in tuberculosis patients in South America.

    Science.gov (United States)

    Ritacco, Viviana; López, Beatriz; Cafrune, Patricia I; Ferrazoli, Lucilaine; Suffys, Philip N; Candia, Norma; Vásquez, Lucy; Realpe, Teresa; Fernández, Jorge; Lima, Karla V; Zurita, Jeannete; Robledo, Jaime; Rossetti, Maria L; Kritski, Afranio L; Telles, Maria A; Palomino, Juan C; Heersma, Herre; van Soolingen, Dick; Kremer, Kristin; Barrera, Lucía

    2008-08-01

    The frequency of the Beijing genotype of Mycobacterium tuberculosis as a cause of tuberculosis (TB) in South America was determined by analyzing genotypes of strains isolated from patients that had been diagnosed with the disease between 1997 and 2003 in seven countries of the subcontinent. In total, 19 of the 1,202 (1.6%) TB cases carried Beijing isolates, including 11 of the 185 patients from Peru (5.9%), five of the 512 patients from Argentina (1.0%), two of the 252 Brazilian cases (0.8%), one of the 166 patients from Paraguay (0.6%) and none of the samples obtained from Chile (35), Colombia (36) and Ecuador (16). Except for two patients that were East Asian immigrants, all cases with Beijing strains were native South Americans. No association was found between carrying a strain with the Beijing genotype and having drug or multi-drug resistant disease. Our data show that presently transmission of M. tuberculosis strains of the Beijing genotype is not frequent in Latin America. In addition, the lack of association of drug resistant TB and infection with M. tuberculosis of the Beijing genotype observed presently demands efforts to define better the contribution of the virulence and lack of response to treatment to the growing spread of Beijing strains observed in other parts of the world.

  16. Mycobacterium tuberculosis strains of the Beijing genotype are rarely observed in tuberculosis patients in South America

    Directory of Open Access Journals (Sweden)

    Viviana Ritacco

    2008-08-01

    Full Text Available The frequency of the Beijing genotype of Mycobacterium tuberculosis as a cause of tuberculosis (TB in South America was determined by analyzing genotypes of strains isolated from patients that had been diagnosed with the disease between 1997 and 2003 in seven countries of the subcontinent. In total, 19 of the 1,202 (1.6% TB cases carried Beijing isolates, including 11 of the 185 patients from Peru (5.9%, five of the 512 patients from Argentina (1.0%, two of the 252 Brazilian cases (0.8%, one of the 166 patients from Paraguay (0.6% and none of the samples obtained from Chile (35, Colombia (36 and Ecuador (16. Except for two patients that were East Asian immigrants, all cases with Beijing strains were native South Americans. No association was found between carrying a strain with the Beijing genotype and having drug or multi-drug resistant disease. Our data show that presently transmission of M. tuberculosis strains of the Beijing genotype is not frequent in Latin America. In addition, the lack of association of drug resistant TB and infection with M. tuberculosis of the Beijing genotype observed presently demands efforts to define better the contribution of the virulence and lack of response to treatment to the growing spread of Beijing strains observed in other parts of the world.

  17. Aptamer from whole-bacterium SELEX as new therapeutic reagent against virulent Mycobacterium tuberculosis

    International Nuclear Information System (INIS)

    Chen, Fan; Zhou, Jing; Luo, Fengling; Mohammed, Al-Bayati; Zhang, Xiao-Lian

    2007-01-01

    Worldwide, tuberculosis (TB) remains the most frequent and important infectious disease causing morbidity and death. One-third of the world's population is infected with Mycobacterium tuberculosis (MTB), the etiologic agent of TB. Because of the global health problems of TB, the development of potent new anti-TB drugs without cross-resistance with known antimycobacterial agents is urgently needed. In this study, we have applied a Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process to identify a single aptamer (NK2) that binds to virulent strain M. tuberculosis (H37Rv) with high affinity and specificity. We have found that this aptamer improves CD4 + T cells to produce IFN-γ after binding to H37Rv. The different component between H37Rv and BCG was identified as some membrane protein. Moreover, the survival rates of mice challenged with i.v. H37Rv have been prolonged after treatment with single injection of aptamer NK2. The bacterial numbers were significantly lower in the spleen of mice treated with aptamer NK2. The histopathological examination of lung biopsy specimens showed lesser pulmonary alveolar fusion and swelling in the presence of the aptamer. These results suggest that aptamer NK2 has inhibitory effects on M. tuberculosis and can be used as antimycobacterial agent

  18. Lipoprotein Processing Is Essential for Resistance of Mycobacterium tuberculosis to Malachite Green▿

    Science.gov (United States)

    Banaei, Niaz; Kincaid, Eleanor Z.; Lin, S.-Y. Grace; Desmond, Edward; Jacobs, William R.; Ernst, Joel D.

    2009-01-01

    Malachite green, a synthetic antimicrobial dye, has been used for over 50 years in mycobacterial culture medium to inhibit the growth of contaminants. The molecular basis of mycobacterial resistance to malachite green is unknown, although the presence of malachite green-reducing enzymes in the cell envelope has been suggested. The objective of this study was to investigate the role of lipoproteins in resistance of Mycobacterium tuberculosis to malachite green. The replication of an M. tuberculosis lipoprotein signal peptidase II (lspA) mutant (ΔlspA::lspAmut) on Middlebrook agar with and without 1 mg/liter malachite green was investigated. The lspA mutant was also compared with wild-type M. tuberculosis in the decolorization rate of malachite green and sensitivity to sodium dodecyl sulfate (SDS) detergent and first-line antituberculosis drugs. The lspA mutant has a 104-fold reduction in CFU-forming efficiency on Middlebrook agar with malachite green. Malachite green is decolorized faster in the presence of the lspA mutant than wild-type bacteria. The lspA mutant is hypersensitive to SDS detergent and shows increased sensitivity to first-line antituberculosis drugs. In summary, lipoprotein processing by LspA is essential for resistance of M. tuberculosis to malachite green. A cell wall permeability defect is likely responsible for the hypersensitivity of lspA mutant to malachite green. PMID:19596883

  19. Lipoprotein processing is essential for resistance of Mycobacterium tuberculosis to malachite green.

    Science.gov (United States)

    Banaei, Niaz; Kincaid, Eleanor Z; Lin, S-Y Grace; Desmond, Edward; Jacobs, William R; Ernst, Joel D

    2009-09-01

    Malachite green, a synthetic antimicrobial dye, has been used for over 50 years in mycobacterial culture medium to inhibit the growth of contaminants. The molecular basis of mycobacterial resistance to malachite green is unknown, although the presence of malachite green-reducing enzymes in the cell envelope has been suggested. The objective of this study was to investigate the role of lipoproteins in resistance of Mycobacterium tuberculosis to malachite green. The replication of an M. tuberculosis lipoprotein signal peptidase II (lspA) mutant (DeltalspA::lspAmut) on Middlebrook agar with and without 1 mg/liter malachite green was investigated. The lspA mutant was also compared with wild-type M. tuberculosis in the decolorization rate of malachite green and sensitivity to sodium dodecyl sulfate (SDS) detergent and first-line antituberculosis drugs. The lspA mutant has a 10(4)-fold reduction in CFU-forming efficiency on Middlebrook agar with malachite green. Malachite green is decolorized faster in the presence of the lspA mutant than wild-type bacteria. The lspA mutant is hypersensitive to SDS detergent and shows increased sensitivity to first-line antituberculosis drugs. In summary, lipoprotein processing by LspA is essential for resistance of M. tuberculosis to malachite green. A cell wall permeability defect is likely responsible for the hypersensitivity of lspA mutant to malachite green.

  20. High functional diversity in Mycobacterium tuberculosis driven by genetic drift and human demography.

    Directory of Open Access Journals (Sweden)

    Ruth Hershberg

    2008-12-01

    Full Text Available Mycobacterium tuberculosis infects one third of the human world population and kills someone every 15 seconds. For more than a century, scientists and clinicians have been distinguishing between the human- and animal-adapted members of the M. tuberculosis complex (MTBC. However, all human-adapted strains of MTBC have traditionally been considered to be essentially identical. We surveyed sequence diversity within a global collection of strains belonging to MTBC using seven megabase pairs of DNA sequence data. We show that the members of MTBC affecting humans are more genetically diverse than generally assumed, and that this diversity can be linked to human demographic and migratory events. We further demonstrate that these organisms are under extremely reduced purifying selection and that, as a result of increased genetic drift, much of this genetic diversity is likely to have functional consequences. Our findings suggest that the current increases in human population, urbanization, and global travel, combined with the population genetic characteristics of M. tuberculosis described here, could contribute to the emergence and spread of drug-resistant tuberculosis.

  1. Putative in vitro expressed gene fragments unique to Mycobacterium avium subspecies para tuberculosis

    DEFF Research Database (Denmark)

    Nielsen, Kirstine Klitgaard; Ahrens, Peter

    2002-01-01

    By a suppression subtractive hybridization based method, nine novel Mycobacterium avium subsp. para tuberculosis (M. para tuberculosis) fragments of between 318 and 596 bp have been identified and characterized. Database search revealed little or no similarity with other mycobacteria. The uniquen......By a suppression subtractive hybridization based method, nine novel Mycobacterium avium subsp. para tuberculosis (M. para tuberculosis) fragments of between 318 and 596 bp have been identified and characterized. Database search revealed little or no similarity with other mycobacteria....... The uniqueness and diagnostic potential of seven of these fragments in relation to M. paratuberculosis closest relative Mycobacterium avium subsp. avium (M. avium) was confirmed by species-specific PCR and Southern blot. Furthermore, RT-PCR indicated that eight of the nine fragments originate from areas...

  2. Genomic diversity among drug sensitive and multidrug resistant isolates of Mycobacterium tuberculosis with identical DNA fingerprints.

    Directory of Open Access Journals (Sweden)

    Stefan Niemann

    2009-10-01

    Full Text Available Mycobacterium tuberculosis complex (MTBC, the causative agent of tuberculosis (TB, is characterized by low sequence diversity making this bacterium one of the classical examples of a genetically monomorphic pathogen. Because of this limited DNA sequence variation, routine genotyping of clinical MTBC isolates for epidemiological purposes relies on highly discriminatory DNA fingerprinting methods based on mobile and repetitive genetic elements. According to the standard view, isolates exhibiting the same fingerprinting pattern are considered direct progeny of the same bacterial clone, and most likely reflect ongoing transmission or disease relapse within individual patients.Here we further investigated this assumption and used massively parallel whole-genome sequencing to compare one drug-susceptible (K-1 and one multidrug resistant (MDR isolate (K-2 of a rapidly spreading M. tuberculosis Beijing genotype clone from a high incidence region (Karakalpakstan, Uzbekistan. Both isolates shared the same IS6110 RFLP pattern and the same allele at 23 out of 24 MIRU-VNTR loci. We generated 23.9 million (K-1 and 33.0 million (K-2 paired 50 bp purity filtered reads corresponding to a mean coverage of 483.5 fold and 656.1 fold respectively. Compared with the laboratory strain H37Rv both Beijing isolates shared 1,209 SNPs. The two Beijing isolates differed by 130 SNPs and one large deletion. The susceptible isolate had 55 specific SNPs, while the MDR variant had 75 specific SNPs, including the five known resistance-conferring mutations.Our results suggest that M. tuberculosis isolates exhibiting identical DNA fingerprinting patterns can harbour substantial genomic diversity. Because this heterogeneity is not captured by traditional genotyping of MTBC, some aspects of the transmission dynamics of tuberculosis could be missed or misinterpreted. Furthermore, a valid differentiation between disease relapse and exogenous reinfection might be impossible using

  3. Genomic diversity among drug sensitive and multidrug resistant isolates of Mycobacterium tuberculosis with identical DNA fingerprints.

    Science.gov (United States)

    Niemann, Stefan; Köser, Claudio U; Gagneux, Sebastien; Plinke, Claudia; Homolka, Susanne; Bignell, Helen; Carter, Richard J; Cheetham, R Keira; Cox, Anthony; Gormley, Niall A; Kokko-Gonzales, Paula; Murray, Lisa J; Rigatti, Roberto; Smith, Vincent P; Arends, Felix P M; Cox, Helen S; Smith, Geoff; Archer, John A C

    2009-10-12

    Mycobacterium tuberculosis complex (MTBC), the causative agent of tuberculosis (TB), is characterized by low sequence diversity making this bacterium one of the classical examples of a genetically monomorphic pathogen. Because of this limited DNA sequence variation, routine genotyping of clinical MTBC isolates for epidemiological purposes relies on highly discriminatory DNA fingerprinting methods based on mobile and repetitive genetic elements. According to the standard view, isolates exhibiting the same fingerprinting pattern are considered direct progeny of the same bacterial clone, and most likely reflect ongoing transmission or disease relapse within individual patients. Here we further investigated this assumption and used massively parallel whole-genome sequencing to compare one drug-susceptible (K-1) and one multidrug resistant (MDR) isolate (K-2) of a rapidly spreading M. tuberculosis Beijing genotype clone from a high incidence region (Karakalpakstan, Uzbekistan). Both isolates shared the same IS6110 RFLP pattern and the same allele at 23 out of 24 MIRU-VNTR loci. We generated 23.9 million (K-1) and 33.0 million (K-2) paired 50 bp purity filtered reads corresponding to a mean coverage of 483.5 fold and 656.1 fold respectively. Compared with the laboratory strain H37Rv both Beijing isolates shared 1,209 SNPs. The two Beijing isolates differed by 130 SNPs and one large deletion. The susceptible isolate had 55 specific SNPs, while the MDR variant had 75 specific SNPs, including the five known resistance-conferring mutations. Our results suggest that M. tuberculosis isolates exhibiting identical DNA fingerprinting patterns can harbour substantial genomic diversity. Because this heterogeneity is not captured by traditional genotyping of MTBC, some aspects of the transmission dynamics of tuberculosis could be missed or misinterpreted. Furthermore, a valid differentiation between disease relapse and exogenous reinfection might be impossible using standard

  4. Mycobacterium tuberculosis TlyA Protein Negatively Regulates T Helper (Th) 1 and Th17 Differentiation and Promotes Tuberculosis Pathogenesis*

    Science.gov (United States)

    Rahman, Md. Aejazur; Sobia, Parveen; Dwivedi, Ved Prakash; Bhawsar, Aakansha; Singh, Dhiraj Kumar; Sharma, Pawan; Moodley, Prashini; Van Kaer, Luc; Bishai, William R; Das, Gobardhan

    2015-01-01

    Mycobacterium tuberculosis, the causative agent of tuberculosis, is an ancient pathogen and a major cause of death worldwide. Although various virulence factors of M. tuberculosis have been identified, its pathogenesis remains incompletely understood. TlyA is a virulence factor in several bacterial infections and is evolutionarily conserved in many Gram-positive bacteria, but its function in M. tuberculosis pathogenesis has not been elucidated. Here, we report that TlyA significantly contributes to the pathogenesis of M. tuberculosis. We show that a TlyA mutant M. tuberculosis strain induces increased IL-12 and reduced IL-1β and IL-10 cytokine responses, which sharply contrasts with the immune responses induced by wild type M. tuberculosis. Furthermore, compared with wild type M. tuberculosis, TlyA-deficient M. tuberculosis bacteria are more susceptible to autophagy in macrophages. Consequently, animals infected with the TlyA mutant M. tuberculosis organisms exhibited increased host-protective immune responses, reduced bacillary load, and increased survival compared with animals infected with wild type M. tuberculosis. Thus, M. tuberculosis employs TlyA as a host evasion factor, thereby contributing to its virulence. PMID:25847237

  5. Comparison of antigen-specific T-cell responses of tuberculosis patients using complex or single antigens of Mycobacterium tuberculosis

    DEFF Research Database (Denmark)

    Mustafa, A S; Amoudy, H A; Wiker, H G

    1998-01-01

    GroES, rPstS, rGroEL and rDnaK) antigens of Mycobacterium tuberculosis. The responses of PBMC to these defined antigens were compared with the corresponding results obtained with complex antigens, such as whole-cell M. tuberculosis, M. tuberculosis culture filtrate (MT-CF) and cell wall antigens, as well...... as the vaccine strain, Mycobacterium bovis bacillus Calmette-Guerin (BCG). In addition, M. tuberculosis and MT-CF-induced T-cell lines were tested in the same assays against the panel of purified and complex antigens. The compiled data from PBMC and T-cell lines tested for antigen-induced proliferation and IFN...

  6. Efficacy of amikacin and ciprofloxacin against clinical isolates of mycobacterium tuberculosis

    International Nuclear Information System (INIS)

    Satti, M.; Faqir, F.; Sattar, A.; Abbasi, S.; Butt, T.; Karamat, K.A.; Abidi, M.

    2010-01-01

    Background: Tuberculosis was a leading cause of death at the turn of the 20 century and continues to be one of the medical scourges of mankind. Before the availability of antimicrobial drugs the cornerstone of treatment was rest in the open air in sanatoria. The major breakthrough in treatment of tuberculosis came with the discovery of Streptomycin. Later, INH, Ethambutol, Pyrazinamide, Rifampicin were added to the arsenal. Objective of this study was to determine the sensitivity of clinical isolates of Mycobacterium tuberculosis against two second-line anti-tuberculosis drugs, Amikacin and Ciprofloxacin. Methods: This cross-sectional study was conducted at Department of Microbiology, Armed Forces Institute of Pathology (AFIP) Rawalpindi. All routine clinical samples received for acid fast bacilli (AFB) in the Department of Microbiology, AFIP, Rawalpindi were processed by modified Petroff's technique and inoculated on Lowenstein Jensen (LJ) medium and Bactec 460 Mycobacterium tuberculosis culture system. After identification of M. tuberculosis sensitivity was performed against first-line anti-tuberculosis drugs. Then susceptibility of M. tuberculosis isolates against Amikacin and Ciprofloxacin was performed on LJ medium. H37Rv was used as control strain. Results: Results were interpreted using resistance ratio method. Out of 100 M. tuberculosis isolates, 98% were sensitive to Amikacin and 97% to Ciprofloxacin. Conclusion: Amikacin and Ciprofloxacin are very effective second line anti-tuberculosis drugs against tuberculosis isolates in our set-up. (author)

  7. Genotyping of rifampin-resistant Mycobacterium tuberculosis isolates from Western Turkey

    International Nuclear Information System (INIS)

    Cavasoglu, Cengiz; Bilgic, Altinay; Durmaz, Riza; Gunal, Selami

    2004-01-01

    Although the rate of multiple drug resistance is high there is no published data on the transmission rate of drug-resistant strains of Mycobacterium tuberculosis in the Aegean region of Western Turkey that are based on molecular methods. IS6110 and pTBN12 restriction fragment lengthpolymorphism (RFLP) methods were used for typing Mycobacterium tuberculosis isolated from 26 sputum samples from 26 patients. 19 of rifampin-resistant isolates (73.1%) contained 6 to 11 copies of 156110. Eighteen different IS6110 DNA fingerprint patterns were observed in the 26 rifampin resistant isolates. 23 of the 26 rifampin-resistant isolates were also resistant to isoniazid. When evaluated together, both methods yielded 21 (80.9%) different banding patterns and the level of clustering was 34.6%. The average number per pattern was 1.23 (26/21). IS6110 fingerprinting suggests that the rifampin-resistant isolates obtained from the Aegean region had a relatively high clustering rate and were clonally related. These findings showed that the rifampin-resistant isolates are actively transmitted between patients. Urgent measures should be taken to prevent the spread of these resistant strains. (author)

  8. Identification of gene targets against dormant phase Mycobacterium tuberculosis infections

    Directory of Open Access Journals (Sweden)

    Murphy Dennis J

    2007-07-01

    Full Text Available Abstract Background Mycobacterium tuberculosis, the causative agent of tuberculosis (TB, infects approximately 2 billion people worldwide and is the leading cause of mortality due to infectious disease. Current TB therapy involves a regimen of four antibiotics taken over a six month period. Patient compliance, cost of drugs and increasing incidence of drug resistant M. tuberculosis strains have added urgency to the development of novel TB therapies. Eradication of TB is affected by the ability of the bacterium to survive up to decades in a dormant state primarily in hypoxic granulomas in the lung and to cause recurrent infections. Methods The availability of M. tuberculosis genome-wide DNA microarrays has lead to the publication of several gene expression studies under simulated dormancy conditions. However, no single model best replicates the conditions of human pathogenicity. In order to identify novel TB drug targets, we performed a meta-analysis of multiple published datasets from gene expression DNA microarray experiments that modeled infection leading to and including the dormant state, along with data from genome-wide insertional mutagenesis that examined gene essentiality. Results Based on the analysis of these data sets following normalization, several genome wide trends were identified and used to guide the selection of targets for therapeutic development. The trends included the significant up-regulation of genes controlled by devR, down-regulation of protein and ATP synthesis, and the adaptation of two-carbon metabolism to the hypoxic and nutrient limited environment of the granuloma. Promising targets for drug discovery were several regulatory elements (devR/devS, relA, mprAB, enzymes involved in redox balance and respiration, sulfur transport and fixation, pantothenate, isoprene, and NAD biosynthesis. The advantages and liabilities of each target are discussed in the context of enzymology, bacterial pathways, target tractability

  9. Updates on antibody functions in Mycobacterium tuberculosis infection and their relevance for developing a vaccine against tuberculosis.

    Science.gov (United States)

    Achkar, Jacqueline M; Prados-Rosales, Rafael

    2018-04-12

    A more effective vaccine to control tuberculosis (TB), a major global public health problem, is urgently needed. Current vaccine candidates focus predominantly on eliciting cell-mediated immunity but other arms of the immune system also contribute to protection against TB. We review here recent studies that enhance our current knowledge of antibody-mediated functions against Mycobacterium tuberculosis. These findings, which contribute to the increasing evidence that antibodies have a protective role against TB, include demonstrations that firstly distinct human antibody Fc glycosylation patterns, found in latent M. tuberculosis infection but not in active TB, influence the efficacy of the host to control M. tuberculosis infection, secondly antibody isotype influences human antibody functions, and thirdly that antibodies targeting M. tuberculosis surface antigens are protective. We discuss these findings in the context of TB vaccine development and highlight the need for further research on antibody-mediated immunity in M. tuberculosis infection. Copyright © 2018 Elsevier Ltd. All rights reserved.

  10. JST Thesaurus Headwords and Synonyms: Mycobacterium tuberculosis [MeCab user dictionary for science technology term[Archive

    Lifescience Database Archive (English)

    Full Text Available MeCab user dictionary for science technology term Mycobacterium tuberculosis 名詞 一般 * * * * 結核...菌 ケッカクキン ケッカクキン Thesaurus2015 200906007893342100 C LS07 UNKNOWN_2 Mycobacterium tuberculosis

  11. Tuberculosis associated factors caused by Mycobacterium tuberculosis of the RDRio genotype

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    Eloise Brasil Moraes

    Full Text Available BACKGROUND Tuberculosis (TB continues to be a disease that affects many countries around the world, including Brazil. Recently, a subtype of Latin American-Mediterranean family strain was identified and characterised by RDRio. The strain has been associated with different characteristics of the disease. OBJECTIVES In the present study we investigated the association of epidemiological, clinical, radiological and bacteriological variables with pulmonary tuberculosis caused by RDRioMycobacterium tuberculosis strain in large regions of São Paulo. METHODS We conducted a cross-sectional study in 530 patients with pulmonary tuberculosis, diagnosed using sputum culture, from two regions of the São Paulo state in Brazil. The samples were brought to São Paulo reference laboratories for epidemiological, clinical, radiological and bacteriological analyses, and the data were obtained from a TB notification system. RDRio genotyping and Spoligotyping of the samples were performed. For the analysis of the categorical variables we used the chi-square test or the Fisher’s exact test, and for the continuous variables, the Mann-Whitney test. In addition, a logistic regression was used for multivariate analysis. Differences with p < 0.05 were considered significant. FINDINGS The RDRio deletion was identified in 152 (28.7% samples. In the univariate analysis, both the age groups above 25 years and alcohol consumption were associated with the RDRio deletion. The multivariate analysis confirmed the association of the RDRio deletion with the age groups: 25-35 years old [OR: 2.28 (1.02-5.07; p = 0.04] and 36-60 years old (OR: 2.36 (1.11-5.05; p = 0.03], and also with alcohol consumption [OR: 1.63 (1.05-2.54; p = 0,03]. MAIN CONCLUSIONS In this study, we identified new factors associated with the M. tuberculosis of the RDRio deletion strains infection.

  12. Molecular diversity of Mycobacterium tuberculosis isolates from patients with tuberculosis in Honduras

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    Ghebremichael Solomon

    2010-08-01

    Full Text Available Abstract Background Tuberculosis persists as a public health problem in Honduras. A better knowledge of the molecular characteristics of Mycobacterium tuberculosis strains will contribute to understand the transmission dynamics of the disease within the country. The aim of this study was to provide an insight of the genetic biodiversity of M. tuberculosis clinical isolates collected in Honduras between 1994 and 2002. Genotyping was performed using spoligotyping and RFLP. The spoligotypes obtained were compared with the SITVIT2 proprietary database of the Pasteur Institute of Guadeloupe. Results Spoligotyping grouped 84% of the isolates into 27 clusters (2 to 43 strains per cluster. Of the 44 shared international types (SITs identified among the Honduran stains, 8 SITs were newly identified either within the present study or after match with an orphan type previously identified in the SITVIT2 database. In addition, 16 patterns corresponded to orphan, previously unreported isolates. The Latin American Mediterranean (LAM lineage was the most common in this study; 55% of the strains belonged to this family. Other genotypes found were Haarlem (16%, T (16%, X-clade (6%, Unknown signature (5% and S (1%. Only one Beijing strain was identified (0.5%. We observed a high degree of diversity after characterizing the 43 isolates belonging to the main spoligotyping cluster (SIT 33, LAM3 with IS6110-RFLP. A total of 35 different RFLP-fingerprints were detected, of which 6 patterns corresponded to the same number of clusters comprising 14 strains. Conclusions The findings obtained in this study show that tuberculosis transmission in Honduras is due to modern M. tuberculosis lineages with high level of biodiversity.

  13. Molecular diversity of Mycobacterium tuberculosis isolates from patients with tuberculosis in Honduras

    Science.gov (United States)

    2010-01-01

    Background Tuberculosis persists as a public health problem in Honduras. A better knowledge of the molecular characteristics of Mycobacterium tuberculosis strains will contribute to understand the transmission dynamics of the disease within the country. The aim of this study was to provide an insight of the genetic biodiversity of M. tuberculosis clinical isolates collected in Honduras between 1994 and 2002. Genotyping was performed using spoligotyping and RFLP. The spoligotypes obtained were compared with the SITVIT2 proprietary database of the Pasteur Institute of Guadeloupe. Results Spoligotyping grouped 84% of the isolates into 27 clusters (2 to 43 strains per cluster). Of the 44 shared international types (SITs) identified among the Honduran stains, 8 SITs were newly identified either within the present study or after match with an orphan type previously identified in the SITVIT2 database. In addition, 16 patterns corresponded to orphan, previously unreported isolates. The Latin American Mediterranean (LAM) lineage was the most common in this study; 55% of the strains belonged to this family. Other genotypes found were Haarlem (16%), T (16%), X-clade (6%), Unknown signature (5%) and S (1%). Only one Beijing strain was identified (0.5%). We observed a high degree of diversity after characterizing the 43 isolates belonging to the main spoligotyping cluster (SIT 33, LAM3) with IS6110-RFLP. A total of 35 different RFLP-fingerprints were detected, of which 6 patterns corresponded to the same number of clusters comprising 14 strains. Conclusions The findings obtained in this study show that tuberculosis transmission in Honduras is due to modern M. tuberculosis lineages with high level of biodiversity. PMID:20678242

  14. Molecular Identification of Mycobacterium Tuberculosis and Analysis of Its Resistance to Rifampin in Sputa from Tuberculosis Suspected Patients

    International Nuclear Information System (INIS)

    Syaifudin, M.

    2010-01-01

    An accurate identification of different species of Mycobacterium provides to allow appropriate treatment for Mycobacterium tuberculosis infection. Beside that, drug resistance of M. tuberculosis strains to rifampin is not clearly understood in contributing to the spread of tuberculosis in Indonesia. To assess the molecular mechanism of rifampin resistance, a number of clinical specimens of M. tuberculosis were analyzed their molecular nature of a part of the rpoB gene using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) methods. DNA's extracted from sputum samples were amplified and 32 P-labeled by PCR with the specific primers and the product was analyzed their mutation conferring resistance by MDE gel electrophoresis. Of the 70 specimens tested, 57 specimens were positive for M. tuberculosis organism only, three specimens contained a mixture of M. tuberculosis and non tuberculosis mycobacteria (NTM), and 10 specimens were negative approved by Duplex PCR. Of these sixty DNA positive samples (thus the sensitivity of PCR was 85.71%), 5 (8.3%) of them suspected to contain mutations in rpoB which were associated with rifampin resistance. Even though the frequency of mutation was low, the results from our study clearly indicate that the molecular mechanism of rifampin resistance in M. tuberculosis isolates from Indonesia involves alterations in the rpoB gene. Molecular diagnosis by PCR which is fast and easy to perform is useful for early and rapid detection of TB in sputum specimen. (author)

  15. Comparative 'omics analyses differentiate Mycobacterium tuberculosis and Mycobacterium bovis and reveal distinct macrophage responses to infection with the human and bovine tubercle bacilli

    Science.gov (United States)

    Malone, Kerri M.; Rue-Albrecht, Kévin; Magee, David A.; Conlon, Kevin; Schubert, Olga T.; Nalpas, Nicolas C.; Browne, John A.; Smyth, Alicia; Gormley, Eamonn; Aebersold, Ruedi; MacHugh, David E.; Gordon, Stephen V.

    2018-01-01

    Members of the Mycobacterium tuberculosis complex (MTBC) are the causative agents of tuberculosis in a range of mammals, including humans. A key feature of MTBC pathogens is their high degree of genetic identity yet distinct host tropism. Notably, while Mycobacterium bovis is highly virulent and pathogenic for cattle, the human pathogen M. tuberculosis is attenuated in cattle. Previous research also suggests that host preference amongst MTBC members has a basis in host innate immune responses. To explore MTBC host tropism, we present in-depth profiling of the MTBC reference strains M. bovis AF2122/97 and M. tuberculosis H37Rv at both the global transcriptional and the translational level via RNA-sequencing and SWATH MS. Furthermore, a bovine alveolar macrophage infection time course model was used to investigate the shared and divergent host transcriptomic response to infection with M. tuberculosis H37Rv or M. bovis AF2122/97. Significant differential expression of virulence-associated pathways between the two bacilli was revealed, including the ESX-1 secretion system. A divergent transcriptional response was observed between M. tuberculosis H37Rv and M. bovis AF2122/97 infection of bovine alveolar macrophages, in particular cytosolic DNA-sensing pathways at 48 h post-infection, and highlights a distinct engagement of M. bovis with the bovine innate immune system. The work presented here therefore provides a basis for the identification of host innate immune mechanisms subverted by virulent host-adapted mycobacteria to promote their survival during the early stages of infection. PMID:29557774

  16. SUSCEPTIBILITY OF RIFAMPICIN-ISONIAZID RESISTANT MYCOBACTERIUM TUBERCULOSIS ISOLATES AGAINST LEVOFLOXACIN

    Directory of Open Access Journals (Sweden)

    A. H. Kurniawan

    2016-01-01

    Full Text Available Background: Tuberculosis (TB is a high burden disease in Indonesia with multidrug-resistant (MDR TB incidence started to increase. Treatment success of MDR-TB globally was low in number than it was targeted which was especially caused by fluoroquinolone resistance. One of the fluoroquinolone is levofloxacin, an antibiotic that has been widely used irrationally as antimicrobial treatment. Therefore, this study investigated the sensitivity and MBC of MDR Mycobacterium tuberculosis isolates against Levofloxacin. Method: The susceptibility test for MDR-Mycobacterium tuberculosis on levofloxacin by standard method with levofloxacin were on concentrations 0,5 μg/ml, 1 μg/ml, and 2 μg/ml. Sample of 8 strains MDR-Mycobacterium tuberculosis were cultured with each concentrations on Middlebrook 7H9 for 1 week incubation. Next, each of the incubated concentration was subcultured on solid media Middlebrook 7H10 for 3 weeks incubation. Colonized agar plates after 3 weeks incubation were confirmed with acid-fast stain. Results: On MB 7H10 with levofloxacin concentration 2 μg/ml showed bactericidal effect 100% by no MDR Mycobacterium tuberculosis colony grew (0/8 while the MB 7H10 with levofloxacin concentration 1 μg/ml and 0,5 μg/ml showed the bactericidal effect 37,5% and 25% respectively. The colonized agar plate implied that the MDR Mycobacterium tuberculosis with levofloxacin concentration 1 μg/ml (5/8 and 0,5 μg/ml (6/8 grew well. Conclusion: Levofloxacin concentration 2 μg/ml was susceptible on MDR Mycobacterium tuberculosis. The concentration 2 μg/ml of levofloxacin could be considered as MBC.

  17. Use of Mycobacterium smegmatis deficient in ADP-ribosyltransferase as surrogate for Mycobacterium tuberculosis in drug testing and mutation analysis.

    Science.gov (United States)

    Agrawal, Priyanka; Miryala, Sandeep; Varshney, Umesh

    2015-01-01

    Rifampicin (Rif) is a first line drug used for tuberculosis treatment. However, the emergence of drug resistant strains has necessitated synthesis and testing of newer analogs of Rif. Mycobacterium smegmatis is often used as a surrogate for M. tuberculosis. However, the presence of an ADP ribosyltransferase (Arr) in M. smegmatis inactivates Rif, rendering it impractical for screening of Rif analogs or other compounds when used in conjunction with them (Rif/Rif analogs). Rifampicin is also used in studying the role of various DNA repair enzymes by analyzing mutations in RpoB (a subunit of RNA polymerase) causing Rif resistance. These analyses use high concentrations of Rif when M. smegmatis is used as model. Here, we have generated M. smegmatis strains by deleting arr (Δarr). The M. smegmatis Δarr strains show minimum inhibitory concentration (MIC) for Rif which is similar to that for M. tuberculosis. The MICs for isoniazid, pyrazinamide, ethambutol, ciprofloxacin and streptomycin were essentially unaltered for M. smegmatis Δarr. The growth profiles and mutation spectrum of Δarr and, Δarr combined with ΔudgB (udgB encodes a DNA repair enzyme that excises uracil) strains were similar to their counterparts wild-type for arr. However, the mutation spectrum of ΔfpgΔarr strain differed somewhat from that of the Δfpg strain (fpg encodes a DNA repair enzyme that excises 8-oxo-G). Our studies suggest M. smegmatis Δarr strain as an ideal model system in drug testing and mutation spectrum determination in DNA repair studies.

  18. Use of Mycobacterium smegmatis deficient in ADP-ribosyltransferase as surrogate for Mycobacterium tuberculosis in drug testing and mutation analysis.

    Directory of Open Access Journals (Sweden)

    Priyanka Agrawal

    Full Text Available Rifampicin (Rif is a first line drug used for tuberculosis treatment. However, the emergence of drug resistant strains has necessitated synthesis and testing of newer analogs of Rif. Mycobacterium smegmatis is often used as a surrogate for M. tuberculosis. However, the presence of an ADP ribosyltransferase (Arr in M. smegmatis inactivates Rif, rendering it impractical for screening of Rif analogs or other compounds when used in conjunction with them (Rif/Rif analogs. Rifampicin is also used in studying the role of various DNA repair enzymes by analyzing mutations in RpoB (a subunit of RNA polymerase causing Rif resistance. These analyses use high concentrations of Rif when M. smegmatis is used as model. Here, we have generated M. smegmatis strains by deleting arr (Δarr. The M. smegmatis Δarr strains show minimum inhibitory concentration (MIC for Rif which is similar to that for M. tuberculosis. The MICs for isoniazid, pyrazinamide, ethambutol, ciprofloxacin and streptomycin were essentially unaltered for M. smegmatis Δarr. The growth profiles and mutation spectrum of Δarr and, Δarr combined with ΔudgB (udgB encodes a DNA repair enzyme that excises uracil strains were similar to their counterparts wild-type for arr. However, the mutation spectrum of ΔfpgΔarr strain differed somewhat from that of the Δfpg strain (fpg encodes a DNA repair enzyme that excises 8-oxo-G. Our studies suggest M. smegmatis Δarr strain as an ideal model system in drug testing and mutation spectrum determination in DNA repair studies.

  19. Distribution of Insertion- and Deletion-Associated Genetic Polymorphisms among Four Mycobacterium tuberculosis Phospholipase C Genes and Associations with Extrathoracic Tuberculosis: a Population-Based Study

    OpenAIRE

    Kong, Y.; Cave, M. D.; Yang, D.; Zhang, L.; Marrs, C. F.; Foxman, B.; Bates, J. H.; Wilson, F.; Mukasa, L. N.; Yang, Z. H.

    2005-01-01

    The Mycobacterium tuberculosis genome contains four phospholipase C (PLC)-encoding genes, designated plcA, plcB, plcC, and plcD, respectively. Each of the four genes contributes to the overall PLC activity of M. tuberculosis. PLC is hypothesized to contribute to M. tuberculosis virulence. Infection of M. tuberculosis strains carrying a truncated plcD gene is associated with the occurrence of extrathoracic tuberculosis. However, whether the other three plc genes are also associated with extrat...

  20. Horizontal acquisition of a hypoxia-responsive molybdenum cofactor biosynthesis pathway contributed to Mycobacterium tuberculosis pathoadaptation.

    Science.gov (United States)

    Levillain, Florence; Poquet, Yannick; Mallet, Ludovic; Mazères, Serge; Marceau, Michael; Brosch, Roland; Bange, Franz-Christoph; Supply, Philip; Magalon, Axel; Neyrolles, Olivier

    2017-11-01

    The unique ability of the tuberculosis (TB) bacillus, Mycobacterium tuberculosis, to persist for long periods of time in lung hypoxic lesions chiefly contributes to the global burden of latent TB. We and others previously reported that the M. tuberculosis ancestor underwent massive episodes of horizontal gene transfer (HGT), mostly from environmental species. Here, we sought to explore whether such ancient HGT played a part in M. tuberculosis evolution towards pathogenicity. We were interested by a HGT-acquired M. tuberculosis-specific gene set, namely moaA1-D1, which is involved in the biosynthesis of the molybdenum cofactor. Horizontal acquisition of this gene set was striking because homologues of these moa genes are present all across the Mycobacterium genus, including in M. tuberculosis. Here, we discovered that, unlike their paralogues, the moaA1-D1 genes are strongly induced under hypoxia. In vitro, a M. tuberculosis moaA1-D1-null mutant has an impaired ability to respire nitrate, to enter dormancy and to survive in oxygen-limiting conditions. Conversely, heterologous expression of moaA1-D1 in the phylogenetically closest non-TB mycobacterium, Mycobacterium kansasii, which lacks these genes, improves its capacity to respire nitrate and grants it with a marked ability to survive oxygen depletion. In vivo, the M. tuberculosis moaA1-D1-null mutant shows impaired survival in hypoxic granulomas in C3HeB/FeJ mice, but not in normoxic lesions in C57BL/6 animals. Collectively, our results identify a novel pathway required for M. tuberculosis resistance to host-imposed stress, namely hypoxia, and provide evidence that ancient HGT bolstered M. tuberculosis evolution from an environmental species towards a pervasive human-adapted pathogen.

  1. MicroRNA signatures from multidrug-resistant Mycobacterium tuberculosis

    Science.gov (United States)

    REN, NA; GAO, GUIJU; SUN, YUE; ZHANG, LING; WANG, HUIZHU; HUA, WENHAO; WAN, KANGLIN; LI, XINGWANG

    2015-01-01

    Tuberculosis (TB) infections, caused by multi-drug-resistant Mycobacterium tuberculosis (MDR MTB), remain a significant public health concern worldwide. The regulatory mechanisms underlying the emergence of MDR MTB strains remain to be fully elucidated, and further investigation is required in order to develop better strategies for TB control. The present study investigated the expression profile of microRNA (miRNA) in MTB strains, and examined the differences between sensitive MTB and MDR MTB using next generation sequencing (NGS) with Illumina Deep Sequencing technology to better understand the mechanisms of resistance in MDR MTB, A total of 5, 785 and 195, and 6, 290 and 595 qualified Illumina reads were obtained from two MDR MTB strains, and 6, 673 and 665, and 7, 210 and 217 qualified Illumina reads were obtained from two sensitive MTB strains. The overall de novo assembly of miRNA sequence data generated 62 and 62, and 95 and 112 miRNAs between the 18 and 30 bp long from sensitive MTB strains and MDR MTB strains, respectively. Comparative miRNA analysis revealed that 142 miRNAs were differentially expressed in the MDR MTB strain, compared with the sensitive MTB strain, of which 48 were upregulated and 94 were downregulated. There were six similarly expressed miRNAs between the MDR and sensitive MTB strains, and 108 miRNAs were expressed only in the MDR MTB strain. The present study acquired miRNA data from sensitive MTB and MDR MTB strains using NGS techniques, and this identification miRNAs may serve as an invaluable resource for revealing the molecular basis of the regulation of expression associated with the mechanism of drug-resistance in MTB. PMID:26324150

  2. MicroRNA signatures from multidrug‑resistant Mycobacterium tuberculosis.

    Science.gov (United States)

    Ren, Na; Gao, Guiju; Sun, Yue; Zhang, Ling; Wang, Huizhu; Hua, Wenhao; Wan, Kanglin; Li, Xingwang

    2015-11-01

    Tuberculosis (TB) infections, caused by multidrug‑resistant Mycobacterium tuberculosis (MDR MTB), remain a significant public health concern worldwide. The regulatory mechanisms underlying the emergence of MDR MTB strains remain to be fully elucidated, and further investigation is required in order to develop better strategies for TB control. The present study investigated the expression profile of microRNA (miRNA) in MTB strains, and examined the differences between sensitive MTB and MDR MTB using next generation sequencing (NGS) with Illumina Deep Sequencing technology to better understand the mechanisms of resistance in MDR MTB, A total of 5, 785 and 195, and 6, 290 and 595 qualified Illumina reads were obtained from two MDR MTB strains, and 6, 673 and 665, and 7, 210 and 217 qualified Illumina reads were obtained from two sensitive MTB strains. The overall de novo assembly of miRNA sequence data generated 62 and 62, and 95 and 112 miRNAs between the 18 and 30 bp long from sensitive MTB strains and MDR MTB strains, respectively. Comparative miRNA analysis revealed that 142 miRNAs were differentially expressed in the MDR MTB strain, compared with the sensitive MTB strain, of which 48 were upregulated and 94 were downregulated. There were six similarly expressed miRNAs between the MDR and sensitive MTB strains, and 108 miRNAs were expressed only in the MDR MTB strain. The present study acquired miRNA data from sensitive MTB and MDR MTB strains using NGS techniques, and this identification miRNAs may serve as an invaluable resource for revealing the molecular basis of the regulation of expression associated with the mechanism of drug‑resistance in MTB.

  3. Loperamide Restricts Intracellular Growth of Mycobacterium tuberculosis in Lung Macrophages.

    Science.gov (United States)

    Juárez, Esmeralda; Carranza, Claudia; Sánchez, Guadalupe; González, Mitzi; Chávez, Jaime; Sarabia, Carmen; Torres, Martha; Sada, Eduardo

    2016-12-01

    New approaches for improving tuberculosis (TB) control using adjunct host-directed cellular and repurposed drug therapies are needed. Autophagy plays a crucial role in the response to TB, and a variety of autophagy-inducing drugs that are currently available for various medical conditions may serve as an adjunct treatment in pulmonary TB. Here, we evaluated the potential of loperamide, carbamazepine, valproic acid, verapamil, and rapamycin to enhance the antimicrobial immune response to Mycobacterium tuberculosis (Mtb). Human monocyte-derived macrophages (MDMs) and murine alveolar cells (MACs) were infected with Mtb and treated with loperamide, carbamazepine, valproic acid, verapamil, and rapamycin in vitro. Balb/c mice were intraperitoneally administered loperamide, valproic acid, and verapamil, and MACs were infected in vitro with Mtb. The induction of autophagy, the containment of Mtb within autophagosomes and the intracellular Mtb burden were determined. Autophagy was induced by all of the drugs in human and mouse macrophages, and loperamide significantly increased the colocalization of microtubule-associated protein 1 light chain 3 with Mtb in MDMs. Carbamazepine, loperamide, and valproic acid induced microtubule-associated protein 1 light chain 3 and autophagy related 16- like protein 1 gene expression in MDMs and in MACs. Loperamide also induced a reduction in TNF-α production. Loperamide and verapamil induced autophagy, which was associated with a significant reduction in the intracellular growth of Mtb in MACs and alveolar macrophages. The intraperitoneal administration of loperamide and valproic acid induced autophagy in freshly isolated MACs. The antimycobacterial activity in MACs was higher after loperamide treatment and was associated with the degradation of p62. In conclusion, loperamide shows potential as an adjunctive therapy for the treatment of TB.

  4. Mesa en cuello por Mycobacterium tuberculosis en un paciente infectado por HIV A case of cervical mass due to mycobacterium tuberculosis in an HIV infected patient

    Directory of Open Access Journals (Sweden)

    Mónica Soto

    1995-02-01

    Full Text Available Se presenta el caso de un paciente de sexo masculino con diagnóstico de infección por HIV quien presentó una masa en cuello de 7 x 10 cm, dolorosa, de consistencia dura, adherida, no pulsátil, con edema de la piel adyacente y sin otros signos asociados. La evolución fue de 6 meses. Los estudios de la masa y el esputo dieron como resultado el aislamiento de M. tuberculosis, lo que permitió un adecuado enfoque terapéutico y por consiguiente una respuesta clínica satisfactoria

    We report on the case of an HIV-infected male with Mycobacterium tuberculosis infection, manifested as both pulmonary involvement and a cervical mass; lung infection was proven by culture; pathological study of the mass revealed both chronic granulomatous infection and acidfast bacilli. Response to treatment was favorable
    and rapid. We suggest that every fluid and tissue obtained from HIV-positive patients be routinely stained and cultured for mycobacteria.

