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Sample records for mycobacterium tuberculosis enoyl

  1. Inhibition of the Mycobacterium tuberculosis enoyl acyl carrier protein reductase InhA by arylamides

    PubMed Central

    He, Xin; Alian, Akram; Ortiz de Montellano, Paul R.

    2007-01-01

    InhA, the enoyl acyl carrier protein reductase (ENR) from Mycobacterium tuberculosis, is one of the key enzymes involved in the type II fatty acid biosynthesis pathway of M. tuberculosis. We report here the discovery, through high-throughput screening, of a series of arylamides as a novel class of potent InhA inhibitors. These direct InhA inhibitors require no mycobacterial enzymatic activation and thus circumvent the resistance mechanism to antitubercular prodrugs such as INH and ETA that is most commonly observed in drug-resistant clinical isolates. The crystal structure of InhA complexed with one representative inhibitor reveals the binding mode of the inhibitor within the InhA active site. Further optimization through a microtiter synthesis strategy followed by in situ activity screening led to the discovery of a potent InhA inhibitor with in vitro IC50 = 90 nM, representing a 34-fold potency improvement over the lead compound. PMID:17723305

  2. Inhibition of the Mycobacterium tuberculosis enoyl acyl carrier protein reductase InhA by arylamides.

    PubMed

    He, Xin; Alian, Akram; Ortiz de Montellano, Paul R

    2007-11-01

    InhA, the enoyl acyl carrier protein reductase (ENR) from Mycobacterium tuberculosis, is one of the key enzymes involved in the type II fatty acid biosynthesis pathway of M. tuberculosis. We report here the discovery, through high-throughput screening, of a series of arylamides as a novel class of potent InhA inhibitors. These direct InhA inhibitors require no mycobacterial enzymatic activation and thus circumvent the resistance mechanism to antitubercular prodrugs such as INH and ETA that is most commonly observed in drug-resistant clinical isolates. The crystal structure of InhA complexed with one representative inhibitor reveals the binding mode of the inhibitor within the InhA active site. Further optimization through a microtiter synthesis strategy followed by in situ activity screening led to the discovery of a potent InhA inhibitor with in vitro IC(50)=90 nM, representing a 34-fold potency improvement over the lead compound.

  3. Identification of the missing trans-acting enoyl reductase required for phthiocerol dimycocerosate and phenolglycolipid biosynthesis in Mycobacterium tuberculosis.

    PubMed

    Siméone, Roxane; Constant, Patricia; Guilhot, Christophe; Daffé, Mamadou; Chalut, Christian

    2007-07-01

    Phthiocerol dimycocerosates (DIM) and phenolglycolipids (PGL) are functionally important surface-exposed lipids of Mycobacterium tuberculosis. Their biosynthesis involves the products of several genes clustered in a 70-kb region of the M. tuberculosis chromosome. Among these products is PpsD, one of the modular type I polyketide synthases responsible for the synthesis of the lipid core common to DIM and PGL. Bioinformatic analyses have suggested that this protein lacks a functional enoyl reductase activity domain required for the synthesis of these lipids. We have identified a gene, Rv2953, that putatively encodes an enoyl reductase. Mutation in Rv2953 prevents conventional DIM formation and leads to the accumulation of a novel DIM-like product. This product is unsaturated between C-4 and C-5 of phthiocerol. Consistently, complementation of the mutant with a functional pks15/1 gene from Mycobacterium bovis BCG resulted in the accumulation of an unsaturated PGL-like substance. When an intact Rv2953 gene was reintroduced into the mutant strain, the phenotype reverted to the wild type. These findings indicate that Rv2953 encodes a trans-acting enoyl reductase that acts with PpsD in phthiocerol and phenolphthiocerol biosynthesis.

  4. Pyrrolidine Carboxamides as a Novel Class of Inhibitors of Enoyl Acyl Carrier Protein Reductase (InhA) from Mycobacterium tuberculosis

    PubMed Central

    He, Xin; Alian, Akram; Stroud, Robert; de Montellano, Paul R. Ortiz

    2008-01-01

    In view of the worldwide spread of multidrug resistance of Mycobacterium tuberculosis, there is an urgent need to discover antituberculosis agent with novel structures. InhA, the enoyl acyl carrier protein reductase (ENR) from Mycobacterium tuberculosis is one of the key enzymes involved in the mycobacterial fatty acid elongation cycle and has been validated as an effective antimicrobial target. We report here discovery through high throughput screening of a series of pyrrolidine carboxamides as a novel class of potent InhA inhibitors. Crystal structures of InhA complexed with three inhibitors have been used to elucidate the inhibitor binding mode. The potency of the lead compound was improved over 160-fold by subsequent optimization through iterative microtiter library synthesis followed by in situ activity screening without purification. Resolution of racemic mixtures of several inhibitors indicate that only one enantiomer is active as an inhibitor of InhA. PMID:17034137

  5. Pyrrolidine carboxamides as a novel class of inhibitors of enoyl acyl carrier protein reductase from Mycobacterium tuberculosis.

    PubMed

    He, Xin; Alian, Akram; Stroud, Robert; Ortiz de Montellano, Paul R

    2006-10-19

    In view of the worldwide spread of multidrug resistance of Mycobacterium tuberculosis, there is an urgent need to discover antituberculosis agent with novel structures. InhA, the enoyl acyl carrier protein reductase (ENR) from M. tuberculosis, is one of the key enzymes involved in the mycobacterial fatty acid elongation cycle and has been validated as an effective antimicrobial target. We report here the discovery, through high-throughput screening, of a series of pyrrolidine carboxamides as a novel class of potent InhA inhibitors. Crystal structures of InhA complexed with three inhibitors have been used to elucidate the inhibitor binding mode. The potency of the lead compound was improved over 160-fold by subsequent optimization through iterative microtiter library synthesis followed by in situ activity screening without purification. Resolution of racemic mixtures of several inhibitors indicate that only one enantiomer is active as an inhibitor of InhA.

  6. Radiolabelling and positron emission tomography of PT70, a time-dependent inhibitor of InhA, the Mycobacterium tuberculosis enoyl-ACP reductase

    DOE PAGES

    Wang, Hui; Liu, Li; Lu, Yang; ...

    2015-07-14

    PT70 is a diaryl ether inhibitor of InhA, the enoyl-ACP reductase in the Mycobacterium tuberculosis fatty acid biosynthesis pathway. It has a residence time of 24 min on the target, and also shows antibacterial activity in a mouse model of tuberculosis infection. Due to the interest in studying target tissue pharmacokinetics of PT70, we developed a method to radiolabel PT70 with carbon-11 and have studied its pharmacokinetics in mice and baboons using positron emission tomography.

  7. Radiolabelling and positron emission tomography of PT70, a time-dependent inhibitor of InhA, the Mycobacterium tuberculosis enoyl-ACP reductase

    SciTech Connect

    Wang, Hui; Liu, Li; Lu, Yang; Pan, Pan; Hooker, Jacob M.; Fowler, Joanna S.; Tonge, Peter J.

    2015-07-14

    PT70 is a diaryl ether inhibitor of InhA, the enoyl-ACP reductase in the Mycobacterium tuberculosis fatty acid biosynthesis pathway. It has a residence time of 24 min on the target, and also shows antibacterial activity in a mouse model of tuberculosis infection. Due to the interest in studying target tissue pharmacokinetics of PT70, we developed a method to radiolabel PT70 with carbon-11 and have studied its pharmacokinetics in mice and baboons using positron emission tomography.

  8. Virtually Designed Triclosan-Based Inhibitors of Enoyl-Acyl Carrier Protein Reductase of Mycobacterium tuberculosis and of Plasmodium falciparum.

    PubMed

    Owono Owono, Luc C; Ntie-Kang, Fidele; Keita, Melalie; Megnassan, Eugene; Frecer, Vladimir; Miertus, Stanislav

    2015-05-01

    We report here new chemical structures of predicted nanomolar triclosan-based inhibitors (TCLs) of Mycobacterium tuberculosis enoyl-acyl carrier protein reductase (InhA) virtually proposed by computer-assisted molecular design. 3D models of InhA-TCL complexes were prepared by in situ modifications of the reference crystal structure (PDB entry 1P45) for a training set of 15 TCLs with known InhA inhibitory activities. A QSAR model was built leading to linear correlation between the calculated free energies of complexation (ΔΔGcom ) and experimental values IC50 (exp) : pIC50 =-0.0657×ΔΔGcom +3.0502, R(2) =0.96. In addition, ligand-based quantitative pharmacophore model (PH4) was built from bound conformations of the training set compounds and confirmed the correlation between molecular models and observed activities: pIC50 (exp=) 0.8929×pIC50 (pre) -0.441, R(2) =0.95. Structural information from both models helped us to propose new TCL analogues. A virtual library of TCLs with known predicted activities against enoyl-acyl carrier protein reductase of Plasmodium falciparum (PfENR) was evaluated, revealing dual target TCLs. Moreover, analysis of binding site interactions suggested enriching substitutions, which led to more potent TCLs with predicted pIC50 (pre) as low as 7 nM. The computational approach, which used both free energy estimated from molecular modeling and 3D-QSAR pharmacophore model, was helpful in virtually proposing the dual-targeted drugs and provided valuable information for the design of novel potential antituberculotic agents.