  5. Whole genome sequencing reveals genomic heterogeneity and antibiotic purification in Mycobacterium tuberculosis isolates

    KAUST Repository

    Black, PA

    2015-10-24

    Background Whole genome sequencing has revolutionised the interrogation of mycobacterial genomes. Recent studies have reported conflicting findings on the genomic stability of Mycobacterium tuberculosis during the evolution of drug resistance. In an age where whole genome sequencing is increasingly relied upon for defining the structure of bacterial genomes, it is important to investigate the reliability of next generation sequencing to identify clonal variants present in a minor percentage of the population. This study aimed to define a reliable cut-off for identification of low frequency sequence variants and to subsequently investigate genetic heterogeneity and the evolution of drug resistance in M. tuberculosis. Methods Genomic DNA was isolated from single colonies from 14 rifampicin mono-resistant M. tuberculosis isolates, as well as the primary cultures and follow up MDR cultures from two of these patients. The whole genomes of the M. tuberculosis isolates were sequenced using either the Illumina MiSeq or Illumina HiSeq platforms. Sequences were analysed with an in-house pipeline. Results Using next-generation sequencing in combination with Sanger sequencing and statistical analysis we defined a read frequency cut-off of 30 % to identify low frequency M. tuberculosis variants with high confidence. Using this cut-off we demonstrated a high rate of genetic diversity between single colonies isolated from one population, showing that by using the current sequencing technology, single colonies are not a true reflection of the genetic diversity within a whole population and vice versa. We further showed that numerous heterogeneous variants emerge and then disappear during the evolution of isoniazid resistance within individual patients. Our findings allowed us to formulate a model for the selective bottleneck which occurs during the course of infection, acting as a genomic purification event. Conclusions Our study demonstrated true levels of genetic diversity

  6. Supplementary Material for: Whole genome sequencing reveals genomic heterogeneity and antibiotic purification in Mycobacterium tuberculosis isolates

    KAUST Repository

    Black, PA

    2015-01-01

    Abstract Background Whole genome sequencing has revolutionised the interrogation of mycobacterial genomes. Recent studies have reported conflicting findings on the genomic stability of Mycobacterium tuberculosis during the evolution of drug resistance. In an age where whole genome sequencing is increasingly relied upon for defining the structure of bacterial genomes, it is important to investigate the reliability of next generation sequencing to identify clonal variants present in a minor percentage of the population. This study aimed to define a reliable cut-off for identification of low frequency sequence variants and to subsequently investigate genetic heterogeneity and the evolution of drug resistance in M. tuberculosis. Methods Genomic DNA was isolated from single colonies from 14 rifampicin mono-resistant M. tuberculosis isolates, as well as the primary cultures and follow up MDR cultures from two of these patients. The whole genomes of the M. tuberculosis isolates were sequenced using either the Illumina MiSeq or Illumina HiSeq platforms. Sequences were analysed with an in-house pipeline. Results Using next-generation sequencing in combination with Sanger sequencing and statistical analysis we defined a read frequency cut-off of 30 % to identify low frequency M. tuberculosis variants with high confidence. Using this cut-off we demonstrated a high rate of genetic diversity between single colonies isolated from one population, showing that by using the current sequencing technology, single colonies are not a true reflection of the genetic diversity within a whole population and vice versa. We further showed that numerous heterogeneous variants emerge and then disappear during the evolution of isoniazid resistance within individual patients. Our findings allowed us to formulate a model for the selective bottleneck which occurs during the course of infection, acting as a genomic purification event. Conclusions Our study demonstrated true levels of genetic

  7. Management of latent Mycobacterium tuberculosis infection: WHO guidelines for low tuberculosis burden countries.

    Science.gov (United States)

    Getahun, Haileyesus; Matteelli, Alberto; Abubakar, Ibrahim; Aziz, Mohamed Abdel; Baddeley, Annabel; Barreira, Draurio; Den Boon, Saskia; Borroto Gutierrez, Susana Marta; Bruchfeld, Judith; Burhan, Erlina; Cavalcante, Solange; Cedillos, Rolando; Chaisson, Richard; Chee, Cynthia Bin-Eng; Chesire, Lucy; Corbett, Elizabeth; Dara, Masoud; Denholm, Justin; de Vries, Gerard; Falzon, Dennis; Ford, Nathan; Gale-Rowe, Margaret; Gilpin, Chris; Girardi, Enrico; Go, Un-Yeong; Govindasamy, Darshini; D Grant, Alison; Grzemska, Malgorzata; Harris, Ross; Horsburgh, C Robert; Ismayilov, Asker; Jaramillo, Ernesto; Kik, Sandra; Kranzer, Katharina; Lienhardt, Christian; LoBue, Philip; Lönnroth, Knut; Marks, Guy; Menzies, Dick; Migliori, Giovanni Battista; Mosca, Davide; Mukadi, Ya Diul; Mwinga, Alwyn; Nelson, Lisa; Nishikiori, Nobuyuki; Oordt-Speets, Anouk; Rangaka, Molebogeng Xheedha; Reis, Andreas; Rotz, Lisa; Sandgren, Andreas; Sañé Schepisi, Monica; Schünemann, Holger J; Sharma, Surender Kumar; Sotgiu, Giovanni; Stagg, Helen R; Sterling, Timothy R; Tayeb, Tamara; Uplekar, Mukund; van der Werf, Marieke J; Vandevelde, Wim; van Kessel, Femke; van't Hoog, Anna; Varma, Jay K; Vezhnina, Natalia; Voniatis, Constantia; Vonk Noordegraaf-Schouten, Marije; Weil, Diana; Weyer, Karin; Wilkinson, Robert John; Yoshiyama, Takashi; Zellweger, Jean Pierre; Raviglione, Mario

    2015-12-01

    Latent tuberculosis infection (LTBI) is characterised by the presence of immune responses to previously acquired Mycobacterium tuberculosis infection without clinical evidence of active tuberculosis (TB). Here we report evidence-based guidelines from the World Health Organization for a public health approach to the management of LTBI in high risk individuals in countries with high or middle upper income and TB incidence of <100 per 100 000 per year. The guidelines strongly recommend systematic testing and treatment of LTBI in people living with HIV, adult and child contacts of pulmonary TB cases, patients initiating anti-tumour necrosis factor treatment, patients receiving dialysis, patients preparing for organ or haematological transplantation, and patients with silicosis. In prisoners, healthcare workers, immigrants from high TB burden countries, homeless persons and illicit drug users, systematic testing and treatment of LTBI is conditionally recommended, according to TB epidemiology and resource availability. Either commercial interferon-gamma release assays or Mantoux tuberculin skin testing could be used to test for LTBI. Chest radiography should be performed before LTBI treatment to rule out active TB disease. Recommended treatment regimens for LTBI include: 6 or 9 month isoniazid; 12 week rifapentine plus isoniazid; 3-4 month isoniazid plus rifampicin; or 3-4 month rifampicin alone. Copyright ©ERS 2015.

  8. Protective and therapeutic efficacy of Mycobacterium smegmatis expressing HBHA-hIL12 fusion protein against Mycobacterium tuberculosis in mice.

    Directory of Open Access Journals (Sweden)

    Shanmin Zhao

    Full Text Available Tuberculosis (TB remains a major worldwide health problem. The only vaccine against TB, Mycobacterium bovis Bacille Calmette-Guerin (BCG, has demonstrated relatively low efficacy and does not provide satisfactory protection against the disease. More efficient vaccines and improved therapies are urgently needed to decrease the worldwide spread and burden of TB, and use of a viable, metabolizing mycobacteria vaccine may be a promising strategy against the disease. Here, we constructed a recombinant Mycobacterium smegmatis (rMS strain expressing a fusion protein of heparin-binding hemagglutinin (HBHA and human interleukin 12 (hIL-12. Immune responses induced by the rMS in mice and protection against Mycobacterium tuberculosis (MTB were investigated. Administration of this novel rMS enhanced Th1-type cellular responses (IFN-γ and IL-2 in mice and reduced bacterial burden in lungs as well as that achieved by BCG vaccination. Meanwhile, the bacteria load in M. tuberculosis infected mice treated with the rMS vaccine also was significantly reduced. In conclusion, the rMS strain expressing the HBHA and human IL-12 fusion protein enhanced immunogencity by improving the Th1-type response against TB, and the protective effect was equivalent to that of the conventional BCG vaccine in mice. Furthermore, it could decrease bacterial load and alleviate histopathological damage in lungs of M. tuberculosis infected mice.

  9. Cellular immune responses to ESAT-6 discriminate between patients with pulmonary disease due to Mycobacterium avium complex and those with pulmonary disease due to Mycobacterium tuberculosis

    DEFF Research Database (Denmark)

    Lein, A D; von Reyn, C F; Ravn, P

    1999-01-01

    ESAT-6 (for 6-kDa early secreted antigenic target) is a secreted antigen found almost exclusively in organisms of the Mycobacterium tuberculosis complex. We compared in vitro gamma interferon (IFN-gamma) responses by peripheral blood mononuclear cells to this antigen in patients with pulmonary...... disease due to either Mycobacterium avium complex (MAC) or Mycobacterium tuberculosis with those in healthy, skin test-negative, control subjects. Significant IFN-gamma responses to ESAT-6 were detected in 16 (59%) of 27 M. tuberculosis pulmonary disease patients, 0 (0%) of 8 MAC disease patients, and 0...... (0%) of 8 controls. Significant IFN-gamma responses to M. tuberculosis purified protein derivative were detected in 23 (85%) of 27 M. tuberculosis disease patients, 2 (25%) of 8 MAC disease patients, and 5 (63%) of 8 healthy controls. M. avium sensitin was recognized in 24 (89%) of 27 M. tuberculosis...

  10. Whole genome sequencing of Mycobacterium tuberculosis SB24 isolated from Sabah, Malaysia

    Directory of Open Access Journals (Sweden)

    Noraini Philip

    2016-09-01

    Full Text Available Mycobacterium tuberculosis (M. tuberculosis is the causative agent of tuberculosis (TB that causes millions of death every year. We have sequenced the genome of M. tuberculosis isolated from cerebrospinal fluid (CSF of a patient diagnosed with tuberculous meningitis (TBM. The isolated strain was referred as M. tuberculosis SB24. Genomic DNA of the M. tuberculosis SB24 was extracted and subjected to whole genome sequencing using PacBio platform. The draft genome size of M. tuberculosis SB24 was determined to be 4,452,489 bp with a G + C content of 65.6%. The whole genome shotgun project has been deposited in NCBI SRA under the accession number SRP076503.

  11. Phylogenetic analysis of vitamin B12-related metabolism in Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Douglas B. Young

    2015-03-01

    Full Text Available Comparison of genome sequences from clinical isolates of Mycobacterium tuberculosis with phylogenetically-related pathogens Mycobacterium marinum, Mycobacterium kansasii and Mycobacterium leprae reveals diversity amongst genes associated with vitamin B12-related metabolism. Diversity is generated by gene deletion events, differential acquisition of genes by horizontal transfer, and single nucleotide polymorphisms with predicted impact on protein function and transcriptional regulation. Differences in the B12 synthesis pathway, methionine biosynthesis, fatty acid catabolism, and DNA repair and replication are consistent with adaptations to different environmental niches and pathogenic lifestyles. While there is no evidence of further gene acquisition during expansion of the M. tuberculosis complex, the emergence of other forms of genetic diversity provides insights into continuing host-pathogen co-evolution and has the potential to identify novel targets for disease intervention.

  12. Mycobacterium tuberculosis population structure and molecular epidemiological analysis in Sucre municipality, Miranda state, Venezuela.

    Science.gov (United States)

    Patiño, Margareth A; Abadía, Edgar; Solalba Gómez; Maes, Mailis; Muñoz, Mariana; Gómez, Daniela; Guzmán, Patricia; Méndez, María Victoria; Ramirez, Carmen; Mercedes, España; de Waard, Jacobus; Takiff, Howard

    2014-12-01

    Sucre municipality is a large, densely populated marginal area in the eastern part of Caracas, Venezuela that consistently has more cases of tuberculosis than other municipalities in the country. To identify the neighborhoods in the municipality with the highest prevalence of tuberculosis, and determine whether the Mycobacterium tuberculosis strain distribution in this municipality is different from that previously found in the western part of Caracas and the rest of Venezuela, we collected data on all tuberculosis cases in the municipality diagnosed in 2005-6. We performed two separate molecular epidemiological studies, spoligotyping 44 strains in a first study, and spoligotyping 131 strains, followed by MIRU-VNTR 15 on 21 clustered isolates in the second. With spoligotyping, the most common patterns were Shared International Type SIT17 (21%); SIT42 (15%); SIT93 (11%); SIT20 (7%); SIT53 (6%), a distribution similar to other parts of Venezuela, except that SIT42 and SIT20 were more common. MIRU-VNTR 15 showed that six of seven SIT17 strains examined belonged to a large cluster previously found circulating in Venezuela, but all of the SIT42 strains were related to a cluster centered in the neighborhoods of Unión and Maca, with a MIRU-VNTR pattern not previously seen in Venezuela. It appears that a large percentage of the tuberculosis in the Sucre municipality is caused by the active transmission of two strain families centered within distinct neighborhoods, one reflecting communication with the rest of the country, and the other suggesting the insular, isolated nature of some sectors.

  13. Kinetics and inhibition of nicotinamidase from Mycobacterium tuberculosis.

    Science.gov (United States)

    Seiner, Derrick R; Hegde, Subray S; Blanchard, John S

    2010-11-09

    Nicotinamidase/pyrazinamidase (PncA) is involved in the NAD+ salvage pathway of Mycobacterium tuberculosis and other bacteria. In addition to hydrolyzing nicotinamide into nicotinic acid, PncA also hydrolyzes the prodrug pyrazinamide to generate the active form of the drug, pyrazinoic acid, which is an essential component of the multidrug treatment of TB. A coupled enzymatic activity assay has been developed for PncA that allows for the spectroscopic observation of enzyme activity. The enzyme activity was essentially pH-independent under the conditions tested; however, the measurement of the pH dependence of iodoacetamide alkylation revealed a pK value of 6.6 for the active site cysteine. Solvent deuterium kinetic isotope effects revealed an inverse value for kcat of 0.64, reconfirming the involvement of a thiol group in the mechanism. A mechanism is proposed for PncA catalysis that is similar to the mechanisms proposed for members of the nitrilase superfamily, in which nucleophilic attack by the active site cysteine generates a tetrahedral intermediate that collapses with the loss of ammonia and subsequent hydrolysis of the thioester bond by water completes the cycle. An inhibitor screen identified the competitive inhibitor 3-pyridine carboxaldehyde with a Ki of 290 nM. Additionally, pyrazinecarbonitrile was found to be an irreversible inactivator of PncA, with a kinact/KI of 975 M(−1) s(−1).

  14. Transcriptional landscape of Mycobacterium tuberculosis infection in macrophages

    KAUST Repository

    Roy, Sugata

    2018-04-24

    Mycobacterium tuberculosis (Mtb) infection reveals complex and dynamic host-pathogen interactions, leading to host protection or pathogenesis. Using a unique transcriptome technology (CAGE), we investigated the promoter-based transcriptional landscape of IFNγ (M1) or IL-4/IL-13 (M2) stimulated macrophages during Mtb infection in a time-kinetic manner. Mtb infection widely and drastically altered macrophage-specific gene expression, which is far larger than that of M1 or M2 activations. Gene Ontology enrichment analysis for Mtb-induced differentially expressed genes revealed various terms, related to host-protection and inflammation, enriched in up-regulated genes. On the other hand, terms related to dis-regulation of cellular functions were enriched in down-regulated genes. Differential expression analysis revealed known as well as novel transcription factor genes in Mtb infection, many of them significantly down-regulated. IFNγ or IL-4/IL-13 pre-stimulation induce additional differentially expressed genes in Mtb-infected macrophages. Cluster analysis uncovered significant numbers, prolonging their expressional changes. Furthermore, Mtb infection augmented cytokine-mediated M1 and M2 pre-activations. In addition, we identified unique transcriptional features of Mtb-mediated differentially expressed lncRNAs. In summary we provide a comprehensive in depth gene expression/regulation profile in Mtb-infected macrophages, an important step forward for a better understanding of host-pathogen interaction dynamics in Mtb infection.

  15. A robust SNP barcode for typing Mycobacterium tuberculosis complex strains

    KAUST Repository

    Coll, Francesc

    2014-09-01

    Strain-specific genomic diversity in the Mycobacterium tuberculosis complex (MTBC) is an important factor in pathogenesis that may affect virulence, transmissibility, host response and emergence of drug resistance. Several systems have been proposed to classify MTBC strains into distinct lineages and families. Here, we investigate single-nucleotide polymorphisms (SNPs) as robust (stable) markers of genetic variation for phylogenetic analysis. We identify ∼92k SNP across a global collection of 1,601 genomes. The SNP-based phylogeny is consistent with the gold-standard regions of difference (RD) classification system. Of the ∼7k strain-specific SNPs identified, 62 markers are proposed to discriminate known circulating strains. This SNP-based barcode is the first to cover all main lineages, and classifies a greater number of sublineages than current alternatives. It may be used to classify clinical isolates to evaluate tools to control the disease, including therapeutics and vaccines whose effectiveness may vary by strain type. © 2014 Macmillan Publishers Limited.

  16. Structure of Ribosomal Silencing Factor Bound to Mycobacterium tuberculosis Ribosome.

    Science.gov (United States)

    Li, Xiaojun; Sun, Qingan; Jiang, Cai; Yang, Kailu; Hung, Li-Wei; Zhang, Junjie; Sacchettini, James C

    2015-10-06

    The ribosomal silencing factor RsfS slows cell growth by inhibiting protein synthesis during periods of diminished nutrient availability. The crystal structure of Mycobacterium tuberculosis (Mtb) RsfS, together with the cryo-electron microscopy (EM) structure of the large subunit 50S of Mtb ribosome, reveals how inhibition of protein synthesis by RsfS occurs. RsfS binds to the 50S at L14, which, when occupied, blocks the association of the small subunit 30S. Although Mtb RsfS is a dimer in solution, only a single subunit binds to 50S. The overlap between the dimer interface and the L14 binding interface confirms that the RsfS dimer must first dissociate to a monomer in order to bind to L14. RsfS interacts primarily through electrostatic and hydrogen bonding to L14. The EM structure shows extended rRNA density that it is not found in the Escherichia coli ribosome, the most striking of these being the extended RNA helix of H54a. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. [Identification of the Mycobacterium tuberculosis Beijing lineage in Ecuador].

    Science.gov (United States)

    Jiménez, Patricia; Calvopiña, Karina; Herrera, Diana; Rojas, Carlos; Pérez-Lago, Laura; Grijalva, Marcelo; Guna, Remedios; García-de Viedma, Darío

    2017-06-01

    Mycobacterium tuberculosis Beijing lineage isolates are considered to be especially virulent, transmissible and prone to acquire resistances. Beijing strains have been reported worldwide, but studies in Latin America are still scarce. The only multinational study performed in the region indicated a heterogeneous distribution for this lineage, which was absent in Chile, Colombia and Ecuador, although further studies found the lineage in Chile and Colombia. To search for the presence of the Beijing lineage in Ecuador, the only country in the region where it remains unreported. We obtained a convenience sample (2006-2012) from two hospitals covering different populations. The isolates were genotyped using 24-MIRU-VNTR. Lineages were assigned by comparing their patterns to those in the MIRU-VNTRplus platform. Isolates belonging to the Beijing lineage were confirmed by allele-specific PCR. We identified the first Beijing isolate in Ecuador in an unexpected epidemiological scenario: A patient was infected in the Andean region, in a population with low mobility and far from the borders of the neighboring countries where Beijing strains had been previously reported. This is the first report of the presence of the Beijing lineage in Ecuador in an unusual epidemiological context that deserves special attention.

  18. Specific T-cell epitopes for immunoassay-based diagnosis of Mycobacterium tuberculosis infection

    DEFF Research Database (Denmark)

    Brock, I; Weldingh, K; Leyten, EM

    2004-01-01

    Specific T-cell epitopes for immunoassay-based diagnosis of Mycobacterium tuberculosis infection.Brock I, Weldingh K, Leyten EM, Arend SM, Ravn P, Andersen P. Department of Infectious Disease Immunology, Statens Serum Institute, Artillerivej 5, DK-2300 Copenhagen S, Denmark. The currently used...... method for immunological detection of tuberculosis infection, the tuberculin skin test, has low specificity. Antigens specific for Mycobacterium tuberculosis to replace purified protein derivative are therefore urgently needed. We have performed a rigorous assessment of the diagnostic potential of four...... recently identified antigens (Rv2653, Rv2654, Rv3873, and Rv3878) from genomic regions that are lacking from the Mycobacterium bovis bacillus Calmette-Guerin (BCG) vaccine strains as well as from the most common nontuberculous mycobacteria. The fine specificity of potential epitopes in these molecules...

  19. Proteasomal control of cytokinin synthesis protects Mycobacterium tuberculosis against nitric oxide

    Science.gov (United States)

    Samanovic, Marie I.; Tu, Shengjiang; Novák, Ondřej; Iyer, Lakshminarayan M.; McAllister, Fiona E.; Aravind, L.; Gygi, Steven P.; Hubbard, Stevan R.; Strnad, Miroslav; Darwin, K. Heran

    2015-01-01

    Summary One of several roles of the Mycobacterium tuberculosis proteasome is to defend against host-produced nitric oxide (NO), a free radical that can damage numerous biological macromolecules. Mutations that inactivate proteasomal degradation in Mycobacterium tuberculosis result in bacteria that are hypersensitive to NO and attenuated for growth in vivo, but it was not known why. To elucidate the link between proteasome function, NO-resistance, and pathogenesis, we screened for suppressors of NO hypersensitivity in a mycobacterial proteasome ATPase mutant and identified mutations in Rv1205. We determined that Rv1205 encodes a pupylated proteasome substrate. Rv1205 is a homologue of the plant enzyme LONELY GUY, which catalyzes the production of hormones called cytokinins. Remarkably, we report for the first time that an obligate human pathogen secretes several cytokinins. Finally, we determined that the Rv1205-dependent accumulation of cytokinin breakdown products is likely responsible for the sensitization of Mycobacterium tuberculosis proteasome-associated mutants to NO. PMID:25728768

  20. Activity of Medicinal Plant Extracts on Multiplication of Mycobacterium tuberculosis under Reduced Oxygen Conditions Using Intracellular and Axenic Assays

    Directory of Open Access Journals (Sweden)

    Purva D. Bhatter

    2016-01-01

    Full Text Available Aim. Test the activity of selected medicinal plant extracts on multiplication of Mycobacterium tuberculosis under reduced oxygen concentration which represents nonreplicating conditions. Material and Methods. Acetone, ethanol and aqueous extracts of the plants Acorus calamus L. (rhizome, Ocimum sanctum L. (leaf, Piper nigrum L. (seed, and Pueraria tuberosa DC. (tuber were tested on Mycobacterium tuberculosis H37Rv intracellularly using an epithelial cell (A549 infection model. The extracts found to be active intracellularly were further studied axenically under reducing oxygen concentrations. Results and Conclusions. Intracellular multiplication was inhibited ≥60% by five of the twelve extracts. Amongst these 5 extracts, in axenic culture, P. nigrum (acetone was active under aerobic, microaerophilic, and anaerobic conditions indicating presence of multiple components acting at different levels and P. tuberosa (aqueous showed bactericidal activity under microaerophilic and anaerobic conditions implying the influence of anaerobiosis on its efficacy. P. nigrum (aqueous and A. calamus (aqueous and ethanol extracts were not active under axenic conditions but only inhibited intracellular growth of Mycobacterium tuberculosis, suggesting activation of host defense mechanisms to mediate bacterial killing rather than direct bactericidal activity.

  1. Anti-mycobacterium tuberculosis activity of polyherbal medicines used for the treatment of tuberculosis in Eastern Cape, South Africa.

    Science.gov (United States)

    Famewo, Elizabeth B; Clarke, Anna M; Wiid, Ian; Ngwane, Andile; van Helden, Paul; Afolayan, Anthony J

    2017-09-01

    The emergence of drug-resistant strains of Mycobacterium tuberculosis has become a global public health problem. Polyherbal medicines offer great hope for developing alternative drugs for the treatment of tuberculosis. To evaluate the anti-tubercular activity of polyherbal medicines used for the treatment of tuberculosis. The remedies were screened against Mycobacterium tuberculosis H37Rv using Middlebrook 7H9 media and MGIT BACTEC 960 system. They were liquid preparations from King Williams Town site A (KWTa), King Williams Town site B (KWTb), King Williams Town site C (KWTc), Hogsback first site (HBfs), Hogsback second site (HBss), Hogsback third site (HBts), East London (EL), Alice (AL) and Fort Beaufort (FB). The susceptibility testing revealed that all the remedies contain anti-tubercular activity with KWTa, KWTb, KWTc, HBfs, HBts, AL and FB exhibiting more activity at a concentration below 25 µl/ml. Furthermore, MIC values exhibited inhibitory activity with the most active remedies from KWTa, HBfs and HBts at 1.562 µg/ml. However, isoniazid showed more inhibitory activity against M. tuberculosis at 0.05 µg/ml when compare to the polyherbal remedies. This study has indicated that these remedies could be potential sources of new anti-mycobacterial agents against M. tuberculosis . However, the activity of these preparations and their active principles still require in vivo study in order to assess their future as new anti-tuberculosis agents.

  2. Molecular epidemiology of Mycobacterium tuberculosis in aboriginal peoples of Taiwan, 2006-2011.

    Science.gov (United States)

    Chen, Yih-Yuan; Chang, Jia-Ru; Huang, Wei-Feng; Kuo, Shu-Chen; Yeh, Jun-Jun; Lee, Jen-Jyh; Jang, Chang-Sheng; Sun, Jun-Ren; Chiueh, Tzong-Shi; Su, Ih-Jen; Dou, Horng-Yunn

    2014-04-01

    Previous research revealed a 6-fold higher incidence of tuberculosis (TB) amongst aborigines compared to Han Chinese in Taiwan. To investigate the reasons for this disparity, we genotyped Mycobacterium tuberculosis (MTB) strains obtained from members of different aboriginal tribes in different geographical regions of Taiwan by using molecular methods. In total, 177 isolates of MTB collected from patients at four hospitals in Taiwan from January 2006 to December 2011 were analysed by spoligotyping, mycobacterial interspersed repetitive unit-variable number tandem-repeat (MIRU-VNTR) typing. The most prevalent strains in the eastern and central regions of Taiwan were Beijing (45.7% in eastern) and Haarlem (39.1% in eastern, 37.1% in central) lineages, whereas in southern regions the most prevalent strains were EAI (47.7%) and Haarlem (20.5%) lineages. The high prevalence of EAI in southern Taiwan aborigines may be closely associated with Austronesian culture. This study provides a first overview of the M. tuberculosis strains circulating in aboriginal populations in Taiwan. The high prevalences of certain MTB lineages within aboriginal sub-populations suggest that transmission of MTB may have been restricted to close contacts. Copyright © 2013 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

  3. Crystal structure of the apurinic/apyrimidinic endonuclease IV from Mycobacterium tuberculosis.

    Science.gov (United States)

    Zhang, Wei; Xu, Yueyang; Yan, Mengrong; Li, Shanshan; Wang, Huiying; Yang, Haitao; Zhou, Weihong; Rao, Zihe

    2018-03-25

    Endonuclease IV is a typical endonuclease of the apurinic-apyrimidinic (AP) or abasic endonuclease superfamily. It repairs damaged DNA through base excision repair by cleaving the DNA backbone immediately 5' of an AP site. In Mycobacterium tuberculosis, endonuclease IV is the major AP endonuclease. This enzyme is absent from mammalian cells, making it an attractive target for anti-tuberculosis drug development. In this study, the structure of the recombinant endonuclease IV from M. tuberculosis (MtbEndo IV) was determined at a high resolution of 1.18 Å. MtbEndo IV was found to have a classical α8β8-fold TIM barrel with loops on its surface connecting the α-helices and β-strands that constitute a groove for DNA binding. Three zinc ions were identified at the active site. A comparison between the structures of MtbEndo IV and Escherichia coli End IV suggested that Gln32 of MtbEndo IV may plays a role in regulating substrate binding. Copyright © 2018. Published by Elsevier Inc.

  4. Elucidation and chemical modulation of sulfolipid-1 biosynthesis in Mycobacterium tuberculosis.

    Science.gov (United States)

    Seeliger, Jessica C; Holsclaw, Cynthia M; Schelle, Michael W; Botyanszki, Zsofia; Gilmore, Sarah A; Tully, Sarah E; Niederweis, Michael; Cravatt, Benjamin F; Leary, Julie A; Bertozzi, Carolyn R

    2012-03-09

    Mycobacterium tuberculosis possesses unique cell-surface lipids that have been implicated in virulence. One of the most abundant is sulfolipid-1 (SL-1), a tetraacyl-sulfotrehalose glycolipid. Although the early steps in SL-1 biosynthesis are known, the machinery underlying the final acylation reactions is not understood. We provide genetic and biochemical evidence for the activities of two proteins, Chp1 and Sap (corresponding to gene loci rv3822 and rv3821), that complete this pathway. The membrane-associated acyltransferase Chp1 accepts a synthetic diacyl sulfolipid and transfers an acyl group regioselectively from one donor substrate molecule to a second acceptor molecule in two successive reactions to yield a tetraacylated product. Chp1 is fully active in vitro, but in M. tuberculosis, its function is potentiated by the previously identified sulfolipid transporter MmpL8. We also show that the integral membrane protein Sap and MmpL8 are both essential for sulfolipid transport. Finally, the lipase inhibitor tetrahydrolipstatin disrupts Chp1 activity in M. tuberculosis, suggesting an avenue for perturbing SL-1 biosynthesis in vivo. These data complete the SL-1 biosynthetic pathway and corroborate a model in which lipid biosynthesis and transmembrane transport are coupled at the membrane-cytosol interface through the activity of multiple proteins, possibly as a macromolecular complex.

  5. Mutations in ppe38 block PE_PGRS secretion and increase virulence of Mycobacterium tuberculosis

    KAUST Repository

    Ates, Louis S.

    2018-01-12

    Mycobacterium tuberculosis requires a large number of secreted and exported proteins for its virulence, immune modulation and nutrient uptake. Most of these proteins are transported by the different type VII secretion systems1,2. The most recently evolved type VII secretion system, ESX-5, secretes dozens of substrates belonging to the PE and PPE families, which are named for conserved proline and glutamic acid residues close to the amino terminus3,4. However, the role of these proteins remains largely elusive1. Here, we show that mutations of ppe38 completely block the secretion of two large subsets of ESX-5 substrates, that is, PPE-MPTR and PE_PGRS, together comprising >80 proteins. Importantly, hypervirulent clinical M. tuberculosis strains of the Beijing lineage have such a mutation and a concomitant loss of secretion5. Restoration of PPE38-dependent secretion partially reverted the hypervirulence phenotype of a Beijing strain, and deletion of ppe38 in moderately virulent M. tuberculosis increased virulence. This indicates that these ESX-5 substrates have an important role in virulence attenuation. Phylogenetic analysis revealed that deletion of ppe38 occurred at the branching point of the ‘modern’ Beijing sublineage and is shared by Beijing outbreak strains worldwide, suggesting that this deletion may have contributed to their success and global distribution6,7.

  6. Mutations in ppe38 block PE_PGRS secretion and increase virulence of Mycobacterium tuberculosis.

    Science.gov (United States)

    Ates, Louis S; Dippenaar, Anzaan; Ummels, Roy; Piersma, Sander R; van der Woude, Aniek D; van der Kuij, Kim; Le Chevalier, Fabien; Mata-Espinosa, Dulce; Barrios-Payán, Jorge; Marquina-Castillo, Brenda; Guapillo, Carolina; Jiménez, Connie R; Pain, Arnab; Houben, Edith N G; Warren, Robin M; Brosch, Roland; Hernández-Pando, Rogelio; Bitter, Wilbert

    2018-02-01

    Mycobacterium tuberculosis requires a large number of secreted and exported proteins for its virulence, immune modulation and nutrient uptake. Most of these proteins are transported by the different type VII secretion systems 1,2 . The most recently evolved type VII secretion system, ESX-5, secretes dozens of substrates belonging to the PE and PPE families, which are named for conserved proline and glutamic acid residues close to the amino terminus 3,4 . However, the role of these proteins remains largely elusive 1 . Here, we show that mutations of ppe38 completely block the secretion of two large subsets of ESX-5 substrates, that is, PPE-MPTR and PE_PGRS, together comprising >80 proteins. Importantly, hypervirulent clinical M. tuberculosis strains of the Beijing lineage have such a mutation and a concomitant loss of secretion 5 . Restoration of PPE38-dependent secretion partially reverted the hypervirulence phenotype of a Beijing strain, and deletion of ppe38 in moderately virulent M. tuberculosis increased virulence. This indicates that these ESX-5 substrates have an important role in virulence attenuation. Phylogenetic analysis revealed that deletion of ppe38 occurred at the branching point of the 'modern' Beijing sublineage and is shared by Beijing outbreak strains worldwide, suggesting that this deletion may have contributed to their success and global distribution 6,7 .

  7. Mutational analysis of the respiratory nitrate transporter NarK2 of Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Michelle M Giffin

    Full Text Available Mycobacterium tuberculosis induces nitrate reductase activity in response to decreasing oxygen levels. This is due to regulation of both the transcription and the activity of the nitrate transporter NarK2. A model of NarK2 structure is proposed containing 12 membrane spanning regions consistent with other members of the major facilitator superfamily. The role of the proton gradient was determined by exposing M. tuberculosis to uncouplers. Nitrite production decreased indicating that the importation of nitrate involved an H(+/nitrate symporter. The addition of nitrite before nitrate had no effect, suggesting no role for a nitrate/nitrite antiporter. In addition the NarK2 knockout mutant showed no defect in nitrite export. NarK2 is proposed to be a Type I H(+/nitrate symporter. Site directed mutagenesis was performed changing 23 amino acids of NarK2. This allowed the identification of important regions and amino acids of this transporter. Five of these mutants were inactive for nitrate transport, seven produced reduced activity and eleven mutants retained wild type activity. NarK2 is inactivated in the presence of oxygen by an unknown mechanism. However none of the mutants, including those with mutated cysteines, were altered in their response to oxygen levels. The assimilatory nitrate transporter NasA of Bacillus subtilis was expressed in the M. tuberculosis NarK2 mutant. It remained active during aerobic incubation showing that the point of oxygen control is NarK2.

  8. NOD2 enhances the innate response of alveolar macrophages to Mycobacterium tuberculosis in humans.

    Science.gov (United States)

    Juárez, Esmeralda; Carranza, Claudia; Hernández-Sánchez, Fernando; León-Contreras, Juan C; Hernández-Pando, Rogelio; Escobedo, Dante; Torres, Martha; Sada, Eduardo

    2012-04-01

    A role for the nucleotide-binding oligomerization domain 2 (NOD2) receptor in pulmonary innate immune responses has recently been explored. In the present study, we investigated the role that NOD2 plays in human alveolar macrophage innate responses and determined its involvement in the response to infection with virulent Mycobacterium tuberculosis. Our results showed that NOD2 was expressed in human alveolar macrophages, and significant amounts of IL-1β, IL-6, and TNF-α were produced upon ligand recognition with muramyldipeptide (MDP). NOD2 ligation induced the transcription and protein expression of the antimicrobial peptide LL37 and the autophagy enzyme IRGM in alveolar macrophages, demonstrating a novel function for this receptor in these cells. MDP treatment of alveolar macrophages improved the intracellular growth control of virulent M. tuberculosis; this was associated with a significant release of TNF-α and IL-6 and overexpression of bactericidal LL37. In addition, the autophagy proteins IRGM, LC3 and ATG16L1 were recruited to the bacteria-containing autophagosome after treatment with MDP. In conclusion, our results suggest that NOD2 can modulate the innate immune response of alveolar macrophages and play a role in the initial control of respiratory M. tuberculosis infections. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Killing of intracellular Mycobacterium tuberculosis by receptor-mediated drug delivery

    International Nuclear Information System (INIS)

    Majumdar, S.; Basu, S.K.

    1991-01-01

    p-Aminosalicylic acid (PAS) conjugated to maleylated bovine serum albumin (MBSA) was taken up efficiently through high-affinity MBSA-binding sites on macrophages. Binding of the radiolabeled conjugate to cultured mouse peritoneal macrophages at 4 degrees C was competed for by MBSA but not by PAS. At 37 degrees C, the radiolabeled conjugate was rapidly degraded by the macrophages, leading to release of acid-soluble degradation products in the medium. The drug conjugate was nearly 100 times as effective as free PAS in killing the intracellular mycobacteria in mouse peritoneal macrophages infected in culture with Mycobacterium tuberculosis. The killing of intracellular mycobacteria mediated by the drug conjugate was effectively prevented by simultaneous addition of excess MBSA (100 micrograms/ml) or chloroquine (3 microM) to the medium, whereas these agents did not affect the microbicidal action of free PAS. These results suggest that (i) uptake of the PAS-MBSA conjugate was mediated by cell surface receptors on macrophages which recognize MBSA and (ii) lysosomal hydrolysis of the internalized conjugate resulted in intracellular release of a pharmacologically active form of the drug, which led to selective killing of the M. tuberculosis harbored by mouse macrophages infected in culture. This receptor-mediated modality of delivering drugs to macrophages could contribute to greater therapeutic efficacy and minimization of toxic side effects in the management of tuberculosis and other intracellular mycobacterial infections

  10. Identification of small molecular ligands as potent inhibitors of fatty acid metabolism in Mycobacterium tuberculosis

    Science.gov (United States)

    Malikanti, Ramesh; Vadija, Rajender; Veeravarapu, Hymavathi; Mustyala, Kiran Kumar; Malkhed, Vasavi; Vuruputuri, Uma

    2017-12-01

    Tuberculosis (Tb) is one of the major health challenges for the global scientific community. The 3-hydroxy butyryl-CoA dehydrogenase (Fad B2) protein belongs to 3-hydroxyl acetyl-CoA dehydrogenase family, which plays a key role in the fatty acid metabolism and β-oxidation in the cell membrane of Mycobacterium tuberculosis (Mtb). In the present study the Fad B2 protein is targeted for the identification of potential drug candidates for tuberculosis. The 3D model of the target protein Fad B2, was generated using homology modeling approach and was validated. The plausible binding site of the Fad B2 protein was identified from computational binding pocket prediction tools, which ranges from ASN120 to VAL150 amino acid residues. Virtual screening was carried out with the databases, Ligand box UOS and hit definder, at the binding site region. 133 docked complex structures were generated as an output. The identified ligands show good glide scores and glide energies. All the ligand molecules contain benzyl amine pharmacophore in common, which show specific and selective binding interactions with the SER122 and ASN146 residues of the Fad B2 protein. The ADME properties of all the ligand molecules were observed to be within the acceptable range. It is suggested from the result of the present study that the docked molecular structures with a benzyl amine pharmacophore act as potential ligands for Fad B2 protein binding and as leads in Tb drug discovery.

  11. Mutations in ppe38 block PE_PGRS secretion and increase virulence of Mycobacterium tuberculosis

    KAUST Repository

    Ates, Louis S.; Dippenaar, Anzaan; Ummels, Roy; Piersma, Sander R.; van der Woude, Aniek D.; van der Kuij, Kim; Le Chevalier, Fabien; Mata-Espinosa, Dulce; Barrios-Payá n, Jorge; Marquina-Castillo, Brenda; Guapillo, Carolina; Jimé nez, Connie R.; Pain, Arnab; Houben, Edith N. G.; Warren, Robin M.; Brosch, Roland; Herná ndez-Pando, Rogelio; Bitter, Wilbert

    2018-01-01

    Mycobacterium tuberculosis requires a large number of secreted and exported proteins for its virulence, immune modulation and nutrient uptake. Most of these proteins are transported by the different type VII secretion systems1,2. The most recently evolved type VII secretion system, ESX-5, secretes dozens of substrates belonging to the PE and PPE families, which are named for conserved proline and glutamic acid residues close to the amino terminus3,4. However, the role of these proteins remains largely elusive1. Here, we show that mutations of ppe38 completely block the secretion of two large subsets of ESX-5 substrates, that is, PPE-MPTR and PE_PGRS, together comprising >80 proteins. Importantly, hypervirulent clinical M. tuberculosis strains of the Beijing lineage have such a mutation and a concomitant loss of secretion5. Restoration of PPE38-dependent secretion partially reverted the hypervirulence phenotype of a Beijing strain, and deletion of ppe38 in moderately virulent M. tuberculosis increased virulence. This indicates that these ESX-5 substrates have an important role in virulence attenuation. Phylogenetic analysis revealed that deletion of ppe38 occurred at the branching point of the ‘modern’ Beijing sublineage and is shared by Beijing outbreak strains worldwide, suggesting that this deletion may have contributed to their success and global distribution6,7.

  12. Mycobacterium tuberculosis infection in cattle from the Eastern Cape Province of South Africa.

    Science.gov (United States)

    Hlokwe, Tiny Motlatso; Said, Halima; Gcebe, Nomakorinte

    2017-10-10

    Mycobacterium tuberculosis is the main causative agent of tuberculosis (TB) in human and Mycobacterium bovis commonly causes tuberculosis in animals. Transmission of tuberculosis caused by both pathogens can occur from human to animals and vice versa. In the current study, M. tuberculosis, as confirmed by polymerase chain reaction (PCR) using primers targeting 3 regions of difference (RD4, RD9 and RD12) on the genomes, was isolated from cattle originating from two epidemiologically unrelated farms in the Eastern Cape (E.C) Province of South Africa. Although the isolates were genotyped with variable number of tandem repeat (VNTR) typing, no detailed epidemiological investigation was carried out on the respective farms to unequivocally confirm or link humans as sources of TB transmission to cattle, a move that would have embraced the 'One Health' concept. In addition, strain comparison with human M. tuberculosis in the database from the E.C Province and other provinces in the country did not reveal any match. This is the first report of cases of M. tuberculosis infection in cattle in South Africa. The VNTR profiles of the M. tuberculosis strains identified in the current study will form the basis for creating M. tuberculosis VNTR database for animals including cattle for future epidemiological studies. Our findings however, call for urgent reinforcement of collaborative efforts between the veterinary and the public health services of the country.

  13. Evolution and diversity of clonal bacteria: the paradigm of Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Tiago Dos Vultos

    Full Text Available BACKGROUND: Mycobacterium tuberculosis complex species display relatively static genomes and 99.9% nucleotide sequence identity. Studying the evolutionary history of such monomorphic bacteria is a difficult and challenging task. PRINCIPAL FINDINGS: We found that single-nucleotide polymorphism (SNP analysis of DNA repair, recombination and replication (3R genes in a comprehensive selection of M. tuberculosis complex strains from across the world, yielded surprisingly high levels of polymorphisms as compared to house-keeping genes, making it possible to distinguish between 80% of clinical isolates analyzed in this study. Bioinformatics analysis suggests that a large number of these polymorphisms are potentially deleterious. Site frequency spectrum comparison of synonymous and non-synonymous variants and Ka/Ks ratio analysis suggest a general negative/purifying selection acting on these sets of genes that may lead to suboptimal 3R system activity. In turn, the relaxed fidelity of 3R genes may allow the occurrence of adaptive variants, some of which will survive. Furthermore, 3R-based phylogenetic trees are a new tool for distinguishing between M. tuberculosis complex strains. CONCLUSIONS/SIGNIFICANCE: This situation, and the consequent lack of fidelity in genome maintenance, may serve as a starting point for the evolution of antibiotic resistance, fitness for survival and pathogenicity, possibly conferring a selective advantage in certain stressful situations. These findings suggest that 3R genes may play an important role in the evolution of highly clonal bacteria, such as M. tuberculosis. They also facilitate further epidemiological studies of these bacteria, through the development of high-resolution tools. With many more microbial genomes being sequenced, our results open the door to 3R gene-based studies of adaptation and evolution of other, highly clonal bacteria.

  14. Role of Granulocyte-Macrophage Colony-Stimulating Factor Production by T Cells during Mycobacterium tuberculosis Infection.

    Science.gov (United States)

    Rothchild, Alissa C; Stowell, Britni; Goyal, Girija; Nunes-Alves, Cláudio; Yang, Qianting; Papavinasasundaram, Kadamba; Sassetti, Christopher M; Dranoff, Glenn; Chen, Xinchun; Lee, Jinhee; Behar, Samuel M

    2017-10-24

    Mice deficient for granulocyte-macrophage colony-stimulating factor (GM-CSF -/- ) are highly susceptible to infection with Mycobacterium tuberculosis , and clinical data have shown that anti-GM-CSF neutralizing antibodies can lead to increased susceptibility to tuberculosis in otherwise healthy people. GM-CSF activates human and murine macrophages to inhibit intracellular M. tuberculosis growth. We have previously shown that GM-CSF produced by iNKT cells inhibits growth of M. tuberculosis However, the more general role of T cell-derived GM-CSF during infection has not been defined and how GM-CSF activates macrophages to inhibit bacterial growth is unknown. Here we demonstrate that, in addition to nonconventional T cells, conventional T cells also produce GM-CSF during M. tuberculosis infection. Early during infection, nonconventional iNKT cells and γδ T cells are the main source of GM-CSF, a role subsequently assumed by conventional CD4 + T cells as the infection progresses. M. tuberculosis -specific T cells producing GM-CSF are also detected in the peripheral blood of infected people. Under conditions where nonhematopoietic production of GM-CSF is deficient, T cell production of GM-CSF is protective and required for control of M. tuberculosis infection. However, GM-CSF is not required for T cell-mediated protection in settings where GM-CSF is produced by other cell types. Finally, using an in vitro macrophage infection model, we demonstrate that GM-CSF inhibition of M. tuberculosis growth requires the expression of peroxisome proliferator-activated receptor gamma (PPARγ). Thus, we identified GM-CSF production as a novel T cell effector function. These findings suggest that a strategy augmenting T cell production of GM-CSF could enhance host resistance against M. tuberculosis IMPORTANCE Mycobacterium tuberculosis is the bacterium that causes tuberculosis, the leading cause of death by any infection worldwide. T cells are critical components of the immune

  15. Diagnosis of intestinal tuberculosis using a monoclonal antibody to Mycobacterium tuberculosis

    Institute of Scientific and Technical Information of China (English)

    Yasushi Ihama; Akira Hokama; Kenji Hibiya; Kazuto Kishimoto; Manabu Nakamoto; Tetsuo Hirata; Nagisa Kinjo

    2012-01-01

    AIM:To investigate the utility of immunohistochemical (IHC) staining with an antibody to Mycobacterium tuberculosis (M.tuberculosis) for the diagnosis of intestinal tuberculosis (TB).METHODS:We retrospectively identified 10 patients (4 males and 6 females; mean age =65.1 ± 13.6 years)with intestinal TB.Clinical characteristics,including age,gender,underlying disease,and symptoms were obtained.Chest radiograph and laboratory tests,including sputum Ziehl-Neelsen (ZN) staining,M.tuberculosis culture,and sputum polymerase chain reaction (PCR)for tubercle bacilli DNA,as well as Tuberculin skin test (TST) and QuantiFERON-TB gold test (QFT),were examined.Colonoscopic records recorded on the basis of Sato's classification were also reviewed,in addition to data from intestinal biopsies examined for histopathological findings,including hematoxylin and eosin staining,and ZN staining,as well as M.tuberculosis culture,and PCR for tubercle bacilli DNA.For the present study,archived formalin-fixed paraffin-embedded (FFPE) intestinal tissue samples were immunohistochemically stained using a commercially available species-specific monoclonal antibody to the 38-kDa antigen of the M.tuberculosis complex.These sections were also stained with the pan-macrophage marker CD68 antibody.RESULTS:From the clinical data,we found that no patients were immunocompromised,and that the main symptoms were diarrhea and weight loss.Three patients displayed active pulmonary TB,six patients (60%) had a positive TST,and 4 patients (40%) had a positive QFT.Colonoscopic findings revealed that all patients had type 1 findings (linear ulcers in a circumferential arrangement or linear ulcers arranged circumferentially with mucosa showing multiple nodules),all of which were located in the right hemicolon and/or terminal ileum.Seven patients (70%) had concomitant healed lesions in the ileocecal area.No acid-fast bacilli were detected with ZN staining of the intestinal tissue samples,and both M.tuberculosis

  16. Bioaktivitas Ekstrak Metanol Daun Pegagan (Centella Asiatica L. Terhadap Pertumbuhan Bakteri Mycobacterium Tuberculosis

    Directory of Open Access Journals (Sweden)

    Yusran Yusran

    2016-01-01

    Full Text Available Plants gotu kola (Centella Asiatica L .Urban is a wild plant that efficacious as remedies traditional cure disease tuberculosis (TB.TB is disease contagious infection caused by bacteria mycobacterium tuberculosis. Research aims to understand the ability extract methanol leaves gotu kola red and leaves gotu kola green and determines the concentration optimal extract methanol leaves gotu kola red and leaves gotu kola green and to know the comparison between extract methanol leaves gotu kola red with an extract methanol leaves gotu kola green in inhibits the activity of mycobacterium tuberculosis.Extraction done with the methods maceration use methanol and continued with evaporation until obtained extract viscous .Testing antibacterial activity done in a microscopic observation drug susceptibility ( mods use plate petri dish 24 hole with the variation of concentration ie 20%,40%, 60%, 80% and 100%.The results of testing show that extracts methanol leaves gotu kola red and leaves gotu kola green positive capable of inhibiting the growth of bacteria mycobacterium tuberculosis with inhibition optimal in concentration 80 % and 100 % characterized by the absence of growth bacteria colonies which are (- or 0 %.Extract methanol leaves gotu kola green capable of inhibiting the growth of bacteria mycobacterium tuberculosis better than extract methanol leaves gotu kola red seen in concentration 40% and 60%.

  17. Specific T-cell epitopes for immunoassay-based diagnosis of Mycobacterium tuberculosis infection

    DEFF Research Database (Denmark)

    Brock, I; Weldingh, K; Leyten, EM

    2004-01-01

    Specific T-cell epitopes for immunoassay-based diagnosis of Mycobacterium tuberculosis infection.Brock I, Weldingh K, Leyten EM, Arend SM, Ravn P, Andersen P. Department of Infectious Disease Immunology, Statens Serum Institute, Artillerivej 5, DK-2300 Copenhagen S, Denmark. The currently used...... method for immunological detection of tuberculosis infection, the tuberculin skin test, has low specificity. Antigens specific for Mycobacterium tuberculosis to replace purified protein derivative are therefore urgently needed. We have performed a rigorous assessment of the diagnostic potential of four...... selected and combined the specific peptide stretches from the four proteins not recognized by M. bovis BCG-vaccinated individuals. These peptide stretches were tested with peripheral blood mononuclear cells obtained from patients with microscopy- or culture-confirmed tuberculosis and from healthy M. bovis...

  18. Insertion sequence typing of Mycobacterium tuberculosis: characterization of a widespread subtype with a single copy of IS6110.

    Science.gov (United States)

    Fomukong, N G; Tang, T H; al-Maamary, S; Ibrahim, W A; Ramayah, S; Yates, M; Zainuddin, Z F; Dale, J W

    1994-12-01

    DNA fingerprinting with the insertion sequence IS6110 (also known as IS986) has become established as a major tool for investigating the spread of tuberculosis. Most strains of Mycobacterium tuberculosis have multiple copies of IS6110, but a small minority carry a single copy only. We have examined selected strains from Malaysia, Tanzania and Oman, in comparison with M. bovis isolates and BCG strains carrying one or two copies of IS6110. The insertion sequence appears to be present in the same position in all these strains, which suggests that in these organisms the element is defective in transposition and that the loss of transposability may have occurred at an early stage in the evolution of the M. tuberculosis complex.

  19. Infections with Mycobacterium tuberculosis and Mycobacterium avium among HIV-infected patients after the introduction of highly active antiretroviral therapy. EuroSIDA Study Group JD

    DEFF Research Database (Denmark)

    Kirk, O; Gatell, J M; Mocroft, A

    2000-01-01

    the introduction of HAART, using data from the EuroSIDA study, a European, multicenter observational cohort of more than 7,000 patients. Overall incidences of Mycobacterium tuberculosis (TB) and Mycobacterium avium complex (MAC) were 0.8 and 1.4 cases/100 person-years of follow-up (PYF), decreasing from 1.8 (TB...

  20. Detection of Mycobacterium bovis and Mycobacterium tuberculosis from Cattle: Possible Public Health Relevance

    DEFF Research Database (Denmark)

    Thakur, Aneesh; Sharma, Mandeep; Katoch, Vipin C.

    2012-01-01

    Mycobacterium bovis and Mycobacterium tuberculosis infect both animals and humans. The disease epidemiology by these agents differs in developed and developing countries due to the differences in the implementation of the prevention and control strategies. The present study describes the detectio...

  1. Usefulness of the secondary probe pTBN12 in DNA fingerprinting of Mycobacterium tuberculosis.

    OpenAIRE

    Chaves, F; Yang, Z; el Hajj, H; Alonso, M; Burman, W J; Eisenach, K D; Dronda, F; Bates, J H; Cave, M D

    1996-01-01

    A comparison was made between DNA fingerprints of Mycobacterium tuberculosis produced with the insertion sequence IS6110 and those produced with the polymorphic GC-rich repetitive sequence contained in the plasmid pTBN12. A total of 302 M. tuberculosis isolates from the prison system in Madrid, Spain, and the Denver Public Health Department (Denver, Colo.) were analyzed with the two probes. Both probes identified the same isolates in the same clusters when the fingerprints had six or more cop...

  2. Optimal Combination of VNTR Typing for Discrimination of Isolated Mycobacterium tuberculosis in Korea

    OpenAIRE

    Lee, Jihye; Kang, Heeyoon; Kim, Sarang; Yoo, Heekyung; Kim, Hee Jin; Park, Young Kil

    2014-01-01

    Background Variable-number tandem repeat (VNTR) typing is a promising method to discriminate the Mycobacterium tuberculosis isolates in molecular epidemiology. The purpose of this study is to determine the optimal VNTR combinations for discriminating isolated M. tuberculosis strains in Korea. Methods A total of 317 clinical isolates collected throughout Korea were genotyped by using the IS6110 restriction fragment length polymorphism (RFLP), and then analysed for the number of VNTR copies fro...

  3. Evolutionary history and global spread of the Mycobacterium tuberculosis Beijing lineage.

    OpenAIRE

    Merker Matthias; Blin Camille; Mona Stefano; Duforet-Frebourg Nicolas; Lecher Sophie; Willery Eve; Blum Michael G B; Rüsch-Gerdes Sabine; Mokrousov Igor; Aleksic Eman; Allix-Béguec Caroline; Antierens Annick; Augustynowicz-Kopec Ewa; Ballif Marie; Barletta Francesca

    2015-01-01

    International audience; Mycobacterium tuberculosis strains of the Beijing lineage are globally distributed and are associated with the massive spread of multidrug-resistant (MDR) tuberculosis in Eurasia. Here we reconstructed the biogeographical structure and evolutionary history of this lineage by genetic analysis of 4,987 isolates from 99 countries and whole-genome sequencing of 110 representative isolates. We show that this lineage initially originated in the Far East, from where it radiat...

  4. Resistance to first-line anti-TB drugs is associated with reduced nitric oxide susceptibility in Mycobacterium tuberculosis

    DEFF Research Database (Denmark)

    Idh, Jonna; Mekonnen, Mekidim; Abate, Ebba

    2012-01-01

    The relative contribution of nitric oxide (NO) to the killing of Mycobacterium tuberculosis in human tuberculosis (TB) is controversial, although this has been firmly established in rodents. Studies have demonstrated that clinical strains of M. tuberculosis differ in susceptibility to NO, but how...

  5. Efficacy of moxifloxacin & econazole against multidrug resistant (MDR Mycobacterium tuberculosis in murine model

    Directory of Open Access Journals (Sweden)

    U D Gupta

    2015-01-01

    Full Text Available Background & objectives: Studies have shown the bactericidal potential of econazole and clotrimazole against Mycobacterium tuberculosis under in vitro and ex vivo conditions along with their synergism with conventional antituberculosis drugs. These molecules were also found to be effective against different multidrug resistant (MDR M. tuberculosis isolates in vitro. Hence the present study was designed to evaluate the in vivo antimycobacterial potential of moxifloxacin and econazole alone and in combination against multidrug resistant tuberculosis (MDR-TB in a mice model. Methods: Mice were infected with 2.5×10 [7] bacilli of MDR strain of M. tuberculosis by aerosol route of infection. After four weeks of infection, chemotherapy was started orally by moxifloxacin 8.0 mg/kg body wt and econazole 3.3 mg/kg alone and in combination, as well as with four first line anti-tuberculosis drugs as a positive control. The animals were sacrificed and the lungs and spleen were excised under aspetic conditions. The tissues were homogenized with sterile normal saline, an aliquot of the homogenate was plated on Middlebrook 7H11 agar supplemented with oleate albumin dextrose catalase (OADC and incubated at 37°C for four weeks. The number of visible and individual colonies were counted. Results: The first line anti-tuberculosis drugs (RIF+INH+EMB+PZA after eight weeks of therapy had no impact as the bacillary load in lungs and spleens remained unchanged. However, econazole, moxifloxacin alone as well as in combination significantly reduced the bacillary load in lungs as well as in spleens of MDR-TB bacilli infected mice. Interpretation & conclusions: Co-administration of the two drugs (econazole and moxifloxacin to MDR-TB strain JAL-7782 infected mice exhibited additive effect, the efficacy of the drugs in combination being higher as compared with ECZ or MOX alone. These results were substantiated by histopathological studies. This study suggests the utility of

  6. Detection and differentiation of Mycobacterium tuberculosis and nontuberculous mycobacterial isolates by real-time PCR.

    Science.gov (United States)

    Shrestha, Nabin K; Tuohy, Marion J; Hall, Gerri S; Reischl, Udo; Gordon, Steven M; Procop, Gary W

    2003-11-01

    Mycobacteria cause a variety of illnesses that differ in severity and public health implications. The differentiation of Mycobacterium tuberculosis from nontuberculous mycobacteria (NTM) is of primary importance for infection control and choice of antimicrobial therapy. Despite advances in molecular diagnostics, the ability to rapidly diagnose M. tuberculosis infections by PCR is still inadequate, largely because of the possibility of false-negative reactions. We designed and validated a real-time PCR for mycobacteria by using the LightCycler system with 18 reference strains and 168 clinical mycobacterial isolates. All clinically significant mycobacteria were detected; the mean melting temperatures (with 99.9% confidence intervals [99.9% CI] in parentheses) for the different mycobacteria were as follows: M. tuberculosis, 64.35 degrees C (63.27 to 65.42 degrees C); M. kansasii, 59.20 degrees C (58.07 to 60.33 degrees C); M. avium, 57.82 degrees C (57.05 to 58.60 degrees C); M. intracellulare, 54.46 degrees C (53.69 to 55.23 degrees C); M. marinum, 58.91 degrees C (58.28 to 59.55 degrees C); rapidly growing mycobacteria, 53.09 degrees C (50.97 to 55.20 degrees C) or 43.19 degrees C (42.19 to 44.49 degrees C). This real-time PCR assay with melting curve analysis consistently accurately detected and differentiated M. tuberculosis from NTM. Detection of an NTM helps ensure that the negative result for M. tuberculosis is a true negative. The specific melting temperature also provides a suggestion of the identity of the NTM present, when the most commonly encountered mycobacterial species are considered. In a parallel comparison, both the LightCycler assay and the COBAS Amplicor M. tuberculosis assay correctly categorized 48 of 50 specimens that were proven by culture to contain M. tuberculosis, and the LightCycler assay correctly characterized 3 of 3 specimens that contained NTM.

  7. Extra pulmonary tuberculosis: Rapid identification of Mycobacterium tuberculosis grown in Mycobacterium growth indicator tube 960 and Lowenstein-Jensen media, employing Standard diagnostics Bioline Mycobacterium tuberculosis protein 64 antigen detection kit

    Directory of Open Access Journals (Sweden)

    G Kandhakumari

    2015-01-01

    Full Text Available Background: Investigation of extra pulmonary tuberculosis (EPTB in and around Pondicherry is being carried out since August 2011 in our tertiary care super specialty hospital. Objectives: To compare the rapid Kit SD Bio-Line MPT 64 Ag with conventional and time consuming biochemical tests. Confirmation of Mycobacterium tuberculosis at a reasonable time frame is the main thrust. Materials and Methods: Thirty three Mycobacterium tuberculosis and four Non-Tuberculous Mycobacteria (NTM grown in MGIT960 system/Lowenstein-Jensen media (LJ were examined by the rapid MPT 64 antigen detection as well as a battery of conventional tests like niacin, nitrate reduction, paraminobenzoic acid susceptibility and cord formation. Results and Conclusion: . Both the rapid kit and conventional tests correctly identified 33 M.tuberculosis isolates. Keeping conventional identification as reference, sensitivity and specificity for rapid kit was 100%. Rapid kit which takes only 15 minutes is accurate, cost effective, and facilitates early treatment for these EPTB patients, whose clinical specimens are paucibacillary.

  8. A Mycobacterium tuberculosis cluster demonstrating the use of genotyping in urban tuberculosis control

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    Burdo Conny CA

    2009-09-01

    Full Text Available Abstract Background DNA fingerprinting of Mycobacterium tuberculosis isolates offers better opportunities to study links between tuberculosis (TB cases and can highlight relevant issues in urban TB control in low-endemic countries. Methods A medium-sized molecular cluster of TB cases with identical DNA fingerprints was used for the development of a visual presentation of epidemiologic links between cases. Results Of 32 cases, 17 (53% were linked to the index case, and 11 (34% to a secondary case. The remaining four (13% could not be linked and were classified as possibly caused by the index patient. Of the 21 cases related to the index case, TB developed within one year of the index diagnosis in 11 patients (52%, within one to two years in four patients (19%, and within two to five years in six patients (29%. Conclusion Cluster analysis underscored several issues for TB control in an urban setting, such as the recognition of the outbreak, the importance of reinfections, the impact of delayed diagnosis, the contribution of pub-related transmissions and its value for decision-making to extend contact investigations. Visualising cases in a cluster diagram was particularly useful in finding transmission locations and the similarities and links between patients.

  9. Potential Inhibitors for Isocitrate Lyase of Mycobacterium tuberculosis and Non-M. tuberculosis: A Summary

    Directory of Open Access Journals (Sweden)

    Yie-Vern Lee

    2015-01-01

    Full Text Available Isocitrate lyase (ICL is the first enzyme involved in glyoxylate cycle. Many plants and microorganisms are relying on glyoxylate cycle enzymes to survive upon downregulation of tricarboxylic acid cycle (TCA cycle, especially Mycobacterium tuberculosis (MTB. In fact, ICL is a potential drug target for MTB in dormancy. With the urge for new antitubercular drug to overcome tuberculosis treat such as multidrug resistant strain and HIV-coinfection, the pace of drug discovery has to be increased. There are many approaches to discovering potential inhibitor for MTB ICL and we hereby review the updated list of them. The potential inhibitors can be either a natural compound or synthetic compound. Moreover, these compounds are not necessary to be discovered only from MTB ICL, as it can also be discovered by a non-MTB ICL. Our review is categorized into four sections, namely, (a MTB ICL with natural compounds; (b MTB ICL with synthetic compounds; (c non-MTB ICL with natural compounds; and (d non-MTB ICL with synthetic compounds. Each of the approaches is capable of overcoming different challenges of inhibitor discovery. We hope that this paper will benefit the discovery of better inhibitor for ICL.

  10. Prevalence and evolution of Mycobacterium tuberculosis infection in tuberculosis case contacts

    Directory of Open Access Journals (Sweden)

    Silvia Paulino Ribeiro Albanese

    2015-06-01

    Full Text Available INTRODUCTION : The tuberculin test is a diagnostic method for detecting latent tuberculosis (TB infection, especially among disease contact cases. The objective of this study was to analyze the prevalence and evolution of Mycobacterium tuberculosis infection among TB contact cases. METHODS : A retrospective cohort study was performed in a reference center for TB. The study population consisted of 2,425 patients who underwent a tuberculin test from 2003 to 2010 and whose results indicated contact with individuals with TB. The data were collected from the registry book of the tuberculin tests, patient files and the Information System Records of Notification Grievance. To verify the evolution of TB, case records through September 2014 were consulted. Data were analyzed using the Statistical Package for the Social Sciences (SPSS. In all hypothesis tests, a significance level of 0.05 was used. RESULTS : From the studied sample, 435 (17.9% contacts did not return for reading. Among the 1,990 contacts that completed the test, the prevalence of latent TB infection was 35.4%. Of these positive cases, 50.6% were referred to treatment; the dropout rate was 42.5%. Among all of the contacts, the TB prevalence was 1.8%, from which 13.2% abandoned treatment. CONCLUSIONS : The collected data indicate the need for more effective public policies to improve TB control, including administering tests that do not require a return visit for reading, enhancing contact tracing and encouraging actions that reinforce full treatment adherence.

  11. IFNG-mediated immune responses enhance autophagy against Mycobacterium tuberculosis antigens in patients with active tuberculosis

    Science.gov (United States)

    Rovetta, Ana I; Peña, Delfina; Hernández Del Pino, Rodrigo E; Recalde, Gabriela M; Pellegrini, Joaquín; Bigi, Fabiana; Musella, Rosa M; Palmero, Domingo J; Gutierrez, Marisa; Colombo, María I; García, Verónica E

    2015-01-01

    Protective immunity against Mycobacterium tuberculosis (Mtb) requires IFNG. Besides, IFNG-mediated induction of autophagy suppresses survival of virulent Mtb in macrophage cell lines. We investigated the contribution of autophagy to the defense against Mtb antigen (Mtb-Ag) in cells from tuberculosis patients and healthy donors (HD). Patients were classified as high responders (HR) if their T cells produced significant IFNG against Mtb-Ag; and low responders (LR) when patients showed weak or no T cell responses to Mtb-Ag. The highest autophagy levels were detected in HD cells whereas the lowest quantities were observed in LR patients. Interestingly, upon Mtb-Ag stimulation, we detected a positive correlation between IFNG and MAP1LC3B-II/LC3-II levels. Actually, blockage of Mtb-Ag-induced IFNG markedly reduced autophagy in HR patients whereas addition of limited amounts of IFNG significantly increased autophagy in LR patients. Therefore, autophagy collaborates with human immune responses against Mtb in close association with specific IFNG secreted against the pathogen. PMID:25426782

  12. Differential Recognition of Mycobacterium tuberculosis-Specific Epitopes as a Function of Tuberculosis Disease History.

    Science.gov (United States)

    Scriba, Thomas J; Carpenter, Chelsea; Pro, Sebastian Carrasco; Sidney, John; Musvosvi, Munyaradzi; Rozot, Virginie; Seumois, Grégory; Rosales, Sandy L; Vijayanand, Pandurangan; Goletti, Delia; Makgotlho, Edward; Hanekom, Willem; Hatherill, Mark; Peters, Bjoern; Sette, Alessandro; Arlehamn, Cecilia S Lindestam

    2017-09-15

    Individuals with a history of tuberculosis (TB) disease are at elevated risk of disease recurrence. The underlying cause is not known, but one explanation is that previous disease results in less-effective immunity against Mycobacterium tuberculosis (Mtb). We hypothesized that the repertoire of Mtb-derived epitopes recognized by T cells from individuals with latent Mtb infection differs as a function of previous diagnosis of active TB disease. T-cell responses to peptide pools in samples collected from an adult screening and an adolescent validation cohort were measured by IFN-γ enzyme-linked immunospot assay or intracellular cytokine staining. We identified a set of "type 2" T-cell epitopes that were recognized at 10-fold-lower levels in Mtb-infected individuals with a history of TB disease less than 6 years ago than in those without previous TB. By contrast, "type 1" epitopes were recognized equally well in individuals with or without previous TB. The differential epitope recognition was not due to differences in HLA class II binding, memory phenotypes, or gene expression in the responding T cells. Instead, "TB disease history-sensitive" type 2 epitopes were significantly (P < 0.0001) more homologous to sequences from bacteria found in the human microbiome than type 1 epitopes. Preferential loss of T-cell reactivity to Mtb epitopes that are homologous to bacteria in the microbiome in persons with previous TB disease may reflect long-term effects of antibiotic TB treatment on the microbiome.

  13. Detection of mutation in isoniazid-resistant Mycobacterium tuberculosis isolates from tuberculosis patients in Belarus

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    Bostanabad S

    2008-01-01

    Full Text Available The aim of this study was to investigate the frequency, location and type of katG mutations in Mycobacterium tuberculosis strains isolated from patients in Belarus. Forty two isoniazid-resistant isolates were identified from sputum of 163 patients with active pulmonary tuberculosis. Drug susceptibility testing was determined by using CDC standard conventional proportional method and BACTEC system. Standard PCR method for detection of isoniazid resistance associated mutations was performed by katG gene amplification and DNA sequencing. Most mutations were found in katG gene codons 315, 316 and 309. Four types of mutations were identified in codon 315: AGC→ACC ( n = 36 85%, AGC→AGG ( n = 1 2.3%, AGC→AAC ( n = 2 4.7%, AGC→GGC ( n = 1 2.3%. One type of mutation was found in codon 316: GGC→AGC ( n = 1841.4%, four types of mutations were detected in codon 309: GGT→GGT ( n = 716.1%, GGT→GCT ( n = 49.2%, GGT→GTC ( n = 36.9%, GGT→GGG ( n = 12.7%. The highest frequency of mutations sharing between primary and secondary infections was found in codon 315.

  14. Mycobacterium tuberculosis Invasion of the Human Lung: First Contact

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    Jeroen Maertzdorf

    2018-06-01

    Full Text Available Early immune responses to Mycobacterium tuberculosis (Mtb invasion of the human lung play a decisive role in the outcome of infection, leading to either rapid clearance of the pathogen or stable infection. Despite their critical impact on health and disease, these early host–pathogen interactions at the primary site of infection are still poorly understood. In vitro studies cannot fully reflect the complexity of the lung architecture and its impact on host–pathogen interactions, while animal models have their own limitations. In this study, we have investigated the initial responses in human lung tissue explants to Mtb infection, focusing primarily on gene expression patterns in different tissue-resident cell types. As first cell types confronted with pathogens invading the lung, alveolar macrophages, and epithelial cells displayed rapid proinflammatory chemokine and cytokine responses to Mtb infection. Other tissue-resident innate cells like gamma/delta T cells, mucosal associated invariant T cells, and natural killer cells showed partially similar but weaker responses, with a high degree of variability across different donors. Finally, we investigated the responses of tissue-resident innate lymphoid cells to the inflammatory milieu induced by Mtb infection. Our infection model provides a unique approach toward host–pathogen interactions at the natural port of Mtb entry and site of its implantation, i.e., the human lung. Our data provide a first detailed insight into the early responses of different relevant pulmonary cells in the alveolar microenvironment to contact with Mtb. These results can form the basis for the identification of host markers that orchestrate early host defense and provide resistance or susceptibility to stable Mtb infection.

  15. Genotypic characterization of multi-drug-resistant Mycobacterium tuberculosis isolates in Myanmar.

    Science.gov (United States)

    Aye, Khin Saw; Nakajima, Chie; Yamaguchi, Tomoyuki; Win, Min Min; Shwe, Mu Mu; Win, Aye Aye; Lwin, Thandar; Nyunt, Wint Wint; Ti, Ti; Suzuki, Yasuhiko

    2016-03-01

    The number of multi-drug-resistant tuberculosis (MDR-TB) cases is rising worldwide. As a countermeasure against this situation, the implementation of rapid molecular tests to identify MDR-TB would be effective. To develop such tests, information on the frequency and distribution of mutations associating with phenotypic drug resistance in Mycobacterium tuberculosis is required in each country. During 2010, the common mutations in the rpoB, katG and inhA of 178 phenotypically MDR M. tuberculosis isolates collected by the National Tuberculosis Control Program (NTP) in Myanmar were investigated by DNA sequencing. Mutations affecting the 81-bp rifampicin (RIF) resistance-determining region (RRDR) of the rpoB were identified in 127 of 178 isolates (71.3%). Two of the most frequently affected codons were 531 and 526, with percentages of 48.3% and 14.0% respectively. For isoniazid (INH) resistance, 114 of 178 MDR-TB isolates (64.0%) had mutations in the katG in which a mutation-conferring amino acid substitution at codon 315 from Ser to Thr was the most common. Mutations in the inhA regulatory region were also detected in 20 (11.2%) isolates, with the majority at position -15. Distinct mutation rate and pattern from surrounding countries might suggest that MDR-TB has developed and spread domestically in Myanmar. Copyright © 2015 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  16. Development and analysis of an in vivo-compatible metabolic network of Mycobacterium tuberculosis

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    Reifman Jaques

    2010-11-01

    Full Text Available Abstract Background During infection, Mycobacterium tuberculosis confronts a generally hostile and nutrient-poor in vivo host environment. Existing models and analyses of M. tuberculosis metabolic networks are able to reproduce experimentally measured cellular growth rates and identify genes required for growth in a range of different in vitro media. However, these models, under in vitro conditions, do not provide an adequate description of the metabolic processes required by the pathogen to infect and persist in a host. Results To better account for the metabolic activity of M. tuberculosis in the host environment, we developed a set of procedures to systematically modify an existing in vitro metabolic network by enhancing the agreement between calculated and in vivo-measured gene essentiality data. After our modifications, the new in vivo network contained 663 genes, 838 metabolites, and 1,049 reactions and had a significantly increased sensitivity (0.81 in predicted gene essentiality than the in vitro network (0.31. We verified the modifications generated from the purely computational analysis through a review of the literature and found, for example, that, as the analysis suggested, lipids are used as the main source for carbon metabolism and oxygen must be available for the pathogen under in vivo conditions. Moreover, we used the developed in vivo network to predict the effects of double-gene deletions on M. tuberculosis growth in the host environment, explore metabolic adaptations to life in an acidic environment, highlight the importance of different enzymes in the tricarboxylic acid-cycle under different limiting nutrient conditions, investigate the effects of inhibiting multiple reactions, and look at the importance of both aerobic and anaerobic cellular respiration during infection. Conclusions The network modifications we implemented suggest a distinctive set of metabolic conditions and requirements faced by M. tuberculosis during

  17. Identification and characterization of the ESAT-6 homologue of Mycobacterium leprae and T-cell cross-reactivity with Mycobacterium tuberculosis

    NARCIS (Netherlands)

    Geluk, Annemieke; van Meijgaarden, Krista E.; Franken, Kees L. M. C.; Subronto, Yanri W.; Wieles, Brigitte; Arend, Sandra M.; Sampaio, Elizabeth P.; de Boer, Tjitske; Faber, William R.; Naafs, Ben; Ottenhoff, Tom H. M.

    2002-01-01

    In this paper we describe identification and characterization of Mycobacterium leprae ESAT-6 (L-ESAT-6), the homologue of M. tuberculosis ESAT-6 (T-ESAT-6). T-ESAT-6 is expressed by all pathogenic strains belonging to the M. tuberculosis complex but is absent from virtually all other mycobacterial

  18. [Tuberculosis caused by Mycobacterium bovis in workers of bovine tuberculosis sanitation farms in Antioquia, Boyacá and Cundinamarca].

    Science.gov (United States)

    Leal-Bohórquez, Andrés F; Castro-Osorio, Claudia M; Wintaco-Martínez, Luz M; Villalobos, Rafael; Puerto-Castro, Gloria M

    2016-01-01

    To perform classic and molecular epidemiological surveillance of human tuberculosis caused by Mycobacterium bovis in bovine supply chains at farms with PPD positive bovines in the departments of Antioquia, Boyacá and Cundinamarca during a one-year period. Livestock farms with PPD positive bovines or buffalos were visited in the study departments according to information obtained in the "Programa Nacional de Tuberculosis bovina" (National program on bovine Tuberculosis) released by ICA (Colombian Agriculture and Livestock Institute). Data on socio-demographic information and tuberculosis risk factors associated to the occupation were collected through a survey applied to all workers at the visited farms. Sputum samples were obtained after informed consent. The sputa underwent microbiological and molecular testing to identify members of the M. tuberculosis complex. Thirty-three livestock farms were visited and information of 164 workers from the bovine supply chain was collected. Staying in a PPD positive farm for more than a year, ignorance about the disease and the presence of possible vectors, like dogs and cats, were identified as possible risk factors for developing tuberculosis. No cases of tuberculosis caused by M. bovis or M. tuberculosis in workers of the visited farms were found. No cases of the disease caused by this zoonotic agent were documented in the departments of Antioquia, Boyacá and Cundinamarca.

  19. The curative activity of thioridazine on mice infected with Mycobacterium tuberculosis

    DEFF Research Database (Denmark)

    Martins, Marta; Viveiros, Miguel; Kristiansen, Jette E

    2007-01-01

    BACKGROUND: The aim of the study was to evaluate the effectiveness of thioridazine (TZ) at different dose levels on mice that had been infected intraperitoneally (i.p.) with a high dose of the Mycobacterium tuberculosis ATCC H37Rv strain. SUBJECTS AND METHODS: Groups of five female BALB/C mice were...

  20. Pathology of the emerging Mycobacterium tuberculosis complex pathogen, M. mungi in the banded mongoose (Mungos mungo)

    Science.gov (United States)

    Wild banded mongooses (Mungos mungo) in northeastern Botswana and Northwest Zimbabwe are infected with a novel Mycobacterium tuberculosis complex pathogen (MTC), M. mungi. This pathogen is transmitted environmentally between mongoose hosts through exposure to infected scent marks used in olfactory c...

  1. Two-Component Systems of Mycobacterium tuberculosis—Structure-Based Approaches

    DEFF Research Database (Denmark)

    Tucker, Paul; Nowak, Elzbieta; Morth, Jens Preben

    2007-01-01

    Mycobacterium tuberculosis contains few two‐component systems compared to many other bacteria, possibly because it has more serine/threonine signaling pathways. Even so, these two‐component systems appear to play an important role in early intracellular survival of the pathogen as well as in aspe...

  2. New Approaches and Therapeutic Options for Mycobacterium tuberculosis in a Dormant State.

    NARCIS (Netherlands)

    Caño-Muñiz, Santiago; Anthony, Richard; Niemann, Stefan; Alffenaar, Jan-Willem C

    We are far away from the days when tuberculosis (TB) accounted for 1 in 4 deaths during the 19th century. However,Mycobacterium tuberculosiscomplex (MTBC) strains are still the leading cause of morbidity and mortality by a single infectious disease, with 9.6 million cases and 1.5 million deaths

  3. Structural and Functional Aspects of the Sensor Histidine Kinase PrrB from Mycobacterium tuberculosis

    DEFF Research Database (Denmark)

    Nowak, E.; Panjikar, S.; Morth, J.P.

    2006-01-01

    We describe the solution structures of two- and three-domain constructs of the sensor histidine kinase PrrB from Mycobacterium tuberculosis, which allow us to locate the HAMP linker relative to the ATP binding and dimerization domains. We show that the three-domain construct is active both...

  4. Novel genetic polymorphisms that further delineate the phylogeny of the Mycobacterium tuberculosis complex.

    NARCIS (Netherlands)

    Huard, Richard C; Fabre, Michel; Haas, Petra de; Lazzarini, Luiz Claudio Oliveira; Soolingen, Dick van; Cousins, Debby; Ho, John L

    2006-01-01

    In a previous report, we described a PCR protocol for the differentiation of the various species of the Mycobacterium tuberculosis complex (MTC) on the basis of genomic deletions (R. C. Huard, L. C. de Oliveira Lazzarini, W. R. Butler, D. van Soolingen, and J. L. Ho, J. Clin. Microbiol.

  5. Mycobacterium tuberculosis Phosphoenolpyruvate Carboxykinase Is Regulated by Redox Mechanisms and Interaction with Thioredoxin

    Czech Academy of Sciences Publication Activity Database

    Machová, Iva; Snášel, Jan; Zimmermann, M.; Laubitz, D.; Plocinski, P.; Oehlmann, W.; Singh, M.; Dostál, Jiří; Sauer, U.; Pichová, Iva

    2014-01-01

    Roč. 289, č. 19 (2014), s. 13066-13078 ISSN 0021-9258 EU Projects: European Commission(XE) 241587 - SYSTEMTB Grant - others:OPPK(CZ) CZ.2.16/3.1.00/24016 Institutional support: RVO:61388963 Keywords : enzyme kinetics * hypoxia * metabolism * Mycobacterium tuberculosis * oxidation-reduction * thioredoxin * Phosphoenolpyruvate carboxykinase Subject RIV: CE - Biochemistry Impact factor: 4.573, year: 2014

  6. Methyltransferase Erm(37) Slips on rRNA to Confer Atypical Resistance in Mycobacterium tuberculosis

    Czech Academy of Sciences Publication Activity Database

    Madsen, Ch. T.; Jakobsen, L.; Buriánková, Karolína; Doucet-Populaire, F.; Perdonet, J. L.; Douthwaite, S.

    2005-01-01

    Roč. 280, č. 47 (2005), s. 38942-38947 ISSN 0021-9258 R&D Projects: GA ČR GA310/03/0292 Institutional research plan: CEZ:AV0Z50200510 Keywords : methyltransferase erm * mycobacterium tuberculosis * rRNA Subject RIV: EE - Microbiology, Virology Impact factor: 5.854, year: 2005

  7. A Rational Approach to Identify Inhibitors of Mycobacterium tuberculosis Enoyl Acyl Carrier Protein Reductase

    Czech Academy of Sciences Publication Activity Database

    Chhabria, M. T.; Parmar, K. B.; Brahmkshatriya, Pathik

    2013-01-01

    Roč. 19, č. 21 (2013), s. 3878-3883 ISSN 1381-6128 Institutional support: RVO:61388963 Keywords : mycobacterium tuberculosis * enoyl acyl carrier protein reductase * pharmacophore modeling * molecular docking * binding interactions Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 3.288, year: 2013

  8. Differential T-cell recognition of native and recombinant Mycobacterium tuberculosis GroES

    DEFF Research Database (Denmark)

    Rosenkrands, I; Weldingh, K; Ravn, P

    1999-01-01

    Mycobacterium tuberculosis GroES was purified from culture filtrate, and its identity was confirmed by immunoblot analysis and N-terminal sequencing. Comparing the immunological recognition of native and recombinant GroES, we found that whereas native GroES elicited a strong proliferative response...

  9. Progression of chronic pulmonary tuberculosis in mice intravenously infected with ethambutol resistant Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Srivastava S

    2008-01-01

    Full Text Available Purpose: Ethambutol (EMB is an important first line drug, however little information on its molecular mechanism of resistance and pathogenicity of resistant isolates is available. Present work was designed to study virulence of the EMB resistant M. tuberculosis strains and the host responses in-vivo on infection of EMB resistant M. tuberculosis using Balb/c mouse model of infection. Methods: Three groups of Balb/c mice (female, age 4-6 wk; 21 mice in each group were infected intravenously with 106 CFU of M. tuberculosis H37Rv and two EMB resistant clinical isolates. Age and sex matched control animals were mock inoculated with Middlebrook 7H9 broth alone. At 10, 20, 30, 40, 50, 60, and 70 days post-infection three animals from each group were sacrificed by cervical dislocation and lung tissue was collected for further analysis. Results: Infection with EMB resistant M. tuberculosis led to progressive and chronic disease with significantly high bacillary load (p=0.02. Massive infiltration and exacerbated lung pathology with increased expression of IFN-γ and TNF-α was observed in lungs of mice infected with EMB resistant strains. The present study suggests that infection with EMB resistant M. tuberculosis leads to chronic infection with subsequent loss of lung function, bacterial persistence with elevated expression of TNF-α resulting in increased lung pathology. Conclusion: These findings highlight that EMB resistant M. tuberculosis regulates host immune response differentially and its pathogenicity is different from drug sensitive strains of M. tuberculosis.

  10. In vitro activities of DA-7157 and DA-7218 against Mycobacterium tuberculosis and Nocardia brasiliensis.

    Science.gov (United States)

    Vera-Cabrera, Lucio; Gonzalez, Eva; Rendon, Adrian; Ocampo-Candiani, Jorge; Welsh, Oliverio; Velazquez-Moreno, Victor M; Choi, Sung Hak; Molina-Torres, Carmen

    2006-09-01

    The in vitro activities of DA-7157, a novel oxazolidinone, against clinical isolates of Nocardia brasiliensis and Mycobacterium tuberculosis were determined. Equal MIC(50)s and MIC(90)s (0.25 and 0.5 microg/ml, respectively) were found for susceptible and multidrug-resistant isolates of M. tuberculosis. The N. brasiliensis isolates showed an MIC(90) of 1 microg/ml and an MIC(50) of 1 microg/ml. The DA-7157 prodrug, DA-7218, exhibited similar MICs for M. tuberculosis but fivefold-higher MICs for N. brasiliensis.

  11. Comprehensive functional analysis of Mycobacterium tuberculosis toxin-antitoxin systems: implications for pathogenesis, stress responses, and evolution.

    Directory of Open Access Journals (Sweden)

    Holly R Ramage

    2009-12-01

    Full Text Available Toxin-antitoxin (TA systems, stress-responsive genetic elements ubiquitous in microbial genomes, are unusually abundant in the major human pathogen Mycobacterium tuberculosis. Why M. tuberculosis has so many TA systems and what role they play in the unique biology of the pathogen is unknown. To address these questions, we have taken a comprehensive approach to identify and functionally characterize all the TA systems encoded in the M. tuberculosis genome. Here we show that 88 putative TA system candidates are present in M. tuberculosis, considerably more than previously thought. Comparative genomic analysis revealed that the vast majority of these systems are conserved in the M. tuberculosis complex (MTBC, but largely absent from other mycobacteria, including close relatives of M. tuberculosis. We found that many of the M. tuberculosis TA systems are located within discernable genomic islands and were thus likely acquired recently via horizontal gene transfer. We discovered a novel TA system located in the core genome that is conserved across the genus, suggesting that it may fulfill a role common to all mycobacteria. By expressing each of the putative TA systems in M. smegmatis, we demonstrate that 30 encode a functional toxin and its cognate antitoxin. We show that the toxins of the largest family of TA systems, VapBC, act by inhibiting translation via mRNA cleavage. Expression profiling demonstrated that four systems are specifically activated during stresses likely encountered in vivo, including hypoxia and phagocytosis by macrophages. The expansion and maintenance of TA genes in the MTBC, coupled with the finding that a subset is transcriptionally activated by stress, suggests that TA systems are important for M. tuberculosis pathogenesis.

  12. A small-molecule allosteric inhibitor of Mycobacterium tuberculosis tryptophan synthase

    Energy Technology Data Exchange (ETDEWEB)

    Wellington, Samantha; Nag, Partha P.; Michalska, Karolina; Johnston, Stephen E.; Jedrzejczak, Robert P.; Kaushik, Virendar K.; Clatworthy, Anne E.; Siddiqi, Noman; McCarren, Patrick; Bajrami, Besnik; Maltseva, Natalia I.; Combs, Senya; Fisher, Stewart L.; Joachimiak, Andrzej; Schreiber, Stuart L.; Hung, Deborah T.

    2017-07-03

    New antibiotics with novel targets are greatly needed. Bacteria have numerous essential functions, but only a small fraction of such processes—primarily those involved in macromolecular synthesis—are inhibited by current drugs. Targeting metabolic enzymes has been the focus of recent interest, but effective inhibitors have been difficult to identify. We describe a synthetic azetidine derivative, BRD4592, that kills Mycobacterium tuberculosis (Mtb) through allosteric inhibition of tryptophan synthase (TrpAB), a previously untargeted, highly allosterically regulated enzyme. BRD4592 binds at the TrpAB a–b-subunit interface and affects multiple steps in the enzyme’s overall reaction, resulting in inhibition not easily overcome by changes in metabolic environment. We show that TrpAB is required for the survival of Mtb and Mycobacterium marinum in vivo and that this requirement may be independent of an adaptive immune response. This work highlights the effectiveness of allosteric inhibition for targeting proteins that are naturally highly dynamic and that are essential in vivo, despite their apparent dispensability under in vitro conditions, and suggests a framework for the discovery of a next generation of allosteric inhibitors.

  13. A small-molecule allosteric inhibitor of Mycobacterium tuberculosis tryptophan synthase

    Energy Technology Data Exchange (ETDEWEB)

    Wellington, Samantha; Nag, Partha P.; Michalska, Karolina; Johnston, Stephen E.; Jedrzejczak, Robert P.; Kaushik, Virendar K.; Clatworthy, Anne E.; Siddiqi, Noman; McCarren, Patrick; Bajrami, Besnik; Maltseva, Natalia I.; Combs, Senya; Fisher, Stewart L.; Joachimiak, Andrzej; Schreiber, Stuart L.; Hung, Deborah T.

    2017-07-03

    New antibiotics with novel targets are greatly needed. Bacteria have numerous essential functions, but only a small fraction of such processes—primarily those involved in macromolecular synthesis—are inhibited by current drugs. Targeting metabolic enzymes has been the focus of recent interest, but effective inhibitors have been difficult to identify. We describe a synthetic azetidine derivative, BRD4592, that kills Mycobacterium tuberculosis (Mtb) through allosteric inhibition of tryptophan synthase (TrpAB), a previously untargeted, highly allosterically regulated enzyme. BRD4592 binds at the TrpAB α–β-subunit interface and affects multiple steps in the enzyme's overall reaction, resulting in inhibition not easily overcome by changes in metabolic environment. We show that TrpAB is required for the survival of Mtb and Mycobacterium marinum in vivo and that this requirement may be independent of an adaptive immune response. This work highlights the effectiveness of allosteric inhibition for targeting proteins that are naturally highly dynamic and that are essential in vivo, despite their apparent dispensability under in vitro conditions, and suggests a framework for the discovery of a next generation of allosteric inhibitors.

  14. Organization of the origins of replication of the chromosomes of Mycobacterium smegmatis, Mycobacterium leprae and Mycobacterium tuberculosis and isolation of a functional origin from M. smegmatis.

    Science.gov (United States)

    Salazar, L; Fsihi, H; de Rossi, E; Riccardi, G; Rios, C; Cole, S T; Takiff, H E

    1996-04-01

    The genus Mycobacterium is composed of species with widely differing growth rates ranging from approximately three hours in Mycobacterium smegmatis to two weeks in Mycobacterium leprae. As DNA replication is coupled to cell duplication, it may be regulated by common mechanisms. The chromosomal regions surrounding the origins of DNA replication from M. smegmatis, M. tuberculosis, and M. leprae have been sequenced, and show very few differences. The gene order, rnpA-rpmH-dnaA-dnaN-recF-orf-gyrB-gyrA, is the same as in other Gram-positive organisms. Although the general organization in M. smegmatis is very similar to that of Streptomyces spp., a closely related genus, M. tuberculosis and M. leprae differ as they lack an open reading frame, between dnaN and recF, which is similar to the gnd gene of Escherichia coli. Within the three mycobacterial species, there is extensive sequence conservation in the intergenic regions flanking dnaA, but more variation from the consensus DnaA box sequence was seen than in other bacteria. By means of subcloning experiments, the putative chromosomal origin of replication of M. smegmatis, containing the dnaA-dnaN region, was shown to promote autonomous replication in M. smegmatis, unlike the corresponding regions from M. tuberculosis or M. leprae.

  15. Comparison of antigen-specific T-cell responses of tuberculosis patients using complex or single antigens of Mycobacterium tuberculosis

    DEFF Research Database (Denmark)

    Mustafa, A S; Amoudy, H A; Wiker, H G

    1998-01-01

    We have screened peripheral blood mononuclear cells (PBMC) from tuberculosis (TB) patients for proliferative reactivity and interferon-gamma (IFN-gamma) secretion against a panel of purified recombinant (r) and natural (n) culture filtrate (rESAT-6, nMPT59, nMPT64 and nMPB70) and somatic-derived (r......GroES, rPstS, rGroEL and rDnaK) antigens of Mycobacterium tuberculosis. The responses of PBMC to these defined antigens were compared with the corresponding results obtained with complex antigens, such as whole-cell M. tuberculosis, M. tuberculosis culture filtrate (MT-CF) and cell wall antigens, as well...... as the vaccine strain, Mycobacterium bovis bacillus Calmette-Guerin (BCG). In addition, M. tuberculosis and MT-CF-induced T-cell lines were tested in the same assays against the panel of purified and complex antigens. The compiled data from PBMC and T-cell lines tested for antigen-induced proliferation and IFN...