  9. Radiolabelling and positron emission tomography of PT70, a time-dependent inhibitor of InhA, the Mycobacterium tuberculosis enoyl-ACP reductase.

    PubMed

    Wang, Hui; Liu, Li; Lu, Yang; Pan, Pan; Hooker, Jacob M; Fowler, Joanna S; Tonge, Peter J

    2015-11-01

    PT70 is a diaryl ether inhibitor of InhA, the enoyl-ACP reductase in the Mycobacterium tuberculosis fatty acid biosynthesis pathway. It has a residence time of 24 min on the target, and also shows antibacterial activity in a mouse model of tuberculosis infection. Due to the interest in studying target tissue pharmacokinetics of PT70, we developed a method to radiolabel PT70 with carbon-11 and have studied its pharmacokinetics in mice and baboons using positron emission tomography. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. A rational approach to identify inhibitors of Mycobacterium tuberculosis enoyl acyl carrier protein reductase.

    PubMed

    Chhabria, Mahesh T; Parmar, Kailash B; Brahmkshatriya, Pathik S

    2013-01-01

    Mycobacterial enoyl acyl carrier protein (ACP) reductase is an attractive target for focused design of novel antitubercular agents. Structural information available on enoyl-ACP reductase in complex with different ligands was used to generate receptor-based pharmacophore model in Discovery Studio (DS). In parallel, pharmacophore models were also generated using ligand-based approach (HypoGen module in DS). Statistically significant models were generated (r(2) = 0.85) which were found to be predictive as indicated from internal and external cross-validations. The model was used as a query tool to search Zinc and Maybridge databases to identify lead compounds and predict their activity in silico. Database searching retrieved many potential lead compounds having better estimated IC50 values than the training set compounds. These compounds were then evaluated for their drug-likeliness and pharmacokinetic properties using DS. Few selected compounds were then docked into the crystal structure of enoyl-ACP reductase using Dock 6.5. Most compounds were found to have high score values, which was found to be consistent with the results from pharmacophore mapping. Additionally, molecular docking provided useful insights into the nature of binding of the identified hit molecules. In summary, we show a useful strategy employing ligand- and structure-based approaches (pharmacophore modeling coupled with molecular docking) to identify new enoyl- ACP reductase inhibitors for antimycobacterial chemotherapy.

  11. Functional, thermodynamics, structural and biological studies of in silico-identified inhibitors of Mycobacterium tuberculosis enoyl-ACP(CoA) reductase enzyme

    PubMed Central

    Martinelli, Leonardo K. B.; Rotta, Mariane; Villela, Anne D.; Rodrigues-Junior, Valnês S.; Abbadi, Bruno L.; Trindade, Rogério V.; Petersen, Guilherme O.; Danesi, Giuliano M.; Nery, Laura R.; Pauli, Ivani; Campos, Maria M.; Bonan, Carla D.; de Souza, Osmar Norberto; Basso, Luiz A.; Santos, Diogenes S.

    2017-01-01

    Novel chemotherapeutics agents are needed to kill Mycobacterium tuberculosis, the main causative agent of tuberculosis (TB). The M. tuberculosis 2-trans-enoyl-ACP(CoA) reductase enzyme (MtInhA) is the druggable bona fide target of isoniazid. New chemotypes were previously identified by two in silico approaches as potential ligands to MtInhA. The inhibition mode was determined by steady-state kinetics for seven compounds that inhibited MtInhA activity. Dissociation constant values at different temperatures were determined by protein fluorescence spectroscopy. van’t Hoff analyses of ligand binding to MtInhA:NADH provided the thermodynamic signatures of non-covalent interactions (ΔH°, ΔS°, ΔG°). Phenotypic screening showed that five compounds inhibited in vitro growth of M. tuberculosis H37Rv strain. Labio_16 and Labio_17 compounds also inhibited the in vitro growth of PE-003 multidrug-resistant strain. Cytotoxic effects on Hacat, Vero and RAW 264.7 cell lines were assessed for the latter two compounds. The Labio_16 was bacteriostatic and Labio_17 bactericidal in an M. tuberculosis-infected macrophage model. In Zebrafish model, Labio_16 showed no cardiotoxicity whereas Labio_17 showed dose-dependent cardiotoxicity. Accordingly, a model was built for the MtInhA:NADH:Labio_16 ternary complex. The results show that the Labio_16 compound is a direct inhibitor of MtInhA, and it may represent a hit for the development of chemotherapeutic agents to treat TB. PMID:28436453

  12. Functional, thermodynamics, structural and biological studies of in silico-identified inhibitors of Mycobacterium tuberculosis enoyl-ACP(CoA) reductase enzyme

    NASA Astrophysics Data System (ADS)

    Martinelli, Leonardo K. B.; Rotta, Mariane; Villela, Anne D.; Rodrigues-Junior, Valnês S.; Abbadi, Bruno L.; Trindade, Rogério V.; Petersen, Guilherme O.; Danesi, Giuliano M.; Nery, Laura R.; Pauli, Ivani; Campos, Maria M.; Bonan, Carla D.; de Souza, Osmar Norberto; Basso, Luiz A.; Santos, Diogenes S.

    2017-04-01

    Novel chemotherapeutics agents are needed to kill Mycobacterium tuberculosis, the main causative agent of tuberculosis (TB). The M. tuberculosis 2-trans-enoyl-ACP(CoA) reductase enzyme (MtInhA) is the druggable bona fide target of isoniazid. New chemotypes were previously identified by two in silico approaches as potential ligands to MtInhA. The inhibition mode was determined by steady-state kinetics for seven compounds that inhibited MtInhA activity. Dissociation constant values at different temperatures were determined by protein fluorescence spectroscopy. van’t Hoff analyses of ligand binding to MtInhA:NADH provided the thermodynamic signatures of non-covalent interactions (ΔH°, ΔS°, ΔG°). Phenotypic screening showed that five compounds inhibited in vitro growth of M. tuberculosis H37Rv strain. Labio_16 and Labio_17 compounds also inhibited the in vitro growth of PE-003 multidrug-resistant strain. Cytotoxic effects on Hacat, Vero and RAW 264.7 cell lines were assessed for the latter two compounds. The Labio_16 was bacteriostatic and Labio_17 bactericidal in an M. tuberculosis-infected macrophage model. In Zebrafish model, Labio_16 showed no cardiotoxicity whereas Labio_17 showed dose-dependent cardiotoxicity. Accordingly, a model was built for the MtInhA:NADH:Labio_16 ternary complex. The results show that the Labio_16 compound is a direct inhibitor of MtInhA, and it may represent a hit for the development of chemotherapeutic agents to treat TB.

  13. Functional, thermodynamics, structural and biological studies of in silico-identified inhibitors of Mycobacterium tuberculosis enoyl-ACP(CoA) reductase enzyme.

    PubMed

    Martinelli, Leonardo K B; Rotta, Mariane; Villela, Anne D; Rodrigues-Junior, Valnês S; Abbadi, Bruno L; Trindade, Rogério V; Petersen, Guilherme O; Danesi, Giuliano M; Nery, Laura R; Pauli, Ivani; Campos, Maria M; Bonan, Carla D; de Souza, Osmar Norberto; Basso, Luiz A; Santos, Diogenes S

    2017-04-24

    Novel chemotherapeutics agents are needed to kill Mycobacterium tuberculosis, the main causative agent of tuberculosis (TB). The M. tuberculosis 2-trans-enoyl-ACP(CoA) reductase enzyme (MtInhA) is the druggable bona fide target of isoniazid. New chemotypes were previously identified by two in silico approaches as potential ligands to MtInhA. The inhibition mode was determined by steady-state kinetics for seven compounds that inhibited MtInhA activity. Dissociation constant values at different temperatures were determined by protein fluorescence spectroscopy. van't Hoff analyses of ligand binding to MtInhA:NADH provided the thermodynamic signatures of non-covalent interactions (ΔH°, ΔS°, ΔG°). Phenotypic screening showed that five compounds inhibited in vitro growth of M. tuberculosis H37Rv strain. Labio_16 and Labio_17 compounds also inhibited the in vitro growth of PE-003 multidrug-resistant strain. Cytotoxic effects on Hacat, Vero and RAW 264.7 cell lines were assessed for the latter two compounds. The Labio_16 was bacteriostatic and Labio_17 bactericidal in an M. tuberculosis-infected macrophage model. In Zebrafish model, Labio_16 showed no cardiotoxicity whereas Labio_17 showed dose-dependent cardiotoxicity. Accordingly, a model was built for the MtInhA:NADH:Labio_16 ternary complex. The results show that the Labio_16 compound is a direct inhibitor of MtInhA, and it may represent a hit for the development of chemotherapeutic agents to treat TB.

  14. The isoniazid-NAD adduct is a slow, tight-binding inhibitor of InhA, the Mycobacterium tuberculosis enoyl reductase: adduct affinity and drug resistance.