  16. Production of antibodies against glycolipids from the Mycobacterium tuberculosis cell wall in aerosol murine models of tuberculosis.

    Science.gov (United States)

    Cardona, P J; Julián, E; Vallès, X; Gordillo, S; Muñoz, M; Luquin, M; Ausina, V

    2002-06-01

    Evolution of antibodies against glycolipids from the Mycobacterium tuberculosis cell wall has been studied for the first time in experimental murine models of tuberculosis induced by aerosol, in which infection, reinfection, reactivation, prophylaxis and treatment with antibiotics have been assayed. Results show a significant humoral response against these antigens, where diacyltrehaloses (DAT) and sulpholipid I (SL-I) elicited higher antibody levels than protein antigens like antigen 85 protein complex (Ag85), culture filtrate proteins (CFP) and purified protein derivative (PPD). Only immunoglobulin M (IgM) antibodies have been detected against DAT and SL-I. Their evolution has a positive correlation with bacillary concentration in tissues.

  17. Population Structure among Mycobacterium tuberculosis Isolates from Pulmonary Tuberculosis Patients in Colombia

    Science.gov (United States)

    Realpe, Teresa; Correa, Nidia; Rozo, Juan Carlos; Ferro, Beatriz Elena; Gomez, Verónica; Zapata, Elsa; Ribon, Wellman; Puerto, Gloria; Castro, Claudia; Nieto, Luisa María; Diaz, Maria Lilia; Rivera, Oriana; Couvin, David; Rastogi, Nalin; Arbelaez, Maria Patricia; Robledo, Jaime

    2014-01-01

    Background Phylogeographic composition of M. tuberculosis populations reveals associations between lineages and human populations that might have implications for the development of strategies to control the disease. In Latin America, lineage 4 or the Euro-American, is predominant with considerable variations among and within countries. In Colombia, although few studies from specific localities have revealed differences in M. tuberculosis populations, there are still areas of the country where this information is lacking, as is a comparison of Colombian isolates with those from the rest of the world. Principal Findings A total of 414 M. tuberculosis isolates from adult pulmonary tuberculosis cases from three Colombian states were studied. Isolates were genotyped using IS6110-restriction fragment length polymorphism (RFLP), spoligotyping, and 24-locus Mycobacterial interspersed repetitive units variable number tandem repeats (MIRU-VNTRs). SIT42 (LAM9) and SIT62 (H1) represented 53.3% of isolates, followed by 8.21% SIT50 (H3), 5.07% SIT53 (T1), and 3.14% SIT727 (H1). Composite spoligotyping and 24-locus MIRU- VNTR minimum spanning tree analysis suggest a recent expansion of SIT42 and SIT62 evolved originally from SIT53 (T1). The proportion of Haarlem sublineage (44.3%) was significantly higher than that in neighboring countries. Associations were found between M. tuberculosis MDR and SIT45 (H1), as well as HIV-positive serology with SIT727 (H1) and SIT53 (T1). Conclusions This study showed the population structure of M. tuberculosis in several regions from Colombia with a dominance of the LAM and Haarlem sublineages, particularly in two major urban settings (Medellín and Cali). Dominant spoligotypes were LAM9 (SIT 42) and Haarlem (SIT62). The proportion of the Haarlem sublineage was higher in Colombia compared to that in neighboring countries, suggesting particular conditions of co-evolution with the corresponding human population that favor the success of this

  18. Design and Construction of a Cloning Vector Containing the hspX Gene of Mycobacterium tuberculosis.

    Science.gov (United States)

    Yaghoubi, Atieh; Aryan, Ehsan; Zare, Hosna; Alami, Shadi; Teimourpour, Roghayeh; Meshkat, Zahra

    2016-10-01

    Tuberculosis (TB) is a major cause of death worldwide. Finding an effective vaccine against TB is the best way to control it. Several vaccines against this disease have been developed but none are completely protective. The aim of this study was to design and construct a cloning vector containing the Mycobacterium tuberculosis (M. tuberculosis) heat shock protein X (hspX) . First, an hspX fragment was amplified by PCR and cloned into plasmid pcDNA3.1(+) and recombinant vector was confirmed. A 435 bp hspX fragment was isolated. The fragment was 100% homologous with hspX of M. tuberculosis strain H37Rv in GenBank. In this study, the cloning vector pcDNA3.1(+), containing a 435-bp hspX fragment of M. tuberculosis , was constructed. This could be used as a DNA vaccine to induce immune responses in animal models in future studies.

  19. Progenitor “Mycobacterium canettii” clone responsible for lymph node tuberculosis epidemic, Djibouti.

    Science.gov (United States)

    Blouin, Yann; Cazajous, Géraldine; Dehan, Céline; Soler, Charles; Vong, Rithy; Hassan, Mohamed Osman; Hauck, Yolande; Boulais, Christian; Andriamanantena, Dina; Martinaud, Christophe; Martin, Émilie; Pourcel, Christine; Vergnaud, Gilles

    2014-01-01

    Mycobacterium canettii,” an opportunistic human pathogen living in an unknown environmental reservoir, is the progenitor species from which Mycobacterium tuberculosis emerged. Since its discovery in 1969, most of the ≈70 known M. canettii strains were isolated in the Republic of Djibouti, frequently from expatriate children and adults. We show here, by whole-genome sequencing, that most strains collected from February 2010 through March 2013, and associated with 2 outbreaks of lymph node tuberculosis in children, belong to a unique epidemic clone within M. canettii. Evolution of this clone, which has been recovered regularly since 1983, may mimic the birth of M. tuberculosis. Thus, recognizing this organism and identifying its reservoir are clinically important.

  20. Intracellular bacillary burden reflects a burst size for Mycobacterium tuberculosis in vivo.

    Directory of Open Access Journals (Sweden)

    Teresa Repasy

    2013-02-01

    Full Text Available We previously reported that Mycobacterium tuberculosis triggers macrophage necrosis in vitro at a threshold intracellular load of ~25 bacilli. This suggests a model for tuberculosis where bacilli invading lung macrophages at low multiplicity of infection proliferate to burst size and spread to naïve phagocytes for repeated cycles of replication and cytolysis. The current study evaluated that model in vivo, an environment significantly more complex than in vitro culture. In the lungs of mice infected with M. tuberculosis by aerosol we observed three distinct mononuclear leukocyte populations (CD11b(- CD11c(+/hi, CD11b(+/lo CD11c(lo/-, CD11b(+/hi CD11c(+/hi and neutrophils hosting bacilli. Four weeks after aerosol challenge, CD11b(+/hi CD11c(+/hi mononuclear cells and neutrophils were the predominant hosts for M. tuberculosis while CD11b(+/lo CD11c(lo/- cells assumed that role by ten weeks. Alveolar macrophages (CD11b(- CD11c(+/hi were a minority infected cell type at both time points. The burst size model predicts that individual lung phagocytes would harbor a range of bacillary loads with most containing few bacilli, a smaller proportion containing many bacilli, and few or none exceeding a burst size load. Bacterial load per cell was enumerated in lung monocytic cells and neutrophils at time points after aerosol challenge of wild type and interferon-γ null mice. The resulting data fulfilled those predictions, suggesting a median in vivo burst size in the range of 20 to 40 bacilli for monocytic cells. Most heavily burdened monocytic cells were nonviable, with morphological features similar to those observed after high multiplicity challenge in vitro: nuclear condensation without fragmentation and disintegration of cell membranes without apoptotic vesicle formation. Neutrophils had a narrow range and lower peak bacillary burden than monocytic cells and some exhibited cell death with release of extracellular neutrophil traps. Our studies suggest

  1. Mechanish of dTTP Inhibition of the Bifunctional dCTP Deaminase:dUTPase Encoded by Mycobacterium tuberculosis

    DEFF Research Database (Denmark)

    Helt, Signe Smedegaard; Thymark, Majbritt; Harris, Pernille

    2008-01-01

    Recombinant deoxycytidine triphosphate (dCTP) deaminase from Mycobacterium tuberculosis was produced in Escherichia coli and purified. The enzyme proved to be a bifunctional dCTP deaminase:deoxyuridine triphosphatase. As such, the M. tuberculosis enzyme is the second bifunctional enzyme to be cha......Recombinant deoxycytidine triphosphate (dCTP) deaminase from Mycobacterium tuberculosis was produced in Escherichia coli and purified. The enzyme proved to be a bifunctional dCTP deaminase:deoxyuridine triphosphatase. As such, the M. tuberculosis enzyme is the second bifunctional enzyme...

  2. Multidrug-Resistant Mycobacterium tuberculosis of the Latin American Mediterranean Lineage, Wrongly Identified as Mycobacterium pinnipedii (Spoligotype International Type 863 [SIT863]), Causing Active Tuberculosis in South Brazil

    KAUST Repository

    Dalla Costa, Elis R.; Vasconcelos, Sidra E. G.; Esteves, Leonardo S.; Gomes, Harrison M.; Gomes, Lia L.; Almeida da Silva, Pedro; Perdigã o, Joã o; Portugal, Isabel; Viveiros, Miguel; McNerney, Ruth; Pain, Arnab; Clark, Taane G.; Rastogi, Nalin; Unis, Gisela; Rossetti, Maria Lucia R.; Suffys, Philip Noel

    2015-01-01

    We recently detected the spoligotype patterns of strains of Mycobacterium pinnipedii, a species of the Mycobacterium tuberculosis complex, in sputum samples from nine cases with pulmonary tuberculosis residing in Porto Alegre, South Brazil. Because this species is rarely encountered in humans, we further characterized these nine isolates by additional genotyping techniques, including 24-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) typing, verification of the loci TbD1, RD9, pks15/1, RDRio, and fbpC, the insertion of IS6110 at a site specific to the M. tuberculosis Latin American Mediterranean (LAM) lineage, and whole-genome sequencing. The combined analysis of these markers revealed that the isolates are in fact M. tuberculosis and more specifically belong to the LAM genotype. Most of these isolates (n = 8) were shown to be multidrug resistant (MDR), which prompted us to perform partial sequencing of the rpoA, rpoB, rpoC, katG, and inhA genes. Seven isolates (77.8%) carried the S315T mutation in katG, and one of these (11%) also presented the C(−17)T single-nucleotide polymorphism (SNP) in inhA. Interestingly, six of the MDR isolates also presented an undescribed insertion of 12 nucleotides (CCA GAA CAA CCC) in codon 516 of rpoB. No putative compensatory mutation was found in either rpoA or rpoC. This is the first report of an M. tuberculosis LAM family strain with a convergent M. pinnipedii spoligotype. These spoligotypes are observed in genotype databases at a modest frequency, highlighting that care must be taken when identifying isolates in the M. tuberculosis complex on the basis of single genetic markers.

  3. Multidrug-Resistant Mycobacterium tuberculosis of the Latin American Mediterranean Lineage, Wrongly Identified as Mycobacterium pinnipedii (Spoligotype International Type 863 [SIT863]), Causing Active Tuberculosis in South Brazil

    KAUST Repository

    Dalla Costa, Elis R.

    2015-09-23

    We recently detected the spoligotype patterns of strains of Mycobacterium pinnipedii, a species of the Mycobacterium tuberculosis complex, in sputum samples from nine cases with pulmonary tuberculosis residing in Porto Alegre, South Brazil. Because this species is rarely encountered in humans, we further characterized these nine isolates by additional genotyping techniques, including 24-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) typing, verification of the loci TbD1, RD9, pks15/1, RDRio, and fbpC, the insertion of IS6110 at a site specific to the M. tuberculosis Latin American Mediterranean (LAM) lineage, and whole-genome sequencing. The combined analysis of these markers revealed that the isolates are in fact M. tuberculosis and more specifically belong to the LAM genotype. Most of these isolates (n = 8) were shown to be multidrug resistant (MDR), which prompted us to perform partial sequencing of the rpoA, rpoB, rpoC, katG, and inhA genes. Seven isolates (77.8%) carried the S315T mutation in katG, and one of these (11%) also presented the C(−17)T single-nucleotide polymorphism (SNP) in inhA. Interestingly, six of the MDR isolates also presented an undescribed insertion of 12 nucleotides (CCA GAA CAA CCC) in codon 516 of rpoB. No putative compensatory mutation was found in either rpoA or rpoC. This is the first report of an M. tuberculosis LAM family strain with a convergent M. pinnipedii spoligotype. These spoligotypes are observed in genotype databases at a modest frequency, highlighting that care must be taken when identifying isolates in the M. tuberculosis complex on the basis of single genetic markers.

  4. Mycobacterium tuberculosis: just desserts for an ungrateful guest.

    Science.gov (United States)

    Halloran, M E

    1994-02-01

    Myobacterium tuberculosis is the most common infectious cause of death in the world, with up to one-third of the population infected. In industrial countries infection with M. tuberculosis and tuberculosis disease has been decreasing since the 19th century. Now, however, tuberculosis disease is on the increase again, with resistance of the bacillus to available drugs spreading rapidly. This resurgence can be seen from the ecological and evolutionary point of view, where human hosts are the niche of the tuberculosis bacillus. Copyright © 1994. Published by Elsevier Ltd.

  5. Evolution and role of corded cell aggregation in Mycobacterium tuberculosis cultures.

    Science.gov (United States)

    Caceres, Neus; Vilaplana, Cristina; Prats, Clara; Marzo, Elena; Llopis, Isaac; Valls, Joaquim; Lopez, Daniel; Cardona, Pere-Joan

    2013-11-01

    The aim of this study was to evaluate the evolution and role of corded cell aggregation in Mycobacterium tuberculosis cultures according to growth time and conditions. Thus, in standard culture using aerated 7H9 Middlebrook broth supplemented with 0.05% Tween 80, a dramatic CFU decrease was observed at the end of the exponential phase. This phase was followed by a stable stationary phase that led to dissociation between the optical density (O.D.) and CFU values, together with the formation of opaque colonies in solid culture. Further analysis revealed that this was due to cording. Scanning electron microscopy showed that cording led to the formation of very stable coiled structures and corded cell aggregations which proved impossible to disrupt by any of the physical means tested. Modulation of cording with a high but non-toxic concentration of Tween 80 led to a slower growth rate, avoidance of a sudden drop-off to the stationary phase, the formation of weaker cording structures and the absence of opaque colonies, together with a lower survival at later time-points. An innovative automated image analysis technique has been devised to characterize the cording process. This analysis has led to important practical consequences for the elaboration of M. tuberculosis inocula and suggests the importance of biofilm formation in survival of the bacilli in the extracellular milieu. Copyright © 2013. Published by Elsevier Ltd.

  6. Advance in Research on Mycobacterium tuberculosis FabG4 and Its Inhibitor

    Directory of Open Access Journals (Sweden)

    Debajyoti Dutta

    2018-06-01

    Full Text Available Increasing evidence from recent reports of drug-resistant mycobacterial strains poses a challenge worldwide. Drug-resistant strains often undergo mutations, adopt alternative pathways, and express drug efflux pumps to reduce or eliminate drug doses. Besides these intrinsic resistance mechanisms, bacteria can evade drug doses by forming biofilms. Biofilms are the concerted growth of adherent microorganisms, which can also be formed at the air-water interface. The growth is supported by the extracellular polymer matrix which is self-produced by the microorganisms. Reduced metabolic activity in a nutrient-deficient environment in the biofilm may cause the microorganisms to take alternative pathways that can make the microorganisms recalcitrant to the drug doses. Recent works have shown that Mycobacterium tuberculosis expresses several proteins during its growth in biofilm, those when deleted, did not show any effect on mycobacterial growth in normal nutrient-sufficient conditions. Studying these unconventional proteins in mycobacterial biofilms is therefore of utmost importance. In this article, I will discuss one such mycobacterial biofilm-related protein FabG4 that is recently shown to be important for mycobacterial survival in the presence of antibiotic stressors and limited nutrient condition. In an attempt to find more effective FabG4 inhibitors and its importance in biofilm forming M. tuberculosis, present knowledge about FabG4 and its known inhibitors are discussed. Based on the existing data, a putative role of FabG4 is also suggested.

  7. MicroRNA expression signatures in lungs of mice infected with Mycobacterium tuberculosis.

    Science.gov (United States)

    Malardo, Thiago; Gardinassi, Luiz Gustavo; Moreira, Bernardo Pereira; Padilha, Éverton; Lorenzi, Júlio César Cetrulo; Soares, Luana Silva; Gembre, Ana Flávia; Fontoura, Isabela Cardoso; de Almeida, Luciana Previato; de Miranda Santos, Isabel Kinney Ferreira; Silva, Célio Lopes; Coelho-Castelo, Arlete Aparecida Martins

    2016-12-01

    Tuberculosis (TB) is a major public health concern worldwide; however the factors that account for resistance or susceptibility to disease are not completely understood. Although some studies suggest that the differential expression of miRNAs in peripheral blood of TB patients could be useful as biomarkers of active disease, their involvement during the inflammatory process in lungs of infected individuals is unknown. Here, we evaluated the global expression of miRNAs in the lungs of mice experimentally infected with Mycobacterium tuberculosis on 30 and 60 days post-infection. We observed that several miRNAs were differentially expressed compared to uninfected mice. Furthermore, we verified that the expression of miR-135b, miR-21, miR-155, miR-146a, and miR-146b was significantly altered in distinct leukocyte subsets isolated from lungs of infected mice, while genes potentially targeted by those miRNAs were associated with a diversity of immune related molecular pathways. Importantly, we validated the inhibition of Pellino 1 expression by miR-135b in vitro. Overall, this study contributes to the understanding of the dynamics of miRNA expression in lungs during experimental TB and adds further perspectives into the role of miRNAs on the regulation of immune processes such as leukocyte activation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Hypoxia promotes Mycobacterium tuberculosis-specific up-regulation of granulysin in human T cells.

    Science.gov (United States)

    Zenk, Sebastian F; Vollmer, Michael; Schercher, Esra; Kallert, Stephanie; Kubis, Jan; Stenger, Steffen

    2016-06-01

    Oxygen tension affects local immune responses in inflammation and infection. In tuberculosis mycobacteria avoid hypoxic areas and preferentially persist and reactivate in the oxygen-rich apex of the lung. Oxygen restriction activates antimicrobial effector mechanisms in macrophages and restricts growth of intracellular Mycobacterium tuberculosis (M.Tb). The effect of oxygen restriction on T cell-mediated antimicrobial effector mechanisms is unknown. Therefore we determined the influence of hypoxia on the expression of granulysin, an antimicrobial peptide of lymphocytes. Hypoxia increased the antigen-specific up-regulation of granulysin mRNA and protein in human CD4(+) and CD8(+) T lymphocytes. This observation was functionally relevant, because oxygen restriction supported the growth-limiting effect of antigen-specific T cells against virulent M.Tb residing in primary human macrophages. Our results provide evidence that oxygen restriction promotes the expression of granulysin and suggest that this effect-in conjunction with additional T cell-mediated immune responses-supports protection against mycobacteria. The therapeutic modulation of oxygen availability may offer a new strategy for the host-directed therapy of infectious diseases with intracellular pathogens.

  9. Mycobacterium tuberculosis: Active disease and latent infection in a renal transplant cohort.

    Science.gov (United States)

    Rafiei, Nastaran; Williams, Jackie; Mulley, William R; Trauer, James M; Jenkin, Grant A; Rogers, Benjamin A

    2018-04-16

    Tuberculosis (TB) is a serious opportunistic infection in renal transplant recipients associated with high mortality. Screening and treatment of latent Mycobacterium tuberculosis infection (LTBI) offers an opportunity to prevent subsequent active disease. We retrospectively reviewed the records of all adult patients who underwent renal transplantation at our centre from 2005 to 2014 to assess current screening practices, the risks for and burden of active TB. A total of 660 individuals underwent renal transplantation during this period, totalling 3647 person years of follow up. Three patients were diagnosed with active TB after renal transplant, resulting in an incidence of 82 per 100,000 person-years. Of 656 transplant recipients, 102 (15.5%) were born in high TB incidence countries and 89 (13.5%) had an interferon gamma release assay (IGRA) at any point. Individuals born in high TB risk countries had a much higher incidence of active TB (530 per 100,000 person-years). Ten individuals had positive IGRA tests, of whom two were treated for active TB, two received chemoprophylaxis and six were not treated. In the absence of formal guidelines, IGRA-based screening for LTBI was infrequently performed. Our data suggests that screening and treatment of renal transplant recipients born in high incidence countries is an important preventive measure. This article is protected by copyright. All rights reserved.

  10. Systematic Survey of Serine Hydrolase Activity in Mycobacterium tuberculosis Defines Changes Associated with Persistence

    Energy Technology Data Exchange (ETDEWEB)

    Ortega, Corrie; Anderson, Lindsey N.; Frando, Andrew; Sadler, Natalie C.; Brown, Robert W.; Smith, Richard D.; Wright, Aaron T.; Grundner, Christoph

    2016-02-01

    The transition between replication and non-replication underlies much of Mycobacterium tuberculosis (Mtb) pathogenicity, as non- or slowly replicating Mtb are responsible for persistence and poor treatment outcomes. Therapeutic targeting of non-replicating, persistent populations is a priority for tuberculosis treatment, but only few drug targets in non-replicating Mtb are currently known. Here, we directly measure the activity of the highly diverse and druggable serine hydrolases (SHs) during active replication and non-replication by activity-based proteomics. We predict serine hydrolase activity for 78 proteins, including 27 proteins with previously unknown function, and identify 37 SHs that remain active even in the absence of replication, providing a set of candidate persistence targets. Non-replication was associated with large shifts in the activity of the majority of SHs. These activity changes were largely independent of SH abundance, indicating extensive post-translational regulation. By probing a large cross-section of druggable Mtb enzyme space during replication and non-replication, we identify new SHs and suggest new persistence targets.

  11. Structure of Mycobacterium tuberculosis Rv2714, a representative of a duplicated gene family in Actinobacteria

    International Nuclear Information System (INIS)

    Graña, Martin; Bellinzoni, Marco; Miras, Isabelle; Fiez-Vandal, Cedric; Haouz, Ahmed; Shepard, William; Buschiazzo, Alejandro; Alzari, Pedro M.

    2009-01-01

    The crystal structure of Rv2714, a protein of unknown function from M. tuberculosis, has been determined at 2.6 Å resolution using single-wavelength anomalous diffraction methods. The gene Rv2714 from Mycobacterium tuberculosis, which codes for a hypothetical protein of unknown function, is a representative member of a gene family that is largely confined to the order Actinomycetales of Actinobacteria. Sequence analysis indicates the presence of two paralogous genes in most mycobacterial genomes and suggests that gene duplication was an ancient event in bacterial evolution. The crystal structure of Rv2714 has been determined at 2.6 Å resolution, revealing a trimer in which the topology of the protomer core is similar to that observed in a functionally diverse set of enzymes, including purine nucleoside phosphorylases, some carboxypeptidases, bacterial peptidyl-tRNA hydrolases and even the plastidic form of an intron splicing factor. However, some structural elements, such as a β-hairpin insertion involved in protein oligomerization and a C-terminal α-helical domain that serves as a lid to the putative substrate-binding (or ligand-binding) site, are only found in Rv2714 bacterial homologues and represent specific signatures of this protein family

  12. Structure of Mycobacterium tuberculosis Rv2714, a representative of a duplicated gene family in Actinobacteria

    Energy Technology Data Exchange (ETDEWEB)

    Graña, Martin; Bellinzoni, Marco [Institut Pasteur, Unité de Biochimie Structurale, URA CNRS 2185, 25 Rue du Dr Roux, 75724 Paris (France); Miras, Isabelle; Fiez-Vandal, Cedric; Haouz, Ahmed; Shepard, William [Institut Pasteur, Plate-forme de Cristallogenèse et Diffraction des Rayons X, 25 Rue du Dr Roux, 75724 Paris (France); Buschiazzo, Alejandro; Alzari, Pedro M., E-mail: alzari@pasteur.fr [Institut Pasteur, Unité de Biochimie Structurale, URA CNRS 2185, 25 Rue du Dr Roux, 75724 Paris (France)

    2009-10-01

    The crystal structure of Rv2714, a protein of unknown function from M. tuberculosis, has been determined at 2.6 Å resolution using single-wavelength anomalous diffraction methods. The gene Rv2714 from Mycobacterium tuberculosis, which codes for a hypothetical protein of unknown function, is a representative member of a gene family that is largely confined to the order Actinomycetales of Actinobacteria. Sequence analysis indicates the presence of two paralogous genes in most mycobacterial genomes and suggests that gene duplication was an ancient event in bacterial evolution. The crystal structure of Rv2714 has been determined at 2.6 Å resolution, revealing a trimer in which the topology of the protomer core is similar to that observed in a functionally diverse set of enzymes, including purine nucleoside phosphorylases, some carboxypeptidases, bacterial peptidyl-tRNA hydrolases and even the plastidic form of an intron splicing factor. However, some structural elements, such as a β-hairpin insertion involved in protein oligomerization and a C-terminal α-helical domain that serves as a lid to the putative substrate-binding (or ligand-binding) site, are only found in Rv2714 bacterial homologues and represent specific signatures of this protein family.

  13. bioA mutant of Mycobacterium tuberculosis shows severe growth defect and imparts protection against tuberculosis in guinea pigs

    Science.gov (United States)

    Kar, Ritika; Nangpal, Prachi; Mathur, Shubhita; Singh, Swati

    2017-01-01

    Owing to the devastation caused by tuberculosis along with the unsatisfactory performance of the Bacillus Calmette–Guérin (BCG) vaccine, a more efficient vaccine than BCG is required for the global control of tuberculosis. A number of studies have demonstrated an essential role of biotin biosynthesis in the growth and survival of several microorganisms, including mycobacteria, through deletion of the genes involved in de novo biotin biosynthesis. In this study, we demonstrate that a bioA mutant of Mycobacterium tuberculosis (MtbΔbioA) is highly attenuated in the guinea pig model of tuberculosis when administered aerogenically as well as intradermally. Immunization with MtbΔbioA conferred significant protection in guinea pigs against an aerosol challenge with virulent M. tuberculosis, when compared with the unvaccinated animals. Booster immunization with MtbΔbioA offered no advantage over a single immunization. These experiments demonstrate the vaccinogenic potential of the attenuated M. tuberculosis bioA mutant against tuberculosis. PMID:28658275

  14. bioA mutant of Mycobacterium tuberculosis shows severe growth defect and imparts protection against tuberculosis in guinea pigs.

    Directory of Open Access Journals (Sweden)

    Ritika Kar

    Full Text Available Owing to the devastation caused by tuberculosis along with the unsatisfactory performance of the Bacillus Calmette-Guérin (BCG vaccine, a more efficient vaccine than BCG is required for the global control of tuberculosis. A number of studies have demonstrated an essential role of biotin biosynthesis in the growth and survival of several microorganisms, including mycobacteria, through deletion of the genes involved in de novo biotin biosynthesis. In this study, we demonstrate that a bioA mutant of Mycobacterium tuberculosis (MtbΔbioA is highly attenuated in the guinea pig model of tuberculosis when administered aerogenically as well as intradermally. Immunization with MtbΔbioA conferred significant protection in guinea pigs against an aerosol challenge with virulent M. tuberculosis, when compared with the unvaccinated animals. Booster immunization with MtbΔbioA offered no advantage over a single immunization. These experiments demonstrate the vaccinogenic potential of the attenuated M. tuberculosis bioA mutant against tuberculosis.

  15. Evidence of presence of Mycobacterium tuberculosis in bovine tissue samples by multiplex PCR: possible relevance to reverse zoonosis.

    Science.gov (United States)

    Mittal, M; Chakravarti, S; Sharma, V; Sanjeeth, B S; Churamani, C P; Kanwar, N S

    2014-04-01

    Bovine tuberculosis, caused by Mycobacterium bovis, remains one of the most important zoonotic health concerns worldwide. The transmission of Mycobacterium tuberculosis from humans to animals also occurs especially in countries where there is close interaction of humans with the animals. In the present study, thirty bovine lung tissue autopsy samples from an organized dairy farm located in North India were screened for the presence of Mycobacterium tuberculosis complex by smear microscopy, histopathological findings and PCR. Differential diagnosis of M. tuberculosis and M. bovis was made based on the deletion of mce-3 operon in M. bovis. The present study found eight of these samples positive for M. tuberculosis by multiplex PCR. Sequencing was performed on two PCR-positive representative samples and on annotation, and BLAST analysis confirmed the presence of gene fragment specific to Mycobacterium tuberculosis. The presence of M. tuberculosis in all the positive samples raises the possibility of human-to-cattle transmission and possible adaptation of this organism in bovine tissues. This study accentuates the importance of screening and differential diagnosis of Mycobacterium tuberculosis complex in humans and livestock for adopting effective TB control and eradication programmes. © 2014 Blackwell Verlag GmbH.

  16. Direct detection of rpoB and katG gene mutations of Mycobacterium tuberculosis in clinical samples

    Directory of Open Access Journals (Sweden)

    Sunil Pandey

    2017-08-01

    Conclusions: We can conclude that genetic mutation in Mycobacterium tuberculosis can be identified directly from the clinical samples. However, we have carried this study in less sample size and to validate research on large number of sample is recommended.

  17. Phenotypic and genotypic analysis of anti-tuberculosis drug resistance in Mycobacterium tuberculosis isolates in Myanmar.

    Science.gov (United States)

    Aung, Wah Wah; Ei, Phyu Win; Nyunt, Wint Wint; Swe, Thyn Lei; Lwin, Thandar; Htwe, Mi Mi; Kim, Kyung Jun; Lee, Jong Seok; Kim, Chang Ki; Cho, Sang Nae; Song, Sun Dae; Chang, Chulhun L

    2015-09-01

    Tuberculosis (TB) is one of the most serious health problems in Myanmar. Because TB drug resistance is associated with genetic mutation(s) relevant to responses to each drug, genotypic methods for detecting these mutations have been proposed to overcome the limitations of classic phenotypic drug susceptibility testing (DST). We explored the current estimates of drug-resistant TB and evaluated the usefulness of genotypic DST in Myanmar. We determined the drug susceptibility of Mycobacterium tuberculosis isolated from sputum smear-positive patients with newly diagnosed pulmonary TB at two main TB centers in Myanmar during 2013 by using conventional phenotypic DST and the GenoType MTBDRplus assay (Hain Lifescience, Germany). Discrepant results were confirmed by sequencing the genes relevant to each type of resistance (rpoB for rifampicin; katG and inhA for isoniazid). Of 191 isolates, phenotypic DST showed that 27.7% (n=53) were resistant to at least one first-line drug and 20.9% (n=40) were resistant to two or more, including 18.3% (n=35) multidrug-resistant TB (MDR-TB) strains. Monoresistant strains accounted for 6.8% (n=13) of the samples. Genotypic assay of 189 isolates showed 17.5% (n=33) MDR-TB and 5.3% (n=10) isoniazid-monoresistant strains. Genotypic susceptibility results were 99.5% (n=188) concordant and agreed almost perfectly with phenotypic DST (kappa=0.99; 95% confidence interval 0.96-1.01). The results highlight the burden of TB drug resistance and prove the usefulness of the genotypic DST in Myanmar.

  18. Tuberculosis

    NARCIS (Netherlands)

    Ankrah, Alfred O; Glaudemans, Andor W J M; Maes, Alex; Van de Wiele, Christophe; Dierckx, Rudi A J O; Vorster, Mariza; Sathekge, Mike M

    Tuberculosis (TB) is currently the world's leading cause of infectious mortality. Imaging plays an important role in the management of this disease. The complex immune response of the human body to Mycobacterium tuberculosis results in a wide array of clinical manifestations, making clinical and

  19. The Mycobacterium tuberculosis Rv2540c DNA sequence encodes a bifunctional chorismate synthase

    Directory of Open Access Journals (Sweden)

    Santos Diógenes S

    2008-04-01

    Full Text Available Abstract Background The emergence of multi- and extensively-drug resistant Mycobacterium tuberculosis strains has created an urgent need for new agents to treat tuberculosis (TB. The enzymes of shikimate pathway are attractive targets to the development of antitubercular agents because it is essential for M. tuberculosis and is absent from humans. Chorismate synthase (CS is the seventh enzyme of this route and catalyzes the NADH- and FMN-dependent synthesis of chorismate, a precursor of aromatic amino acids, naphthoquinones, menaquinones, and mycobactins. Although the M. tuberculosis Rv2540c (aroF sequence has been annotated to encode a chorismate synthase, there has been no report on its correct assignment and functional characterization of its protein product. Results In the present work, we describe DNA amplification of aroF-encoded CS from M. tuberculosis (MtCS, molecular cloning, protein expression, and purification to homogeneity. N-terminal amino acid sequencing, mass spectrometry and gel filtration chromatography were employed to determine identity, subunit molecular weight and oligomeric state in solution of homogeneous recombinant MtCS. The bifunctionality of MtCS was determined by measurements of both chorismate synthase and NADH:FMN oxidoreductase activities. The flavin reductase activity was characterized, showing the existence of a complex between FMNox and MtCS. FMNox and NADH equilibrium binding was measured. Primary deuterium, solvent and multiple kinetic isotope effects are described and suggest distinct steps for hydride and proton transfers, with the former being more rate-limiting. Conclusion This is the first report showing that a bacterial CS is bifunctional. Primary deuterium kinetic isotope effects show that C4-proS hydrogen is being transferred during the reduction of FMNox by NADH and that hydride transfer contributes significantly to the rate-limiting step of FMN reduction reaction. Solvent kinetic isotope effects and

  20. Diagnosis of latent Mycobacterium tuberculosis infection: is the demise of the Mantoux test imminent?

    Science.gov (United States)

    Rothel, James S; Andersen, Peter

    2005-12-01

    Tuberculosis is responsible for more then 2 million deaths worldwide each year and vies with HIV as the world's most fatal infectious disease. In many developing countries, attempts to control the spread of infection rely solely on identification and treatment of those with active disease, ignoring subclinical infection. However, in developed countries, large efforts are also expended to identify and give prophylactic drugs to people with latent tuberculosis infection. Until recently, the 100-year-old tuberculin skin test (Mantoux) has been the only available diagnostic test for latent tuberculosis infection, despite its many well-known limitations. Advances in scientific knowledge have led to the development of tests for tuberculosis that measure the production of interferon-gamma by T-cells stimulated in vitro with Mycobacterium tuberculosis-specific antigens. These interferon-gamma tests are highly specific and unaffected by prior Bacille Calmette-Guérin vaccination or immune reactivity to most atypical mycobacteria. They are more sensitive than the tuberculin skin test in detecting people with active tuberculosis, and their results correlate more closely with M. tuberculosis exposure risk factors than the tuberculin skin test in people likely to have latent tuberculosis infection. Science has caught up with one of the oldest diagnostic tests still in use worldwide, and the adoption of new, tuberculosis-specific interferon-gamma-based tests should move us one step closer to better control of this insidious pathogen.

  1. The biological and structural characterization of Mycobacterium tuberculosis UvrA provides novel insights into its mechanism of action.

    Science.gov (United States)

    Rossi, Franca; Khanduja, Jasbeer Singh; Bortoluzzi, Alessio; Houghton, Joanna; Sander, Peter; Güthlein, Carolin; Davis, Elaine O; Springer, Burkhard; Böttger, Erik C; Relini, Annalisa; Penco, Amanda; Muniyappa, K; Rizzi, Menico

    2011-09-01

    Mycobacterium tuberculosis is an extremely well adapted intracellular human pathogen that is exposed to multiple DNA damaging chemical assaults originating from the host defence mechanisms. As a consequence, this bacterium is thought to possess highly efficient DNA repair machineries, the nucleotide excision repair (NER) system amongst these. Although NER is of central importance to DNA repair in M. tuberculosis, our understanding of the processes in this species is limited. The conserved UvrABC endonuclease represents the multi-enzymatic core in bacterial NER, where the UvrA ATPase provides the DNA lesion-sensing function. The herein reported genetic analysis demonstrates that M. tuberculosis UvrA is important for the repair of nitrosative and oxidative DNA damage. Moreover, our biochemical and structural characterization of recombinant M. tuberculosis UvrA contributes new insights into its mechanism of action. In particular, the structural investigation reveals an unprecedented conformation of the UvrB-binding domain that we propose to be of functional relevance. Taken together, our data suggest UvrA as a potential target for the development of novel anti-tubercular agents and provide a biochemical framework for the identification of small-molecule inhibitors interfering with the NER activity in M. tuberculosis.

  2. Inhibition of apoptosis by Rv2456c through nuclear factor-κB extends the survival of Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Kristen L. Jurcic Smith

    2016-01-01

    Full Text Available Mycobacterium tuberculosis, the causative agent of tuberculosis, is an intracellular pathogen with several survival mechanisms aimed at subverting the host immune system. Apoptosis has been shown to be mycobactericidal, to activate CD8+ T cells, and to be modulated by mycobacterial proteins. Since few mycobacterial proteins have so far been directly implicated in the interactions between M. Tuberculosis and host cell apoptosis, we screened M. Tuberculosis H37Rv transposon mutants to identify mutants that fail to inhibit cell death (FID. One of these FID mutants, FID19, had a transposon insertion in Rv2456c and is important for survival in host cells. The lack of the protein resulted in enhanced caspase-3 mediated apoptosis, which is probably due to an inability to activate nuclear factor-κB. Additionally, FID19 infection enhanced polyfunctional CD8+ T cells and induced a higher frequency of interferon-γ secreting immune cells in a murine model. Taken together, our data suggest that Rv2456c is important for the survival of H37Rv by subduing the innate and ultimately adaptive immune responses of its host by preventing apoptosis of the infected cell. Better understanding of the host-mycobacterial interactions may be beneficial to develop novel drug targets and engineer more efficacious vaccine strains against tuberculosis.

  3. Different Transcriptional Profiles of Human Monocyte-Derived Dendritic Cells Infected with Distinct Strains of Mycobacterium tuberculosis and Mycobacterium bovis Bacillus Calmette-Guérin

    Directory of Open Access Journals (Sweden)

    Nunzia Sanarico

    2011-01-01

    Full Text Available In order to analyze dendritic cells (DCs activation following infection with different mycobacterial strains, we studied the expression profiles of 165 genes of human monocyte-derived DCs infected with H37Rv, a virulent Mycobacterium tuberculosis (MTB laboratory strain, CMT97, a clinical MTB isolate, Mycobacterium bovis bacillus Calmette-Guérin (BCG, Aventis Pasteur, and BCG Japan, both employed as vaccine against tuberculosis. The analysis of the gene expression reveals that, despite a set of genes similarly modulated, DCs response resulted strain dependent. In particular, H37Rv significantly upregulated EBI3 expression compared with BCG Japan, while it was the only strain that failed to release a significant IL-10 amount. Of note, BCG Japan showed a marked increase in CCR7 and TNF-α expression regarding both MTB strains and it resulted the only strain failing in exponential intracellular growth. Our results suggest that DCs display the ability to elicit a tailored strain-specific immune response.

  4. Cytokine responses in relation to age, gender, body mass index, Mycobacterium tuberculosis infection, and otitis media among inuit in greenland

    DEFF Research Database (Denmark)

    Nielsen, Nina Odgaard; Soborg, Bolette; Børresen, Malene

    2013-01-01

    To evaluate the cytokine response pattern in Inuit in Greenland in relation to age, gender, body mass index (BMI), Mycobacterium tuberculosis infection (MTI), and otitis media (OM) to assess whether Inuit may have signs of impaired immune responsiveness to infection.......To evaluate the cytokine response pattern in Inuit in Greenland in relation to age, gender, body mass index (BMI), Mycobacterium tuberculosis infection (MTI), and otitis media (OM) to assess whether Inuit may have signs of impaired immune responsiveness to infection....

  5. MenA Is a Promising Drug Target for Developing Novel Lead Molecules to Combat Mycobacterium tuberculosis

    OpenAIRE

    Kurosu, Michio; Crick, Dean C.

    2009-01-01

    Potent inhibitors of MenA (1,4-dihydroxy-2-naphtoate prenyltrasferase) in Mycobacterium tuberculosis are identified, and are also effective in inhibiting growth of Mycobacterium tuberculosis at low concentrations. The MenA inhibitors possess common chemical structural features of ((alkylamino)alkoxyphenyl)(phenyl)methanones. Significantly, the MenA inhibitors can be synthesized in a few steps with high overall yields. The representative MenA inhibitors are highly effective in killing nonrepli...

  6. Molecular Strain Typing of Mycobacterium tuberculosis: a Review of Frequently Used Methods

    Science.gov (United States)

    2016-01-01

    Tuberculosis, caused by the bacterium Mycobacterium tuberculosis, remains one of the most serious global health problems. Molecular typing of M. tuberculosis has been used for various epidemiologic purposes as well as for clinical management. Currently, many techniques are available to type M. tuberculosis. Choosing the most appropriate technique in accordance with the existing laboratory conditions and the specific features of the geographic region is important. Insertion sequence IS6110-based restriction fragment length polymorphism (RFLP) analysis is considered the gold standard for the molecular epidemiologic investigations of tuberculosis. However, other polymerase chain reaction-based methods such as spacer oligonucleotide typing (spoligotyping), which detects 43 spacer sequence-interspersing direct repeats (DRs) in the genomic DR region; mycobacterial interspersed repetitive units–variable number tandem repeats, (MIRU-VNTR), which determines the number and size of tandem repetitive DNA sequences; repetitive-sequence-based PCR (rep-PCR), which provides high-throughput genotypic fingerprinting of multiple Mycobacterium species; and the recently developed genome-based whole genome sequencing methods demonstrate similar discriminatory power and greater convenience. This review focuses on techniques frequently used for the molecular typing of M. tuberculosis and discusses their general aspects and applications. PMID:27709842

  7. Orchestration of pulmonary T cell immunity during Mycobacterium tuberculosis infection: immunity interruptus

    Science.gov (United States)

    Behar, Samuel M.; Carpenter, Stephen M.; Booty, Matthew G.; Barber, Daniel L.; Jayaraman, Pushpa

    2014-01-01

    Despite the introduction almost a century ago of Mycobacterium bovis BCG (BCG), an attenuated form of M. bovis that is used as a vaccine against Mycobacterium tuberculosis, tuberculosis remains a global health threat and kills more than 1.5 million people each year. This is mostly because BCG fails to prevent pulmonary disease – the contagious form of tuberculosis. Although there have been significant advances in understanding how the immune system responds to infection, the qualities that define protective immunity against M. tuberculosis remain poorly characterized. The ability to predict who will maintain control over the infection and who will succumb to clinical disease would revolutionize our approach to surveillance, control, and treatment. Here we review the current understanding of pulmonary T cell responses following M. tuberculosis infection. While infection elicits a strong immune response that contains infection, M. tuberculosis evades eradication. Traditionally, its intracellular lifestyle and alteration of macrophage function are viewed as the dominant mechanisms of evasion. Now we appreciate that chronic inflammation leads to T cell dysfunction. While this may arise as the host balances the goals of bacterial sterilization and avoidance of tissue damage, it is becoming clear that T cell dysfunction impairs host resistance. Defining the mechanisms that lead to T cell dysfunction is crucial as memory T cell responses are likely to be subject to the same subject to the same pressures. Thus, success of T cell based vaccines is predicated on memory T cells avoiding exhaustion while at the same time not promoting overt tissue damage. PMID:25311810

  8. Transmission of Mycobacterium tuberculosis from patients who are nucleic acid amplification test- negative.

    Science.gov (United States)

    Xie, Yingda L; Cronin, Wendy A; Proschan, Michael; Oatis, Richard; Cohn, Silvia; Curry, Scott R; Golub, Jonathan E; Barry Iii, Clifton E; Dorman, Susan E

    2018-04-24

    Among adults with signs and symptoms of pulmonary tuberculosis (TB), recognition of transmissible TB has implications for airborne infection isolation and public health activities. Sputum smear-negative TB patients account for around one-fifth of tuberculosis transmission. The tuberculosis transmission risk of TB patients with negative results on nucleic acid amplification (NAA) testing of respiratory specimens has not been established. We sought to estimate the tuberculosis transmission risk of NAA test-negative TB patients. We retrospectively reviewed Maryland TB program data from 2004 to 2009 during which NAA testing by the Mycobacterium Tuberculosis Direct Test (MTD) was performed routinely. Patients with sputum Mycobacterium tuberculosis (M.tb) isolates having matching genotypes were assigned to clusters. Transmission sequence was approximated by collection order of individuals' first culture-positive specimens. Minimum transmission risks of NAA (MTD)-negative TB patients and of smear-negative TB patients were estimated based on individuals' positions within clusters. Among 809 patients with culture-confirmed TB, M.tb genotypes were available for 782 (96.7%). For NAA-negative TB patients the minimum transmission risk estimate was 5.1% (95% CI 0-11.4). For smear-negative TB patients the minimum transmission risk estimate was 11.2% (95% CI 7.2-15.3). Minimum transmission risk of NAA-negative TB patients was lower than that of smear-negative TB patients. However, transmission risk of NAA-negative TB patients appears to not be negligible.

  9. The response of Mycobacterium tuberculosis to reactive oxygen and nitrogen species

    Directory of Open Access Journals (Sweden)

    Martin I Voskuil

    2011-05-01

    Full Text Available The bacteriostatic and bacteriocidal effects and the transcriptional response of Mycobacterium tuberculosis to representative oxidative and nitrosative stresses were investigated by growth and survival studies and whole genome expression analysis. The M. tuberculosis reaction to a range of hydrogen peroxide (H2O2 concentrations fell into three distinct categories: (1 low level exposure resulted in induction of a few highly sensitive H2O2-responsive genes, (2 intermediate exposure resulted in massive transcriptional changes without an effect on growth or survival, and (3 high exposure resulted in a muted transcriptional response and eventual death. M. tuberculosis appears highly resistant to DNA damage-dependent, mode-one killing caused by low millimolar levels of H2O2 and only succumbs to overwhelming levels of oxidative stress observed in mode-two killing. Nitric oxide (NO exposure initiated much the same transcriptional response as H2O2. However, unlike H2O2 exposure, NO exposure induced dormancy-related genes and caused dose-dependent bacteriostatic activity without killing. Included in the large shared response to H2O2 and NO was the induction of genes encoding iron-sulfur cluster repair functions including iron acquisition. Stress regulons controlled by IdeR, Sigma H, Sigma E, and FurA comprised a large portion of the response to both stresses. Expression of several oxidative stress defense genes was constitutive, or increased moderately from an already elevated constitutive level, suggesting that bacilli are continually primed for oxidative stress defense.

  10. Cytosolic proteome profiling of aminoglycosides resistant Mycobacterium tuberculosis clinical isolates using MALDI-TOF/MS

    Directory of Open Access Journals (Sweden)

    Divakar Sharma

    2016-11-01

    Full Text Available Emergence of extremely drug resistant tuberculosis (XDR-TB is the consequence of the failure of second line TB treatment. Aminoglycosides are the important second line anti-TB drugs used to treat the multi drug resistant tuberculosis (MDR-TB. Main known mechanism of action of aminoglycosides is to inhibit the protein synthesis by inhibiting the normal functioning of ribosome. Primary target of aminoglycosides are the ribosomal RNA and its associated proteins. Various mechanisms have been proposed for aminoglycosides resistance but still some are unsolved. As proteins are involved in most of the biological processes, these act as a potential diagnostic markers and drug targets. In the present study we analyzed the purely cytosolic proteome of amikacin (AK and kanamycin (KM resistant Mycobacterium tuberculosis isolates by proteomic and bioinformatic approaches. Twenty protein spots were found to have over expressed in resistant isolates and were identified. Among these Rv3208A, Rv2623, Rv1360, Rv2140c, Rv1636 and Rv2185c are six proteins with unknown functions or undefined role. Docking results showed that AK and KM binds to the conserved domain (DUF, USP-A, Luciferase, PEBP and Polyketidecyclase/dehydrase domain of these hypothetical proteins and over expression of these proteins might neutralize/modulate the effect of drug molecules. TBPred and GPS-PUP predicted cytoplasmic nature and potential pupylation sites within these identified proteins respectively. String analysis also suggested that over expressed proteins along with their interactive partners might be involved in aminoglycosides resistance. Cumulative effect of these over expressed proteins could be involved in AK and KM resistance by mitigating the toxicity, repression of drug target and neutralizing affect. These findings need further exploitation for the expansion of newer therapeutics or diagnostic markers against AK and KM resistance so that an extreme condition like XDR-TB can

  11. Cytosolic Proteome Profiling of Aminoglycosides Resistant Mycobacterium tuberculosis Clinical Isolates Using MALDI-TOF/MS.

    Science.gov (United States)

    Sharma, Divakar; Lata, Manju; Singh, Rananjay; Deo, Nirmala; Venkatesan, Krishnamurthy; Bisht, Deepa

    2016-01-01

    Emergence of extensively drug resistant tuberculosis (XDR-TB) is the consequence of the failure of second line TB treatment. Aminoglycosides are the important second line anti-TB drugs used to treat the multi drug resistant tuberculosis (MDR-TB). Main known mechanism of action of aminoglycosides is to inhibit the protein synthesis by inhibiting the normal functioning of ribosome. Primary target of aminoglycosides are the ribosomal RNA and its associated proteins. Various mechanisms have been proposed for aminoglycosides resistance but still some are unsolved. As proteins are involved in most of the biological processes, these act as a potential diagnostic markers and drug targets. In the present study we analyzed the purely cytosolic proteome of amikacin (AK) and kanamycin (KM) resistant Mycobacterium tuberculosis isolates by proteomic and bioinformatic approaches. Twenty protein spots were found to have over expressed in resistant isolates and were identified. Among these Rv3208A, Rv2623, Rv1360, Rv2140c, Rv1636, and Rv2185c are six proteins with unknown functions or undefined role. Docking results showed that AK and KM binds to the conserved domain (DUF, USP-A, Luciferase, PEBP and Polyketidecyclase/dehydrase domain) of these hypothetical proteins and over expression of these proteins might neutralize/modulate the effect of drug molecules. TBPred and GPS-PUP predicted cytoplasmic nature and potential pupylation sites within these identified proteins, respectively. String analysis also suggested that over expressed proteins along with their interactive partners might be involved in aminoglycosides resistance. Cumulative effect of these over expressed proteins could be involved in AK and KM resistance by mitigating the toxicity, repression of drug target and neutralizing affect. These findings need further exploitation for the expansion of newer therapeutics or diagnostic markers against AK and KM resistance so that an extreme condition like XDR-TB can be prevented.

  12. L-form transformation phenomenon in Mycobacterium tuberculosis associated with drug tolerance to ethambutol

    Directory of Open Access Journals (Sweden)

    Georgi Slavchev

    2016-01-01

    Full Text Available Objective/background: Cell wall-deficient bacterial forms (L-forms may occur along with resistance to factors that trigger their appearance. It is of interest to study the relationship between the L-form transformation of Mycobacterium tuberculosis and the exhibition of drug tolerance to ethambutol (EMB, an inhibitor of cell wall synthesis. Methods: L-form variant was produced from a sensitive EMB strain of M. Tuberculosis through a cryogenic stress treatment protocol and was subsequently cultivated in Middlebrook 7H9 semisolid medium, containing EMB in a minimal inhibitory concentration of 2 mg/L. Susceptibility to EMB of the parental strain and its L-form variant was evaluated phenotypically and using polymerase chain reaction-restriction fragment length polymorphism assay targeting a mutation in the embB306 gene fragment. Results: In contrast to the sensitivity to EMB of the parental strain, its L-form variant showed phenotypic resistance to high concentrations of EMB (16 mg/L, but the mutation in embB306 was not found. Electron microscopy observation of the L-form variant showed a heterogenic population of bacteria, with different degrees of cell wall deficiency, as well as cells of protoplastic type without cell walls. Of special interest were the observed capsule-like structures around the L-form cells and the biofilm-like matrix produced by the L-form population. Conclusion: We suggest that the expression of phenotypic resistance to EMB in M. Tuberculosis can be associated with alterations or loss of cell walls in L-form bacteria, respectively, which results in a lack of a specific target for EMB action. In addition, production of capsule-like structures and biofilm matrix by L-forms could contribute to their resistance and survival in the presence of antibacterial agents.

  13. Tuberculosis

    International Nuclear Information System (INIS)

    Latorre Tortello, Pablo

    1998-01-01

    The tuberculosis is an infection bacterial chronicle of world distribution. Three organisms of the family of the mycobacterium, the m. tuberculosis, the m. bovis and m. africanum, phenotypic and genetically similar, produce it, but only the m. tuberculosis has importance; the others rarely produce illness in the human. By definition, the lung tuberculosis is the localization of the m. tuberculosis in the breathing tract, the most common and main form in the affection and the only able to contaminate to other people. The koch bacillus, transmits the illness directly person to person. The paper Includes topics like pathogenesis, natural history, epidemiology, diagnose, symptomatology and treatment

  14. TUBERCULOSIS

    OpenAIRE

    Tarik Bajrović; Mahmud Nurkić; Šukrija Zvizdić

    2013-01-01

    Tuberculosis, known as the "White Plague" in the early 19th century, is the infectious disease, which is being researched today even in some of the most developed countries in the world. Epidemiological- epizootiological research points to the importance of pasteurizing milk as well as the transmission in aerosolized droplets in humans and animals. Mycobacterium tuberculosis (Mtb), M. bovis, M. africanum and M. microti are the mycobacteria that cause tuberculosis. Other mycobacteria cause dis...

  15. Ex vivo expansion of alveolar macrophages with Mycobacterium tuberculosis from the resected lungs of patients with pulmonary tuberculosis

    Science.gov (United States)

    Petrunina, Ekaterina; Umpeleva, Tatiana; Karskanova, Svetlana; Bayborodin, Sergey; Vakhrusheva, Diana; Kravchenko, Marionella; Skornyakov, Sergey

    2018-01-01

    Tuberculosis (TB), with the Mycobacterium tuberculosis (Mtb) as the causative agent, remains to be a serious world health problem. Traditional methods used for the study of Mtb in the lungs of TB patients do not provide information about the number and functional status of Mtb, especially if Mtb are located in alveolar macrophages. We have developed a technique to produce ex vivo cultures of cells from different parts of lung tissues surgically removed from patients with pulmonary TB and compared data on the number of cells with Mtb inferred by the proposed technique to the results of bacteriological and histological analyses used for examination of the resected lungs. The ex vivo cultures of cells obtained from the resected lungs of all patients were largely composed of CD14-positive alveolar macrophages, foamy or not, with or without Mtb. Lymphocytes, fibroblasts, neutrophils, and multinucleate Langhans giant cells were also observed. We found alveolar macrophages with Mtb in the ex vivo cultures of cells from the resected lungs of even those TB patients, whose sputum smears and lung tissues did not contain acid-fast Mtb or reveal growing Mtb colonies on dense medium. The detection of alveolar macrophages with Mtb in ex vivo culture as soon as 16–18 h after isolation of cells from the resected lungs of all TB patients suggests that the technique proposed for assessing the level of infection in alveolar macrophages of TB patients has higher sensitivity than do prolonged bacteriological or pathomorphological methods. The proposed technique allowed us to rapidly (in two days after surgery) determine the level of infection with Mtb in the cells of the resected lungs of TB patients and, by the presence or absence of Mtb colonies, including those with cording morphology, the functional status of the TB agent at the time of surgery. PMID:29401466

  16. Aspectos relevantes del uso de Mycobacterium´habana´ como candidato vacunal contra la tuberculosis

    Directory of Open Access Journals (Sweden)

    Iliana Valdés

    2011-12-01

    Full Text Available La eficacia protectora de la actual vacuna (BCG contra la tuberculosis, para contrarrestar las formas pulmonares de esta enfermedad y su reactivación, resulta variable o poco eficiente, lo cual impone la búsqueda urgente de nuevas alternativas profilácticas contra esta enfermedad. Basados en las ventajas inmunogénicas que ofrece el uso de vacunas vivas, se han encaminado diferentes estrategias de este tipo empleando mutantes auxotróficos de Mycobacterium tuberculosis , BCG recombinante o micobacterias no tuberculosas. Existen evidencias experimentales acerca de la protección conferida tras la vacunación con cepas vivas, inactivadas o fracciones proteicas de Mycobacterium'habana' TMC-5135 contra la infección por Mycobacterium tuberculosis. Esta respuesta protectora parece ir aparejada de escasos signos de virulencia en los modelos animales ensayados, lo cual coloca a M.´habana´ dentro de los posibles candidatos vacunales contra la tuberculosis al ajustarse a la condición que impone una vacuna clásica de reproducir la infección y los eventos inmunes que le suceden lo más fielmente posible a como ocurren de manera natural, sin causar extensos daños en el receptor.

  17. Pulmonary tuberculosis

    Science.gov (United States)

    TB; Tuberculosis - pulmonary; Mycobacterium - pulmonary ... Pulmonary TB is caused by the bacterium Mycobacterium tuberculosis (M tuberculosis) . TB is contagious. This means the bacteria is easily spread from an infected person ...

  18. [A study on genotype of 271 mycobacterium tuberculosis isolates in 6 prefectures in Yunnan Province].

    Science.gov (United States)

    Chen, L Y; Yang, X; Ru, H H; Yang, H J; Yan, S Q; Ma, L; Chen, J O; Yang, R; Xu, L

    2018-01-06

    Objective: To understand the characteristics of genotypes of Mycobacterium tuberculosis isolates in Yunnan province, and provide the molecular epidemiological evidence for prevention and control of tuberculosis in Yunnan Province. Methods: Mycobacterium Tuberculosis isolates were collected from 6 prefectures of Yunnan province in 2014 and their Genetypes of Mycobacterium tuberculosis isolates were obtained using spoligotyping and multiple locus variable numbers of tandem repeats analysis (MLVA). The results of spoligotyping were entered into the SITVITWEB database to obtain the Spoligotyping International Type (SIT) patterns and the sublineages of MTB isolates. The genoyping patterns were clustered with BioNumerics (version 5.0). Results: A total of 271 MTB isolates represented patients were collected from six prefectures in Yunnan province. Out of these patients, 196 (72.3%) were male. The mean age of the patients was (41.9±15.1) years. The most MTB isolates were from Puer, totally 94 iusolates(34.69%). Spoligotyping analysis revealed that 151 (55.72%) MTB isolates belonged to the Beijing genotype, while the other 120 (44.28%) were from non-Beijing genotype; 40 genotypes were consisted of 24 unique genotypes and 16 clusters. The 271 isolates were differentiated into 30 clusters (2 to 17 isolates per cluster) and 177 unique genotypes, showing a clustering rate of 23.62%. Beijing genotype strains showed higher clustering rate than non-Beijing genotype strains (29.14% vs 16.67%). The HGI of 12-locus VNTR in total MTB strains, Beijing genotype strains and non-Beijing genotype was 0.993, 0.982 and 0.995 respectively. Conclusion: The Beijing genotype was the predominant genotype in Yunnan Province, the characteristics of Mycobacterium tuberculosis showed high genetic diversity. The genotyping data reflect the potential recent ongoing transmission in some area, which highlights the urgent need for early diagnosis and treatment of the infectious TB cases, to cut off the

  19. Molecular identification of Mycobacterium tuberculosis complex isolates from Kermanshah Province, Iran

    Directory of Open Access Journals (Sweden)

    Roghieh Moghaddam

    2016-01-01

    Full Text Available Tuberculosis is one of the most important zoonotic diseases in the world. Rapid diagnosis of the disease and identification of species is extremely important for proper treatment of the disease as some species of the complex are resistant to the first-line of tuberculosis drugs. The aim of present study was molecular identification of Mycobacterium tuberculosis (MTB complex isolates from Kermanshah Province, Iran, which were submitted to the Tuberculosis Reference Laboratory at Razi Vaccine and Serum Research Institute (Tehran, Iran. To identify the genus Mycobacterium, all isolates were subjected to 16S rRNA polymerase chain reaction (PCR, and PCR-IS6110 was subsequently used to confirm that the isolates belonged to MTB complex. Finally, region of difference (RD typing was used to identify the species in the complex. The results of 16S rRNA and IS6110 PCR analysis showed the presence of 543-bp and 245-bp bands, respectively. Furthermore, 146bp, 172bp, 235bp, and 369bp at RD1, RD4, RD9, and RD12, respectively, were observed during RD typing. Thus, based on the results, all isolates were identified as MTB. It is worth mentioning that most tuberculosis cases are identified on the basis of acid-fast bacilli detection, and antibiotic therapy is immediately initiated subsequently. Moreover, it should be noted that some of these acid-fast positive cases might not be of genus Mycobacterium, and thus, the antibiotics prescribed might threaten the health of the patients. Additionally, if the identified bacilli are not within MTB complex, the drug therapy would differ. However, Mycobacterium bovis, which is a member of MTB complex and is resistant to pyrazinamide, requires exact strain identification. Based on the findings, individual isolates should be identified by RD typing methods, which could clearly discriminate the species from each other.

  20. Molecular modeling and in silico characterization of Mycobacterium tuberculosis TlyA: Possible misannotation of this tubercle bacilli-hemolysin

    Directory of Open Access Journals (Sweden)

    Vizcaíno Carolina

    2011-03-01

    Full Text Available Abstract Background The TlyA protein has a controversial function as a virulence factor in Mycobacterium tuberculosis (M. tuberculosis. At present, its dual activity as hemolysin and RNA methyltransferase in M. tuberculosis has been indirectly proposed based on in vitro results. There is no evidence however for TlyA relevance in the survival of tubercle bacilli inside host cells or whether both activities are functionally linked. A thorough analysis of structure prediction for this mycobacterial protein in this study shows the need for reevaluating TlyA's function in virulence. Results Bioinformatics analysis of TlyA identified a ribosomal protein binding domain (S4 domain, located between residues 5 and 68 as well as an FtsJ-like methyltranferase domain encompassing residues 62 and 247, all of which have been previously described in translation machinery-associated proteins. Subcellular localization prediction showed that TlyA lacks a signal peptide and its hydrophobicity profile showed no evidence of transmembrane helices. These findings suggested that it may not be attached to the membrane, which is consistent with a cytoplasmic localization. Three-dimensional modeling of TlyA showed a consensus structure, having a common core formed by a six-stranded β-sheet between two α-helix layers, which is consistent with an RNA methyltransferase structure. Phylogenetic analyses showed high conservation of the tlyA gene among Mycobacterium species. Additionally, the nucleotide substitution rates suggested purifying selection during tlyA gene evolution and the absence of a common ancestor between TlyA proteins and bacterial pore-forming proteins. Conclusion Altogether, our manual in silico curation suggested that TlyA is involved in ribosomal biogenesis and that there is a functional annotation error regarding this protein family in several microbial and plant genomes, including the M. tuberculosis genome.

  1. Description of polymerase chain reaction and sequencing DNA Mycobacterium tuberculosis from specimen sputum of tuberculosis patients in Medan

    Science.gov (United States)

    Lily; Siregar, Y.; Ilyas, S.

    2018-03-01

    This study purposed to describe the product Polymerase Chain Reaction (PCR) and sequencing of DNA Mycobacterium (M.) tuberculosis from sputum of tuberculosis (TB) patients in Medan. Sputum was collected from patients that diagnosed with pulmonary TB by a physician. Specimen processed by PCR method of Li et al. and sequencing at Macrogen Laboratory. All of 12 product PCR were showed brightness bands at 126 base pair (bp). These results indicated similarity to the study of Li et al. Sequencing analysis showed the presence of a mutation and non-mutation groups of M. tuberculosis. The reference and outcome berange of the mutation and non-mutation of M. tuberculosis were 56-107, 59-85, 60-120 and 63-94, respectively. The percentage bp difference between the outcome and references for mutation and non-mutation were 3.448-6.569and 3.278-7.428%, respectively. In conclusion, the successful amplification of PCR products in a 1.5% agarose gel electrophoresis where all 12 sputa contained rpoB-positive M. tuberculosis and 0.644% difference was found between the outcome with reference bp of the mutation and non-mutation M. tuberculosis groups.

  2. Whole genome sequencing reveals genomic heterogeneity and antibiotic purification in Mycobacterium tuberculosis isolates

    KAUST Repository

    Black, PA; de Vos, M.; Louw, GE; van der Merwe, RG; Dippenaar, A.; Streicher, EM; Abdallah, A. M.; Sampson, SL; Victor, TC; Dolby, T.; Simpson, JA; van Helden, PD; Warren, RM; Pain, Arnab

    2015-01-01

    Our study demonstrated true levels of genetic diversity within an M. tuberculosis population and showed that genetic diversity may be re-defined when a selective pressure, such as drug exposure, is imposed on M. tuberculosis populations during the course of infection. This suggests that the genome of M. tuberculosis is more dynamic than previously thought, suggesting preparedness to respond to a changing environment.

  3. Deficiency of double-strand DNA break repair does not impair Mycobacterium tuberculosis virulence in multiple animal models of infection.

    Science.gov (United States)

    Heaton, Brook E; Barkan, Daniel; Bongiorno, Paola; Karakousis, Petros C; Glickman, Michael S

    2014-08-01

    Mycobacterium tuberculosis persistence within its human host requires mechanisms to resist the effector molecules of host immunity, which exert their bactericidal effects through damaging pathogen proteins, membranes, and DNA. Substantial evidence indicates that bacterial pathogens, including M. tuberculosis, require DNA repair systems to repair the DNA damage inflicted by the host during infection, but the role of double-strand DNA break (DSB) repair systems is unclear. Double-strand DNA breaks are the most cytotoxic form of DNA damage and must be repaired for chromosome replication to proceed. M. tuberculosis elaborates three genetically distinct DSB repair systems: homologous recombination (HR), nonhomologous end joining (NHEJ), and single-strand annealing (SSA). NHEJ, which repairs DSBs in quiescent cells, may be particularly relevant to M. tuberculosis latency. However, very little information is available about the phenotype of DSB repair-deficient M. tuberculosis in animal models of infection. Here we tested M. tuberculosis strains lacking NHEJ (a Δku ΔligD strain), HR (a ΔrecA strain), or both (a ΔrecA Δku strain) in C57BL/6J mice, C3HeB/FeJ mice, guinea pigs, and a mouse hollow-fiber model of infection. We found no difference in bacterial load, histopathology, or host mortality between wild-type and DSB repair mutant strains in any model of infection. These results suggest that the animal models tested do not inflict DSBs on the mycobacterial chromosome, that other repair pathways can compensate for the loss of NHEJ and HR, or that DSB repair is not required for M. tuberculosis pathogenesis. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  4. Conspicuous multidrug-resistant Mycobacterium tuberculosis cluster strains do not trespass country borders in Latin America and Spain.

    Science.gov (United States)

    Ritacco, Viviana; Iglesias, María-José; Ferrazoli, Lucilaine; Monteserin, Johana; Dalla Costa, Elis R; Cebollada, Alberto; Morcillo, Nora; Robledo, Jaime; de Waard, Jacobus H; Araya, Pamela; Aristimuño, Liselotte; Díaz, Raúl; Gavin, Patricia; Imperiale, Belen; Simonsen, Vera; Zapata, Elsa M; Jiménez, María S; Rossetti, Maria L; Martin, Carlos; Barrera, Lucía; Samper, Sofia

    2012-06-01

    Multidrug-resistant Mycobacterium tuberculosis strain diversity in Ibero-America was examined by comparing extant genotype collections in national or state tuberculosis networks. To this end, genotypes from over 1000 patients with multidrug-resistant tuberculosis diagnosed from 2004 through 2008 in Argentina, Brazil, Chile, Colombia, Venezuela and Spain were compared in a database constructed ad hoc. Most of the 116 clusters identified by IS6110 restriction fragment length polymorphism were small and restricted to individual countries. The three largest clusters, of 116, 49 and 25 patients, were found in Argentina and corresponded to previously documented locally-epidemic strains. Only 13 small clusters involved more than one country, altogether accounting for 41 patients, of whom 13 were, in turn, immigrants from Latin American countries different from those participating in the study (Peru, Ecuador and Bolivia). Most of these international clusters belonged either to the emerging RD(Rio) LAM lineage or to the Haarlem family of M. tuberculosis and four were further split by country when analyzed with spoligotyping and rifampin resistance-conferring mutations, suggesting that they did not represent ongoing transnational transmission events. The Beijing genotype accounted for 1.3% and 10.2% of patients with multidrug-resistant tuberculosis in Latin America and Spain, respectively, including one international cluster of two cases. In brief, Euro-American genotypes were widely predominant among multidrug-resistant M. tuberculosis strains in Ibero-America, reflecting closely their predominance in the general M. tuberculosis population in the region, and no evidence was found of acknowledged outbreak strains trespassing country borders. Copyright © 2011 Elsevier B.V. All rights reserved.

  5. Cyclic GMP-AMP Synthase Is an Innate Immune DNA Sensor for Mycobacterium tuberculosis.

    Science.gov (United States)

    Collins, Angela C; Cai, Haocheng; Li, Tuo; Franco, Luis H; Li, Xiao-Dong; Nair, Vidhya R; Scharn, Caitlyn R; Stamm, Chelsea E; Levine, Beth; Chen, Zhijian J; Shiloh, Michael U

    2015-06-10

    Activation of the DNA-dependent cytosolic surveillance pathway in response to Mycobacterium tuberculosis infection stimulates ubiquitin-dependent autophagy and inflammatory cytokine production, and plays an important role in host defense against M. tuberculosis. However, the identity of the host sensor for M. tuberculosis DNA is unknown. Here we show that M. tuberculosis activated cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) in macrophages to produce cGAMP, a second messenger that activates the adaptor protein stimulator of interferon genes (STING) to induce type I interferons and other cytokines. cGAS localized with M. tuberculosis in mouse and human cells and in human tuberculosis lesions. Knockdown or knockout of cGAS in human or mouse macrophages blocked cytokine production and induction of autophagy. Mice deficient in cGAS were more susceptible to lethality caused by infection with M. tuberculosis. These results demonstrate that cGAS is a vital innate immune sensor of M. tuberculosis infection. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Nitazoxanide stimulates autophagy and inhibits mTORC1 signaling and intracellular proliferation of Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Karen K Y Lam

    Full Text Available Tuberculosis, caused by Mycobacterium tuberculosis infection, is a major cause of morbidity and mortality in the world today. M. tuberculosis hijacks the phagosome-lysosome trafficking pathway to escape clearance from infected macrophages. There is increasing evidence that manipulation of autophagy, a regulated catabolic trafficking pathway, can enhance killing of M. tuberculosis. Therefore, pharmacological agents that induce autophagy could be important in combating tuberculosis. We report that the antiprotozoal drug nitazoxanide and its active metabolite tizoxanide strongly stimulate autophagy and inhibit signaling by mTORC1, a major negative regulator of autophagy. Analysis of 16 nitazoxanide analogues reveals similar strict structural requirements for activity in autophagosome induction, EGFP-LC3 processing and mTORC1 inhibition. Nitazoxanide can inhibit M. tuberculosis proliferation in vitro. Here we show that it inhibits M. tuberculosis proliferation more potently in infected human THP-1 cells and peripheral monocytes. We identify the human quinone oxidoreductase NQO1 as a nitazoxanide target and propose, based on experiments with cells expressing NQO1 or not, that NQO1 inhibition is partly responsible for mTORC1 inhibition and enhanced autophagy. The dual action of nitazoxanide on both the bacterium and the host cell response to infection may lead to improved tuberculosis treatment.

  7. Nitazoxanide stimulates autophagy and inhibits mTORC1 signaling and intracellular proliferation of Mycobacterium tuberculosis.

    Science.gov (United States)

    Lam, Karen K Y; Zheng, Xingji; Forestieri, Roberto; Balgi, Aruna D; Nodwell, Matt; Vollett, Sarah; Anderson, Hilary J; Andersen, Raymond J; Av-Gay, Yossef; Roberge, Michel

    2012-01-01

    Tuberculosis, caused by Mycobacterium tuberculosis infection, is a major cause of morbidity and mortality in the world today. M. tuberculosis hijacks the phagosome-lysosome trafficking pathway to escape clearance from infected macrophages. There is increasing evidence that manipulation of autophagy, a regulated catabolic trafficking pathway, can enhance killing of M. tuberculosis. Therefore, pharmacological agents that induce autophagy could be important in combating tuberculosis. We report that the antiprotozoal drug nitazoxanide and its active metabolite tizoxanide strongly stimulate autophagy and inhibit signaling by mTORC1, a major negative regulator of autophagy. Analysis of 16 nitazoxanide analogues reveals similar strict structural requirements for activity in autophagosome induction, EGFP-LC3 processing and mTORC1 inhibition. Nitazoxanide can inhibit M. tuberculosis proliferation in vitro. Here we show that it inhibits M. tuberculosis proliferation more potently in infected human THP-1 cells and peripheral monocytes. We identify the human quinone oxidoreductase NQO1 as a nitazoxanide target and propose, based on experiments with cells expressing NQO1 or not, that NQO1 inhibition is partly responsible for mTORC1 inhibition and enhanced autophagy. The dual action of nitazoxanide on both the bacterium and the host cell response to infection may lead to improved tuberculosis treatment.

  8. Overproduction, purification and preliminary X-ray diffraction analysis of a sulfotransferase from Mycobacterium tuberculosis H37Rv

    International Nuclear Information System (INIS)

    Tanaka, Shotaro; Moriizumi, Yuuji; Kimura, Makoto; Kakuta, Yoshimitsu

    2004-01-01

    A sulfotransferase from M. tuberculosis was crystallized and preliminarily analyzed using X-ray diffraction. Sulfotransferase STF1 from the Mycobacterium tuberculosis H37Rv genome was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystals diffract to 1.5 Å resolution using synchrotron radiation at SPring-8. The crystals are monoclinic and belong to space group P2 1 , with unit-cell parameters a = 40.86, b = 95.76, c = 48.04 Å, β = 106.43°. The calculated Matthews coefficient is approximately 2.1 Å 3 Da −1 assuming the presence of one molecule of STF1 in the asymmetric unit. A substrate-binding assay using a PAP–agarose column suggests that STF1 exhibits sulfotransferase activity

  9. Increasing incidence of fluoroquinolone-resistant Mycobacterium tuberculosis in Mumbai, India.

    Science.gov (United States)

    Agrawal, D; Udwadia, Z F; Rodriguez, C; Mehta, A

    2009-01-01

    Tertiary referral centre, private hospital, Mumbai, India. To analyse the incidence of fluoroquinolone (FQ) resistant Mycobacterium tuberculosis (TB) in our laboratory from 1995 to 2004. Retrospective review and analysis of the drug susceptibility test records of all M. tuberculosis culture-positive samples from our Microbiology Department from 1995 to 2004. FQ resistance has increased exponentially in our laboratory, from 3% in 1996 to 35% in 2004. The incidence of multidrug-resistant tuberculosis has also increased during the same period, from 33% in 1995 to 56% in 2004. The incidence of FQ-resistant M. tuberculosis is gradually increasing to alarming levels. This may be due to widespread use of this vital group of drugs in the treatment of community-acquired infections. We urge that these broad spectrum antibiotics be used judiciously, and ideally be reserved for treatment of resistant TB in TB-endemic areas.

  10. Protection against Mycobacterium tuberculosis infection by adoptive immunotherapy. Requirement for T cell-deficient recipients

    International Nuclear Information System (INIS)

    Orme, I.M.; Collins, F.M.

    1983-01-01

    The results of this study demonstrate that spleen cells taken from mice at the height of the primary immune response to intravenous infection with Mycobacterium tuberculosis possess the capacity to transfer adoptive protection to M. tuberculosis-infected recipients, but only if these recipients are first rendered T cell-deficient, either by thymectomy and gamma irradiation, or by sublethal irradiation. A similar requirement was necessary to demonstrate the adoptive protection of the lungs after exposure to an acute aerosol-delivered M. tuberculosis infection. In both infectious models successful adoptive immunotherapy was shown to be mediated by T lymphocytes, which were acquired in the donor animals in response to the immunizing infection. It is proposed that the results of this study may serve as a basic model for the subsequent analysis of the nature of the T cell-mediated immune response to both systemic and aerogenic infections with M. tuberculosis

  11. Inhibition of Glutamine Synthetase: A Potential Drug Target in Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Sherry L. Mowbray

    2014-08-01

    Full Text Available Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis. Globally, tuberculosis is second only to AIDS in mortality and the disease is responsible for over 1.3 million deaths each year. The impractically long treatment schedules (generally 6–9 months and unpleasant side effects of the current drugs often lead to poor patient compliance, which in turn has resulted in the emergence of multi-, extensively- and totally-drug resistant strains. The development of new classes of anti-tuberculosis drugs and new drug targets is of global importance, since attacking the bacterium using multiple strategies provides the best means to prevent resistance. This review presents an overview of the various strategies and compounds utilized to inhibit glutamine synthetase, a promising target for the development of drugs for TB therapy.

  12. Substituted aminopyrimidine protein kinase B (PknB) inhibitors show activity against Mycobacterium tuberculosis

    Science.gov (United States)

    Chapman, Timothy M.; Bouloc, Nathalie; Buxton, Roger S.; Chugh, Jasveen; Lougheed, Kathryn E.A.; Osborne, Simon A.; Saxty, Barbara; Smerdon, Stephen J.; Taylor, Debra L.; Whalley, David

    2012-01-01

    A high-throughput screen against PknB, an essential serine–threonine protein kinase present in Mycobacterium tuberculosis (M. tuberculosis), allowed the identification of an aminoquinazoline inhibitor which was used as a starting point for SAR investigations. Although a significant improvement in enzyme affinity was achieved, the aminoquinazolines showed little or no cellular activity against M. tuberculosis. However, switching to an aminopyrimidine core scaffold and the introduction of a basic amine side chain afforded compounds with nanomolar enzyme binding affinity and micromolar minimum inhibitory concentrations against M. tuberculosis. Replacement of the pyrazole head group with pyridine then allowed equipotent compounds with improved selectivity against a human kinase panel to be obtained. PMID:22469702

  13. The Capacity of Mycobacterium tuberculosis To Survive Iron Starvation Might Enable It To Persist in Iron-Deprived Microenvironments of Human Granulomas.

    Science.gov (United States)

    Kurthkoti, Krishna; Amin, Hamel; Marakalala, Mohlopheni J; Ghanny, Saleena; Subbian, Selvakumar; Sakatos, Alexandra; Livny, Jonathan; Fortune, Sarah M; Berney, Michael; Rodriguez, G Marcela

    2017-08-15

    This study was conducted to investigate the role of iron deprivation in the persistence of Mycobacterium tuberculosis We present evidence of iron restriction in human necrotic granulomas and demonstrate that under iron starvation M. tuberculosis persists, refractive to antibiotics and capable of restarting replication when iron is made available. Transcriptomics and metabolomic analyses indicated that the persistence of M. tuberculosis under iron starvation is dependent on strict control of endogenous Fe utilization and is associated with upregulation of pathogenicity and intrinsic antibiotic resistance determinants. M. tuberculosis mutants compromised in their ability to survive Fe starvation were identified. The findings of this study advance the understanding of the physiological settings that may underpin the chronicity of human tuberculosis (TB) and are relevant to the design of effective antitubercular therapies. IMPORTANCE One-third of the world population may harbor persistent M. tuberculosis , causing an asymptomatic infection that is refractory to treatment and can reactivate to become potentially lethal tuberculosis disease. However, little is known about the factors that trigger and maintain M. tuberculosis persistence in infected individuals. Iron is an essential nutrient for M. tuberculosis growth. In this study, we show, first, that in human granulomas the immune defense creates microenvironments in which M. tuberculosis likely experiences drastic Fe deprivation and, second, that Fe-starved M. tuberculosis is capable of long-term persistence without growth. Together, these observations suggest that Fe deprivation in the lung might trigger a state of persistence in M. tuberculosis and promote chronic TB. We also identified vulnerabilities of iron-restricted persistent M. tuberculosis , which can be exploited for the design of new antitubercular therapies. Copyright © 2017 Kurthkoti et al.

  14. Tuberculosis Caused by Mycobacterium orygis in Dairy Cattle and Captured Monkeys in Bangladesh: a New Scenario of Tuberculosis in South Asia.

    Science.gov (United States)

    Rahim, Z; Thapa, J; Fukushima, Y; van der Zanden, A G M; Gordon, S V; Suzuki, Y; Nakajima, C

    2017-12-01

    Mycobacterium orygis, commonly known as the oryx bacillus and a newly proposed Mycobacterium tuberculosis complex subspecies, was isolated from 18 cattle in a dairy farm and two captured rhesus monkeys in a zoo in Bangladesh. All the infected animals had tuberculosis lesions in their lungs, suggesting transmission and infection with M. orygis by an airborne route. The 20 isolates were analysed using a range of conventional and molecular typing methods, and RD-deletion typing and sequencing of selected genes confirmed the isolates as M. orygis. Multiple-locus variable-number tandem repeat analysis (MLVA) allowed the isolates to be divided into three clusters based on the relatedness of their MLVA profiles. The two monkey isolates shared the same MLVA pattern with 15 of the cattle isolates, whereas the remaining three cattle isolates had different patterns, even though the latter animals had been kept in the same dairy farm. The diversity observed among isolates may suggest the bacteria have been established in this area for a long period. This study along with other recent findings that report the detection of M. orygis from animals as well as humans originating from South Asia potentially indicate endemic distribution of M. orygis in South Asia. © 2016 Blackwell Verlag GmbH.

  15. Mycobacterium tuberculosis, but not vaccine BCG, specifically upregulates matrix metalloproteinase-1.

    Science.gov (United States)

    Elkington, Paul T G; Nuttall, Robert K; Boyle, Joseph J; O'Kane, Cecilia M; Horncastle, Donna E; Edwards, Dylan R; Friedland, Jon S

    2005-12-15

    Pulmonary cavitation is fundamental to the global success of Mycobacterium tuberculosis. However, the mechanisms of this lung destruction are poorly understood. The biochemistry of lung matrix predicts matrix metalloproteinase (MMP) involvement in immunopathology. We investigated gene expression of all MMPs, proteins with a disintegrin and metalloproteinase domain, and tissue inhibitors of metalloproteinases in M. tuberculosis-infected human macrophages by real-time polymerase chain reaction. MMP secretion was measured by zymography and Western analysis, and expression in patients with pulmonary tuberculosis was localized by immunohistochemistry. MMP-1 and MMP-7 gene expression and secretion are potently upregulated by M. tuberculosis, and no increase in tissue inhibitor of metalloproteinase expression occurs to oppose their activity. Dexamethasone completely suppresses MMP-1 but not MMP-7 gene expression and secretion. In patients with active tuberculosis, macrophages express MMP-1 and MMP-7 adjacent to areas of tissue destruction. MMP-1 but not MMP-7 expression and secretion are relatively M. tuberculosis specific, are not upregulated by tuberculosis-associated cytokines, and are prostaglandin dependent. In contrast, the vaccine M. bovis bacillus Calmette-Guérin (BCG) does not stimulate MMP-1 secretion from human macrophages, although M. tuberculosis and BCG do upregulate MMP-7 equally. BCG-infected macrophages secrete reduced prostaglandin E2 concentrations compared with M. tuberculosis-infected macrophages, and prostaglandin pathway supplementation augments MMP-1 secretion from BCG-infected cells. M. tuberculosis specifically upregulates MMP-1 in a cellular model of human infection and in patients with tuberculosis. In contrast, vaccine BCG, which does not cause lung cavitation, does not upregulate prostaglandin E2-dependent MMP-1 secretion.

  16. Added value of IP-10 as a read-out of Mycobacterium tuberculosis

    DEFF Research Database (Denmark)

    Jenum, Synne; Dhanasekaran, Sivmakumaran; Ritz, Christian

    2016-01-01

    We have explored the added value of IFNγ-inducible protein 10 as a read-out of Mycobacterium tuberculosis specific immunity in young Indian children where the sensitivity of the IGRA for tuberculosis (TB) is poor. Reduced frequency of indeterminate results and an increased sensitivity for TB, sug...

  17. A broad set of different llama antibodies specific for a 16 kDa heat shock protein of Mycobacterium tuberculosis

    NARCIS (Netherlands)

    Trilling, Anke K.; de Ronde, Hans; Noteboom, Linda; van Houwelingen, Adèle; Roelse, Margriet; Srivastava, Saurabh K.; Haasnoot, Willem; Jongsma, Maarten A.; Kolk, Arend; Zuilhof, Han; Beekwilder, Jules

    2011-01-01

    Recombinant antibodies are powerful tools in engineering of novel diagnostics. Due to the small size and stable nature of llama antibody domains selected antibodies can serve as a detection reagent in multiplexed and sensitive assays for M. tuberculosis. Antibodies for Mycobacterium tuberculosis (M.

  18. Clinical implications of molecular drug resistance testing for Mycobacterium tuberculosis: a TBNET/RESIST-TB consensus statement

    NARCIS (Netherlands)

    Domínguez, J.; Boettger, E. C.; Cirillo, D.; Cobelens, F.; Eisenach, K. D.; Gagneux, S.; Hillemann, D.; Horsburgh, R.; Molina-Moya, B.; Niemann, S.; Tortoli, E.; Whitelaw, A.; Lange, C.

    2016-01-01

    The emergence of drug-resistant strains of Mycobacterium tuberculosis is a challenge to global tuberculosis (TB) control. Although culture-based methods have been regarded as the gold standard for drug susceptibility testing (DST), molecular methods provide rapid information on mutations in the M.

  19. Identification of MHC class II restricted T‐cell‐mediated reactivity against MHC class I binding Mycobacterium tuberculosis peptides

    DEFF Research Database (Denmark)

    Wang, Mingjun; Tang, Sheila Tuyet; Stryhn, Anette

    2011-01-01

    Major histocompatibility complex (MHC) class I restricted cytotoxic T lymphocytes (CTL) are known to play an important role in the control of Mycobacterium tuberculosis infection so identification of CTL epitopes from M. tuberculosis is of importance for the development of effective peptide...

  20. Overview of errors in the reference sequence and annotation of Mycobacterium tuberculosis H37Rv, and variation amongst its isolates

    KAUST Repository

    Kö ser, Claudio U.; Niemann, Stefan; Summers, David K.; Archer, John A.C.

    2012-01-01

    Since its publication in 1998, the genome sequence of the Mycobacterium tuberculosis H37Rv laboratory strain has acted as the cornerstone for the study of tuberculosis. In this review we address some of the practical aspects that have come to light

  1. Evaluation of highly conserved hsp65-specific nested PCR primers for diagnosing Mycobacterium tuberculosis.

    Science.gov (United States)

    Priyadarshini, P; Tiwari, K; Das, A; Kumar, D; Mishra, M N; Desikan, P; Nath, G

    2017-02-01

    To evaluate the sensitivity and specificity of a new nested set of primers designed for the detection of Mycobacterium tuberculosis complex targeting a highly conserved heat shock protein gene (hsp65). The nested primers were designed using multiple sequence alignment assuming the nucleotide sequence of the M. tuberculosis H37Rv hsp65 genome as base. Multidrug-resistant Mycobacterium species along with other non-mycobacterial and fungal species were included to evaluate the specificity of M. tuberculosis hsp65 gene-specific primers. The sensitivity of the primers was determined using serial 10-fold dilutions, and was 100% as shown by the bands in the case of M. tuberculosis complex. None of the other non M. tuberculosis complex bacterial and fungal species yielded any band on nested polymerase chain reaction (PCR). The first round of amplification could amplify 0.3 ng of the template DNA, while nested PCR could detect 0.3 pg. The present hsp65-specific primers have been observed to be sensitive, specific and cost-effective, without requiring interpretation of biochemical tests, real-time PCR, sequencing or high-performance liquid chromatography. These primer sets do not have the drawbacks associated with those protocols that target insertion sequence 6110, 16S rDNA, rpoB, recA and MPT 64.

  2. First insights into the genetic diversity of Mycobacterium tuberculosis strains in Salvador, Bahia State, Brazil Primeiro ensaio sobre diversidade genética das cepas de Mycobacterium tuberculosis em Salvador, Bahia, Brasil

    Directory of Open Access Journals (Sweden)

    Aída Cristina do Nascimento Silva

    2011-09-01

    Full Text Available This study constitutes a first attempt to describe the genetic population structure of Mycobacterium tuberculosis circulating in Salvador, Bahia State, Brazil. A total of 56 confirmed cases of pulmonary tuberculosis, identified between March and June 2008, were analyzed using restriction fragment length polymorphism (IS6110-RFLP. The study population was characterized by a predominance of males (71.43% over 30 years of age (68.75%. Forty-one isolates were found to belong to a single pattern (73.2%, while 15 (26.7% were found in group patterns, forming six clusters. The higher level of diversity observed is much more suggestive of endogenous reactivation than recent transmission.Este é o primeiro estudo realizado na Bahia, Brasil, visando à descrição da estrutura da população genética circulante do Mycobacterium tuberculosis na cidade de Salvador. Um total de 56 casos confirmados de tuberculose pulmonar, identificados entre março e junho de 2008, foi analisado pelo método Restriction Fragment Lenght Polymorphism (IS6110-RFLP. A população de estudo foi caracterizada como a maioria do sexo masculino (71,43 %, idade acima de 30 anos (68,75%. Quarenta e um isolados (73,21% com padrão único, enquanto 15 (26,75% apresentaram padrões agrupáveis, formando seis clusters. A alta taxa de diversidade das cepas de M. tuberculosis observada é mais sugestiva de reativação endógena do que transmissão recente.

  3. Purification, crystallization and preliminary X-ray crystallographic studies of Rv3705c from Mycobacterium tuberculosis

    International Nuclear Information System (INIS)

    Lu, Feifei; Gao, Feng; Li, Honglin; Gong, Weimin; Zhou, Lin; Bi, Lijun

    2014-01-01

    The cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of Rv3705c from M. tuberculosis are described. The conserved protein Rv3705c from Mycobacterium tuberculosis has been cloned, expressed, purified and crystallized by the sitting-drop vapour-diffusion method using PEG 3350 as a precipitant. The Rv3705c crystals exhibited space group P6 1 22 or P6 5 22, with unit-cell parameters a = b = 198.0, c = 364.1 Å, α = β = 90, γ = 120°, and diffracted to a resolution of 3.3 Å

  4. Purification, crystallization and preliminary X-ray crystallographic studies of Rv3705c from Mycobacterium tuberculosis

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Feifei [East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, People’s Republic of (China); Gao, Feng [Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, People’s Republic of (China); Li, Honglin [East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, People’s Republic of (China); Gong, Weimin [Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, People’s Republic of (China); Zhou, Lin, E-mail: gdtb-bg@vip.163.com [Center for Tuberculosis Control of Guangdong Province, Guangzhou, People’s Republic of (China); Bi, Lijun, E-mail: gdtb-bg@vip.163.com [East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, People’s Republic of (China)

    2014-07-23

    The cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of Rv3705c from M. tuberculosis are described. The conserved protein Rv3705c from Mycobacterium tuberculosis has been cloned, expressed, purified and crystallized by the sitting-drop vapour-diffusion method using PEG 3350 as a precipitant. The Rv3705c crystals exhibited space group P6{sub 1}22 or P6{sub 5}22, with unit-cell parameters a = b = 198.0, c = 364.1 Å, α = β = 90, γ = 120°, and diffracted to a resolution of 3.3 Å.

  5. Thermostability of IFN-γ and IP-10 release assays for latent infection with Mycobacterium tuberculosis

    DEFF Research Database (Denmark)

    Blauenfeldt, Thomas; Wagner, Dirk; Aabye, Martine Grosos

    2016-01-01

    accuracy of IP-10 release assay and IGRAs. RESULTS: We included 65 patients with confirmed pulmonary tuberculosis and 160 healthy controls from 6 European centres collaborating in the TBnet. In patients, IP-10 responses increased 1.07 (IQR 0.90-1.36) fold and IFN-γ responses decreased 0.88 (IQR 0......INTRODUCTION: Interferon-γ (IFN-γ) inducible protein 10kD (IP-10) and IFN-γ release assays (IGRAs) are immunodiagnostic tests aiming to identify the presence of specific cellular immune responses, interpreted as markers for latent infection with Mycobacterium tuberculosis. Incubation at higher...