    PubMed

    Rawat, Richa; Whitty, Adrian; Tonge, Peter J

    2003-11-25

    Isoniazid (INH), a frontline antitubercular drug, inhibits InhA, the enoyl reductase from Mycobacterium tuberculosis, by forming a covalent adduct with the NAD cofactor. Here, we report that the INH-NAD adduct is a slow, tight-binding competitive inhibitor of InhA. Demonstration that the adduct binds to WT InhA by a two-step enzyme inhibition mechanism, with initial, weak binding (K(-1) = 16 +/- 11 nM) followed by slow conversion to a final inhibited complex (EI*) with overall Ki = 0.75 +/- 0.08 nM, reconciles existing contradictory values for the inhibitory potency of INH-NAD for InhA. The first order rate constant for conversion of the initial EI complex to EI* (k2 = 0.13 +/- 0.01 min(-1)) is similar to the maximum rate constant observed for InhA inhibition in reaction mixtures containing InhA, INH, NADH, and the INH-activating enzyme KatG (catalase/peroxidase from M. tuberculosis), consistent with an inhibition mechanism in which the adduct forms in solution rather than on the enzyme. Importantly, three mutations that correlate with INH resistance, I21V, I47T, and S94A, have little impact on the inhibition constants. Thus, drug resistance does not result simply from a reduction in affinity of INH-NAD for pure InhA. Instead, we hypothesize that protein-protein interactions within the FASII complex are critical to the mechanism of INH action. Finally, for M161V, an InhA mutation that correlates with resistance to the common biocide triclosan in Mycobacterium smegmatis, binding to form the initial EI complex is significantly weakened, explaining why this mutant inactivates more slowly than WT InhA when incubated with INH, NADH, and KatG.

  15. Observed crowding effects on Mycobacterium tuberculosis 2-trans-enoyl-ACP (CoA) reductase enzyme activity are not due to excluded volume only.

    PubMed

    Rotta, Mariane; Timmers, Luis F S M; Sequeiros-Borja, Carlos; Bizarro, Cristiano V; de Souza, Osmar N; Santos, Diogenes S; Basso, Luiz A

    2017-07-28

    The cellular milieu is a complex and crowded aqueous solution. Macromolecular crowding effects are commonly studied in vitro using crowding agents. The aim of the present study was to evaluate the effects, if any, of macromolecular synthetic crowding agents on the apparent steady-state kinetic parameters (K m , k cat , and k cat /K m ) of Mycobacterium tuberculosis 2-trans-enoyl-ACP (CoA) reductase (InhA). Negligible effects on InhA activity were observed for ficoll 70, ficoll 400 and dextran 70. A complex effect was observed for PEG 6000. Glucose and sucrose showed, respectively, no effect on InhA activity and decreased k cat /K m for NADH and k cat for 2-trans-dodecenoyl-CoA. Molecular dynamics results suggest that InhA adopts a more compact conformer in sucrose solution. The effects of the crowding agents on the energy (E a and E η ), enthalpy (∆H (#) ), entropy (∆S (#) ), and Gibbs free energy (∆G (#) ) of activation were determined. The ∆G (#) values for all crowding agents were similar to buffer, suggesting that excluded volume effects did not facilitate stable activated ES (#) complex formation. Nonlinear Arrhenius plot for PEG 6000 suggests that "soft" interactions play a role in crowding effects. The results on InhA do not unequivocally meet the criteria for crowding effect due to exclude volume only.

  16. Computer-Aided Design of Orally Bioavailable Pyrrolidine Carboxamide Inhibitors of Enoyl-Acyl Carrier Protein Reductase of Mycobacterium tuberculosis with Favorable Pharmacokinetic Profiles.

    PubMed

    Kouassi, Affiba Florance; Kone, Mawa; Keita, Melalie; Esmel, Akori; Megnassan, Eugene; N'Guessan, Yao Thomas; Frecer, Vladimir; Miertus, Stanislav

    2015-12-12

    We have carried out a computational structure-based design of new potent pyrrolidine carboxamide (PCAMs) inhibitors of enoyl-acyl carrier protein reductase (InhA) of Mycobacterium tuberculosis (MTb). Three-dimensional (3D) models of InhA-PCAMx complexes were prepared by in situ modification of the crystal structure of InhA-PCAM1 (Protein Data Bank (PDB) entry code: 4U0J), the reference compound of a training set of 20 PCAMs with known experimental inhibitory potencies (IC50(exp)). First, we built a gas phase quantitative structure-activity relationships (QSAR) model, linearly correlating the computed enthalpy of the InhA-PCAM complex formation and the IC50(exp). Further, taking into account the solvent effect and loss of inhibitor entropy upon enzyme binding led to a QSAR model with a superior linear correlation between computed Gibbs free energies (ΔΔGcom) of InhA-PCAM complex formation and IC50(exp) (pIC50(exp) = -0.1552·ΔΔGcom + 5.0448, R² = 0.94), which was further validated with a 3D-QSAR pharmacophore model generation (PH4). Structural information from the models guided us in designing of a virtual combinatorial library (VL) of more than 17 million PCAMs. The VL was adsorption, distribution, metabolism and excretion (ADME) focused and reduced down to 1.6 million drug like orally bioavailable analogues and PH4 in silico screened to identify new potent PCAMs with predicted IC50(pre) reaching up to 5 nM. Combining molecular modeling and PH4 in silico screening of the VL resulted in the proposed novel potent antituberculotic agent candidates with favorable pharmacokinetic profiles.

  17. Computer-Aided Design of Orally Bioavailable Pyrrolidine Carboxamide Inhibitors of Enoyl-Acyl Carrier Protein Reductase of Mycobacterium tuberculosis with Favorable Pharmacokinetic Profiles

    PubMed Central

    Kouassi, Affiba Florance; Kone, Mawa; Keita, Melalie; Esmel, Akori; Megnassan, Eugene; N’Guessan, Yao Thomas; Frecer, Vladimir; Miertus, Stanislav

    2015-01-01

    We have carried out a computational structure-based design of new potent pyrrolidine carboxamide (PCAMs) inhibitors of enoyl-acyl carrier protein reductase (InhA) of Mycobacterium tuberculosis (MTb). Three-dimensional (3D) models of InhA-PCAMx complexes were prepared by in situ modification of the crystal structure of InhA-PCAM1 (Protein Data Bank (PDB) entry code: 4U0J), the reference compound of a training set of 20 PCAMs with known experimental inhibitory potencies (IC50exp). First, we built a gas phase quantitative structure-activity relationships (QSAR) model, linearly correlating the computed enthalpy of the InhA-PCAM complex formation and the IC50exp. Further, taking into account the solvent effect and loss of inhibitor entropy upon enzyme binding led to a QSAR model with a superior linear correlation between computed Gibbs free energies (ΔΔGcom) of InhA-PCAM complex formation and IC50exp (pIC50exp = −0.1552·ΔΔGcom + 5.0448, R2 = 0.94), which was further validated with a 3D-QSAR pharmacophore model generation (PH4). Structural information from the models guided us in designing of a virtual combinatorial library (VL) of more than 17 million PCAMs. The VL was adsorption, distribution, metabolism and excretion (ADME) focused and reduced down to 1.6 million drug like orally bioavailable analogues and PH4 in silico screened to identify new potent PCAMs with predicted IC50pre reaching up to 5 nM. Combining molecular modeling and PH4 in silico screening of the VL resulted in the proposed novel potent antituberculotic agent candidates with favorable pharmacokinetic profiles. PMID:26703572

  18. Binding of the tautomeric forms of isoniazid-NAD adducts to the active site of the Mycobacterium tuberculosis enoyl-ACP reductase (InhA): a theoretical approach.

    PubMed

    Stigliani, Jean-Luc; Arnaud, Philippe; Delaine, Tamara; Bernardes-Génisson, Vania; Meunier, Bernard; Bernadou, Jean

    2008-11-01

    The front-line antituberculosis drug isoniazid (INH) inhibits InhA, the NADH-dependent fatty acid biosynthesis enoyl ACP-reductase from Mycobacterium tuberculosis, via formation of covalent adducts with NAD (INH-NAD adducts). While ring tautomers were found the main species formed in solution, only the 4S chain INH-NAD tautomer was evidenced in the crystallized InhA:INH-NAD complex. In this study we attempted to explore the modes of interaction and energy binding of the different isomers placed in the active site of InhA with the help of various molecular modelling techniques. Ligand and enzyme models were generated with the help of the Vega ZZ program package. Resulting ligands were then docked into the InhA active site individually using computational automated docking package AUTODOCK 3.0.5. The more relevant docked conformations were then used to compute the interaction energy between the ligands and the InhA cavity. The AM1 Hamiltonian and the QM/MM ONIOM methodologies were used and the results compared. The various tautomers were found docked in almost the same place where INH-NAD was present as predicted by earlier X-ray crystallographic studies. However, some changes of ligand conformation and of the interactions ligand-protein were evidenced. The lower binding energy was observed for the 4S chain adduct that probably represents the effective active form of the INH-NAD adducts, as compared to the 4R epimer. The two 4S,7R and 4R,7S ring tautomers show intermediate and similar binding energies contrasting with their different experimental inhibitory potency on InhA. As a possible explanation based on calculated conformations, we formulated the hypothesis of an initial binding of the two ring tautomers to InhA followed by opening of only the ring hemiamidal 4S,7R tautomer (possibly catalyzed by Tyr158 phenolate basic group) to give the 4S chain INH-NAD tight-binding inhibitor. The predictions of ligand-protein interactions at the molecular level can be of

  19. Crystallographic studies on the binding of isonicotinyl-NAD adduct to wild-type and isoniazid resistant 2-trans-enoyl-ACP (CoA) reductase from Mycobacterium tuberculosis.