  6. Detection of 123 bp fragment of insertion element IS6110 Mycobacterium tuberculosis for diagnosis of extrapulmonary tuberculosis

    Directory of Open Access Journals (Sweden)

    A K Maurya

    2012-01-01

    Full Text Available Purpose: Extrapulmonary tuberculosis (EPTB is emerging problem in developing and developed countries. The diagnosis of EPTB in its different clinical presentations remains a true challenge. IS6110-based polymerase chain reaction (PCR is used for rapid identification and positivity rate of the Mycobacterium tuberculosis complex in clinical isolates of different sites of EPTB. The present study was carried out to study the prevalence of M. tuberculosis complex in clinical isolates of EPTB at tertiary care centres in Lucknow. Materials and Methods: Seven hundred fifty-six specimens were collected from the suspected cases of EPTB which were processed for Mycobacteria by Ziehl Neelson (ZN staining and BACTEC culture. All the specimens were also processed for IS6110-based PCR amplification with primers targeting 123 bp fragment of insertion element IS6110 of the M. tuberculosis complex. Results: Of these 756 specimens, 71(9.3% were positive for acid fast bacilli (AFB by ZN staining, 227(30.1% were positive for mycobacteria by BACTEC culture and IS6110 PCR were positive for M. tuberculosis complex in 165 (20.7% isolates. We found a significant difference in sensitivities of different tests (P<0.05. Conclusions: This study reveals the positivity of M. tuberculosis complex in clinical isolates of EPTB case in tertiary care hospitals in Northern India. 72.7% of M. tuberculosis complex was confirmed by IS6110-PCR in culture isolates from different sites of EPTB. The high prevalence of the M. tuberculosis complex was seen in lymph node aspirate and synovial fluid. However, utility of PCR may play a potentially significant role in strengthening the diagnosis of EPTB especially targeting IS6110.

  7. Crystallization and preliminary X-ray characterization of phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv

    International Nuclear Information System (INIS)

    Mathur, Divya; Anand, Kanchan; Mathur, Deepika; Jagadish, Nirmala; Suri, Anil; Garg, Lalit C.

    2007-01-01

    The phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv was crystallized and diffraction data were collected to 2.8 Å resolution. Phosphoglucose isomerase is a ubiquitous enzyme that catalyzes the isomerization of d-glucopyranose-6-phosphate to d-fructofuranose-6-phosphate. The present investigation reports the expression, purification, crystallization and preliminary crystallographic studies of the phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv, which shares 46% sequence identity with that of its human host. The recombinant protein, which was prepared using an Escherichia coli expression system, was crystallized by the hanging-drop vapour-diffusion method. The crystals diffracted to a resolution of 2.8 Å and belonged to the orthorhombic space group I2 1 2 1 2 1 , with unit-cell parameters a = 109.0, b = 119.8, c = 138.9 Å

  8. Tuberculosis transmission of predominant genotypes of Mycobacterium tuberculosis in Northern suburbs of Buenos Aires city region Transmisión de la tuberculosis por genotipos predominantes de Mycobacterium tuberculosis en la región Gran Buenos Aires Norte

    Directory of Open Access Journals (Sweden)

    N. Morcillo

    2007-09-01

    Full Text Available In 2003, the incidence of tuberculosis in Argentina showed an increase compared to 2002. The severe national crisis at the end of the 90s has probably strongly contributed to this situation. The goal of this work was to estimate the extent of the spread of the most predominant Mycobacterium tuberculosis strains and to assess the spread of predominant M. tuberculosis clusters as determined by spoligotyping and IS6110 RFLP. The study involved 590 pulmonary, smear-positive TB cases receiving medical attention at health centers and hospitals in Northern Buenos Aires (NBA suburbs, from October 2001 to December 2002. From a total of 208 clinical isolates belonging to 6 major clusters, 63 (30.2% isolates had identical spoligotyping and IS6110 RFLP pattern. Only 22.2% were shown to have epidemiological connections with another member of their respective cluster. In these major clusters, 30.2% of the 208 TB cases studied by both molecular techniques and contact tracing could be convincingly attributable to a recently acquired infection. This knowledge may be useful to assess the clonal distribution of predominant M. tuberculosis clusters in Argentina, which may make an impact on TB control strategies.La incidencia de la tuberculosis en Argentina mostró en 2003 un incremento en comparación con 2002. La grave crisis nacional a fines de los 90 ha probablemente contribuido en gran medida a esta situación. El objetivo del presente trabajo fue determinar la diversidad genética de aislamientos de Mycobacterium tuberculosis y el grado de dispersión de algunas cepas mayoritarias genéticamente relacionadas. El estudio involucró 590 aislamientos clínicos provenientes de muestras respiratorias con examen directo positivo, de pacientes atendidos en los hospitales y centros de salud que conforman la región Gran Buenos Aires Norte (NBA, de octubre de 2001 a diciembre de 2002. De 208 aislamientos que se encontraron en los 6 mayores clusters, 63 (30,2% ten

  9. Tuberculosis

    Science.gov (United States)

    Friend, Milton

    1999-01-01

    Avian tuberculosis is usually caused by the bacterium Mycobacterium avium. At least 20 different types of M. avium have been identified, only three of which are known to cause disease in birds. Other types of Mycobacterium rarely cause tuberculosis in most avian species; however, parrots, macaws, and other large perching birds are susceptible to human and bovine types of tuberculosis bacilli. Avian tuberculosis generally is transmitted by direct contact with infected birds, ingestion of contaminated feed and water, or contact with a contaminated environment. Inhalation of the bacterium can cause respiratory tract infections. Wild bird studies in the Netherlands disclosed tuberculosis-infected puncture-type injuries in birds of prey that fight at the nest site (kestrels) or on the ground (buteo-type buzzards), but tuberculosisinfected injuries were not found in accipiters (falco

  10. The ada operon of Mycobacterium tuberculosis encodes two DNA methyltransferases for inducible repair of DNA alkylation damage.

    Science.gov (United States)

    Yang, Mingyi; Aamodt, Randi M; Dalhus, Bjørn; Balasingham, Seetha; Helle, Ina; Andersen, Pernille; Tønjum, Tone; Alseth, Ingrun; Rognes, Torbjørn; Bjørås, Magnar

    2011-06-10

    The ada operon of Mycobacterium tuberculosis, which encodes a composite protein of AdaA and AlkA and a separate AdaB/Ogt protein, was characterized. M. tuberculosis treated with N-methyl-N'-nitro-N-nitrosoguanidine induced transcription of the adaA-alkA and adaB genes, suggesting that M. tuberculosis mount an inducible response to methylating agents. Survival assays of the methyltransferase defective Escherichia coli mutant KT233 (ada ogt), showed that expression of the adaB gene rescued the alkylation sensitivity. Further, adaB but not adaA-alkA complemented the hypermutator phenotype of KT233. Purified AdaA-AlkA and AdaB possessed methyltransferase activity. These data suggested that AdaB counteract the cytotoxic and mutagenic effect of O(6)-methylguanine, while AdaA-AlkA most likely transfers methyl groups from innocuous methylphosphotriesters. AdaA-AlkA did not possess alkylbase DNA glycosylase activity nor rescue the alkylation sensitivity of the E. coli mutant BK2118 (tag alkA). We propose that AdaA-AlkA is a positive regulator of the adaptive response in M. tuberculosis. It thus appears that the ada operon of M. tuberculosis suppresses the mutagenic effect of alkylation but not the cytotoxic effect of lesions such as 3-methylpurines. Collectively, these data indicate that M. tuberculosis hypermutator strains with defective adaptive response genes might sustain robustness to cytotoxic alkylation DNA damage and confer a selective advantage contributing to host adaptation. Copyright © 2011 Elsevier B.V. All rights reserved.

  11. Mycobacterium tuberculosis lipolytic enzymes as potential biomarkers for the diagnosis of active tuberculosis.

    Directory of Open Access Journals (Sweden)

    Belinda Brust

    Full Text Available BACKGROUND: New diagnosis tests are urgently needed to address the global tuberculosis (TB burden and to improve control programs especially in resource-limited settings. An effective in vitro diagnostic of TB based on serological methods would be regarded as an attractive progress because immunoassays are simple, rapid, inexpensive, and may offer the possibility to detect cases missed by standard sputum smear microscopy. However, currently available serology tests for TB are highly variable in sensitivity and specificity. Lipolytic enzymes have recently emerged as key factors in lipid metabolization during dormancy and/or exit of the non-replicating growth phase, a prerequisite step of TB reactivation. The focus of this study was to analyze and compare the potential of four Mycobacterium tuberculosis lipolytic enzymes (LipY, Rv0183, Rv1984c and Rv3452 as new markers in the serodiagnosis of active TB. METHODS: Recombinant proteins were produced and used in optimized ELISA aimed to detect IgG and IgM serum antibodies against the four lipolytic enzymes. The capacity of the assays to identify infection was evaluated in patients with either active TB or latent TB and compared with two distinct control groups consisting of BCG-vaccinated blood donors and hospitalized non-TB individuals. RESULTS: A robust humoral response was detected in patients with active TB whereas antibodies against lipolytic enzymes were infrequently detected in either uninfected groups or in subjects with latent infection. High specifity levels, ranging from 93.9% to 97.5%, were obtained for all four antigens with sensitivity values ranging from 73.4% to 90.5%, with Rv3452 displaying the highest performances. Patients with active TB usually exhibited strong IgG responses but poor IgM responses. CONCLUSION: These results clearly indicate that the lipolytic enzymes tested are strongly immunogenic allowing to distinguish active from latent TB infections. They appear as potent

  12. Genomic diversity of drug-resistant Mycobacterium tuberculosis isolates in Lisbon Portugal: Towards tuberculosis genomic epidemiology

    Directory of Open Access Journals (Sweden)

    João Perdigão

    2015-01-01

    Full Text Available Multidrug- (MDR and extensively drug-resistant (XDR tuberculosis (TB present a challenge to disease control and elimination goals. Lisbon, Portugal, has a high TB incidencerate and unusual and successful XDR-TB strains that have been found in circulation foralmost two decades. For the last 20 years, a continued circulation of two phylogenetic clades, Lisboa3 and Q1, which are highly associated with MDR and XDR, have been observed. In recent years, these strains have been well characterized regarding the molecular basis of drug resistance and have been inclusively subjected to whole genome sequencing (WGS. Researchers have been studying the genomic diversity of strains circulating in Lisbon and its genomic determinants through cutting-edge next generation sequencing. An enormous amount of whole genome sequence data are now available for the most prevalent and clinically relevant strains circulating in Lisbon. It is the persistence, prevalence and rapid evolution towards drug resistance that has prompted researchers to investigate the properties of these strains at the genomic level and in the future at a global transcriptomic level. Seventy Mycobacterium tuberculosis (MTB isolates, mostly recovered in Lisbon, were genotyped by 24-loci Mycobacterial Interspersed Repetitive Unit – Variable Number of Tandem Repeats (MIRU-VNTR and the genomes sequenced using a next generation sequencing platform – Illumina HiSeq 2000. The genotyping data revealed three major clusters associated with MDR-TB (Lisboa3-A, Lisboa3-B and Q1, two of which are associated with XDR-TB (Lisboa3-B and Q1, whilst the genomic data contributed to elucidating the phylogenetic positioning of circulating MDR-TB strains, showing a high predominance of a single SNP cluster group 5. Furthermore, a genome-wide phylogeny analysis from these strains, together with 19 publicly available genomes of MTB clinical isolates, revealed two major clades responsible for MDR/XDR-TB in the region

  13. Genomic diversity of drug-resistant Mycobacterium tuberculosis isolates in Lisbon Portugal: Towards tuberculosis genomic epidemiology

    KAUST Repository

    Perdigã o, Joã o; Silva, Hugo; Machado, Diana; Macedo, Rita; Maltez, Fernando; Silva, Carla; Jordao, Luisa; Couto, Isabel; Mallard, Kim; Coll, Francesc; Hill-Cawthorne, Grant A.; McNerney, Ruth; Pain, Arnab; Clark, Taane G.; Viveiros, Miguel; Portugal, Isabel

    2015-01-01

    Multidrug- (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) present a challenge to disease control and elimination goals. Lisbon, Portugal, has a high TB incidence rate and unusual and successful XDR-TB strains that have been found in circulation for almost two decades. For the last 20. years, a continued circulation of two phylogenetic clades, Lisboa3 and Q1, which are highly associated with MDR and XDR, have been observed. In recent years, these strains have been well characterized regarding the molecular basis of drug resistance and have been inclusively subjected to whole genome sequencing (WGS). Researchers have been studying the genomic diversity of strains circulating in Lisbon and its genomic determinants through cutting-edge next generation sequencing. An enormous amount of whole genome sequence data are now available for the most prevalent and clinically relevant strains circulating in Lisbon.It is the persistence, prevalence and rapid evolution towards drug resistance that has prompted researchers to investigate the properties of these strains at the genomic level and in the future at a global transcriptomic level. Seventy Mycobacterium tuberculosis (MTB) isolates, mostly recovered in Lisbon, were genotyped by 24-. loci Mycobacterial Interspersed Repetitive Unit - Variable Number of Tandem Repeats (MIRU-VNTR) and the genomes sequenced using a next generation sequencing platform - Illumina HiSeq 2000.The genotyping data revealed three major clusters associated with MDR-TB (Lisboa3-A, Lisboa3-B and Q1), two of which are associated with XDR-TB (Lisboa3-B and Q1), whilst the genomic data contributed to elucidating the phylogenetic positioning of circulating MDR-TB strains, showing a high predominance of a single SNP cluster group 5. Furthermore, a genome-wide phylogeny analysis from these strains, together with 19 publicly available genomes of MTB clinical isolates, revealed two major clades responsible for MDR/XDR-TB in the region: Lisboa3 and Q

  14. Genomic diversity of drug-resistant Mycobacterium tuberculosis isolates in Lisbon Portugal: Towards tuberculosis genomic epidemiology

    KAUST Repository

    Perdigão, João

    2015-03-01

    Multidrug- (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) present a challenge to disease control and elimination goals. Lisbon, Portugal, has a high TB incidence rate and unusual and successful XDR-TB strains that have been found in circulation for almost two decades. For the last 20. years, a continued circulation of two phylogenetic clades, Lisboa3 and Q1, which are highly associated with MDR and XDR, have been observed. In recent years, these strains have been well characterized regarding the molecular basis of drug resistance and have been inclusively subjected to whole genome sequencing (WGS). Researchers have been studying the genomic diversity of strains circulating in Lisbon and its genomic determinants through cutting-edge next generation sequencing. An enormous amount of whole genome sequence data are now available for the most prevalent and clinically relevant strains circulating in Lisbon.It is the persistence, prevalence and rapid evolution towards drug resistance that has prompted researchers to investigate the properties of these strains at the genomic level and in the future at a global transcriptomic level. Seventy Mycobacterium tuberculosis (MTB) isolates, mostly recovered in Lisbon, were genotyped by 24-. loci Mycobacterial Interspersed Repetitive Unit - Variable Number of Tandem Repeats (MIRU-VNTR) and the genomes sequenced using a next generation sequencing platform - Illumina HiSeq 2000.The genotyping data revealed three major clusters associated with MDR-TB (Lisboa3-A, Lisboa3-B and Q1), two of which are associated with XDR-TB (Lisboa3-B and Q1), whilst the genomic data contributed to elucidating the phylogenetic positioning of circulating MDR-TB strains, showing a high predominance of a single SNP cluster group 5. Furthermore, a genome-wide phylogeny analysis from these strains, together with 19 publicly available genomes of MTB clinical isolates, revealed two major clades responsible for MDR/XDR-TB in the region: Lisboa3 and Q

  15. Coinfección por Mycobacterium malmoense y Mycobacterium tuberculosis en paciente con el síndrome de inmunodeficiencia humana

    Directory of Open Access Journals (Sweden)

    Lilian María Mederos Cuervo

    Full Text Available Se presenta un caso de coinfección por Mycobacterium malmoense y Mycobacterium tuberculosis en un paciente cubano con síndrome de inmunodeficiencia adquirida (sida, que producía enfermedad respiratoria y hepática respectivamente. Los cultivos realizados a partir de las muestras de esputo demostraron la presencia de una cepa micobacteriana no pigmentada de crecimiento lento perteneciente al grupo III de Runyon e identificada como Mycobacterium malmoense. A partir de los cultivos del tejido hepático extraído laparoscópicamente se aisló una cepa posteriormente identificada como Mycobacterium tuberculosis. El estudio anatomopatológico confirmó el diagnóstico de tuberculosis, el paciente recibió tratamiento específico y evolucionó clínicamente bien. Se reporta un caso infrecuente de coinfección por Mycobacterium, el cual describe el primer reporte de tuberculosis hepática en una paciente con sida en Cuba.

  16. DNA repair systems and the pathogenesis of Mycobacterium tuberculosis: varying activities at different stages of infection

    OpenAIRE

    Gorna, AE; Bowater, RP; Dziadek, J

    2010-01-01

    Mycobacteria, including most of all MTB (Mycobacterium tuberculosis), cause pathogenic infections in humans and, during the infectious process, are exposed to a range of environmental insults, including the host's immune response. From the moment MTB is exhaled by infected individuals, through an active and latent phase in the body of the new host, until the time they reach the reactivation stage, MTB is exposed to many types of DNA-damaging agents. Like all cellular organisms, MTB has effici...

  17. Decreasing prevalence of multi-drugs resistant Mycobacterium tuberculosis in Nashik City, India

    OpenAIRE

    More, Arun Punaji; Nagdawane, Ramkrishna Panchamrao; Gangurde, Aniket K

    2013-01-01

    Objective: In India, increasing prevalence of multi-drug resistant tuberculosis (MDR) has aggravated the control oftuberculosis problem. In many urban and semi-urban regions of India, no surveillance data of multidrug resistance inMycobacterium tuberculosisis available.Methods: A surveillance study on multidrug resistance was carried out in semi-urban and rural regions in and aroundNashik City of Maharashtra, India. The surveillance study was conducted in this region found that the prevalence...

  18. Diterpenes Synthesized from the Natural Serrulatane Leubethanol and Their in Vitro Activities against Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Ricardo Escarcena

    2015-04-01

    Full Text Available Seventeen new derivatives of the natural diterpene leubethanol, including some potential pro-drugs, with changes in the functionality of the aliphatic chain or modifications of aromatic ring and the phenolic group, were synthesized and tested in vitro by the MABA technique for their activity against the H37Rv strain of Mycobacterium tuberculosis. Some compounds showed antimycobacterial selectivity indices higher than leubethanol.

  19. Preparation and biological evaluation of ethionamide-mesoporous silicon nanoparticles against Mycobacterium tuberculosis

    OpenAIRE

    Vale, Nuno; Correia, Alexandra; Silva, Sara; Figueiredo, Patrícia; Mäkilä, , Ermei; Salonen, Jarno; Hirvonen, Jouni; Pedrosa, Jorge; Santos Hélder A.; Fraga, Alexandra

    2017-01-01

    Ethionamide (ETH) is an important second-line antituberculosis drug used for the treatment of patients infected with multidrug-resistant Mycobacterium tuberculosis. Recently, we reported that the loading of ETH into thermally carbonized-porous silicon (TCPSi) nanoparticles enhanced the solubility and permeability of ETH at different pH-values and also increased its metabolization process. Based on these results, we synthesized carboxylic acid functionalized thermally hydrocarbonized porous si...

  20. Biochemická charakterizace fosfoenolpyruvát karboxykinasy z Mycobacterium tuberculosis

    Czech Academy of Sciences Publication Activity Database

    Machová, Iva; Snášel, Jan; Pichová, Iva

    2013-01-01

    Roč. 107, č. 5 (2013), s. 425-425 ISSN 0009-2770. [Mezioborové setkání mladých biologů, biochemiků a chemiků /13./. 14.5.2013-17.5.2013, Žďár nad Sázavou] Grant - others:European Research Council(XE) FP7-245187 Institutional support: RVO:61388963 Keywords : Mycobacterium tuberculosis * Pepck * GDP/GTP Subject RIV: CE - Biochemistry

  1. Polymorphisms in Isoniazid and Prothionamide Resistance Genes of the Mycobacterium tuberculosis Complex

    KAUST Repository

    Projahn, M.; Koser, C. U.; Homolka, S.; Summers, D. K.; Archer, John A.C.; Niemann, S.

    2011-01-01

    Sequence analyses of 74 strains that encompassed major phylogenetic lineages of the Mycobacterium tuberculosis complex revealed 10 polymorphisms in mshA (Rv0486) and four polymorphisms in inhA (Rv1484) that were not responsible for isoniazid or prothionamide resistance. Instead, some of these mutations were phylogenetically informative. This genetic diversity must be taken into consideration for drug development and for the design of molecular tests for drug resistance.

  2. High-contrast imaging of mycobacterium tuberculosis using third-harmonic generation microscopy

    Science.gov (United States)

    Kim, Bo Ram; Lee, Eungjang; Park, Seung-Han

    2015-07-01

    Nonlinear optical microcopy has become an important tool in investigating biomaterials due to its various advantages such as label-free imaging capabilities. In particular, it has been shown that third-harmonic generation (THG) signals can be produced at interfaces between an aqueous medium (e.g. cytoplasm, interstitial fluid) and a mineralized lipidic surface. In this work, we have demonstrated that label-free high-contrast THG images of the mycobacterium tuberculosis can be obtained using THG microscopy.

  3. Identification of the translational start site of codon-optimized mCherry in Mycobacterium tuberculosis

    OpenAIRE

    Carroll, Paul; Muwanguzi-Karugaba, Julian; Melief, Eduard; Files, Megan; Parish, Tanya

    2014-01-01

    Background Fluorescent proteins are used widely as reporter genes in many organisms. We previously codon-optimized mCherry for Mycobacterium tuberculosis and generated expression constructs with high level expression in mycobacteria with multiple uses in vitro and in vivo. However, little is known about the expression of fluorescent proteins in mycobacteria and the translational start codon for mCherry has not been experimentally determined. Results We determined the translational start site ...

  4. Polymorphisms in Isoniazid and Prothionamide Resistance Genes of the Mycobacterium tuberculosis Complex

    KAUST Repository

    Projahn, M.

    2011-06-27

    Sequence analyses of 74 strains that encompassed major phylogenetic lineages of the Mycobacterium tuberculosis complex revealed 10 polymorphisms in mshA (Rv0486) and four polymorphisms in inhA (Rv1484) that were not responsible for isoniazid or prothionamide resistance. Instead, some of these mutations were phylogenetically informative. This genetic diversity must be taken into consideration for drug development and for the design of molecular tests for drug resistance.

  5. Identification of potential leads against 4-hydroxytetrahydrodipicolinate synthase from Mycobacterium tuberculosis

    OpenAIRE

    Rehman, Ajijur; Akhtar, Salman; Siddiqui, Mohd Haris; Sayeed, Usman; Ahmad, Syed Sayeed; Arif, Jamal M.; Khan, M. Kalim A.

    2016-01-01

    4-hydroxy-tetrahydrodipicolinate synthase (DHDPS) is an important enzyme needed for the biosynthesis of lysine and many more key metabolites in Mycobacterium tuberculosis (Mtb). Inhibition of DHDPS is supposed to a promising therapeutic target due to its specific role in sporulation, cross-linking of the peptidiglycan polymers and biosynthesis of amino acids. In this work, a known inhibitor-based similarity search was carried out against a natural products database (Super Natural II) towards ...

  6. Mycobacterium tuberculosis infection causes different levels of apoptosis and necrosis in human macrophages and alveolar epithelial cells.

    Science.gov (United States)

    Danelishvili, Lia; McGarvey, Jeffery; Li, Yong-Jun; Bermudez, Luiz E

    2003-09-01

    Mycobacterium tuberculosis interacts with macrophages and epithelial cells in the alveolar space of the lung, where it is able to invade and replicate in both cell types. M. tuberculosis-associated cytotoxicity to these cells has been well documented, but the mechanisms of host cell death are not well understood. We examined the induction of apoptosis and necrosis of human macrophages (U937) and type II alveolar epithelial cells (A549) by virulent (H37Rv) and attenuated (H37Ra) M. tuberculosis strains. Apoptosis was determined by both enzyme-linked immunosorbent assay (ELISA) and TdT-mediated dUTP nick end labelling (TUNEL) assay, whereas necrosis was evaluated by the release of lactate dehydrogenase (LDH). Both virulent and attenuated M. tuberculosis induced apoptosis in macrophages; however, the attenuated strain resulted in significantly more apoptosis than the virulent strain after 5 days of infection. In contrast, cytotoxicity of alveolar cells was the result of necrosis, but not apoptosis. Although infection with M. tuberculosis strains resulted in apoptosis of 14% of the cells on the monolayer, cell death associated with necrosis was observed in 59% of alveolar epithelial cells after 5 days of infection. Infection with M. tuberculosis suppressed apoptosis of alveolar epithelial cells induced by the kinase inhibitor, staurosporine. Because our findings suggest that M. tuberculosis can modulate the apoptotic response of macrophages and epithelial cells, we carried out an apoptosis pathway-specific cDNA microarray analysis of human macrophages and alveolar epithelial cells. Whereas the inhibitors of apoptosis, bcl-2 and Rb, were upregulated over 2.5-fold in infected (48 h) alveolar epithelial cells, the proapoptotic genes, bad and bax, were downregulated. The opposite was observed when U937 macrophages were infected with M. tuberculosis. Upon infection of alveolar epithelial cells with M. tuberculosis, the generation of apoptosis, as determined by the

  7. Clinical Concentrations of Thioridazine Kill Intracellular Multidrug-Resistant Mycobacterium tuberculosis

    Science.gov (United States)

    Ordway, Diane; Viveiros, Miguel; Leandro, Clara; Bettencourt, Rosário; Almeida, Josefina; Martins, Marta; Kristiansen, Jette E.; Molnar, Joseph; Amaral, Leonard

    2003-01-01

    The phenothiazines chlorpromazine (CPZ) and thioridazine (TZ) have equal in vitro activities against antibiotic-sensitive and -resistant Mycobacterium tuberculosis. These compounds have not been used as anti-M. tuberculosis agents because their in vitro activities take place at concentrations which are beyond those that are clinically achievable. In addition, chronic administration of CPZ produces frequent severe side effects. Because CPZ has been shown to enhance the killing of intracellular M. tuberculosis at concentrations in the medium that are clinically relevant, we have investigated whether TZ, a phenothiazine whose negative side effects are less frequent and serious than those associated with CPZ, kills M. tuberculosis organisms that have been phagocytosed by human macrophages, which have nominal killing activities against these bacteria. Both CPZ and TZ killed intracellular antibiotic-sensitive and -resistant M. tuberculosis organisms when they were used at concentrations in the medium well below those present in the plasma of patients treated with these agents. These concentrations in vitro were not toxic to the macrophage, nor did they affect in vitro cellular immune processes. TZ thus appears to be a serious candidate for the management of a freshly diagnosed infection of pulmonary tuberculosis or as an adjunct to conventional antituberculosis therapy if the patient originates from an area known to have a high prevalence of multidrug-resistant M. tuberculosis isolates. Nevertheless, we must await the outcomes of clinical trials to determine whether TZ itself may be safely and effectively used as an antituberculosis agent. PMID:12604522

  8. Cutaneous squamous cell carcinoma in lupus vulgaris caused by drug resistant Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Muthu S Kumaran

    2017-01-01

    Full Text Available Tuberculosis (TB is still a major public health problem in the world, with many factors contributing to this burden, including poor living conditions, overcrowding, poverty, malnutrition, illiteracy, and rapid spread of human immunodeficiency virus infection. Cutaneous tuberculosis is a less common form of extrapulmonary tuberculosis, and in this paucibacillary form the diagnosis depends on histopathology, tuberculin positivity, and response to treatment. The diagnosis is even more difficult in cases with drug resistant Mycobacterium tuberculosis due to lack of awareness and lack of facilities to diagnose drug resistant tuberculosis. In this article, we describe an unusual case of multidrug resistant lupus vulgaris (LV, in a 34-year-old male who responded to anti-tubercular treatment (ATT initially, but developed recurrent disease which failed to respond to standard four-drug ATT; subsequently, tissue culture showed growth of multidrug resistant M. tuberculosis. Subsequently, he also developed cutaneous squamous cell carcinoma. This article aims to exemplify a grave complication that can occur in long-standing case of LV, the limitations faced by clinicians in developing countries where tuberculosis is endemic, and classical methods of proving drug resistance are generally unavailable or fail.

  9. Mycobacterium tuberculosis population in northwestern Russia: an update from Russian-EU/Latvian border region.

    Directory of Open Access Journals (Sweden)

    Igor Mokrousov

    Full Text Available This study aimed to characterize the population structure of Mycobacterium tuberculosis in Pskov oblast in northwestern Russia, to view it in the geographical context, to compare drug resistance properties across major genetic families. Ninety M. tuberculosis strains from tuberculosis (TB patients, permanent residents in Pskov oblast were subjected to LAM-specific IS6110-PCR and spoligotyping, followed by comparison with SITVITWEB and MIRU-VNTRplus databases. The Beijing genotype (n = 40 was found the most prevalent followed by LAM (n = 18, T (n = 13, Haarlem (n = 10, Ural (n = 5, and Manu2 (n = 1; the family status remained unknown for 3 isolates. The high rate of Beijing genotype and prevalence of LAM family are similar to those in the other Russian settings. A feature specific for M. tuberculosis population in Pskov is a relatively higher rate of Haarlem and T types. Beijing strains were further typed with 12-MIRU (followed by comparison with proprietary global database and 3 hypervariable loci QUB-3232, VNTR-3820, VNTR-4120. The 12-MIRU typing differentiated 40 Beijing strains into 14 types (HGI = 0.82 while two largest types were M2 (223325153533 prevalent throughout former USSR and M11 (223325173533 prevalent in Russia and East Asia. The use of 3 hypervariable loci increased a discrimination of the Beijing strains (18 profiles, HGI = 0.89. Both major families Beijing and LAM had similar rate of MDR strains (62.5 and 55.6%, respectively that was significantly higher than in other strains (21.9%; P = 0.001 and 0.03, respectively. The rpoB531 mutations were more frequently found in Beijing strains while LAM drug resistant strains mainly harbored rpoB516 and inhA -15 mutations. Taken together with a high rate of multidrug resistance among Beijing strains from new TB cases (79.3% versus 44.4% in LAM, these findings suggest the critical impact of the Beijing genotype on the current situation with MDR-TB in the

  10. Sensitivity Pattern of Second Line Anti-Tuberculosis Drugs against Clinical Isolates of Multidrug Resistant Mycobacterium Tuberculosis

    International Nuclear Information System (INIS)

    Ghafoor, T.; Ikram, A.; Abbasi, S. A.; Zaman, G.; Ayyub, M.; Palomino, J. C.; Vandamme, P.; Martin, A.

    2015-01-01

    Objective:To determine the current sensitivity pattern of second line anti-tuberculosis drugs against clinical isolates of Multidrug Resistant Mycobacterium tuberculosis (MDR-TB). Study Design: A cross-sectional study. Place and Duration of Study: Department of Microbiology, Armed Forces Institute of Pathology (AFIP), Rawalpindi, from November 2011 to April 2013. Methodology: Samples received during the study period were processed on BACTEC MGIT 960 system for Mycobacterium tuberculosis (MTB) culture followed by first line drugs susceptibility testing of culture proven MTB isolates. On the basis of resistance to rifampicin and isoniazid, 100 clinical isolates of MDR-TB were further subjected to susceptibility testing against amikacin (AMK), capreomycin (CAP), ofloxacin (OFL) and ethionamide (ETH) as per standard BACTEC MGIT 960 instructions. Results: Out of 100 MDR-TB isolates, 62% were from male patients and 38% from female patients. 97% were sensitive to AMK, 53% to OFL, 87% to CAP; and 87% were sensitive to ETH. Conclusion: The majority of the MDR-TB isolates showed excellent sensitivity against AMK, CAP and ETH. However, sensitivity of MDR-TB isolates against fluoroquinolones like OFL was not encouraging. (author)

  11. Mycobacterium tuberculosis has diminished capacity to counteract redox stress induced by elevated levels of endogenous superoxide.

    Science.gov (United States)

    Tyagi, Priyanka; Dharmaraja, Allimuthu T; Bhaskar, Ashima; Chakrapani, Harinath; Singh, Amit

    2015-07-01

    Mycobacterium tuberculosis (Mtb) has evolved protective and detoxification mechanisms to maintain cytoplasmic redox balance in response to exogenous oxidative stress encountered inside host phagocytes. In contrast, little is known about the dynamic response of this pathogen to endogenous oxidative stress generated within Mtb. Using a noninvasive and specific biosensor of cytoplasmic redox state of Mtb, we for first time discovered a surprisingly high sensitivity of this pathogen to perturbation in redox homeostasis induced by elevated endogenous reactive oxygen species (ROS). We synthesized a series of hydroquinone-based small molecule ROS generators and found that ATD-3169 permeated mycobacteria to reliably enhance endogenous ROS including superoxide radicals. When Mtb strains including multidrug-resistant (MDR) and extensively drug-resistant (XDR) patient isolates were exposed to this compound, a dose-dependent, long-lasting, and irreversible oxidative shift in intramycobacterial redox potential was detected. Dynamic redox potential measurements revealed that Mtb had diminished capacity to restore cytoplasmic redox balance in comparison with Mycobacterium smegmatis (Msm), a fast growing nonpathogenic mycobacterial species. Accordingly, Mtb strains were extremely susceptible to inhibition by ATD-3169 but not Msm, suggesting a functional linkage between dynamic redox changes and survival. Microarray analysis showed major realignment of pathways involved in redox homeostasis, central metabolism, DNA repair, and cell wall lipid biosynthesis in response to ATD-3169, all consistent with enhanced endogenous ROS contributing to lethality induced by this compound. This work provides empirical evidence that the cytoplasmic redox poise of Mtb is uniquely sensitive to manipulation in steady-state endogenous ROS levels, thus revealing the importance of targeting intramycobacterial redox metabolism for controlling TB infection. Copyright © 2015 The Authors. Published by

  12. Development of a three component complex to increase isoniazid efficacy against isoniazid resistant and nonresistant Mycobacterium tuberculosis.

    Science.gov (United States)

    Manning, Thomas; Plummer, Sydney; Baker, Tess; Wylie, Greg; Clingenpeel, Amy C; Phillips, Dennis

    2015-10-15

    The bacterium responsible for causing tuberculosis has evolved resistance to antibiotics used to treat the disease, resulting in new multidrug resistant Mycobacterium tuberculosis (MDR-TB) and extensively drug resistant M. tuberculosis (XDR-TB) strains. Analytical techniques (1)H and (13)C Nuclear Magnetic Resonance (NMR), Fourier Transform-Ion Cyclotron Resonance with Electrospray Ionization (FT-ICR/ESI), and Matrix Assisted Laser Desorption Ionization-Mass Spectrometry (MALDI-TOF-MS) were used to study different aspects of the Cu(II)-polyethylene glycol (PEG-3350)-sucrose-isoniazid and Cu(II)-polyethylene glycol (PEG3350)-glucose-isoniazid complexes. The Cu(II) cation, sucrose or glucose, and the aggregate formed by PEG primarily serve as a composite drug delivery agent for the frontline antibiotic, however the improvement in MIC values produced with the CU-PEG-SUC-INH complex suggest an additional effect. Several Cu-PEG-SUC-INH complex variations were tested against INH resistant and nonresistant strains of M. tuberculosis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Removing the bottleneck in whole genome sequencing of Mycobacterium tuberculosis for rapid drug resistance analysis: a call to action

    Directory of Open Access Journals (Sweden)

    Ruth McNerney

    2017-03-01

    Full Text Available Whole genome sequencing (WGS can provide a comprehensive analysis of Mycobacterium tuberculosis mutations that cause resistance to anti-tuberculosis drugs. With the deployment of bench-top sequencers and rapid analytical software, WGS is poised to become a useful tool to guide treatment. However, direct sequencing from clinical specimens to provide a full drug resistance profile remains a serious challenge. This article reviews current practices for extracting M. tuberculosis DNA and possible solutions for sampling sputum. Techniques under consideration include enzymatic digestion, physical disruption, chemical degradation, detergent solubilization, solvent extraction, ligand-coated magnetic beads, silica columns, and oligonucleotide pull-down baits. Selective amplification of genomic bacterial DNA in sputum prior to WGS may provide a solution, and differential lysis to reduce the levels of contaminating human DNA is also being explored. To remove this bottleneck and accelerate access to WGS for patients with suspected drug-resistant tuberculosis, it is suggested that a coordinated and collaborative approach be taken to more rapidly optimize, compare, and validate methodologies for sequencing from patient samples.

  14. Human Mycobacterium bovis infection in the south-west of Ireland 1983-1992: a comparison with M. tuberculosis.

    LENUS (Irish Health Repository)

    Cotter, T P

    2012-02-03

    Epidemiological and bacteriological aspects of human Mycobacterium bovis disease were investigated in south-west Ireland (counties Cork & Kerry, population 536,000) over the years 1983-92 inclusive and compared to M. tuberculosis. Results showed a small, stable incidence of culture positive M. bovis human disease, mean annual incidence 0.56 per 100,000 population compared to a higher but declining incidence of culture positive M. tuberculosis (15.3 per 100,000 in 1983, 9.0 per 100,000 in 1992). Male patients were the majority, 63.4 per cent of M. bovis; 62.4% of M. tuberculosis (p = 0.03). Fifty three per cent of M. bovis cases (n = 30) were pulmonary, compared to 85% of M. tuberculosis (n = 626; p = 0.0001). M. bovis patients were older (p = 0.02), mean age 58.4 years (SD 18.9) compared to 48.5 (SD 22.2). The mycobacterial smear positive rate was similar in both groups taken as a whole. No rural-urban difference in incidence was found in either disease, suggesting in the case of M. bovis initial infection in childhood via contaminated milk in the pre-pasteurisation era.

  15. Genetic Diversity and Transmission Characteristics of Beijing Family Strains of Mycobacterium tuberculosis in Peru

    Science.gov (United States)

    Iwamoto, Tomotada; Grandjean, Louis; Arikawa, Kentaro; Nakanishi, Noriko; Caviedes, Luz; Coronel, Jorge; Sheen, Patricia; Wada, Takayuki; Taype, Carmen A.; Shaw, Marie-Anne; Moore, David A. J.; Gilman, Robert H.

    2012-01-01

    Beijing family strains of Mycobacterium tuberculosis have attracted worldwide attention because of their wide geographical distribution and global emergence. Peru, which has a historical relationship with East Asia, is considered to be a hotspot for Beijing family strains in South America. We aimed to unveil the genetic diversity and transmission characteristics of the Beijing strains in Peru. A total of 200 Beijing family strains were identified from 2140 M. tuberculosis isolates obtained in Lima, Peru, between December 2008 and January 2010. Of them, 198 strains were classified into sublineages, on the basis of 10 sets of single nucleotide polymorphisms (SNPs). They were also subjected to variable number tandem-repeat (VNTR) typing using an international standard set of 15 loci (15-MIRU-VNTR) plus 9 additional loci optimized for Beijing strains. An additional 70 Beijing family strains, isolated between 1999 and 2006 in Lima, were also analyzed in order to make a longitudinal comparison. The Beijing family was the third largest spoligotyping clade in Peru. Its population structure, by SNP typing, was characterized by a high frequency of Sequence Type 10 (ST10), which belongs to a modern subfamily of Beijing strains (178/198, 89.9%). Twelve strains belonged to the ancient subfamily (ST3 [n = 3], ST25 [n = 1], ST19 [n = 8]). Overall, the polymorphic information content for each of the 24 loci values was low. The 24 loci VNTR showed a high clustering rate (80.3%) and a high recent transmission index (RTIn−1 = 0.707). These strongly suggest the active and on-going transmission of Beijing family strains in the survey area. Notably, 1 VNTR genotype was found to account for 43.9% of the strains. Comparisons with data from East Asia suggested the genotype emerged as a uniquely endemic clone in Peru. A longitudinal comparison revealed the genotype was present in Lima by 1999. PMID:23185395

  16. Regulation Mechanism of the ald Gene Encoding Alanine Dehydrogenase in Mycobacterium smegmatis and Mycobacterium tuberculosis by the Lrp/AsnC Family Regulator AldR.

    Science.gov (United States)

    Jeong, Ji-A; Hyun, Jaekyung; Oh, Jeong-Il

    2015-10-01

    In the presence of alanine, AldR, which belongs to the Lrp/AsnC family of transcriptional regulators and regulates ald encoding alanine dehydrogenase in Mycobacterium smegmatis, changes its quaternary structure from a homodimer to an octamer with an open-ring conformation. Four AldR-binding sites (O2, O1, O4, and O3) with a consensus sequence of GA/T-N2-NWW/WWN-N2-A/TC were identified upstream of the M. smegmatis ald gene by means of DNase I footprinting analysis. O2, O1, and O4 are required for the induction of ald expression by alanine, while O3 is directly involved in the repression of ald expression. In addition to O3, both O1 and O4 are also necessary for full repression of ald expression in the absence of alanine, due to cooperative binding of AldR dimers to O1, O4, and O3. Binding of a molecule of the AldR octamer to the ald control region was demonstrated to require two AldR-binding sites separated by three helical turns between their centers and one additional binding site that is in phase with the two AldR-binding sites. The cooperative binding of AldR dimers to DNA requires three AldR-binding sites that are aligned with a periodicity of three helical turns. The aldR gene is negatively autoregulated independently of alanine. Comparative analysis of ald expression of M. smegmatis and Mycobacterium tuberculosis in conjunction with sequence analysis of both ald control regions led us to suggest that the expression of the ald genes in both mycobacterial species is regulated by the same mechanism. In mycobacteria, alanine dehydrogenase (Ald) is the enzyme required both to utilize alanine as a nitrogen source and to grow under hypoxic conditions by maintaining the redox state of the NADH/NAD(+) pool. Expression of the ald gene was reported to be regulated by the AldR regulator that belongs to the Lrp/AsnC (feast/famine) family, but the underlying mechanism was unknown. This study revealed the regulation mechanism of ald in Mycobacterium smegmatis and

  17. Acute and persistent Mycobacterium tuberculosis infections depend on the thiol peroxidase TpX.

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    Yanmin Hu

    Full Text Available The macrophage is the natural niche of Mycobacterium tuberculosis infection. In order to combat oxidative and nitrosative stresses and persist in macrophages successfully, M. tuberculosis is endowed with a very efficient antioxidant complex. Amongst these antioxidant enzymes, TpX is the only one in M. tuberculosis with sequence homology to thiol peroxidase. Previous reports have demonstrated that the M. tuberculosis TpX protein functions as a peroxidase in vitro. It is the dominant antioxidant which protects M. tuberculosis against oxidative and nitrosative stresses. The level of the protein increases in oxidative stress. To determine the roles of tpx gene in M. tuberculosis survival and virulence in vivo, we constructed an M. tuberculosis strain lacking the gene. The characteristics of the mutant were examined in an in vitro stationary phase model, in response to stresses; in murine bone marrow derived macrophages and in an acute and an immune resistant model of murine tuberculosis. The tpx mutant became sensitive to H(2O(2 and NO compared to the wild type strain. Enzymatic analysis using bacterial extracts from the WT and the tpx mutant demonstrated that the mutant contains reduced peroxidase activity. As a result of this, the mutant failed to grow and survive in macrophages. The growth deficiency in macrophages became more pronounced after interferon-gamma activation. In contrast, its growth was significantly restored in the macrophages of inducible nitric oxide synthase (iNOS or NOS2 knockout mice. Moreover, the tpx mutant was impaired in its ability to initiate an acute infection and to maintain a persistent infection. Its virulence was attenuated. Our results demonstrated that tpx is required for M. tuberculosis to deal with oxidative and nitrosative stresses, to survive in macrophages and to establish acute and persistent infections in animal tuberculosis models.

  18. Generalized Tuberculosis in Llamas (Lama glama) Due to Mycobacterium microti

    Science.gov (United States)

    Oevermann, A.; Pfyffer, G. E.; Zanolari, P.; Meylan, M.; Robert, N.

    2004-01-01

    Necropsy of two llamas revealed numerous caseous nodules containing abundant acid-fast bacilli (AFB) in various organs. The AFB were identified by spoligotyping as Mycobacterium microti, vole type. Infection caused by M. microti should be considered in the differential diagnosis of debilitating diseases in New World camelids. PMID:15071059

  19. Case of Mycobacterium tuberculosis meningitis: Gram staining as a useful initial diagnostic clue for tuberculous meningitis.

    Science.gov (United States)

    Kawakami, Sayoko; Kawamura, Yasuyosi; Nishiyama, Kyouhei; Hatanaka, Hiroki; Fujisaki, Ryuichi; Ono, Yasuo; Miyazawa, Yukihisa; Nishiya, Hajime

    2012-12-01

    A 32-year-old man was admitted to our hospital because of fever, headache, and loss of consciousness. Four days before admission, he had had difficulty speaking. On the day of admission, his colleague had found him to be unconscious and lying on his back. He was admitted to our hospital. The temperature at the eardrum was 35.2°C. Neurologic evaluation was negative. Computed tomography (CT) scan of the brain showed slight ventricular enlargement bilaterally. An X-ray film of the chest showed no abnormality. On the second hospital day, neck stiffness was noted. The cerebrospinal fluid (CSF) contained 870 white cells/μl, most of which were neutrophils; the glucose level in the CSF was 10 mg/dl, and the protein level was 140 mg/dl. Stained smears of the CSF, including Gram staining and India-ink preparations, disclosed no microorganisms. Capsular antigen tests for several bacteria were negative. Antimicrobial agents were started. However, by changing the microscope focus slightly while viewing Gram stains of the CSF, we could see brightened and Gram-positive bacilli that had been phagocytosed by neutrophils. This finding suggested the presence of Mycobacterium tuberculosis. Ziehl-Neelsen staining of the CSF and gastric juice revealed anti-acid bacilli. Polymerase chain reaction for M. tuberculosis in the gastric juice was positive. This case showed that Gram staining could be useful as an initial adjunct for the diagnosis of tuberculous meningitis, particularly when the CSF shows predominantly neutrocytic pleocytosis, but no other evidence of bacterial meningitis.

  20. Chromosomal rearrangements and protein globularity changes in Mycobacterium tuberculosis isolates from cerebrospinal fluid

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    Seow Hoon Saw

    2016-09-01

    Full Text Available Background Meningitis is a major cause of mortality in tuberculosis (TB. It is not clear what factors promote central nervous system invasion and pathology but it has been reported that certain strains of Mycobacterium tuberculosis (Mtb might have genetic traits associated with neurotropism. Methods In this study, we generated whole genome sequences of eight clinical strains of Mtb that were isolated from the cerebrospinal fluid (CSF of patients presenting with tuberculous meningitis (TBM in Malaysia, and compared them to the genomes of H37Rv and other respiratory Mtb genomes either downloaded from public databases or extracted from local sputum isolates. We aimed to find genomic features that might be distinctly different between CSF-derived and respiratory Mtb. Results Genome-wide comparisons revealed rearrangements (translocations, inversions, insertions and deletions and non-synonymous SNPs in our CSF-derived strains that were not observed in the respiratory Mtb genomes used for comparison. These rearranged segments were rich in genes for PE (proline-glutamate/PPE (proline-proline-glutamate, transcriptional and membrane proteins. Similarly, most of the ns SNPs common in CSF strains were noted in genes encoding PE/PPE proteins. Protein globularity differences were observed among mycobacteria from CSF and respiratory sources and in proteins previously reported to be associated with TB meningitis. Transcription factors and other transcription regulators featured prominently in these proteins. Homologs of proteins associated with Streptococcus pneumoniae meningitis and Neisseria meningitidis virulence were identified in neuropathogenic as well as respiratory mycobacterial spp. examined in this study. Discussion The occurrence of in silico genetic differences in CSF-derived but not respiratory Mtb suggests their possible involvement in the pathogenesis of TBM. However, overall findings in this comparative analysis support the postulation that TB

  1. Protein kinase G confers survival advantage to Mycobacterium tuberculosis during latency-like conditions.

    Science.gov (United States)

    Khan, Mehak Zahoor; Bhaskar, Ashima; Upadhyay, Sandeep; Kumari, Pooja; Rajmani, Raju S; Jain, Preeti; Singh, Amit; Kumar, Dhiraj; Bhavesh, Neel Sarovar; Nandicoori, Vinay Kumar

    2017-09-29

    Protein kinase G (PknG), a thioredoxin-fold-containing eukaryotic-like serine/threonine protein kinase, is a virulence factor in Mycobacterium tuberculosis , required for inhibition of phagolysosomal fusion. Here, we unraveled novel functional facets of PknG during latency-like conditions. We found that PknG mediates persistence under stressful conditions like hypoxia and abets drug tolerance. PknG mutant displayed minimal growth in nutrient-limited conditions, suggesting its role in modulating cellular metabolism. Intracellular metabolic profiling revealed that PknG is necessary for efficient metabolic adaptation during hypoxia. Notably, the PknG mutant exhibited a reductive shift in mycothiol redox potential and compromised stress response. Exposure to antibiotics and hypoxic environment resulted in higher oxidative shift in mycothiol redox potential of PknG mutant compared with the wild type. Persistence during latency-like conditions required kinase activity and thioredoxin motifs of PknG and is mediated through phosphorylation of a central metabolic regulator GarA. Finally, using a guinea pig model of infection, we assessed the in vivo role of PknG in manifestation of disease pathology and established a role for PknG in the formation of stable granuloma, hallmark structures of latent tuberculosis. Taken together, PknG-mediated GarA phosphorylation is important for maintenance of both mycobacterial physiology and redox poise, an axis that is dispensable for survival under normoxic conditions but is critical for non-replicating persistence of mycobacteria. In conclusion, we propose that PknG probably acts as a modulator of latency-associated signals. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Enhanced Anti-Mycobacterium tuberculosis Immunity over Time with Combined Drug and Immunotherapy Treatment

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    Sasha E. Larsen

    2018-05-01

    Full Text Available It is estimated that one third of the world’s population is infected with Mycobacterium tuberculosis (Mtb. This astounding statistic, in combination with costly and lengthy treatment regimens make the development of therapeutic vaccines paramount for controlling the global burden of tuberculosis. Unlike prophylactic vaccination, therapeutic immunization relies on the natural pulmonary infection with Mtb as the mucosal prime that directs boost responses back to the lung. The purpose of this work was to determine the protection and safety profile over time following therapeutic administration of our lead Mtb vaccine candidate, ID93 with a synthetic TLR4 agonist (glucopyranosyl lipid adjuvant in a stable emulsion (GLA-SE, in combination with rifampicin, isoniazid, and pyrazinamide (RHZ drug treatment. We assessed the host inflammatory immune responses and lung pathology 7–22 weeks post infection, and determined the therapeutic efficacy of combined treatment by enumeration of the bacterial load and survival in the SWR/J mouse model. We show that drug treatment alone, or with immunotherapy, tempered the inflammatory responses measured in brochoalveolar lavage fluid and plasma compared to untreated cohorts. RHZ combined with therapeutic immunizations significantly enhanced TH1-type cytokine responses in the lung over time, corresponding to decreased pulmonary pathology evidenced by a significant decrease in the percentage of lung lesions and destructive lung inflammation. These data suggest that bacterial burden assessment alone may miss important correlates of lung architecture that directly contribute to therapeutic vaccine efficacy in the preclinical mouse model. We also confirmed our previous finding that in combination with antibiotics therapeutic immunizations provide an additive survival advantage. Moreover, therapeutic immunizations with ID93/GLA-SE induced differential T cell immune responses over the course of infection that correlated

  3. Protective Effect of a Lipid-Based Preparation from Mycobacterium smegmatis in a Murine Model of Progressive Pulmonary Tuberculosis

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    Maria de los Angeles García

    2014-01-01

    Full Text Available A more effective vaccine against tuberculosis (TB is urgently needed. Based on its high genetic homology with Mycobacterium tuberculosis (Mtb, the nonpathogenic mycobacteria, Mycobacterium smegmatis (Ms, could be an attractive source of potential antigens to be included in such a vaccine. We evaluated the capability of lipid-based preparations obtained from Ms to provide a protective response in Balb/c mice after challenge with Mtb H37Rv strain. The intratracheal model of progressive pulmonary TB was used to assess the level of protection in terms of bacterial load as well as the pathological changes in the lungs of immunized Balb/c mice following challenge with Mtb. Mice immunized with the lipid-based preparation from Ms either adjuvanted with Alum (LMs-AL or nonadjuvanted (LMs showed significant reductions in bacterial load (P<0.01 compared to the negative control group (animals immunized with phosphate buffered saline (PBS. Both lipid formulations showed the same level of protection as Bacille Calmette and Guerin (BCG. Regarding the pathologic changes in the lungs, mice immunized with both lipid formulations showed less pneumonic area when compared with the PBS group (P<0.01 and showed similar results compared with the BCG group. These findings suggest the potential of LMs as a promising vaccine candidate against TB.

  4. The Association between Mycobacterium Tuberculosis Genotype and Drug Resistance in Peru.

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    Louis Grandjean

    Full Text Available The comparison of Mycobacterium tuberculosis bacterial genotypes with phenotypic, demographic, geospatial and clinical data improves our understanding of how strain lineage influences the development of drug-resistance and the spread of tuberculosis.To investigate the association of Mycobacterium tuberculosis bacterial genotype with drug-resistance. Drug susceptibility testing together with genotyping using both 15-loci MIRU-typing and spoligotyping, was performed on 2,139 culture positive isolates, each from a different patient in Lima, Peru. Demographic, geospatial and socio-economic data were collected using questionnaires, global positioning equipment and the latest national census.The Latin American Mediterranean (LAM clade (OR 2.4, p<0.001 was significantly associated with drug-resistance and alone accounted for more than half of all drug resistance in the region. Previously treated patients, prisoners and genetically clustered cases were also significantly associated with drug-resistance (OR's 2.5, 2.4 and 1.8, p<0.001, p<0.05, p<0.001 respectively.Tuberculosis disease caused by the LAM clade was more likely to be drug resistant independent of important clinical, genetic and socio-economic confounding factors. Explanations for this include; the preferential co-evolution of LAM strains in a Latin American population, a LAM strain bacterial genetic background that favors drug-resistance or the "founder effect" from pre-existing LAM strains disproportionately exposed to drugs.

  5. In vitro efficacy of ethionamide and clarithromycin in mycobacterium tuberculosis isolates

    International Nuclear Information System (INIS)

    Satti, M.S.; Abbasi, S.; Rafi, S.; Butt, T.; Karamat, K.A.

    2010-01-01

    To determine the sensitivity of clinical isolates of Mycobacterium tuberculosis isolates against ethionamide, and clarithromycin. Department of Microbiology, Armed Forces institute of Pathology (AFIP) Rawalpindi from June 2003 to June 2004. Materials and Methods: All routine clinical samples received for acid fast bacilli (AFB) culture and yielding positive growth on Lowenstien Jensen medium and Bactec 460 were include in the study. The isolates were from sputum (n=70), bronchioalveolar lavage (n=10), fine needle aspiration (n=6), lymph nodes (n=7), pleural fluid (n=4), endometrium (n=3). After the identification of M. tuberculosis (MTB) sensitivity was performed against first-line antituberculosis drugs. Then susceptibility of M. tuberculosis isolates against ethionamide and clarithromycih was performed on LJ medium. Mycobacterium H37Rv was used as control strain. Results were interpreted using resistance ratio method. Out of 100 M. tuberculosis isolates, sensitivity to ethionamide was 93% and 9% to clarithromycih. Clarithromycin when used alone is ineffective as antituberculosis drug but its efficacy in combination needs to be tested. However ethionamide may be used as an alternative antituberculosis drug. (author)

  6. Capsular glucan and intracellular glycogen of Mycobacterium tuberculosis: biosynthesis and impact on the persistence in mice

    DEFF Research Database (Denmark)

    Sambou, Tounkang; Dinadayala, Premkumar; Stadthagen, Gustavo

    2008-01-01

    Mycobacterium tuberculosis and other pathogenic mycobacterial species produce large amounts of a glycogen-like alpha-glucan that represents the major polysaccharide of their outermost capsular layer. To determine the role of the surface-exposed glucan in the physiology and virulence of these bact......Mycobacterium tuberculosis and other pathogenic mycobacterial species produce large amounts of a glycogen-like alpha-glucan that represents the major polysaccharide of their outermost capsular layer. To determine the role of the surface-exposed glucan in the physiology and virulence...... of these bacteria, orthologues of the glg genes involved in the biosynthesis of glycogen in Escherichia coli were identified in M. tuberculosis H37Rv and inactivated by allelic replacement. Biochemical analyses of the mutants and complemented strains indicated that the synthesis of glucan and glycogen involves...... the alpha-1,4-glucosyltransferases Rv3032 and GlgA (Rv1212c), the ADP-glucose pyrophosphorylase GlgC (Rv1213) and the branching enzyme GlgB (Rv1326c). Disruption of glgC reduced by half the glucan and glycogen contents of M. tuberculosis, whereas the inactivation of glgA and Rv3032 affected the production...

  7. The conserved Lysine69 residue plays a catalytic role in Mycobacterium tuberculosis shikimate dehydrogenase

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    Rodrigues Valnês

    2009-01-01

    Full Text Available Abstract Background The shikimate pathway is an attractive target for the development of antitubercular agents because it is essential in Mycobacterium tuberculosis, the causative agent of tuberculosis, but absent in humans. M. tuberculosis aroE-encoded shikimate dehydrogenase catalyzes the forth reaction in the shikimate pathway. Structural and functional studies indicate that Lysine69 may be involved in catalysis and/or substrate binding in M. tuberculosis shikimate dehydrogenase. Investigation of the kinetic properties of mutant enzymes can bring important insights about the role of amino acid residues for M. tuberculosis shikimate dehydrogenase. Findings We have performed site-directed mutagenesis, steady-state kinetics, equilibrium binding measurements and molecular modeling for both the wild-type M. tuberculosis shikimate dehydrogenase and the K69A mutant enzymes. The apparent steady-state kinetic parameters for the M. tuberculosis shikimate dehydrogenase were determined; the catalytic constant value for the wild-type enzyme (50 s-1 is 68-fold larger than that for the mutant K69A (0.73 s-1. There was a modest increase in the Michaelis-Menten constant for DHS (K69A = 76 μM; wild-type = 29 μM and NADPH (K69A = 30 μM; wild-type = 11 μM. The equilibrium dissociation constants for wild-type and K69A mutant enzymes are 32 (± 4 μM and 134 (± 21, respectively. Conclusion Our results show that the residue Lysine69 plays a catalytic role and is not involved in substrate binding for the M. tuberculosis shikimate dehydrogenase. These efforts on M. tuberculosis shikimate dehydrogenase catalytic mechanism determination should help the rational design of specific inhibitors, aiming at the development of antitubercular drugs.

  8. Association between passive smoking and Mycobacterium tuberculosis infection in children with household TB contact

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    Novaily Zuliartha

    2015-03-01

    Full Text Available Background Tuberculosis (TB and cigarette consumption are relatively high in Indonesia. Passive smoking may increase the risk of infection and disease in adults and children exposed to TB. An association between passive smoking and Mycobacterium tuberculosis infection in children has not been well documented. Objective To assess for an association between passive smoking and M. tuberculosis infection in children who had household contact with a TB patient. Methods This cross-sectional study was conducted in February and March 2011. Children aged 5 to 18 years who had household contact with a TB patient underwent tuberculin testing for M. tuberculosis infection. Subjects were divided into two groups: those exposed to passive smoke and those not exposed to passive smoke. Chi-square test was used to assess for an association between passive smoking and M. tuberculosis infection. Results There were 140 children enrolled in this study, with 70 exposed to passive smoke and 70 not exposed to passive smoke. Prevalence of M. tuberculosis infection was significantly higher in the passive smoking group than in those not exposed to passive smoke [81.4% and 52.9%, respectively, (P= 0.0001]. In the passive smoking group there were significant associations between nutritional state, paternal and maternal education, and M. tuberculosis infection. But no associations were found between M. tuberculosis infection and familial income or BCG vaccination. Conclusion Among children who had household contact with a TB patient, they who exposed to passive smoke are more likely to have M. tuberculosis infection compared to they who not exposed to passive smoke.

  9. Structural measurements and cell line studies of the copper-PEG-Rifampicin complex against Mycobacterium tuberculosis.

    Science.gov (United States)

    Manning, Thomas; Mikula, Rachel; Wylie, Greg; Phillips, Dennis; Jarvis, Jackie; Zhang, Fengli

    2015-02-01

    The bacterium responsible for tuberculosis is increasing its resistance to antibiotics resulting in new multidrug-resistant Mycobacterium tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB). In this study, several analytical techniques including NMR, FT-ICR, MALDI-MS, LC-MS and UV/Vis are used to study the copper-Rifampicin-Polyethylene glycol (PEG-3350) complex. The copper (II) cation is a carrier for the antibiotic Rifampicin as well as nutrients for the bacterium. The NIH-NIAID cell line containing several Tb strains (including antibiotic resistant strains) is tested against seven copper-PEG-RIF complex variations. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. RAPD Analysis and Antibiotic Susceptibility for Mycobacterium tuberculosis Strains Isolated from Different Locations in Egypt

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    Ali, A. M.

    2011-01-01

    Full Text Available The routine identification of mycobacterial strains isolated from patients in different locations in Egypt was confirmed by specific DNA fragment amplification. The susceptibilities of 72 Mycobacterium tuberculosis strains against the four antibiotics used in tuberculosis treatment (Isoniazid, INH; Rifampicin, Rif; Streptomycin, St and Ethambutol, E were examined. Our results indicated that, multi drug resistant tuberculosis (MDR-TB represents about 19.5% of the tested strains, whereas sensitive strains represented 26.4%. The genetic polymorphism of the tested strains was examined using RAPD analysis. Six selected strains represent the different antibiotic susceptibility groups were examined using RAPD fingerprinting. No difference between the strains was recorded using the RFLP analysis of amplified specific fragment. The discrimination power of RAPD analysis was inadequate to clarify the genetic correlation between the tested strains. MDR-TB was approximately double time in 2008 compared with the value in 2007. Most of the new MDRTB was correlated with resident dense population regions.

  11. Determination of in vitro susceptibility of Mycobacterium tuberculosis to cephalosporins by radiometric and conventional methods

    International Nuclear Information System (INIS)

    Heifets, L.B.; Iseman, M.D.; Cook, J.L.; Lindholm-Levy, P.J.; Drupa, I.

    1985-01-01

    Among eight cephalosporins and cephamycins tested in preliminary in vitro screening against Mycobacterium tuberculosis, the most promising for further study was found to be ceforanide, followed by ceftizoxime, cephapirin, and cefotaxime. Moxalactam, cefoxitin, cefamandole, and cephalothin were found to be not active enough against M. tuberculosis to be considered for further in vitro studies. The antibacterial activity of various ceforanide concentrations was investigated by three methods: (i) the dynamics of radiometric readings (growth index) in 7H12 broth; (ii) the number of CFU in the same medium; and (iii) the proportion method on 7H11 agar plates. There was a good correlation among the results obtained with these methods. The MIC for most strains ranged from 6.0 to 25.0 micrograms/ml. The BACTEC radiometric method is a reliable, rapid, and convenient method for preliminary screening and determination of the level of antibacterial activity of drugs not commonly used against M. tuberculosis

  12. Fluoromycobacteriophages for Rapid, Specific, and Sensitive Antibiotic Susceptibility Testing of Mycobacterium tuberculosis

    Science.gov (United States)

    Piuri, Mariana; Jacobs, William R.; Hatfull, Graham F.

    2009-01-01

    Rapid antibiotic susceptibility testing of Mycobacterium tuberculosis is of paramount importance as multiple- and extensively- drug resistant strains of M. tuberculosis emerge and spread. We describe here a virus-based assay in which fluoromycobacteriophages are used to deliver a GFP or ZsYellow fluorescent marker gene to M. tuberculosis, which can then be monitored by fluorescent detection approaches including fluorescent microscopy and flow cytometry. Pre-clinical evaluations show that addition of either Rifampicin or Streptomycin at the time of phage addition obliterates fluorescence in susceptible cells but not in isogenic resistant bacteria enabling drug sensitivity determination in less than 24 hours. Detection requires no substrate addition, fewer than 100 cells can be identified, and resistant bacteria can be detected within mixed populations. Fluorescence withstands fixation by paraformaldehyde providing enhanced biosafety for testing MDR-TB and XDR-TB infections. PMID:19300517

  13. Delayed bactericidal response of Mycobacterium tuberculosis to bedaquiline involves remodelling of bacterial metabolism

    DEFF Research Database (Denmark)

    Koul, A.; Vranckx, L.; Dhar, N.

    2014-01-01

    Bedaquiline (BDQ), an ATP synthase inhibitor, is the first drug to be approved for treatment of multidrug-resistant tuberculosis in decades. Though BDQ has shown excellent efficacy in clinical trials, its early bactericidal activity during the first week of chemotherapy is minimal. Here, using...... microfluidic devices and time-lapse microscopy of Mycobacterium tuberculosis, we confirm the absence of significant bacteriolytic activity during the first 3-4 days of exposure to BDQ. BDQ-induced inhibition of ATP synthesis leads to bacteriostasis within hours after drug addition. Transcriptional...... and proteomic analyses reveal that M. tuberculosis responds to BDQ by induction of the dormancy regulon and activation of ATP-generating pathways, thereby maintaining bacterial viability during initial drug exposure. BDQ-induced bacterial killing is significantly enhanced when the mycobacteria are grown on non...

  14. Proteomic profiling of Mycobacterium tuberculosis identifies nutrient-starvation-responsive toxin-antitoxin systems

    DEFF Research Database (Denmark)

    Albrethsen, Jakob; Agner, Jeppe; Piersma, Sander R

    2013-01-01

    In order to successfully enter the latent stage, Mycobacterium tuberculosis must adapt to conditions such as nutrient limitation and hypoxia. In vitro models that mimic latent infection are valuable tools for describing the changes in metabolism that occur when the bacterium exists in a non......-growing form. We used two complementary proteomic approaches, label-free LC-MS/MS analysis and two-dimensional difference gel electrophoresis, to determine the proteome profile of extracellular proteins from M. tuberculosis cultured under nutrient starvation. Through the label-free LC-MS/MS analysis......, significant differences in the overall metabolism during nutrient starvation were detected. Notably, members of the toxin-antitoxin systems were present in larger quantities in nutrient-starved cultures, supporting a role for these global modules as M. tuberculosis switches its metabolism into dormancy...

  15. Comparative molecular study of Mycobacterium tuberculosis strains, in times of antimicrobial drug resistance

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    G. Varela

    2005-03-01

    Full Text Available Strains of Mycobacterium tuberculosis were compared using two DNA fingerprinting techniques: Restriction Fragment Length Polymorphism (RFLP and Double-Repetitive-Element-PCR (DRE-PCR. Two of these strains: IH1 (susceptible to isoniazid and IH2 (resistant to isoniazid were recovered from cases of pulmonary tuberculosis which occurred in two brothers who lived together. The first one was recognized on July 1999, and the second was diagnosed one year later. IH1 and IH2 showed the same pattern of bands with both molecular tests. These results suggest that single drug chemoprophylaxis may occasionally select resistant strains for that drug, which can eventually cause disease and be recognized through these tests. Strains IH3, IH4 and IH5 were obtained from sputum samples of 3 different patients, and intra-laboratory cross-contamination was suspected when it was realized that the 3 positive materials had been consecutively processed the same day by the same worker in the same biological safety cabinet. Again, the 3 strains revealed identical band patterns with RFLP and DRE-PCR, confirming the posed suspicion. The results with DRE-PCR were obtained after only 8 hours of work, without the need for subcultures. This procedure allows quick correction of treatment conducts, avoiding unnecessary exposure of people and bacteria to antimicrobial drugs.Se compararon cepas de Mycobacterium tuberculosis utilizando 2 procedimientos de ADN fingerprinting: polimorfismo de los fragmentos de restricción (RFLP y Double-Repetitive-Element-PCR (DRE-PCR. Dos de las cepas: IH1 (susceptible a isoniazida e IH2 (resistente a isoniazida se recuperaron a partir de casos de tuberculosis pulmonar que ocurrieron en dos hermanos convivientes. La primera fue aislada en julio de 1999 y la segunda un año después. IH1 e IH2 mostraron el mismo patrón de bandas por ambos procedimientos. Estos resultados sugieren que la quimioprofilaxis con una sola droga puede ocasionalmente

  16. T-cell recognition of Mycobacterium tuberculosis culture filtrate fractions in tuberculosis patients and their household contacts

    DEFF Research Database (Denmark)

    Demissie, A; Ravn, P; Olobo, J

    1999-01-01

    We examined the immune responses of patients with active pulmonary tuberculosis (TB) and their healthy household contacts to short-term culture filtrate (ST-CF) of Mycobacterium tuberculosis or molecular mass fractions derived from it. Our goal was to identify fractions strongly recognized...... to the antigens between the two groups was also found. In general, while all fractions stimulated immune responses, the highest activity was seen with the low-molecular-mass fractions, which include well-defined TB antigens such as ESAT-6. Leukocytes from contacts of TB patients with severe disease produced...... higher levels of antigen-specific IFN-gamma than those from contacts of patients with minimal disease. Both groups of contacts exhibited higher cell-mediated responses than the patients themselves. The enhanced immune response of healthy contacts, especially those of patients with severe disease...

  17. T-cell recognition of Mycobacterium tuberculosis culture filtrate fractions in tuberculosis patients and their household contacts

    DEFF Research Database (Denmark)

    Demissie, A; Ravn, P; Olobo, J

    1999-01-01

    We examined the immune responses of patients with active pulmonary tuberculosis (TB) and their healthy household contacts to short-term culture filtrate (ST-CF) of Mycobacterium tuberculosis or molecular mass fractions derived from it. Our goal was to identify fractions strongly recognized...... antigens and immune responses were determined. Household contacts produced significantly higher levels of gamma interferon (IFN-gamma) than the TB patients in response to antigens present in ST-CF and the 10 narrow-molecular-mass fractions. A similar difference in leukocyte proliferative responses...... to the antigens between the two groups was also found. In general, while all fractions stimulated immune responses, the highest activity was seen with the low-molecular-mass fractions, which include well-defined TB antigens such as ESAT-6. Leukocytes from contacts of TB patients with severe disease produced...

  18. Crystal structure of DNA polymerase III β sliding clamp from Mycobacterium tuberculosis.

    Science.gov (United States)

    Gui, Wen-Jun; Lin, Shi-Qiang; Chen, Yuan-Yuan; Zhang, Xian-En; Bi, Li-Jun; Jiang, Tao

    2011-02-11

    The sliding clamp is a key component of DNA polymerase III (Pol III) required for genome replication. It is known to function with diverse DNA repair proteins and cell cycle-control proteins, making it a potential drug target. To extend our understanding of the structure/function relationship of the sliding clamp, we solved the crystal structure of the sliding clamp from Mycobacterium tuberculosis (M. tuberculosis), a human pathogen that causes most cases of tuberculosis (TB). The sliding clamp from M. tuberculosis forms a ring-shaped head-to-tail dimer with three domains per subunit. Each domain contains two α helices in the inner ring that lie against two β sheets in the outer ring. Previous studies have indicated that many Escherichia coli clamp-binding proteins have a conserved LF sequence, which is critical for binding to the hydrophobic region of the sliding clamp. Here, we analyzed the binding affinities of the M. tuberculosis sliding clamp and peptides derived from the α and δ subunits of Pol III, which indicated that the LF motif also plays an important role in the binding of the α and δ subunits to the sliding clamp of M. tuberculosis. Copyright © 2011 Elsevier Inc. All rights reserved.

  19. Limited variation of DNA fingerprints (IS6110 and IS1081) in Korean strains of Mycobacterium tuberculosis.

    Science.gov (United States)

    Huh, Y J; Ahn, D I; Kim, S J

    1995-08-01

    To establish the usefulness of DNA fingerprinting for the epidemiology of Mycobacterium tuberculosis isolated from Korean tuberculosis patients. Comparison of restriction fragment length polymorphism (RFLP) patterns produced by southern hybridization of PvuII-digested chromosomal DNA. IS6110-associated banding patterns of 41 isolates varied considerably, containing 1-13 copies. The RFLP pattern of the epidemiologically related M. tuberculosis isolates was identical in 8 of 10 groups of close contact patients. No noticeable differences in RFLP were observed between drug-sensitive and drug-resistant isolates. IS1081-containing restriction fragment analysis of 52 isolates showed 6 different banding patterns, and the C type was found dominant in Korea. Identification of G type M. tuberculosis, which has a 8.0 kb IS1081-containing PvuII fragment, is unusual because it has been observed only in M. bovis BCG so far. IS6110 was a very useful tool for tracing the transmission route of tuberculosis; IS1081 was also useful for subdividing M. tuberculosis into several groups.

  20. Immunological response to Mycobacterium tuberculosis infection in blood from type 2 diabetes patients.

    Science.gov (United States)

    Raposo-García, Sara; Guerra-Laso, José Manuel; García-García, Silvia; Juan-García, Javier; López-Fidalgo, Eduardo; Diez-Tascón, Cristina; Nebreda-Mayoral, Teresa; López-Medrano, Ramiro; Rivero-Lezcano, Octavio Miguel

    2017-06-01

    The convergence of tuberculosis and diabetes represents a co-epidemic that threatens progress against tuberculosis. We have investigated type 2 diabetes as a risk factor for tuberculosis susceptibility, and have used as experimental model whole blood infected in vitro with Mycobacterium tuberculosis. Blood samples from diabetic patients were found to have a higher absolute neutrophil count that non-diabetic controls, but their immune functionality seemed impaired because they displayed a lower capacity to phagocytose M. tuberculosis, a finding that had been previously reported only for monocytes. In contrast, an increased production of TNFα was detected in infected blood from diabetic patients. Despite the altered phagocytic capacity showed by cells from these patients, the antimicrobial activity measured in both whole blood and monocyte derived macrophages was similar to that of controls. This unexpected result prompts further improvements in the whole blood model to analyze the immune response of diabetes patients to tuberculosis. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  1. Mycobacterium tuberculosis nuoG is a virulence gene that inhibits apoptosis of infected host cells.

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    Kamalakannan Velmurugan

    2007-07-01

    Full Text Available The survival and persistence of Mycobacterium tuberculosis depends on its capacity to manipulate multiple host defense pathways, including the ability to actively inhibit the death by apoptosis of infected host cells. The genetic basis for this anti-apoptotic activity and its implication for mycobacterial virulence have not been demonstrated or elucidated. Using a novel gain-of-function genetic screen, we demonstrated that inhibition of infection-induced apoptosis of macrophages is controlled by multiple genetic loci in M. tuberculosis. Characterization of one of these loci in detail revealed that the anti-apoptosis activity was attributable to the type I NADH-dehydrogenase of M. tuberculosis, and was mainly due to the subunit of this multicomponent complex encoded by the nuoG gene. Expression of M. tuberculosis nuoG in nonpathogenic mycobacteria endowed them with the ability to inhibit apoptosis of infected human or mouse macrophages, and increased their virulence in a SCID mouse model. Conversely, deletion of nuoG in M. tuberculosis ablated its ability to inhibit macrophage apoptosis and significantly reduced its virulence in mice. These results identify a key component of the genetic basis for an important virulence trait of M. tuberculosis and support a direct causal relationship between virulence of pathogenic mycobacteria and their ability to inhibit macrophage apoptosis.

  2. Disinfectant-susceptibility of multi-drug-resistant Mycobacterium tuberculosis isolated in Japan

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    Noriko Shinoda

    2016-02-01

    Full Text Available Abstract Background Multi-drug-resistant Mycobacterium tuberculosis has been an important problem in public health around the world. However, limited information about disinfectant-susceptibility of multi-drug-resistant strain of M. tuberculosis was available. Findings We studied susceptibility of several Japanese isolates of multi-drug-resistant M. tuberculosis against disinfectants, which are commonly used in clinical and research laboratories. We selected a laboratory reference strain (H37Rv and eight Japanese isolates, containing five drug-susceptible strains and three multi-drug-resistant strains, and determined profiles of susceptibility against eight disinfectants. The M. tuberculosis strains were distinguished into two groups by the susceptibility profile. There was no relationship between multi-drug-resistance and disinfectant-susceptibility in the M. tuberculosis strains. Cresol soap and oxydol were effective against all strains we tested, regardless of drug resistance. Conclusions Disinfectant-resistance is independent from multi-drug-resistance in M. tuberculosis. Cresol soap and oxydol were effective against all strains we tested, regardless of drug resistance.

  3. Pyrosequencing for Rapid Detection of Mycobacterium tuberculosis Resistance to Rifampin, Isoniazid, and Fluoroquinolones ▿

    Science.gov (United States)

    Bravo, Lulette Tricia C.; Tuohy, Marion J.; Ang, Concepcion; Destura, Raul V.; Mendoza, Myrna; Procop, Gary W.; Gordon, Steven M.; Hall, Geraldine S.; Shrestha, Nabin K.

    2009-01-01

    After isoniazid and rifampin (rifampicin), the next pivotal drug class in Mycobacterium tuberculosis treatment is the fluoroquinolone class. Mutations in resistance-determining regions (RDR) of the rpoB, katG, and gyrA genes occur with frequencies of 97%, 50%, and 85% among M. tuberculosis isolates resistant to rifampin, isoniazid, and fluoroquinolones, respectively. Sequences are highly conserved, and certain mutations correlate well with phenotypic resistance. We developed a pyrosequencing assay to determine M. tuberculosis genotypic resistance to rifampin, isoniazid, and fluoroquinolones. We characterized 102 M. tuberculosis clinical isolates from the Philippines for susceptibility to rifampin, isoniazid, and ofloxacin by using the conventional submerged-disk proportion method and validated our pyrosequencing assay using these isolates. DNA was extracted and amplified by using PCR primers directed toward the RDR of the rpoB, katG, and gyrA genes, and pyrosequencing was performed on the extracts. The M. tuberculosis H37Rv strain (ATCC 25618) was used as the reference strain. The sensitivities and specificities of pyrosequencing were 96.7% and 97.3%, 63.8% and 100%, and 70.0% and 100% for the detection of resistance to rifampin, isoniazid, and ofloxacin, respectively. Pyrosequencing is thus a rapid and accurate method for detecting M. tuberculosis resistance to these three drugs. PMID:19846642

  4. Characterisation of Mycobacterium tuberculosis isolates lacking IS6110 in Viet Nam.

    Science.gov (United States)

    Huyen, M N T; Tiemersma, E W; Kremer, K; de Haas, P; Lan, N T N; Buu, T N; Sola, C; Cobelens, F G J; van Soolingen, D

    2013-11-01

    The molecular diagnosis of tuberculosis (TB) in Viet Nam is often based on the detection of insertion sequence (IS) 6110 in Mycobacterium tuberculosis. However, 8-11% of M. tuberculosis strains in South-East Asia do not contain this target and this undermines the validity of these molecular tests. We quantified the frequency of M. tuberculosis strains lacking IS6110 in rural Viet Nam and studied their epidemiological and clinical characteristics. Consecutively diagnosed adult TB patients in rural Southern Viet Nam submitted two sputum samples for culture, IS6110 restriction fragment length polymorphism (RFLP) spoligotyping and 15-loci variable number tandem repeat typing. Polymerase chain reaction (PCR) was performed to confirm the absence of IS6110 elements in strains lacking IS6110 hybridisation in RFLP. Among 2664 TB patient isolates examined, 109 (4.1%) had no IS6110 element. Compared to other strains, these no-copy strains were less often resistant to anti-tuberculosis drugs, particularly to streptomycin (adjusted OR 0.2, 95%CI 0.1-0.5), and showed significant geographic variation. No associations with TB history or demographic factors were found. Strains without the IS6110 target pose a problem in Viet Nam as regards false-negative molecular TB diagnosis in PCR. Compared to other strains circulating in Viet Nam, no-copy strains are more susceptible to anti-tuberculosis drugs.

  5. Mycobacterium tuberculosis complex lipid virulence factors preserved in the 17,000-year-old skeleton of an extinct bison, Bison antiquus.

    Directory of Open Access Journals (Sweden)

    Oona Y-C Lee

    Full Text Available Tracing the evolution of ancient diseases depends on the availability and accessibility of suitable biomarkers in archaeological specimens. DNA is potentially information-rich but it depends on a favourable environment for preservation. In the case of the major mycobacterial pathogens, Mycobacterium tuberculosis and Mycobacterium leprae, robust lipid biomarkers are established as alternatives or complements to DNA analyses. A DNA report, a decade ago, suggested that a 17,000-year-old skeleton of extinct Bison antiquus, from Natural Trap Cave, Wyoming, was the oldest known case of tuberculosis. In the current study, key mycobacterial lipid virulence factor biomarkers were detected in the same two samples from this bison. Fluorescence high-performance liquid chromatography (HPLC indicated the presence of mycolic acids of the mycobacterial type, but they were degraded and could not be precisely correlated with tuberculosis. However, pristine profiles of C(29, C(30 and C(32 mycocerosates and C(27 mycolipenates, typical of the Mycobacterium tuberculosis complex, were recorded by negative ion chemical ionization gas chromatography mass spectrometry of pentafluorobenzyl ester derivatives. These findings were supported by the detection of C(34 and C(36 phthiocerols, which are usually esterified to the mycocerosates. The existence of Pleistocene tuberculosis in the Americas is confirmed and there are many even older animal bones with well-characterised tuberculous lesions similar to those on the analysed sample. In the absence of any evidence of tuberculosis in human skeletons older than 9,000 years BP, the hypothesis that this disease evolved as a zoonosis, before transfer to humans, is given detailed consideration and discussion.

  6. Evaluation of rapid immuno chromatographic assay kit using monoclonal mpt64 antibodies for identification of mycobacterium tuberculosis complex

    International Nuclear Information System (INIS)

    Satti, L.; Ikram, A.; Malik, N.

    2010-01-01

    To evaluate the performance of rapid immuno chromatographic kit MPT64 Ag for the identification of Mycobacterium tuberculosis complex from various Mycobacterium tuberculosis culture positive specimens. Department of Microbiology, Armed Forces Institute of Pathology Rawalpindi, from August 2008 through March 2009. Eighty four Mycobacterium tuberculosis positive cultures on I BACTEC 460 and MGIT 960, one ATCC 25177 MTB strain, three institutional control MTB strains, two institutional control MOTT strains and 20 different bacterial isolates were tested. Tests were performed according to the instructional manual. Out of total 84 tested samples, MPT64 showed positive result in 80 cultures. Only four positive cultures did not display any band on MPT64 kit. These four strains were reconfirmed as Mycobacterium tuberculosis by PCR method. MOTT control strains and all the 20 bacterial isolates were negative for band. The sensitivity and specificity of ICT assay in our study was 95.2% and 100% respectively. Rapid MPT64 Kit is a good diagnostic tool to differentiate between Mycobacterium tuberculosis complex and MOTT with 100% specificity. The technique is simple and can provide prompt information to the clinicians to initiate early and appropriate antituberculosis therapy. (author)

  7. Evaluation of selected Indian traditional folk medicinal plants against Mycobacterium tuberculosis with antioxidant and cytotoxicity study.

    Directory of Open Access Journals (Sweden)

    Tawde K. V

    2012-10-01

    Full Text Available Objective: To evaluate different solvent extracts of selected Indian traditional medicinal plant against Mycobacterium tuberculosis, its antioxidant potential and cytotoxicity. Methods: Acacia catechu (L. Willd (Root extract and Ailanthus excelsa Roxb., leaf extracts of Aegle marmelos Corr., Andrographis paniculata Nees. and Datura metel L. were sequentially extracted in water, ethanol, chloroform and hexane and evaluated for their anti-tuberculosis (TB activity against Mycobacterium tuberculosis using agar diffusion assay. The zone of inhibition ( at 20 and 40 mg/ ml was measured and MIC were calculated. The results were compared with Rifampicin as a standard anti TB drug. The extracts were also evaluated for DPPH and OH radical scavenging activities to understand their antioxidant potential. MTT based cytotoxicity assay was used for evaluating cytotoxicity of the selected samples against Chang liver cells. Results: The selected botanicals were sequentially extracted in water, ethanol, chloroform and hexane and tested for growth inhibition of M. tuberculosi. The hexane extract of A. catechu root and ethanol extract of A. paniculata leaf showed promising activity against M. tuberculosis while remaining extracts showed moderate anti TB activity. The samples were found to possess considerable DPPH and OH radical scavenging activities with no demonstrable cytotoxicity against Chang liver cells. Conclusions: Five traditional medicinal plants were selected for the present study. The selection of medicinal plants was based on their traditional usage for the treatment of tuberculosis, asthma and chronic respiratory diseases. Herein we report for the first time, the anti TB activity of root extracts of Acacia catechu and Ailanthus excelsa while leaf extract of Andrographis paniculata, Aegle marmelos and Datura metel. The study holds importance in the midst of multi drug resistance (MDR crisis in the TB management, since it unravels the scientific basis

  8. Crowd Sourcing a New Paradigm for Interactome Driven Drug Target Identification in Mycobacterium tuberculosis

    Science.gov (United States)

    Rohira, Harsha; Bhat, Ashwini G.; Passi, Anurag; Mukherjee, Keya; Choudhary, Kumari Sonal; Kumar, Vikas; Arora, Anshula; Munusamy, Prabhakaran; Subramanian, Ahalyaa; Venkatachalam, Aparna; S, Gayathri; Raj, Sweety; Chitra, Vijaya; Verma, Kaveri; Zaheer, Salman; J, Balaganesh; Gurusamy, Malarvizhi; Razeeth, Mohammed; Raja, Ilamathi; Thandapani, Madhumohan; Mevada, Vishal; Soni, Raviraj; Rana, Shruti; Ramanna, Girish Muthagadhalli; Raghavan, Swetha; Subramanya, Sunil N.; Kholia, Trupti; Patel, Rajesh; Bhavnani, Varsha; Chiranjeevi, Lakavath; Sengupta, Soumi; Singh, Pankaj Kumar; Atray, Naresh; Gandhi, Swati; Avasthi, Tiruvayipati Suma; Nisthar, Shefin; Anurag, Meenakshi; Sharma, Pratibha; Hasija, Yasha; Dash, Debasis; Sharma, Arun; Scaria, Vinod; Thomas, Zakir; Chandra, Nagasuma; Brahmachari, Samir K.; Bhardwaj, Anshu

    2012-01-01

    A decade since the availability of Mycobacterium tuberculosis (Mtb) genome sequence, no promising drug has seen the light of the day. This not only indicates the challenges in discovering new drugs but also suggests a gap in our current understanding of Mtb biology. We attempt to bridge this gap by carrying out extensive re-annotation and constructing a systems level protein interaction map of Mtb with an objective of finding novel drug target candidates. Towards this, we synergized crowd sourcing and social networking methods through an initiative ‘Connect to Decode’ (C2D) to generate the first and largest manually curated interactome of Mtb termed ‘interactome pathway’ (IPW), encompassing a total of 1434 proteins connected through 2575 functional relationships. Interactions leading to gene regulation, signal transduction, metabolism, structural complex formation have been catalogued. In the process, we have functionally annotated 87% of the Mtb genome in context of gene products. We further combine IPW with STRING based network to report central proteins, which may be assessed as potential drug targets for development of drugs with least possible side effects. The fact that five of the 17 predicted drug targets are already experimentally validated either genetically or biochemically lends credence to our unique approach. PMID:22808064

  9. Structural plasticity in Mycobacterium tuberculosis uracil-DNA glycosylase (MtUng) and its functional implications.

    Science.gov (United States)

    Arif, S M; Geethanandan, K; Mishra, P; Surolia, A; Varshney, U; Vijayan, M

    2015-07-01

    17 independent crystal structures of family I uracil-DNA glycosylase from Mycobacterium tuberculosis (MtUng) and its complexes with uracil and its derivatives, distributed among five distinct crystal forms, have been determined. Thermodynamic parameters of binding in the complexes have been measured using isothermal titration calorimetry. The two-domain protein exhibits open and closed conformations, suggesting that the closure of the domain on DNA binding involves conformational selection. Segmental mobility in the enzyme molecule is confined to a 32-residue stretch which plays a major role in DNA binding. Uracil and its derivatives can bind to the protein in two possible orientations. Only one of them is possible when there is a bulky substituent at the 5' position. The crystal structures of the complexes provide a reasonable rationale for the observed thermodynamic parameters. In addition to providing fresh insights into the structure, plasticity and interactions of the protein molecule, the results of the present investigation provide a platform for structure-based inhibitor design.

  10. Crowd sourcing a new paradigm for interactome driven drug target identification in Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Rohit Vashisht

    Full Text Available A decade since the availability of Mycobacterium tuberculosis (Mtb genome sequence, no promising drug has seen the light of the day. This not only indicates the challenges in discovering new drugs but also suggests a gap in our current understanding of Mtb biology. We attempt to bridge this gap by carrying out extensive re-annotation and constructing a systems level protein interaction map of Mtb with an objective of finding novel drug target candidates. Towards this, we synergized crowd sourcing and social networking methods through an initiative 'Connect to Decode' (C2D to generate the first and largest manually curated interactome of Mtb termed 'interactome pathway' (IPW, encompassing a total of 1434 proteins connected through 2575 functional relationships. Interactions leading to gene regulation, signal transduction, metabolism, structural complex formation have been catalogued. In the process, we have functionally annotated 87% of the Mtb genome in context of gene products. We further combine IPW with STRING based network to report central proteins, which may be assessed as potential drug targets for development of drugs with least possible side effects. The fact that five of the 17 predicted drug targets are already experimentally validated either genetically or biochemically lends credence to our unique approach.

  11. Mycobacterium tuberculosis infection induces non-apoptotic cell death of human dendritic cells

    LENUS (Irish Health Repository)

    Ryan, Ruth CM

    2011-10-24

    Abstract Background Dendritic cells (DCs) connect innate and adaptive immunity, and are necessary for an efficient CD4+ and CD8+ T cell response after infection with Mycobacterium tuberculosis (Mtb). We previously described the macrophage cell death response to Mtb infection. To investigate the effect of Mtb infection on human DC viability, we infected these phagocytes with different strains of Mtb and assessed viability, as well as DNA fragmentation and caspase activity. In parallel studies, we assessed the impact of infection on DC maturation, cytokine production and bacillary survival. Results Infection of DCs with live Mtb (H37Ra or H37Rv) led to cell death. This cell death proceeded in a caspase-independent manner, and without nuclear fragmentation. In fact, substrate assays demonstrated that Mtb H37Ra-induced cell death progressed without the activation of the executioner caspases, 3\\/7. Although the death pathway was triggered after infection, the DCs successfully underwent maturation and produced a host-protective cytokine profile. Finally, dying infected DCs were permissive for Mtb H37Ra growth. Conclusions Human DCs undergo cell death after infection with live Mtb, in a manner that does not involve executioner caspases, and results in no mycobactericidal effect. Nonetheless, the DC maturation and cytokine profile observed suggests that the infected cells can still contribute to TB immunity.

  12. Intranasal IFNγ extends passive IgA antibody protection of mice against Mycobacterium tuberculosis lung infection

    Science.gov (United States)

    Reljic, R; Clark, S O; Williams, A; Falero-Diaz, G; Singh, M; Challacombe, S; Marsh, P D; Ivanyi, J

    2006-01-01

    Intranasal inoculation of mice with monoclonal IgA against the α-crystallin (acr1) antigen can diminish the tuberculous infection in the lungs. As this effect has been observed only over a short-term, we investigated if it could be extended by inoculation of IFNγ 3 days before infection, and further coinoculations with IgA, at 2 h before and 2 and 7 days after aerosol infection with Mycobacterium tuberculosis H37Rv. This treatment reduced the lung infection at 4 weeks more than either IgA or IFNγ alone (i.e. 17-fold, from 4·2 × 107 to 2·5 × 106 CFU, P = 0·006), accompanied also by lower granulomatous infiltration of the lungs. IFNγ added prior to infection of mouse peritoneal macrophages with IgA-opsonized bacilli resulted in a synergistic increase of nitric oxide and TNFα production and a 2–3 fold decrease in bacterial counts. Our improved results suggest, that combined treatment with IFNγ and IgA could be developed towards prophylactic treatment of AIDS patients, or as an adjunct to chemotherapy. PMID:16487246

  13. Isolation and molecular characterization of Mycobacterium bovis causing pulmonary tuberculosis and epistaxis in a Thoroughbred horse.