    PubMed

    Dias, Marcio Vinicius Bertacine; Vasconcelos, Igor Bordin; Prado, Adriane Michele Xavier; Fadel, Valmir; Basso, Luiz Augusto; de Azevedo, Walter Filgueira; Santos, Diógenes Santiago

    2007-09-01

    The resumption of tuberculosis led to an increased need to understand the molecular mechanisms of drug action and drug resistance, which should provide significant insight into the development of newer compounds. Isoniazid (INH), the most prescribed drug to treat TB, inhibits an NADH-dependent enoyl-acyl carrier protein reductase (InhA) that provides precursors of mycolic acids, which are components of the mycobacterial cell wall. InhA is the major target of the mode of action of isoniazid. INH is a pro-drug that needs activation to form the inhibitory INH-NAD adduct. Missense mutations in the inhA structural gene have been identified in clinical isolates of Mycobacterium tuberculosis resistant to INH. To understand the mechanism of resistance to INH, we have solved the structure of two InhA mutants (I21V and S94A), identified in INH-resistant clinical isolates, and compare them to INH-sensitive WT InhA structure in complex with the INH-NAD adduct. We also solved the structure of unliganded INH-resistant S94A protein, which is the first report on apo form of InhA. The salient features of these structures are discussed and should provide structural information to improve our understanding of the mechanism of action of, and resistance to, INH in M. tuberculosis. The unliganded structure of InhA allows identification of conformational changes upon ligand binding and should help structure-based drug design of more potent antimycobacterial agents.

  20. Biological evaluation of potent triclosan-derived inhibitors of the enoyl-acyl carrier protein reductase InhA in drug-sensitive and drug-resistant strains of Mycobacterium tuberculosis.

    PubMed

    Stec, Jozef; Vilchèze, Catherine; Lun, Shichun; Perryman, Alexander L; Wang, Xin; Freundlich, Joel S; Bishai, William; Jacobs, William R; Kozikowski, Alan P

    2014-11-01

    New triclosan (TRC) analogues were evaluated for their activity against the enoyl-acyl carrier protein reductase InhA in Mycobacterium tuberculosis (Mtb). TRC is a well-known inhibitor of InhA, and specific modifications to its positions 5 and 4' afforded 27 derivatives; of these compounds, seven derivatives showed improved potency over that of TRC. These analogues were active against both drug-susceptible and drug-resistant Mtb strains. The most active compound in this series, 4-(n-butyl)-1,2,3-triazolyl TRC derivative 3, had an MIC value of 0.6 μg mL(-1) (1.5 μM) against wild-type Mtb. At a concentration equal to its MIC, this compound inhibited purified InhA by 98 %, and showed an IC50 value of 90 nM. Compound 3 and the 5-methylisoxazole-modified TRC 14 were able to inhibit the biosynthesis of mycolic acids. Furthermore, mc(2) 4914, an Mtb strain overexpressing inhA, was found to be less susceptible to compounds 3 and 14, supporting the notion that InhA is the likely molecular target of the TRC derivatives presented herein.

  1. Biological Evaluation of Potent Triclosan-Derived Inhibitors of the Enoyl-Acyl Carrier Protein Reductase InhA in Drug-sensitive and Drug-resistant Strains of Mycobacterium tuberculosis

    PubMed Central

    Vilchèze, Catherine; Lun, Shichun; Perryman, Alexander L.; Wang, Xin; Freundlich, Joel S.; Bishai, William; Jacobs, William R.

    2014-01-01

    New triclosan (TRC) analogs were evaluated for their activity against the enoyl-acyl carrier protein reductase InhA in Mycobacterium tuberculosis (Mtb). TRC is a well-known inhibitor of InhA and specific modifications to its positions 5 and 4′ afforded twenty-seven derivatives; of these compounds seven derivatives showed an improved potency in comparison to TRC. These analogs were active against both drug-susceptible and drug-resistant Mtb strains. The most active compound in this series, 3, had an MIC value of 0.6 μg/mL (1.5 μM) against wild-type Mtb. At a concentration equal to its MIC, this molecule inhibited the purified InhA enzyme to the extent of 98%, and it showed an IC50 value of 90 nM. Compounds 3 and 14 were able to inhibit the biosynthesis of mycolic acids. Furthermore, mc24914, an Mtb strain overexpressing inhA, was resistant to the compounds 3 and 14, supporting the notion that InhA is the likely molecular target of the TRC derivatives presented herein. PMID:25165007

  2. Cloning of an ORF with homology to Mycobacterium echA1, encoding the enoyl-CoA hydratase, in Rhodococcus fascians.

    PubMed

    Humanes, L; García-Fernández, J M; Roldán, J M; Diez, J

    1999-01-01

    An open reading frame encoding a polypeptide of significant homology (55.7% identity) with the enoyl-CoA hydratase encoded by the gene echA1 from Mycobacterium tuberculosis has been found in the genome of the plant-pathogen bacteria Rhodococcus fascians strain NRRL-B-15096. Sequence alignments showed that it possesses several conserved blocks common to E. coli, M. tuberculosis and human mitochondria. One of such blocks includes a glutamate residue located at position 149, corresponding to the glutamate 139 of Escherichia coli. This glutamate was previously shown to be the catalytic residue of enoyl-CoA hydratase in the multienzyme complex of fatty acid oxidation from E. coli. Our results provide additional information on the conserved domains of this enzyme. Significant homologies in other genome regions between R. fascians and M. tuberculosis confirm their phylogenetic relationship.

  3. Mycobacterium tuberculosis Metabolism

    PubMed Central

    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

  4. Mycobacterium tuberculosis septic shock.

    PubMed

    Kethireddy, Shravan; Light, R Bruce; Mirzanejad, Yazdan; Maki, Dennis; Arabi, Yaseen; Lapinsky, Stephen; Simon, David; Kumar, Aseem; Parrillo, Joseph E; Kumar, Anand

    2013-08-01

    Septic shock due to Mycobacterium tuberculosis (MTB) is an uncommon but well-recognized clinical syndrome. The objective of this study was to describe the unique clinical characteristics, epidemiologic risk factors, and covariates of survival of patients with MTB septic shock in comparison with other bacterial septic shock. A retrospective nested cohort study was conducted of patients given a diagnosis of MTB septic shock derived from a trinational, 8,670-patient database of patients with septic shock between 1996 and 2007. In the database, 53 patients had been given a diagnosis of MTB shock compared with 5,419 with septic shock associated with isolation of more common bacterial pathogens. Patients with MTB and other bacterial septic shock had in-hospital mortality rates of 79.2% and 49.7%, respectively (P < .0001). Of the cases of MTB shock, all but five patients had recognized respiratory tract involvement. Fifty-five percent of patients (29 of 53) were documented (by direct culture or stain) as having disseminated extrapulmonary involvement. Inappropriate and appropriate initial empirical therapy was delivered in 28 patients (52.8%) and 25 patients (47.2%); survival was 7.1% and 36.0%, respectively (P = .0114). Ten patients (18.9%) did not receive anti-MTB therapy; all died. The median time to appropriate antimicrobial therapy for MTB septic shock was 31.0 h (interquartile range, 18.9-71.9 h). Only 11 patients received anti-MTB therapy within 24 h of documentation of hypotension; six of these (54.5%) survived. Only one of 21 patients (4.8%) who started anti-MTB therapy after 24 h survived (P = .0003 vs < 24 h). Survival differences between these time intervals are not significantly different from those seen with bacterial septic shock due to more common bacterial pathogens. MTB septic shock behaves similarly to bacterial septic shock. As with bacterial septic shock, early appropriate antimicrobial therapy appears to improve mortality.

  5. Mycobacterium Tuberculosis in Spinal Tuberculosis

    PubMed Central

    Kim, Sung-Sim; Moon, Han-Lim; Kim, Dong-Hyeon

    2017-01-01

    Even in an era of remarkable medical advances, there is an issue of why tuberculosis remains in the list of disastrous diseases, afflicting humans and causing suffering. There has not been a plausible answer to this, and it has been suggested that clinicians and medical scientists could presently not win the war against the tubercle bacilli. With regards to this issue, based on the authors' own clinical and research experiences, in this review, the available literature was revisited in order to address the raised questions and to provide recent information on characteristics of tubercle bacilli and possible ways to more effectively treat tuberculosis. PMID:28243382

  6. Immune Responses in Cattle Inoculated with Mycobacterium bovis, Mycobacterium tuberculosis, or Mycobacterium kansasii

    USDA-ARS?s Scientific Manuscript database

    Cattle were inoculated with Mycobacterium bovis, Mycobacterium tuberculosis, or Mycobacterium kansasii to compare antigen-specific immune responses to varied patterns of mycobacterial disease. Disease expression ranged from colonization with associated pathology (M. bovis), colonization without path...

  7. Targeting phenotypically tolerant Mycobacterium tuberculosis

    PubMed Central

    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

  8. Tuberculosis due to Mycobacterium bovis and Mycobacterium caprae in sheep.

    PubMed

    Muñoz Mendoza, Marta; Juan, Lucía de; Menéndez, Santiago; Ocampo, Antón; Mourelo, Jorge; Sáez, José L; Domínguez, Lucas; Gortázar, Christian; García Marín, Juan F; Balseiro, Ana

    2012-02-01

    Tuberculosis was diagnosed in three flocks of sheep in Galicia, Spain, in 2009 and 2010. Two flocks were infected with Mycobacterium bovis and one flock was infected with Mycobacterium caprae. Infection was confirmed by the comparative intradermal tuberculin test, bacteriology, molecular analysis and histopathology. Sheep have the potential to act as a reservoir for tuberculosis. Copyright © 2011 Elsevier Ltd. All rights reserved.