    Science.gov (United States)

    Hlokwe, Tiny Motlatso; Sutton, David; Page, Patrick; Michel, Anita Luise

    2016-09-02

    Tuberculosis caused by Mycobacterium bovis (M. bovis) is very uncommon in horses worldwide. In the current study, an eight-year-old male Thoroughbred in good body condition was admitted to the Equine Clinic at the Onderstepoort Veterinary Academic Hospital in 2005 due to bilateral epistaxis accompanied by coughing. Routine examinations were conducted to determine the cause of the condition. Endoscopic examination revealed the major source of the epistaxis as the trachea, whereas thoracic radiography indicated the presence of a primary pulmonary mass. M. bovis was isolated from a broncho-alveolar lavage (BAL) sample collected. The pulmonary mass reduced in size three months later following an oral administration of enrofloxacin (7.5 mg/kg PO SID). Genetic fingerprinting by spoligotyping identified the M. bovis isolate as spoligotype SB0868 strain. This M. bovis strain type was never described previously in South Africa (SA). This is the first case of M. bovis infection in a horse in SA which has been fully documented including clinical findings, isolation and genetic characterisation of the causative pathogen. This report indicates that horses may contract and harbour M. bovis despite their lower susceptibility compared to other domestic animals. It also suggests that the infection may be more easily contained and eliminated from the host.

  14. Genome-wide analysis of multi- and extensively drug-resistant Mycobacterium tuberculosis

    KAUST Repository

    Coll, Francesc

    2018-01-16

    To characterize the genetic determinants of resistance to antituberculosis drugs, we performed a genome-wide association study (GWAS) of 6,465 Mycobacterium tuberculosis clinical isolates from more than 30 countries. A GWAS approach within a mixed-regression framework was followed by a phylogenetics-based test for independent mutations. In addition to mutations in established and recently described resistance-associated genes, novel mutations were discovered for resistance to cycloserine, ethionamide and para-aminosalicylic acid. The capacity to detect mutations associated with resistance to ethionamide, pyrazinamide, capreomycin, cycloserine and para-aminosalicylic acid was enhanced by inclusion of insertions and deletions. Odds ratios for mutations within candidate genes were found to reflect levels of resistance. New epistatic relationships between candidate drug-resistance-associated genes were identified. Findings also suggest the involvement of efflux pumps (drrA and Rv2688c) in the emergence of resistance. This study will inform the design of new diagnostic tests and expedite the investigation of resistance and compensatory epistatic mechanisms.

  15. Incident Mycobacterium tuberculosis infection in household contacts of infectious tuberculosis patients in Brazil.

    Science.gov (United States)

    Jones-López, Edward C; Acuña-Villaorduña, Carlos; Fregona, Geisa; Marques-Rodrigues, Patricia; White, Laura F; Hadad, David Jamil; Dutra-Molina, Lucilia Pereira; Vinhas, Solange; McIntosh, Avery I; Gaeddert, Mary; Ribeiro-Rodrigues, Rodrigo; Salgame, Padmini; Palaci, Moises; Alland, David; Ellner, Jerrold J; Dietze, Reynaldo

    2017-08-18

    In household contact investigations of tuberculosis (TB), a second tuberculin skin test (TST) obtained several weeks after a first negative result consistently identifies individuals that undergo TST conversion. It remains unclear whether this delay in M. tuberculosis infection is related to differences in the infectious exposure, TST boosting, partial host resistance, or some other factor. We conducted a household contact study Vitória, Brazil. Between 2008 and 2013, we identified culture-positive pulmonary TB patients and evaluated their household contacts with both a TST and interferon gamma release assay (IGRA), and identified TST converters at 8-12 weeks post study enrollment. Contacts were classified as TST-positive (≥10 mm) at baseline, TST converters, or persistently TST-negative. We compared TST converters to TST-positive and to TST-negative contacts separately, using generalized estimating equations. We enrolled 160 index patients and 838 contacts; 523 (62.4%) were TST+, 62 (7.4%) TST converters, and 253 (30.2%) TST-. TST converters were frequently IGRA- at 8-12 weeks. In adjusted analyses, characteristics distinguishing TST converters from TST+ contacts (no contact with another TB patient and residence ownership) were different than those differentiating them from TST- contacts (stronger cough in index patient and contact BCG scar). The individual risk and timing of M. tuberculosis infection within households is variable and dependent on index patient, contact and environmental factors within the household, and the surrounding community. Our findings suggest a threshold effect in the risk of infection in humans.

  16. Application of Pulsed Field Gel Electrophoresis for Study of Genetic Diversity in Mycobacterium tuberculosis Strains Isolated From Tuberculosis Patients.

    Science.gov (United States)

    Khosravi, Azar Dokht; Vatani, Shideh; Feizabadi, Mohammad Mehdi; Abasi Montazeri, Effat; Jolodar, Abbas

    2014-05-01

    Mycobacterium tuberculosis genotyping can effectively improve tuberculosis (TB) control programs by controlling disease transmission. Pulsed field gel electrophoresis (PFGE) is a particularly powerful tool for determination of clonal identity of bacteria providing information for understanding and controlling the spread of disease. The aim of present study was to investigate the genetic diversity of M. tuberculosis strains in Khuzestan province by the PFGE technique. In total, 80 M. tuberculosis positive cultures were obtained from tuberculosis patients. PFGE was performed on 60 PCR-confirmed isolates by using DraI and XbaI restriction enzymes according to standard protocols. Plugs containing digested DNA were then loaded on agarose gels and run using contour-clamped homogenous electric fields. Fifty distinct DNA banding patterns were obtained by digestion of DNA with DraI and 38 DNA banding patterns by digestion with XbaI restriction enzymes. The patterns comprised of 17 different clusters in which cluster I was the major one, containing six strains. Three clusters contained three strains each and the 13 remaining clusters comprised of two strains each. Digestion with DraI yielded 15-20 DNA fragments with 50-485 kb size, while digestion by XbaI produced DNA fragments with a size smaller than 50-242 kb. Despite the ability of PFGE for study of genetic diversity of many mycobacterial species and it being considered as a robust and useful tool, in this study we only found a 15% epidemiological relationship amongst the isolates. Thus, for higher discrimination of genotypic clusters among M. tuberculosis clinical isolates, the application of more sophisticated complementary techniques is required.

  17. Mycobacterium tuberculosis drug-resistance in previously treated ...

    African Journals Online (AJOL)

    Corresponding to: Professor Lassana Sangaré, Department of Bacteriology and Virology, University Hospital Centre. Yalgado Ouedraogo, 03 BP 7022 Ouagadougou 03, Burkina Faso. E-mail: sangarel@hotmail.com. Abstract. Background: Tuberculosis drug-resistance becomes common in sub-Saharan Africa; however, ...

  18. Genetic diversity of Mycobacterium tuberculosis in Mbarara, South ...

    African Journals Online (AJOL)

    Background: We determined the genetic diversity of mycobacteria isolated from tuberculosis patients in Mbarara Uganda, using region of difference (RD) analysis and spacer oligonucleotide typing (spoligotyping). Methods: Sputum samples were cultured on Lowenstein Jensen media. The isolates were characterized using ...

  19. Genetic diversity of Mycobacterium tuberculosis isolates from central India

    Directory of Open Access Journals (Sweden)

    Prabha Desikan

    2016-01-01

    Interpretation & conclusions: Well established SITs were found to be predominant in our study. SIT26/CAS1_DEL was the most predominant type. However, the occurrence of a substantial number of orphan isolates may indicate the presence of active spatial and temporal evolutionary dynamics within the isolates of M. tuberculosis.

  20. Evaluation of the efficacy of valproic acid and suberoylanilide hydroxamic acid (vorinostat in enhancing the effects of first-line tuberculosis drugs against intracellular Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Martin Rao

    2018-04-01

    Full Text Available Background: New tuberculosis (TB drug treatment regimens are urgently needed. This study evaluated the potential of the histone deacetylase inhibitors (HDIs valproic acid (VPA and suberoylanilide hydroxamic acid (SAHA to enhance the effects of first-line anti-TB drugs against intracellular Mycobacterium tuberculosis. Methods: M. tuberculosis H37Rv cultures were exposed to VPA or SAHA over 6 days, in the presence or absence of isoniazid (INH and rifampicin (RIF. The efficacy of VPA and SAHA against intracellular M. tuberculosis with and without INH or RIF was tested by treating infected macrophages. Bactericidal activity was assessed by counting mycobacterial colony-forming units (CFU. Results: VPA treatment exhibited superior bactericidal activity to SAHA (2-log CFU reduction, while both HDIs moderately improved the activity of RIF against extracellular M. tuberculosis. The bactericidal effect of VPA against intracellular M. tuberculosis was greater than that of SAHA (1-log CFU reduction and equalled that of INH (1.5-log CFU reduction. INH/RIF and VPA/SAHA combination treatment inhibited intracellular M. tuberculosis survival in a shorter time span than monotherapy (3 days vs. 6 days. Conclusions: VPA and SAHA have adjunctive potential to World Health Organization-recommended TB treatment regimens. Clinical evaluation of the two drugs with regard to reducing the treatment duration and improving treatment outcomes in TB is warranted. Keywords: Mycobacterium tuberculosis, Adjunct host-directed therapy, Tuberculosis, Histone deacetylase inhibitors, Repurposed drugs

  1. Phosphorylation of KasB Regulates Virulence and Acid-Fastness in Mycobacterium tuberculosis

    Science.gov (United States)

    Vilchèze, Catherine; Molle, Virginie; Carrère-Kremer, Séverine; Leiba, Jade; Mourey, Lionel; Shenai, Shubhada; Baronian, Grégory; Tufariello, Joann; Hartman, Travis; Veyron-Churlet, Romain; Trivelli, Xavier; Tiwari, Sangeeta; Weinrick, Brian; Alland, David; Guérardel, Yann; Jacobs, William R.; Kremer, Laurent

    2014-01-01

    Mycobacterium tuberculosis bacilli display two signature features: acid-fast staining and the capacity to induce long-term latent infections in humans. However, the mechanisms governing these two important processes remain largely unknown. Ser/Thr phosphorylation has recently emerged as an important regulatory mechanism allowing mycobacteria to adapt their cell wall structure/composition in response to their environment. Herein, we evaluated whether phosphorylation of KasB, a crucial mycolic acid biosynthetic enzyme, could modulate acid-fast staining and virulence. Tandem mass spectrometry and site-directed mutagenesis revealed that phosphorylation of KasB occurred at Thr334 and Thr336 both in vitro and in mycobacteria. Isogenic strains of M. tuberculosis with either a deletion of the kasB gene or a kasB_T334D/T336D allele, mimicking constitutive phosphorylation of KasB, were constructed by specialized linkage transduction. Biochemical and structural analyses comparing these mutants to the parental strain revealed that both mutant strains had mycolic acids that were shortened by 4–6 carbon atoms and lacked trans-cyclopropanation. Together, these results suggested that in M. tuberculosis, phosphorylation profoundly decreases the condensing activity of KasB. Structural/modeling analyses reveal that Thr334 and Thr336 are located in the vicinity of the catalytic triad, which indicates that phosphorylation of these amino acids would result in loss of enzyme activity. Importantly, the kasB_T334D/T336D phosphomimetic and deletion alleles, in contrast to the kasB_T334A/T336A phosphoablative allele, completely lost acid-fast staining. Moreover, assessing the virulence of these strains indicated that the KasB phosphomimetic mutant was attenuated in both immunodeficient and immunocompetent mice following aerosol infection. This attenuation was characterized by the absence of lung pathology. Overall, these results highlight for the first time the role of Ser/Thr kinase

  2. Phosphorylation of KasB regulates virulence and acid-fastness in Mycobacterium tuberculosis.

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    Catherine Vilchèze

    2014-05-01

    Full Text Available Mycobacterium tuberculosis bacilli display two signature features: acid-fast staining and the capacity to induce long-term latent infections in humans. However, the mechanisms governing these two important processes remain largely unknown. Ser/Thr phosphorylation has recently emerged as an important regulatory mechanism allowing mycobacteria to adapt their cell wall structure/composition in response to their environment. Herein, we evaluated whether phosphorylation of KasB, a crucial mycolic acid biosynthetic enzyme, could modulate acid-fast staining and virulence. Tandem mass spectrometry and site-directed mutagenesis revealed that phosphorylation of KasB occurred at Thr334 and Thr336 both in vitro and in mycobacteria. Isogenic strains of M. tuberculosis with either a deletion of the kasB gene or a kasB_T334D/T336D allele, mimicking constitutive phosphorylation of KasB, were constructed by specialized linkage transduction. Biochemical and structural analyses comparing these mutants to the parental strain revealed that both mutant strains had mycolic acids that were shortened by 4-6 carbon atoms and lacked trans-cyclopropanation. Together, these results suggested that in M. tuberculosis, phosphorylation profoundly decreases the condensing activity of KasB. Structural/modeling analyses reveal that Thr334 and Thr336 are located in the vicinity of the catalytic triad, which indicates that phosphorylation of these amino acids would result in loss of enzyme activity. Importantly, the kasB_T334D/T336D phosphomimetic and deletion alleles, in contrast to the kasB_T334A/T336A phosphoablative allele, completely lost acid-fast staining. Moreover, assessing the virulence of these strains indicated that the KasB phosphomimetic mutant was attenuated in both immunodeficient and immunocompetent mice following aerosol infection. This attenuation was characterized by the absence of lung pathology. Overall, these results highlight for the first time the role of

  3. Immunogenic Properties of Lactobacillus plantarum Producing Surface-Displayed Mycobacterium tuberculosis Antigens.

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    Kuczkowska, Katarzyna; Kleiveland, Charlotte R; Minic, Rajna; Moen, Lars F; Øverland, Lise; Tjåland, Rannei; Carlsen, Harald; Lea, Tor; Mathiesen, Geir; Eijsink, Vincent G H

    2017-01-15

    Tuberculosis (TB) remains among the most deadly diseases in the world. The only available vaccine against tuberculosis is the bacille Calmette-Guérin (BCG) vaccine, which does not ensure full protection in adults. There is a global urgency for the development of an effective vaccine for preventing disease transmission, and it requires novel approaches. We are exploring the use of lactic acid bacteria (LAB) as a vector for antigen delivery to mucosal sites. Here, we demonstrate the successful expression and surface display of a Mycobacterium tuberculosis fusion antigen (comprising Ag85B and ESAT-6, referred to as AgE6) on Lactobacillus plantarum The AgE6 fusion antigen was targeted to the bacterial surface using two different anchors, a lipoprotein anchor directing the protein to the cell membrane and a covalent cell wall anchor. AgE6-producing L. plantarum strains using each of the two anchors induced antigen-specific proliferative responses in lymphocytes purified from TB-positive donors. Similarly, both strains induced immune responses in mice after nasal or oral immunization. The impact of the anchoring strategies was reflected in dissimilarities in the immune responses generated by the two L. plantarum strains in vivo The present study comprises an initial step toward the development of L. plantarum as a vector for M. tuberculosis antigen delivery. This work presents the development of Lactobacillus plantarum as a candidate mucosal vaccine against tuberculosis. Tuberculosis remains one of the top infectious diseases worldwide, and the only available vaccine, bacille Calmette-Guérin (BCG), fails to protect adults and adolescents. Direct antigen delivery to mucosal sites is a promising strategy in tuberculosis vaccine development, and lactic acid bacteria potentially provide easy, safe, and low-cost delivery vehicles for mucosal immunization. We have engineered L. plantarum strains to produce a Mycobacterium tuberculosis fusion antigen and to anchor this

  4. Genetic diversity of drug and multidrug-resistant Mycobacterium tuberculosis circulating in Veracruz, Mexico

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    Munro-Rojas, Daniela; Fernandez-Morales, Esdras; Zarrabal-Meza, José; Martínez-Cazares, Ma. Teresa; Parissi-Crivelli, Aurora; Fuentes-Domínguez, Javier; Séraphin, Marie Nancy; Lauzardo, Michael; González-y-Merchand, Jorge Alberto; Rivera-Gutierrez, Sandra

    2018-01-01

    Background Mexico is one of the most important contributors of drug and multidrug-resistant tuberculosis in Latin America; however, knowledge of the genetic diversity of drug-resistant tuberculosis isolates is limited. Methods In this study, the genetic structure of 112 Mycobacterium tuberculosis strains from the southeastern Mexico was determined by spoligotyping and 24-loci MIRU-VNTRs. Findings The results show eight major lineages, the most of which was T1 (24%), followed by LAM (16%) and H (15%). A total of 29 (25%) isolates were identified as orphan. The most abundant SITs were SIT53/T1 and SIT42/LAM9 with 10 isolates each and SIT50/H3 with eight isolates. Fifty-two spoligotype patterns, twenty-seven clusters and ten clonal complexes were observed, demonstrating an important genetic diversity of drug and multidrug-resistant tuberculosis isolates in circulation and transmission level of these aggravated forms of tuberculosis. Being defined as orphan or as part of an orphan cluster, was a risk factor for multidrug resistant-tuberculosis (OR 2.5, IC 1.05–5.86 and OR 3.3, IC 1–11.03, respectively). Multiple correspondence analyses showed association of some clusters and SITs with specific geographical locations. Conclusions Our study provides one of the most detailed description of the genetic structure of drug and multidrug-resistant tuberculosis strains in southeast Mexico, establishing for the first time a baseline of the genotypes observed in resistant isolates circulating, however further studies are required to better elucidate the genetic structure of tuberculosis in region and the factors that could be participating in their dispersion. PMID:29543819

  5. DNA fingerprinting of Mycobacterium tuberculosis: from phage typing to whole-genome sequencing.

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    Schürch, Anita C; van Soolingen, Dick

    2012-06-01

    Current typing methods for Mycobacterium tuberculosis complex evolved from simple phenotypic approaches like phage typing and drug susceptibility profiling to DNA-based strain typing methods, such as IS6110-restriction fragment length polymorphisms (RFLP) and variable number of tandem repeats (VNTR) typing. Examples of the usefulness of molecular typing are source case finding and epidemiological linkage of tuberculosis (TB) cases, international transmission of MDR/XDR-TB, the discrimination between endogenous reactivation and exogenous re-infection as a cause of relapses after curative treatment of tuberculosis, the evidence of multiple M. tuberculosis infections, and the disclosure of laboratory cross-contaminations. Simultaneously, phylogenetic analyses were developed based on single nucleotide polymorphisms (SNPs), genomic deletions usually referred to as regions of difference (RDs) and spoligotyping which served both strain typing and phylogenetic analysis. National and international initiatives that rely on the application of these typing methods have brought significant insight into the molecular epidemiology of tuberculosis. However, current DNA fingerprinting methods have important limitations. They can often not distinguish between genetically closely related strains and the turn-over of these markers is variable. Moreover, the suitability of most DNA typing methods for phylogenetic reconstruction is limited as they show a high propensity of convergent evolution or misinfer genetic distances. In order to fully explore the possibilities of genotyping in the molecular epidemiology of tuberculosis and to study the phylogeny of the causative bacteria reliably, the application of whole-genome sequencing (WGS) analysis for all M. tuberculosis isolates is the optimal, although currently still a costly solution. In the last years WGS for typing of pathogens has been explored and yielded important additional information on strain diversity in comparison to the

  6. Human tuberculosis caused by Mycobacterium bovis: a retrospective comparison with Mycobacterium tuberculosis in a Mexican tertiary care centre, 2000–2015

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    Pedro Torres-Gonzalez

    2016-11-01

    Full Text Available Abstract Background Human tuberculosis caused by Mycobacterium bovis is believed to be frequent in developing countries. Transmission is usually through ingestion of unpasteurized dairy products, although airborne contagion is possible. Disease caused by M. tuberculosis or M. bovis is clinically indistinguishable from each other. The aim of this study was to determine the factors associated with M. bovis disease. Methods Retrospective analysis of all culture-positive cases of M. bovis and M. tuberculosis from 2000 to 2015, in a Mexican tertiary-care centre. Sociodemographic, clinical, and radiographic data from medical records were compared. Disease site was classified as pulmonary, extrapulmonary, or pulmonary and extrapulmonary, based on cultures. Results We evaluated 533 cases, 372 (69.7 % of which were caused by M. tuberculosis and 161 (30.2 % by M. bovis. Characteristics associated with M. bovis disease were: younger age (aOR 0.97, 95 % CI 0.95–0.98, glucocorticoid use (aOR 2.27, 95 % CI 1.42–3.63, and extrapulmonary disease (aOR 1.80, 95 % CI 1.21–2.69. M. tuberculosis was associated with lower socioeconomic status (aOR 0.52, 95 % CI 0.28–0.97. When we analysed only pulmonary cases, younger age (aOR 0.97, 95 % CI 0.96–0.99, glucocorticoid use (aOR 2.41, 95 % CI 1.30–4.46, and smoking (aOR 1.94, CI 95 % 1.15–3.27 were associated with M. bovis. Both groups showed similar proportions of direct microscopy smear results (respiratory samples and chest X-ray cavitations. Conclusions Younger age, glucocorticoid use, and extrapulmonary disease were associated with M. bovis as the causative agent of tuberculosis in a group of patients from a tertiary care centre in a country where bovine tuberculosis is endemic. Further studies must be conducted in the general population to determine pathogen-specific associated factors and outcomes.

  7. Human tuberculosis caused by Mycobacterium bovis: a retrospective comparison with Mycobacterium tuberculosis in a Mexican tertiary care centre, 2000-2015.

    Science.gov (United States)

    Torres-Gonzalez, Pedro; Cervera-Hernandez, Miguel E; Martinez-Gamboa, Areli; Garcia-Garcia, Lourdes; Cruz-Hervert, Luis P; Bobadilla-Del Valle, Miriam; Ponce-de Leon, Alfredo; Sifuentes-Osornio, Jose

    2016-11-08

    Human tuberculosis caused by Mycobacterium bovis is believed to be frequent in developing countries. Transmission is usually through ingestion of unpasteurized dairy products, although airborne contagion is possible. Disease caused by M. tuberculosis or M. bovis is clinically indistinguishable from each other. The aim of this study was to determine the factors associated with M. bovis disease. Retrospective analysis of all culture-positive cases of M. bovis and M. tuberculosis from 2000 to 2015, in a Mexican tertiary-care centre. Sociodemographic, clinical, and radiographic data from medical records were compared. Disease site was classified as pulmonary, extrapulmonary, or pulmonary and extrapulmonary, based on cultures. We evaluated 533 cases, 372 (69.7 %) of which were caused by M. tuberculosis and 161 (30.2 %) by M. bovis. Characteristics associated with M. bovis disease were: younger age (aOR 0.97, 95 % CI 0.95-0.98), glucocorticoid use (aOR 2.27, 95 % CI 1.42-3.63), and extrapulmonary disease (aOR 1.80, 95 % CI 1.21-2.69). M. tuberculosis was associated with lower socioeconomic status (aOR 0.52, 95 % CI 0.28-0.97). When we analysed only pulmonary cases, younger age (aOR 0.97, 95 % CI 0.96-0.99), glucocorticoid use (aOR 2.41, 95 % CI 1.30-4.46), and smoking (aOR 1.94, CI 95 % 1.15-3.27) were associated with M. bovis. Both groups showed similar proportions of direct microscopy smear results (respiratory samples) and chest X-ray cavitations. Younger age, glucocorticoid use, and extrapulmonary disease were associated with M. bovis as the causative agent of tuberculosis in a group of patients from a tertiary care centre in a country where bovine tuberculosis is endemic. Further studies must be conducted in the general population to determine pathogen-specific associated factors and outcomes.

  8. Mycobacterium tuberculosis Universal Stress Protein Rv2623 Regulates Bacillary Growth by ATP Binding: Requirement for Establishing Chronic Persistent Infection

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    Drumm, J.; Mi, K; Bilder, P; Sun, M; Lim, J; Bielefeldt-Ohmann, H; Basaraba, R; So, M; Zhu, G; et. al.

    2009-01-01

    Tuberculous latency and reactivation play a significant role in the pathogenesis of tuberculosis, yet the mechanisms that regulate these processes remain unclear. The Mycobacterium tuberculosisuniversal stress protein (USP) homolog, rv2623, is among the most highly induced genes when the tubercle bacillus is subjected to hypoxia and nitrosative stress, conditions thought to promote latency. Induction of rv2623 also occurs when M. tuberculosis encounters conditions associated with growth arrest, such as the intracellular milieu of macrophages and in the lungs of mice with chronic tuberculosis. Therefore, we tested the hypothesis that Rv2623 regulates tuberculosis latency. We observed that an Rv2623-deficient mutant fails to establish chronic tuberculous infection in guinea pigs and mice, exhibiting a hypervirulence phenotype associated with increased bacterial burden and mortality. Consistent with this in vivo growth-regulatory role, constitutive overexpression of rv2623 attenuates mycobacterial growth in vitro. Biochemical analysis of purified Rv2623 suggested that this mycobacterial USP binds ATP, and the 2.9-A-resolution crystal structure revealed that Rv2623 engages ATP in a novel nucleotide-binding pocket. Structure-guided mutagenesis yielded Rv2623 mutants with reduced ATP-binding capacity. Analysis of mycobacteria overexpressing these mutants revealed that the in vitro growth-inhibitory property of Rv2623 correlates with its ability to bind ATP. Together, the results indicate that i M. tuberculosis Rv2623 regulates mycobacterial growth in vitro and in vivo, and ii Rv2623 is required for the entry of the tubercle bacillus into the chronic phase of infection in the host; in addition, iii Rv2623 binds ATP; and iv the growth-regulatory attribute of this USP is dependent on its ATP-binding activity. We propose that Rv2623 may function as an ATP-dependent signaling intermediate in a pathway that promotes persistent infection.

  9. Differential Effect of Viable Versus Necrotic Neutrophils on Mycobacterium tuberculosis Growth and Cytokine Induction in Whole Blood

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    David M. Lowe

    2018-04-01

    Full Text Available Neutrophils exert both positive and negative influences on the host response to tuberculosis, but the mechanisms by which these differential effects are mediated are unknown. We studied the impact of live and dead neutrophils on the control of Mycobacterium tuberculosis using a whole blood bioluminescence-based assay, and assayed supernatant cytokine concentrations using Luminex™ technology and ELISA. CD15+ granulocyte depletion from blood prior to infection with M. tuberculosis-lux impaired control of mycobacteria by 96 h, with a greater effect than depletion of CD4+, CD8+, or CD14+ cells (p < 0.001. Augmentation of blood with viable granulocytes significantly improved control of mycobacteria by 96 h (p = 0.001, but augmentation with necrotic granulocytes had the opposite effect (p = 0.01. Both augmentations decreased supernatant concentrations of tumor necrosis factor and interleukin (IL-12 p40/p70, but necrotic granulocyte augmentation also increased concentrations of IL-10, G-CSF, GM-CSF, and CCL2. Necrotic neutrophil augmentation reduced phagocytosis of FITC-labeled M. bovis BCG by all phagocytes, whereas viable neutrophil augmentation specifically reduced early uptake by CD14+ cells. The immunosuppressive effect of dead neutrophils required necrotic debris rather than supernatant. We conclude that viable neutrophils enhance control of M. tuberculosis in blood, but necrotic neutrophils have the opposite effect—the latter associated with induction of IL-10, growth factors, and chemoattractants. Our findings suggest a mechanism by which necrotic neutrophils may exert detrimental effects on the host response in active tuberculosis.

  10. Wild-type MIC distributions for aminoglycoside and cyclic polypeptide antibiotics used for treatment of Mycobacterium tuberculosis infections.

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    Juréen, P; Angeby, K; Sturegård, E; Chryssanthou, E; Giske, C G; Werngren, J; Nordvall, M; Johansson, A; Kahlmeter, G; Hoffner, S; Schön, T

    2010-05-01

    The aminoglycosides and cyclic polypeptides are essential drugs in the treatment of multidrug-resistant tuberculosis, underscoring the need for accurate and reproducible drug susceptibility testing (DST). The epidemiological cutoff value (ECOFF) separating wild-type susceptible strains from non-wild-type strains is an important but rarely used tool for indicating susceptibility breakpoints against Mycobacterium tuberculosis. In this study, we established wild-type MIC distributions on Middlebrook 7H10 medium for amikacin, kanamycin, streptomycin, capreomycin, and viomycin using 90 consecutive clinical isolates and 21 resistant strains. Overall, the MIC variation between and within runs did not exceed +/-1 MIC dilution step, and validation of MIC values in Bactec 960 MGIT demonstrated good agreement. Tentative ECOFFs defining the wild type were established for all investigated drugs, including amikacin and viomycin, which currently lack susceptibility breakpoints for 7H10. Five out of seven amikacin- and kanamycin-resistant isolates were classified as susceptible to capreomycin according to the current critical concentration (10 mg/liter) but were non-wild type according to the ECOFF (4 mg/liter), suggesting that the critical concentration may be too high. All amikacin- and kanamycin-resistant isolates were clearly below the ECOFF for viomycin, and two of them were below the ECOFF for streptomycin, indicating that these two drugs may be considered for treatment of amikacin-resistant strains. Pharmacodynamic indices (peak serum concentration [Cmax]/MIC) were more favorable for amikacin and viomycin compared to kanamycin and capreomycin. In conclusion, our data emphasize the importance of establishing wild-type MIC distributions for improving the quality of drug susceptibility testing against Mycobacterium tuberculosis.

  11. Wild-Type MIC Distributions for Aminoglycoside and Cyclic Polypeptide Antibiotics Used for Treatment of Mycobacterium tuberculosis Infections▿

    Science.gov (United States)

    Juréen, P.; Ängeby, K.; Sturegård, E.; Chryssanthou, E.; Giske, C. G.; Werngren, J.; Nordvall, M.; Johansson, A.; Kahlmeter, G.; Hoffner, S.; Schön, T.

    2010-01-01

    The aminoglycosides and cyclic polypeptides are essential drugs in the treatment of multidrug-resistant tuberculosis, underscoring the need for accurate and reproducible drug susceptibility testing (DST). The epidemiological cutoff value (ECOFF) separating wild-type susceptible strains from non-wild-type strains is an important but rarely used tool for indicating susceptibility breakpoints against Mycobacterium tuberculosis. In this study, we established wild-type MIC distributions on Middlebrook 7H10 medium for amikacin, kanamycin, streptomycin, capreomycin, and viomycin using 90 consecutive clinical isolates and 21 resistant strains. Overall, the MIC variation between and within runs did not exceed ±1 MIC dilution step, and validation of MIC values in Bactec 960 MGIT demonstrated good agreement. Tentative ECOFFs defining the wild type were established for all investigated drugs, including amikacin and viomycin, which currently lack susceptibility breakpoints for 7H10. Five out of seven amikacin- and kanamycin-resistant isolates were classified as susceptible to capreomycin according to the current critical concentration (10 mg/liter) but were non-wild type according to the ECOFF (4 mg/liter), suggesting that the critical concentration may be too high. All amikacin- and kanamycin-resistant isolates were clearly below the ECOFF for viomycin, and two of them were below the ECOFF for streptomycin, indicating that these two drugs may be considered for treatment of amikacin-resistant strains. Pharmacodynamic indices (peak serum concentration [Cmax]/MIC) were more favorable for amikacin and viomycin compared to kanamycin and capreomycin. In conclusion, our data emphasize the importance of establishing wild-type MIC distributions for improving the quality of drug susceptibility testing against Mycobacterium tuberculosis. PMID:20237102

  12. Association between Mycobacterium tuberculosis complex phylogenetic lineage and acquired drug resistance.

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    Courtney M Yuen

    Full Text Available BACKGROUND: Development of resistance to antituberculosis drugs during treatment (i.e., acquired resistance can lead to emergence of resistant strains and consequent poor clinical outcomes. However, it is unknown whether Mycobacterium tuberculosis complex species and lineage affects the likelihood of acquired resistance. METHODS: We analyzed data from the U.S. National Tuberculosis Surveillance System and National Tuberculosis Genotyping Service for tuberculosis cases during 2004-2011 with assigned species and lineage and both initial and final drug susceptibility test results. We determined univariate associations between species and lineage of Mycobacterium tuberculosis complex bacteria and acquired resistance to isoniazid, rifamycins, fluoroquinolones, and second-line injectables. We used Poisson regression with backward elimination to generate multivariable models for acquired resistance to isoniazid and rifamycins. RESULTS: M. bovis was independently associated with acquired resistance to isoniazid (adjusted prevalence ratio = 8.46, 95% CI 2.96-24.14 adjusting for HIV status, and with acquired resistance to rifamycins (adjusted prevalence ratio = 4.53, 95% CI 1.29-15.90 adjusting for homelessness, HIV status, initial resistance to isoniazid, site of disease, and administration of therapy. East Asian lineage was associated with acquired resistance to fluoroquinolones (prevalence ratio = 6.10, 95% CI 1.56-23.83. CONCLUSIONS: We found an association between mycobacterial species and lineage and acquired drug resistance using U.S. surveillance data. Prospective clinical studies are needed to determine the clinical significance of these findings, including whether rapid genotyping of isolates at the outset of treatment may benefit patient management.

  13. Identification of Mycobacterium tuberculosis BioA inhibitors by using structure-based virtual screening

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    Singh S

    2018-05-01

    Full Text Available Swati Singh,1 Garima Khare,1 Ritika Kar Bahal,1 Prahlad C Ghosh,1 Anil K Tyagi1,2 1Department of Biochemistry, University of Delhi South Campus, New Delhi, India; 2Guru Gobind Singh Indraprastha University, New Delhi, India Background: 7,8-Diaminopelargonic acid synthase (BioA, an enzyme of biotin biosynthesis pathway, is a well-known promising target for anti-tubercular drug development. Methods: In this study, structure-based virtual screening was employed against the active site of BioA to identify new chemical entities for BioA inhibition and top ranking compounds were evaluated for their ability to inhibit BioA enzymatic activity. Results: Seven compounds inhibited BioA enzymatic activity by greater than 60% at 100 µg/mL with most potent compounds being A36, A35 and A65, displaying IC50 values of 10.48 µg/mL (28.94 µM, 33.36 µg/mL (88.16 µM and 39.17 µg/mL (114.42 µM, respectively. Compounds A65 and A35 inhibited Mycobacterium tuberculosis (M. tuberculosis growth with MIC90 of 20 µg/mL and 80 µg/mL, respectively, whereas compound A36 exhibited relatively weak inhibition of M. tuberculosis growth (83% inhibition at 200 µg/mL. Compound A65 emerged as the most potent compound identified in our study that inhibited BioA enzymatic activity and growth of the pathogen and possessed drug-like properties. Conclusion: Our study has identified a few hit molecules against M. tuberculosis BioA that can act as potential candidates for further development of potent anti-tubercular therapeutic agents. Keywords: Mycobacterium tuberculosis, BioA, virtual screening, drug discovery

  14. LytB1 and LytB2 of Mycobacterium tuberculosis Are Not Genetically Redundant.

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    Amanda Claire Brown

    Full Text Available Mycobacterium tuberculosis synthesises isoprenoid precursors via the MEP/DOXP pathway and at least five enzymes in the pathway (Dxs1, Dxr/IspC, IspD, IspF, and GcpE/IspG are required for growth in vitro. We investigated the role of LytB (IspH in M. tuberculosis; M. tuberculosis is unusual in that it has two homologs-LytB1 and LytB2. We were unable to delete the lytB2 gene unless we provided an additional copy elsewhere, demonstrating that this is the essential homolog. We expressed lytB1 from the lytB2 promoter and confirmed that this could not complement for loss of function of lytB2, despite LytB1 possessing all the previously described conserved critical residues. Interestingly the sole LytB homolog of Mycobacterium smegmatis was able to compensate for loss of LytB2 in M. tuberculosis. We tested translational fusions of LytB1 and LytB2 for functionality in M. tuberculosis, but only a fusion with 90% N-terminal LytB2 and 10% C-terminal LytB1 was functional. In order to identify the key difference between the two proteins, site directed mutagenesis was used to change LytB2 residues into their counterparts in LytB1. None of these amino acid substitutions was essential for function and all lytB2 mutant alleles were functional. In contrast, mutation of the key residues for [Fe4S4] cluster formation, as well as a catalytic residue in LytB1 did not result in functional complementation. Thus, although LytB1 and LytB2 are not genetically redundant, this is not dependent on small amino acid changes, but is likely to be a result of major overall structural differences.

  15. Toxin-antitoxin systems and regulatory mechanisms in Mycobacterium tuberculosis.

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    Slayden, Richard A; Dawson, Clinton C; Cummings, Jason E

    2018-06-01

    There has been a significant reduction in annual tuberculosis incidence since the World Health Organization declared tuberculosis a global health threat. However, treatment of M. tuberculosis infections requires lengthy multidrug therapeutic regimens to achieve a durable cure. The development of new drugs that are active against resistant strains and phenotypically diverse organisms continues to present the greatest challenge in the future. Numerous phylogenomic analyses have revealed that the Mtb genome encodes a significantly expanded repertoire of toxin-antitoxin (TA) loci that makes up the Mtb TA system. A TA loci is a two-gene operon encoding a 'toxin' protein that inhibits bacterial growth and an interacting 'antitoxin' partner that neutralizes the inhibitory activity of the toxin. The presence of multiple chromosomally encoded TA loci in Mtb raises important questions in regard to expansion, regulation and function. Thus, the functional roles of TA loci in Mtb pathogenesis have received considerable attention over the last decade. The cumulative results indicate that they are involved in regulating adaptive responses to stresses associated with the host environment and drug treatment. Here we review the TA families encoded in Mtb, discuss the duplication of TA loci in Mtb, regulatory mechanism of TA loci, and phenotypic heterogeneity and pathogenesis.

  16. Deciphering the recent phylogenetic expansion of the originally deeply rooted Mycobacterium tuberculosis lineage 7.

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    Yimer, Solomon A; Namouchi, Amine; Zegeye, Ephrem Debebe; Holm-Hansen, Carol; Norheim, Gunnstein; Abebe, Markos; Aseffa, Abraham; Tønjum, Tone

    2016-06-30

    A deeply rooted phylogenetic lineage of Mycobacterium tuberculosis (M. tuberculosis) termed lineage 7 was discovered in Ethiopia. Whole genome sequencing of 30 lineage 7 strains from patients in Ethiopia was performed. Intra-lineage genome variation was defined and unique characteristics identified with a focus on genes involved in DNA repair, recombination and replication (3R genes). More than 800 mutations specific to M. tuberculosis lineage 7 strains were identified. The proportion of non-synonymous single nucleotide polymorphisms (nsSNPs) in 3R genes was higher after the recent expansion of M. tuberculosis lineage 7 strain started. The proportion of nsSNPs in genes involved in inorganic ion transport and metabolism was significantly higher before the expansion began. A total of 22346 bp deletions were observed. Lineage 7 strains also exhibited a high number of mutations in genes involved in carbohydrate transport and metabolism, transcription, energy production and conversion. We have identified unique genomic signatures of the lineage 7 strains. The high frequency of nsSNP in 3R genes after the phylogenetic expansion may have contributed to recent variability and adaptation. The abundance of mutations in genes involved in inorganic ion transport and metabolism before the expansion period may indicate an adaptive response of lineage 7 strains to enable survival, potentially under environmental stress exposure. As lineage 7 strains originally were phylogenetically deeply rooted, this may indicate fundamental adaptive genomic pathways affecting the fitness of M. tuberculosis as a species.

  17. Mycobacterium tuberculosis exploits asparagine to assimilate nitrogen and resist acid stress during infection.

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    Alexandre Gouzy

    2014-02-01

    Full Text Available Mycobacterium tuberculosis is an intracellular pathogen. Within macrophages, M. tuberculosis thrives in a specialized membrane-bound vacuole, the phagosome, whose pH is slightly acidic, and where access to nutrients is limited. Understanding how the bacillus extracts and incorporates nutrients from its host may help develop novel strategies to combat tuberculosis. Here we show that M. tuberculosis employs the asparagine transporter AnsP2 and the secreted asparaginase AnsA to assimilate nitrogen and resist acid stress through asparagine hydrolysis and ammonia release. While the role of AnsP2 is partially spared by yet to be identified transporter(s, that of AnsA is crucial in both phagosome acidification arrest and intracellular replication, as an M. tuberculosis mutant lacking this asparaginase is ultimately attenuated in macrophages and in mice. Our study provides yet another example of the intimate link between physiology and virulence in the tubercle bacillus, and identifies a novel pathway to be targeted for therapeutic purposes.

  18. Differential expression of miRNAs by macrophages infected with virulent and avirulent Mycobacterium tuberculosis.

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    Das, Kishore; Saikolappan, Sankaralingam; Dhandayuthapani, Subramanian

    2013-12-01

    MicroRNAs (miRNAs) are small non-coding RNAs which post-transcriptionally regulate a wide range of biological processes that include cellular differentiation, development, immunity and apoptosis. There is a growing body of evidences that bacteria modulate immune responses by altering the expression of host miRNAs. Since macrophages are immune cells associated with innate and adaptive immunity, we investigated whether Mycobacterium tuberculosis infection affects miRNAs of macrophages. THP-1 macrophages infected with virulent (H37Rv) and avirulent (H37Ra) strains of M. tuberculosis were analyzed for changes in miRNAs' expression using microarray. This revealed that nine miRNA genes (miR-30a, miR-30e, miR-155, miR-1275, miR-3665, miR-3178, miR-4484, miR-4668-5p and miR-4497) were differentially expressed between THP-1cells infected with M. tuberculosis H37Rv and M. tuberculosis H37Ra strains. Additional characterization of these genes is likely to provide insights into their role in the pathogenesis of tuberculosis. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. An insight into the regulation of mce4 operon of Mycobacterium tuberculosis.

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    Rathor, Nisha; Chandolia, Amita; Saini, Neeraj Kumar; Sinha, Rajesh; Pathak, Rakesh; Garima, Kushal; Singh, Satendra; Varma-Basil, Mandira; Bose, Mridula

    2013-07-01

    The mce4 operon is reported to be involved in cholesterol utilization and intracellular survival of Mycobacterium tuberculosis (M. tuberculosis). The regulatory mechanism of this important operon was unknown so far. Here we report detection of the promoter region and regulatory factors of the mce4 operon. The in silico analyzed putative promoter region was cloned in promoter selection vector and promoter strength was measured by O-Nitrophenyl-β-D-galactopyranosidase (ONPG) assay. The transcription start site was determined by 5' Rapid amplification of C terminal end (5'RACE). Surface stress, hypoxia and presence of cholesterol, were found to be stimulatory for mce4 operon promoter induction. Pull down assay coupled with 2D gel electrophoresis resolved many proteins; few prominent spots were processed for identification. MALDI TOF-TOF identified proteins of M. tuberculosis which supported the regulatory function of the identified promoter region and cholesterol utilization of mce4 operon. Since mce4 operon is involved in cholesterol utilization and intracellular survival of M. tuberculosis in the later phase of infection, identification of the promoter sequence as reported in the present communication may facilitate development of effective inhibitors to regulate expression of mce4 operon which may prove to be a good drug target to prevent latency in tuberculosis. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. [Study on VNTR diversity of clinical Mycobacterium tuberculosis isolates from Qinghai].

    Science.gov (United States)

    Li, Bin; Liu, Haican; Wang, Zhaofen; Ma, Yongcheng; Su, Xiaodong; Jiang, Mingxia; Wan, Kanglin; Liu, Shou; Zhao, Xiuqin; Qu, Shugen

    2015-10-01

    To investigate the variable number tandem repeats (VNTR) genetic polymorphisms, genotyping and distribution pattern of clinical Mycobacterium (M.) tuberculosis isolates from Qinghai province. The clinical M. tuberculosis strains isolated from the patients with tuberculosis and related background data were collected from Qinghai Provincial Center for Disease Control and Prevention from 2009 to 2012. Genotyping was conducted by using multiple locus VNTR analysis (MLVA). Genomic DNA was extracted and 15 VNTR loci were amplified with PCR and the PCR products were detected with gel electrophoresis. The VNTR diversity and clusters of genotyping were analyzed with BioNumerics (Version 5.0). A total of 251 clinical M. tuberculosis isolates were analyzed with 15 VNTR loci showing that there were great genetic diversity in these isolates. Six of the 15 VNTR loci, showed that the Hunter-Gaston index (HGI) were higher than 0.6, in which the highest resolution was MIRU26. The clusters of genotyping showed that these isolates could be categorized into four gene clusters and 238 genotypes. The four gene clusters accounted for 4.9%, 91.9%, 1.6% and 1.6% of the clinical isolates, respectively. The results showed that there is great variety of VNTR genetic polymorphisms in clinical M. tuberculosis isolates in Qinghai province.

  1. Molecular Genetic Analysis of Multi-drug Resistance in Indian Isolates of Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Noman Siddiqi

    1998-09-01

    Full Text Available A total of 116 isolates from patients attending the out-patient department at the All India Institute of Medical Sciences, New Delhi and the New Delhi Tuberculosis Centre, New Delhi, India were collected. They were analyzed for resistance to drugs prescribed in the treatment for tuberculosis. The drug resistance was initially determined by microbiological techniques. The Bactec 460TB system was employed to determine the type and level of resistance in each isolate. The isolates were further characterized at molecular level. The multi-drug loci corresponding to rpo b, gyr A, kat G were studied for mutation(s by the polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP technique. The SSCP positive samples were sequenced to characterize the mutations in rpo b, and gyr A loci. While previously reported mutations in the gyr A and rpo b loci were found to be present, several novel mutations were also scored in the rpo b locus. Interestingly, analysis of the gyr A locus showed the presence of point mutation(s that could not be detected by PCR-SSCP. Furthermore, rifampicin resistance was found to be an important marker for checking multi-drug resistance (MDR in clinical isolates of Mycobacterium tuberculosis. This is the first report on molecular genetic analysis of MDR tuberculosis one from India, highlights the increasing incidence of MDR in the Indian isolates of M. tuberculosis.

  2. Structural and functional features of enzymes of Mycobacterium tuberculosis peptidoglycan biosynthesis as targets for drug development.

    Science.gov (United States)

    Moraes, Gleiciane Leal; Gomes, Guelber Cardoso; Monteiro de Sousa, Paulo Robson; Alves, Cláudio Nahum; Govender, Thavendran; Kruger, Hendrik G; Maguire, Glenn E M; Lamichhane, Gyanu; Lameira, Jerônimo

    2015-03-01

    Tuberculosis (TB) is the second leading cause of human mortality from infectious diseases worldwide. The WHO reported 1.3 million deaths and 8.6 million new cases of TB in 2012. Mycobacterium tu