  9. 21 CFR 866.3370 - Mycobacterium tuberculosis immunofluorescent reagents.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... in the diagnosis of tuberculosis and provides epidemiological information on this disease. Mycobacterium tuberculosis is the common causative organism in human tuberculosis, a chronic infectious disease...

  10. 21 CFR 866.3370 - Mycobacterium tuberculosis immunofluorescent reagents.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... in the diagnosis of tuberculosis and provides epidemiological information on this disease. Mycobacterium tuberculosis is the common causative organism in human tuberculosis, a chronic infectious disease...

  11. 21 CFR 866.3370 - Mycobacterium tuberculosis immunofluorescent reagents.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... in the diagnosis of tuberculosis and provides epidemiological information on this disease. Mycobacterium tuberculosis is the common causative organism in human tuberculosis, a chronic infectious disease...

  12. Enoyl-Coenzyme A Hydratase and Antigen 85B of Mycobacterium habana Are Specifically Recognized by Antibodies in Sera from Leprosy Patients ▿

    PubMed Central

    Serafín-López, J.; Talavera-Paulin, M.; Amador-Molina, J. C.; Alvarado-Riverón, M.; Vilchis-Landeros, M. M.; Méndez-Ortega, P.; Fafutis-Morris, M.; Paredes-Cervantes, V.; López-Santiago, R.; León, C. I.; Guerrero, M. I.; Ribas-Aparicio, R. M.; Mendoza-Hernández, G.; Carreño-Martínez, C.; Estrada-Parra, S.; Estrada-García, I.

    2011-01-01

    Leprosy is an infectious disease caused by Mycobacterium leprae, which is a noncultivable bacterium. One of the principal goals of leprosy research is to develop serological tests that will allow identification and early treatment of leprosy patients. M. habana is a cultivable nonpathogenic mycobacterium and candidate vaccine for leprosy, and several antigens that cross-react between M. leprae and M. habana have been discovered. The aim of the present study was to extend the identification of cross-reactive antigens by identifying M. habana proteins that reacted by immunoblotting with antibodies in serum samples from leprosy patients but not with antibodies in sera from tuberculosis (TB) patients or healthy donors (HDs). A 28-kDa antigen that specifically reacted with sera from leprosy patients was identified. To further characterize this antigen, protein spots were aligned in two-dimensional polyacrylamide gels and Western blots. Spots cut out from the gels were then analyzed by mass spectrometry. Two proteins were identified: enoyl-coenzyme A hydratase (lipid metabolism; ML2498) and antigen 85B (Ag85B; mycolyltransferase; ML2028). These proteins represent promising candidates for the design of a reliable tool for the serodiagnosis of lepromatous leprosy, which is the most frequent form in Mexico. PMID:21613461

  13. DNA Replication in Mycobacterium tuberculosis

    PubMed Central

    DITSE, ZANELE; LAMERS, MEINDERT H.; WARNER, DIGBY F.

    2017-01-01

    Faithful replication and maintenance of the genome are essential to the ability of any organism to survive and propagate. For an obligate pathogen such as Mycobacterium tuberculosis that has to complete successive cycles of transmission, infection, and disease in order to retain a foothold in the human population, this requires that genome replication and maintenance must be accomplished under the metabolic, immune, and antibiotic stresses encountered during passage through variable host environments. Comparative genomic analyses have established that chromosomal mutations enable M. tuberculosis to adapt to these stresses: the emergence of drug-resistant isolates provides direct evidence of this capacity, so too the well-documented genetic diversity among M. tuberculosis lineages across geographic loci, as well as the microvariation within individual patients that is increasingly observed as whole-genome sequencing methodologies are applied to clinical samples and tuberculosis (TB) disease models. However, the precise mutagenic mechanisms responsible for M. tuberculosis evolution and adaptation are poorly understood. Here, we summarize current knowledge of the machinery responsible for DNA replication in M. tuberculosis, and discuss the potential contribution of the expanded complement of mycobacterial DNA polymerases to mutagenesis. We also consider briefly the possible role of DNA replication—in particular, its regulation and coordination with cell division—in the ability of M. tuberculosis to withstand antibacterial stresses, including host immune effectors and antibiotics, through the generation at the population level of a tolerant state, or through the formation of a subpopulation of persister bacilli—both of which might be relevant to the emergence and fixation of genetic drug resistance. PMID:28361736

  14. Copper Homeostasis in Mycobacterium tuberculosis

    PubMed Central

    Shi, Xiaoshan; Darwin, K. Heran

    2015-01-01

    Copper (Cu) is a trace element essential for the growth and development of almost all organisms, including bacteria. However, Cu overload in most systems is toxic. Studies show Cu accumulates in macrophage phagosomes infected with bacteria, suggesting Cu provides an innate immune mechanism to combat invading pathogens. To counteract the host-supplied Cu, increasing evidence suggests that bacteria have evolved Cu resistance mechanisms to facilitate their pathogenesis. In particular, Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, has evolved multiple pathways to respond to Cu. Here, we summarize what is currently known about Cu homeostasis in Mtb and discuss potential sources of Cu encountered by this and other pathogens in a mammalian host. PMID:25614981

  15. Multiple receptor conformers based molecular docking study of fluorine enhanced ethionamide with mycobacterium enoyl ACP reductase (InhA).

    PubMed

    Khan, Akib Mahmud; Shawon, Jakaria; Halim, Mohammad A

    2017-09-14

    A major limitation in current molecular docking method is that of failure to account for receptor flexibility. Herein we report multiple receptor conformers based molecular docking as a practical alternative to account for the receptor flexibility. Multiple (forty) conformers of Mycobacterium Enoyl ACP Reductase (InhA) are generated from Molecular Dynamics simulation and twenty crystallographic structures of InhA bound to different inhibitors are obtained from the Protein Data Bank. Fluorine directed modifications are performed to currently available anti-tuberculosis drug ethionamide. The modified drugs are optimized using B3LYP 6-31G (d,p) level of theory. Dipole moment, frontier orbital gap and thermodynamical properties such as electronic energy, enthalpy and Gibbs free energy of these optimized drugs are investigated. These drugs are subsequently docked against the conformers of InhA. Molecular docking against multiple InhA conformations show variation in ligand binding affinity and suggest that Ser94, Gly96, Lys165 and Ile194 amino acids play critical role on strong drug-InhA interaction. Modified drug N1 showed greater binding affinity compared to EN in most conformations. Structure of PDB ID: 2NSD and snapshot conformer at 5.5ns show most favorable binding with N1 compared to other conformers. Fluorine participates in forming fluorine bonds and contributes significantly in increasing binding affinity. Our study reveal that addition of trifluoromethyl group explicitly shows promise in improving thermodynamic properties and in enhancing hydrogen bonding and non-bonded interactions. Molecular dynamics (MD) simulation show that EN and N1 remained in the binding pocket similar to the docked pose of EN-InhA and E1-InhA complexes and also suggested that InhA binds to its inhibitor in inhibitor-induced folding manner. ADMET calculations predict modified drugs to have improved pharmacokinetic properties. Our study concludes that multiple receptor conformers based

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

    SciTech Connect

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

    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.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  18. Osteoarticular manifestations of Mycobacterium tuberculosis infection.

    PubMed

    Zychowicz, Michael E

    2010-01-01

    Mycobacterium tuberculosis has affected humans for much of our existence. The incidence of global tuberculosis infection continues to rise, especially in concert with HIV coinfection. Many disease processes, such as diabetes, increase the likelihood of tuberculosis infection. Tuberculosis bacteria can infect any bone, joint, tendon, or bursa; however, the most common musculoskeletal site for infection includes the spine and weight-bearing joints of the hip and knee. Many patients who present with osteoarticular tuberculosis infection will have a gradual onset of pain at the site of infection. Many patients who develop a musculoskeletal tuberculosis infection will have no evidence of a pulmonary tuberculosis infection on x-ray film and many will have very mild symptoms with the initial infection. Healthcare providers must remember that many patients who develop tuberculosis infection do not progress to active tuberculosis disease; however, the latent infection may become active with immune compromise.

  19. Multidrug-Resistant Mycobacterium tuberculosis, Southwestern Colombia

    PubMed Central

    Nieto, Luisa Maria; Rozo, Juan C.; Forero, Liliana; van Soolingen, Dick

    2011-01-01

    Using spoligotyping, we identified 13 genotypes and 17 orphan types among 160 Mycobacterium tuberculosis isolates from patients in Valle del Cauca, Colombia. The Beijing genotype represented 15.6% of the isolates and was correlated with multidrug-resistant tuberculosis, female sex of the patients, and residence in Buenaventura and may represent a new public health threat. PMID:21762581

  20. Mycobacterium tuberculosis and the host response

    PubMed Central

    Kaufmann, Stefan H.E.; Cole, Stewart T.; Mizrahi, Valerie; Rubin, Eric; Nathan, Carl

    2005-01-01

    Mycobacterium tuberculosis remains a leading cause of morbidity and mortality worldwide. Advances reported at a recent international meeting highlight insights and controversies in the genetics of M. tuberculosis and the infected host, the nature of protective immune responses, adaptation of the bacillus to host-imposed stresses, animal models, and new techniques. PMID:15939785

  1. Mycobacterium tuberculosis prevents inflammasome activation.

    PubMed

    Master, Sharon S; Rampini, Silvana K; Davis, Alexander S; Keller, Christine; Ehlers, Stefan; Springer, Burkhard; Timmins, Graham S; Sander, Peter; Deretic, Vojo

    2008-04-17

    Mycobacterium tuberculosis (Mtb) parasitizes host macrophages and subverts host innate and adaptive immunity. Several cytokines elicited by Mtb are mediators of mycobacterial clearance or are involved in tuberculosis pathology. Surprisingly, interleukin-1beta (IL-1beta), a major proinflammatory cytokine, has not been implicated in host-Mtb interactions. IL-1beta is activated by processing upon assembly of the inflammasome, a specialized inflammatory caspase-activating protein complex. Here, we show that Mtb prevents inflammasome activation and IL-1beta processing. An Mtb gene, zmp1, which encodes a putative Zn(2+) metalloprotease, is required for this process. Infection of macrophages with zmp1-deleted Mtb triggered activation of the inflammasome, resulting in increased IL-1beta secretion, enhanced maturation of Mtb containing phagosomes, improved mycobacterial clearance by macrophages, and lower bacterial burden in the lungs of aerosol-infected mice. Thus, we uncovered a previously masked role for IL-1beta in the control of Mtb and a mycobacterial system that prevents inflammasome and, therefore, IL-1beta activation.

  2. Mycobacterium tuberculosis: Success through dormancy

    PubMed Central

    Gengenbacher, Martin; Kaufmann, Stefan H. E.

    2012-01-01

    Tuberculosis (TB) remains a major health threat, killing near to 2 million individuals around this globe, annually. The sole vaccine developed almost a century ago, provides limited protection only during childhood. After decades without the introduction of new antibiotics, several candidates are currently undergoing clinical investigation. Curing TB requires prolonged combination chemotherapy with several drugs. Moreover, monitoring the success of therapy is questionable due to the lack of reliable biomarkers. To substantially improve the situation, a detailed understanding of the crosstalk between human host and the pathogen Mycobacterium tuberculosis (Mtb) is vital. Principally, Mtb’s enormous success is based on three capacities: First, reprogramming of macrophages after primary infection/phagocytosis in order to prevent its own destruction; second, initiating the formation of well-organized granulomas, comprising different immune cells to create a confined environment for the host–pathogen standoff; third, the capability to shut down its own central metabolism, terminate replication and thereby transit into a stage of dormancy rendering itself extremely resistant to host defense and drug treatment. Here we review the molecular mechanisms underlying these processes, draw conclusions in a working model of mycobacterial dormancy and highlight gaps in our understanding to be addressed in future research. PMID:22320122

  3. The pathology of Mycobacterium tuberculosis infection.

    PubMed

    Sakamoto, K

    2012-05-01

    Mycobacterium tuberculosis is an old enemy of the human race, with evidence of infection observed as early as 5000 years ago. Although more host-restricted than Mycobacterium bovis, which can infect all warm-blooded vertebrates, M. tuberculosis can infect, and cause morbidity and mortality in, several veterinary species as well. As M. tuberculosis is one of the earliest described bacterial pathogens, the literature describing this organism is vast and overwhelming. This review strives to distill what is currently known about this bacterium and the disease it causes for the veterinary pathologist.

  4. Pathway Profiling in Mycobacterium tuberculosis

    PubMed Central

    Thomas, Suzanne T.; VanderVen, Brian C.; Sherman, David R.; Russell, David G.; Sampson, Nicole S.

    2011-01-01

    Mycobacterium tuberculosis, the bacterium that causes tuberculosis, imports and metabolizes host cholesterol during infection. This ability is important in the chronic phase of infection. Here we investigate the role of the intracellular growth operon (igr), which has previously been identified as having a cholesterol-sensitive phenotype in vitro and which is important for intracellular growth of the mycobacteria. We have employed isotopically labeled low density lipoproteins containing either [1,7,15,22,26-14C]cholesterol or [1,7,15,22,26-13C]cholesterol and high resolution LC/MS as tools to profile the cholesterol-derived metabolome of an igr operon-disrupted mutant (Δigr) of M. tuberculosis. A partially metabolized cholesterol species accumulated in the Δigr knock-out strain that was absent in the complemented and parental wild-type strains. Structural elucidation by multidimensional 1H and 13C NMR spectroscopy revealed the accumulated metabolite to be methyl 1β-(2′-propanoate)-3aα-H-4α-(3′-propanoic acid)-7aβ-methylhexahydro-5-indanone. Heterologously expressed and purified FadE28-FadE29, an acyl-CoA dehydrogenase encoded by the igr operon, catalyzes the dehydrogenation of 2′-propanoyl-CoA ester side chains in substrates with structures analogous to the characterized metabolite. Based on the structure of the isolated metabolite, enzyme activity, and bioinformatic annotations, we assign the primary function of the igr operon to be degradation of the 2′-propanoate side chain. Therefore, the igr operon is necessary to completely metabolize the side chain of cholesterol metabolites. PMID:22045806

  5. Aqueous Molecular Dynamics Simulations of the M. tuberculosis Enoyl-ACP Reductase-NADH System and Its Complex with a Substrate Mimic or Diphenyl Ethers Inhibitors

    PubMed Central

    da Silva Lima, Camilo Henrique; de Alencastro, Ricardo Bicca; Kaiser, Carlos Roland; de Souza, Marcus Vinícius Nora; Rodrigues, Carlos Rangel; Albuquerque, Magaly Girão

    2015-01-01

    Molecular dynamics (MD) simulations of 12 aqueous systems of the NADH-dependent enoyl-ACP reductase from Mycobacterium tuberculosis (InhA) were carried out for up to 20–40 ns using the GROMACS 4.5 package. Simulations of the holoenzyme, holoenzyme-substrate, and 10 holoenzyme-inhibitor complexes were conducted in order to gain more insight about the secondary structure motifs of the InhA substrate-binding pocket. We monitored the lifetime of the main intermolecular interactions: hydrogen bonds and hydrophobic contacts. Our MD simulations demonstrate the importance of evaluating the conformational changes that occur close to the active site of the enzyme-cofactor complex before and after binding of the ligand and the influence of the water molecules. Moreover, the protein-inhibitor total steric (ELJ) and electrostatic (EC) interaction energies, related to Gly96 and Tyr158, are able to explain 80% of the biological response variance according to the best linear equation, pKi = 7.772 − 0.1885 × Gly96 + 0.0517 × Tyr158 (R2 = 0.80; n = 10), where interactions with Gly96, mainly electrostatic, increase the biological response, while those with Tyr158 decrease. These results will help to understand the structure-activity relationships and to design new and more potent anti-TB drugs. PMID:26457706

  6. Use of enoyl coenzyme A hydratase of Mycobacterium avium subsp. paratuberculosis for the serological diagnosis of Johne's disease.

    PubMed

    Nagata, Reiko; Kawaji, Satoko; Mori, Yasuyuki

    2013-10-01

    Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP), remains difficult to control because of the lack of specific and sensitive diagnostic tests. In order to improve the specificity of sero-diagnosis for JD, the phage display library derived from genomic DNA of MAP was immunoscreened to identify novel antigenic targets. We selected a clone using antibodies from MAP experimentally infected cattle, and annotated its coding sequence as MAP1197 in the MAP genome, which encoded "echA12_2" in the MAP protein (Map-echA) belonging to Enoyl-CoA hydratase, known as a crotonase enzyme. The Map-echA was expressed in Esherichia coli and purified as a histidine-tag recombinant protein (rMap-echA), and the diagnostic potential of the protein was further evaluated by enzyme-linked immunosorbent assays (ELISA). Antibody responses to rMap-echA were higher in MAP-infected cattle than in uninfected cattle. The specificity of the Map-echA ELISA was also confirmed by evaluation with hyper-immune sera against various kinds of Mycobacterium species. Furthermore, in all experimentally infected cattle the antibody against rMap-echA was detected 2-7months earlier than by a commercially available ELISA kit. These results suggested that Map-echA can be used as a specific and sensitive serological diagnostic antigen for the detection of MAP infection.

  7. Macrophage infection models for Mycobacterium tuberculosis.

    PubMed

    Johnson, Benjamin K; Abramovitch, Robert B

    2015-01-01

    Mycobacterium tuberculosis colonizes, survives, and grows inside macrophages. In vitro macrophage infection models, using both primary macrophages and cell lines, enable the characterization of the pathogen response to macrophage immune pressure and intracellular environmental cues. We describe methods to propagate and infect primary murine bone marrow-derived macrophages and J774 and THP-1 macrophage-like cell lines. We also present methods on the characterization of M. tuberculosis intracellular survival and the preparation of infected macrophages for imaging.

  8. Mycobacterium tuberculosis wears what it eats.

    PubMed

    Russell, David G; VanderVen, Brian C; Lee, Wonsik; Abramovitch, Robert B; Kim, Mi-jeong; Homolka, Susanne; Niemann, Stefan; Rohde, Kyle H

    2010-07-22

    Mycobacterium tuberculosis remains one of the most pernicious of human pathogens. Current vaccines are ineffective, and drugs, although efficacious, require prolonged treatment with constant medical oversight. Overcoming these problems requires a greater appreciation of M. tuberculosis in the context of its host. Upon infection of either macrophages in culture or animal models, the bacterium realigns its metabolism in response to the new environments it encounters. Understanding these environments, and the stresses that they place on M. tuberculosis, should provide insights invaluable for the development of new chemo- and immunotherapeutic strategies.

  9. Novel Mycobacterium tuberculosis complex pathogen, M. mungi.

    PubMed

    Alexander, Kathleen A; Laver, Pete N; Michel, Anita L; Williams, Mark; van Helden, Paul D; Warren, Robin M; Gey van Pittius, Nicolaas C

    2010-08-01

    Seven outbreaks involving increasing numbers of banded mongoose troops and high death rates have been documented. We identified a Mycobacterium tuberculosis complex pathogen, M. mungi sp. nov., as the causative agent among banded mongooses that live near humans in Chobe District, Botswana. Host spectrum and transmission dynamics remain unknown.

  10. [Mycobacterium tuberculosis infection following organ transplantation].

    PubMed

    Haas, Charles; Le Jeunne, Claire

    2006-11-01

    In transplant recipients, immunosuppressive treatment affects cell-mediated immunity and increases the risk of tuberculosis. Tuberculosis may be transmitted by the donor organ or occur de novo, but such cases are rare. The vast majority of cases of active tuberculosis in transplant recipients result from reactivation of latent Mycobacterium tuberculosis infection. The incidence varies from one region of the globe to another, from 0.5-1.0% in North America, to 0.36-5.5% in Europe and 7.0-11.8% in India. The incidence of tuberculosis among transplant recipients is much higher than in the general population. Diabetes mellitus, renal impairment, systemic lupus erythematosus, chronic liver disease and AIDS all increase the risk of post-transplant tuberculosis. Extrapulmonary and disseminated forms are frequent in this setting. The diagnosis of tuberculosis in transplant recipients is often difficult, and treatment is frequently delayed. Tuberculosis can be life-threatening in such cases. Treatment is difficult because rifampicin is a cytochrome P450 inducer (leading to reduced levels of cyclosporine), and because the hepatotoxicity of isoniazid, rifampin and pyrazinamide is frequently increased in transplant recipients. Treatment of latent tuberculosis before transplantation markedly reduces the risk of developing active tuberculosis after transplantation.

  11. Mechanisms of fluoroquinolone resistance in Mycobacterium tuberculosis.

    PubMed

    Yujiao, Zhang; Xiaojing, Li; Kaixia, Mi

    2016-10-20

    Tuberculosis, caused by the pathogen Mycobacterium tuberculosis, is one of the world's deadliest bacterial infectious disease. It is still a global-health threat, particularly because of the drug-resistant forms. Fluoroquinolones, with target of gyrase, are among the drugs used to treat tuberculosis. However, their widespread use has led to bacterial resistance. The molecular mechanisms of fluoroquinolone resistance in mycobacterium tuberculosis have been reported, such as DNA gyrase mutations, drug efflux pumps system, bacterial cell wall thickness and pentapeptide proteins (MfpA) mediated regulation of gyrase. Mutations in gyrase conferring quinolone resistance play important roles and have been extensively studied. Recent studies have shown that the regulation of DNA gyrase affects mycobacterial drug resistance, but the mechanisms, especially by post-translational modification and regulatory proteins, are poorly understood. In this review, we summarize the fluoroquinolone drug development, and the molecular genetics of fluoroquinolone resistance in mycobacteria. Comprehensive understanding of the mechanisms of fluoroquinolone resistance in Mycobacterium tuberculosis will open a new view on understanding drug resistance in mycobacteria and lead to novel strategies to develop new accurate diagnosis methods.

  12. Cytokinins beyond plants: synthesis by Mycobacterium tuberculosis

    PubMed Central

    Samanovic, Marie I.; Darwin, K. H.

    2015-01-01

    Mycobacterium tuberculosis (M. tuberculosis) resides mainly inside macrophages, which produce nitric oxide (NO) to combat microbial infections. Earlier studies revealed that proteasome-associated genes are required for M. tuberculosis to resist NO via a previously uncharacterized mechanism. Twelve years later, we elucidated the link between proteasome function and NO resistance in M. tuberculosis in Molecular Cell, 57 (2015), pp. 984-994. In a proteasome degradation-defective mutant, Rv1205, a homologue of the plant enzyme LONELY GUY (LOG) that is involved in the synthesis of phytohormones called cytokinins, accumulates and as a consequence results in the overproduction of cytokinins. Cytokinins break down into aldehydes that kill mycobacteria in the presence of NO. Importantly, this new discovery reveals for the first time that a mammalian bacterial pathogen produces cytokinins and leaves us with the question: why is M. tuberculosis, an exclusively human pathogen, producing cytokinins? PMID:28357289

  13. Interference of Mycobacterium tuberculosis with macrophage responses.

    PubMed

    Scherr, Nicole; Jayachandran, Rajesh; Mueller, Philipp; Pieters, Jean

    2009-06-01

    Tuberculosis, caused by Mycobacterium tuberculosis, has become an important health and economic burden, with more than four thousand people succumbing to the disease every day. Thus, there is an urgent need to understand the molecular basis of this pathogen's success in causing disease in humans, in order to develop new drugs superior to conventional drugs available at present. One reason why M. tuberculosis is such a dangerous microbe lies within its ability to survive within infected hosts, thereby efficiently circumventing host immune responses. Over the past few years, a number of mechanisms have been unravelled that are utilized by M. tuberculosis to survive within hosts and to avoid immune defence mechanisms. Several of these mechanisms have been described in this communication that may be useful for the development of novel compounds to treat tuberculosis.

  14. Neurons Are Host Cells for Mycobacterium tuberculosis

    PubMed Central

    Randall, Philippa J.; Hsu, Nai-Jen; Lang, Dirk; Cooper, Susan; Sebesho, Boipelo; Allie, Nasiema; Keeton, Roanne; Francisco, Ngiambudulu M.; Salie, Sumayah; Labuschagné, Antoinette; Quesniaux, Valerie; Ryffel, Bernhard; Kellaway, Lauriston

    2014-01-01

    Mycobacterium tuberculosis infection of the central nervous system is thought to be initiated once the bacilli have breached the blood brain barrier and are phagocytosed, primarily by microglial cells. In this study, the interactions of M. tuberculosis with neurons in vitro and in vivo were investigated. The data obtained demonstrate that neurons can act as host cells for M. tuberculosis. M. tuberculosis bacilli were internalized by murine neuronal cultured cells in a time-dependent manner after exposure, with superior uptake by HT22 cells compared to Neuro-2a cells (17.7% versus 9.8%). Internalization of M. tuberculosis bacilli by human SK-N-SH cultured neurons suggested the clinical relevance of the findings. Moreover, primary murine hippocampus-derived neuronal cultures could similarly internalize M. tuberculosis. Internalized M. tuberculosis bacilli represented a productive infection with retention of bacterial viability and replicative potential, increasing 2- to 4-fold within 48 h. M. tuberculosis bacillus infection of neurons was confirmed in vivo in the brains of C57BL/6 mice after intracerebral challenge. This study, therefore, demonstrates neurons as potential new target cells for M. tuberculosis within the central nervous system. PMID:24566619

  15. Neurons are host cells for Mycobacterium tuberculosis.

    PubMed

    Randall, Philippa J; Hsu, Nai-Jen; Lang, Dirk; Cooper, Susan; Sebesho, Boipelo; Allie, Nasiema; Keeton, Roanne; Francisco, Ngiambudulu M; Salie, Sumayah; Labuschagné, Antoinette; Quesniaux, Valerie; Ryffel, Bernhard; Kellaway, Lauriston; Jacobs, Muazzam

    2014-05-01

    Mycobacterium tuberculosis infection of the central nervous system is thought to be initiated once the bacilli have breached the blood brain barrier and are phagocytosed, primarily by microglial cells. In this study, the interactions of M. tuberculosis with neurons in vitro and in vivo were investigated. The data obtained demonstrate that neurons can act as host cells for M. tuberculosis. M. tuberculosis bacilli were internalized by murine neuronal cultured cells in a time-dependent manner after exposure, with superior uptake by HT22 cells compared to Neuro-2a cells (17.7% versus 9.8%). Internalization of M. tuberculosis bacilli by human SK-N-SH cultured neurons suggested the clinical relevance of the findings. Moreover, primary murine hippocampus-derived neuronal cultures could similarly internalize M. tuberculosis. Internalized M. tuberculosis bacilli represented a productive infection with retention of bacterial viability and replicative potential, increasing 2- to 4-fold within 48 h. M. tuberculosis bacillus infection of neurons was confirmed in vivo in the brains of C57BL/6 mice after intracerebral challenge. This study, therefore, demonstrates neurons as potential new target cells for M. tuberculosis within the central nervous system.

  16. Porins Increase Copper Susceptibility of Mycobacterium tuberculosis

    PubMed Central

    Speer, Alexander; Rowland, Jennifer L.; Haeili, Mehri; Niederweis, Michael

    2013-01-01

    Copper resistance mechanisms are crucial for many pathogenic bacteria, including Mycobacterium tuberculosis, during infection because the innate immune system utilizes copper ions to kill bacterial intruders. Despite several studies detailing responses of mycobacteria to copper, the pathways by which copper ions cross the mycobacterial cell envelope are unknown. Deletion of porin genes in Mycobacterium smegmatis leads to a severe growth defect on trace copper medium but simultaneously increases tolerance for copper at elevated concentrations, indicating that porins mediate copper uptake across the outer membrane. Heterologous expression of the mycobacterial porin gene mspA reduced growth of M. tuberculosis in the presence of 2.5 μM copper by 40% and completely suppressed growth at 15 μM copper, while wild-type M. tuberculosis reached its normal cell density at that copper concentration. Moreover, the polyamine spermine, a known inhibitor of porin activity in Gram-negative bacteria, enhanced tolerance of M. tuberculosis for copper, suggesting that copper ions utilize endogenous outer membrane channel proteins of M. tuberculosis to gain access to interior cellular compartments. In summary, these findings highlight the outer membrane as the first barrier against copper ions and the role of porins in mediating copper uptake in M. smegmatis and M. tuberculosis. PMID:24013632

  17. Porins increase copper susceptibility of Mycobacterium tuberculosis.

    PubMed

    Speer, Alexander; Rowland, Jennifer L; Haeili, Mehri; Niederweis, Michael; Wolschendorf, Frank

    2013-11-01

    Copper resistance mechanisms are crucial for many pathogenic bacteria, including Mycobacterium tuberculosis, during infection because the innate immune system utilizes copper ions to kill bacterial intruders. Despite several studies detailing responses of mycobacteria to copper, the pathways by which copper ions cross the mycobacterial cell envelope are unknown. Deletion of porin genes in Mycobacterium smegmatis leads to a severe growth defect on trace copper medium but simultaneously increases tolerance for copper at elevated concentrations, indicating that porins mediate copper uptake across the outer membrane. Heterologous expression of the mycobacterial porin gene mspA reduced growth of M. tuberculosis in the presence of 2.5 μM copper by 40% and completely suppressed growth at 15 μM copper, while wild-type M. tuberculosis reached its normal cell density at that copper concentration. Moreover, the polyamine spermine, a known inhibitor of porin activity in Gram-negative bacteria, enhanced tolerance of M. tuberculosis for copper, suggesting that copper ions utilize endogenous outer membrane channel proteins of M. tuberculosis to gain access to interior cellular compartments. In summary, these findings highlight the outer membrane as the first barrier against copper ions and the role of porins in mediating copper uptake in M. smegmatis and M. tuberculosis.

  18. [Strategical use of genotyping of Mycobacterium tuberculosis in tuberculosis control].

    PubMed

    David, Susana

    2008-01-01

    The tuberculosis situation in Portugal justifies the use of a strategy for the genotyping of Mycobacterium tuberculosis, particularly as Portugal is part of the global backdrop of human mobility, something which has a knock-on effect on the pandemic. Several international studies have placed spoligotyping and MIRU- VNTR typing as first line techniques for the molecular epidemiology of Mycobacterium tuberculosis as these techniques rely on simple technologies (PCR) and produce patterns which are easily translated into a direct interpretation numerical code. Spoligotyping has been accordingly proposed for all the isolates, while MIRU-VNTR typing should be applied to isolates with a common spoliotype. Other techniques, including IS6110-RFLP, should be reserved for use ill accordance with selected criteria. Previous studies in Portugal using spoligotyping have underlined the advantages of a strategy based on sampling consecutive patient isolates with no prior selection criteria. This allows characterisation of the M. tuberculosis population structure through monitoring the distribution of the genotypes geographically over time and within the various risk groups. On the other hand, the association of spoligotyping, MIRU-VNTF (typing and, possibly, other techniques, needs evaluating as part of bigger pictures, including identifying recent transmission situations, distinguishing between reinfection and relapse episodes and mapping the size and dynamics of disease transmission. The solution to the tuberculosis problem in Portugal implies structuring genotyping's role in tuberculosis prevention and control and its evaluation through concrete examples and results.

  19. Virulence factors of the Mycobacterium tuberculosis complex

    PubMed Central

    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

  20. Molecular identification of Mycobacterium tuberculosis in cattle.

    PubMed

    Sweetline Anne, N; Ronald, B S M; Kumar, T M A Senthil; Kannan, P; Thangavelu, A

    2017-01-01

    Bovine tuberculosis continued to be a re-emerging problem in some countries especially in endemic areas due to the fact that human and animal health surveillance system is not adopted to diagnose the infection. This crisis can be attributed due to sharing of the same habitat especially in rural areas. In the present study, a total of 148 samples were collected from cattle for isolation over a period of 3 years from cattle with and without lesions, of which 67 isolates were obtained by culture. Fifty one isolates were identified as Mycobacterium tuberculosis complex (MTBC) by IS6110 PCR of which 43 (84.3%) were identified as M. tuberculosis and 08 (15.6%) were identified as M. bovis by using 12.7kb fragment multiplex PCR. Among this, 31 isolates which were positive for IS6110 PCR were subjected to spoligotyping and revealed 28 isolates belonging to MANU1 strain of M. tuberculosis. This study clearly indicates that high prevalence of M. tuberculosis than M. bovis in bovine was identified by means of culture and by molecular methods M. tuberculosis can affect cattle producing lesion in contradiction to the earlier thoughts. This study speculates that M. tuberculosis MANU1 strain infection in cattle could be due to spill over from human or other non specific hosts in tuberculosis endemic areas. Though bovine tuberculosis due to M. tuberculosis in cattle is not considered a serious threat worldwide, in countries where human TB is endemic, M. tuberculosis infection of cattle needs to be considered. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Mycobacterium tuberculosis effectors interfering host apoptosis signaling.

    PubMed

    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.

  2. Global Distribution of Mycobacterium tuberculosis Spoligotypes

    PubMed Central

    Filliol, Ingrid; Driscoll, Jeffrey R.; van Soolingen, Dick; Kreiswirth, Barry N.; Kremer, Kristin; Valétudie, Georges; Anh, Dang Duc; Barlow, Rachael; Banerjee, Dilip; Bifani, Pablo J.; Brudey, Karin; Cataldi, Angel; Cooksey, Robert C.; Cousins, Debby V.; Dale, Jeremy W.; Dellagostin, Odir A.; Drobniewski, Francis; Engelmann, Guido; Ferdinand, Séverine; Gascoyne-Binzi, Deborah; Gordon, Max; Gutierrez, M. Cristina; Haas, Walter H.; Heersma, Herre; Källenius, Gunilla; Kassa-Kelembho, Eric; Koivula, Tuija; Ly, Ho Minh; Makristathis, Athanasios; Mammina, Caterina; Martin, Gerald; Moström, Peter; Mokrousov, Igor; Narbonne, Valérie; Narvskaya, Olga; Nastasi, Antonino; Niobe-Eyangoh, Sara Ngo; Pape, Jean W; Rasolofo-Razanamparany, Voahangy; Ridell, Malin; Rossetti, M. Lucia; Stauffer, Fritz; Suffys, Philip N.; Takiff, Howard; Texier-Maugein, Jeanne; Vincent, Véronique; de Waard, Jacobus H.

    2002-01-01

    We present a short summary of recent observations on the global distribution of the major clades of the Mycobacterium tuberculosis complex, the causative agent of tuberculosis. This global distribution was defined by data-mining of an international spoligotyping database, SpolDB3. This database contains 11,708 patterns from as many clinical isolates originating from more than 90 countries. The 11,708 spoligotypes were clustered into 813 shared types. A total of 1,300 orphan patterns (clinical isolates showing a unique spoligotype) were also detected. PMID:12453368

  3. Triclosan Derivatives: Towards Potent Inhibitors of Drug-Sensitive and Drug-Resistant Mycobacterium tuberculosis

    SciTech Connect

    Freundlich, Joel S.; Wang, Feng; Vilchèze, Catherine; Gulten, Gulcin; Langley, Robert; Schiehser, Guy A.; Jacobus, David P.; Jacobs, Jr., William R.; Sacchettini, James C.

    2009-06-30

    Isoniazid (INH) is a frontline antitubercular drug that inhibits the enoyl acyl carrier protein reductase InhA. Novel inhibitors of InhA that are not cross-resistant to INH represent a significant goal in antitubercular chemotherapy. The design, synthesis, and biological activity of a series of triclosan-based inhibitors is reported, including their promising efficacy against INH-resistant strains of M. tuberculosis. Triclosan has been previously shown to inhibit InhA, an essential enoyl acyl carrier protein reductase involved in mycolic acid biosynthesis, the inhibition of which leads to the lysis of Mycobacterium tuberculosis. Using a structure-based drug design approach, a series of 5-substituted triclosan derivatives was developed. Two groups of derivatives with alkyl and aryl substituents, respectively, were identified with dramatically enhanced potency against purified InhA. The most efficacious inhibitor displayed an IC{sub 50} value of 21 nM, which was 50-fold more potent than triclosan. X-ray crystal structures of InhA in complex with four triclosan derivatives revealed the structural basis for the inhibitory activity. Six selected triclosan derivatives were tested against isoniazid-sensitive and resistant strains of M. tuberculosis. Among those, the best inhibitor had an MIC value of 4.7 {mu}g mL{sup -1} (13 {mu}M), which represents a tenfold improvement over the bacteriocidal activity of triclosan. A subset of these triclosan analogues was more potent than isoniazid against two isoniazid-resistant M. tuberculosis strains, demonstrating the significant potential for structure-based design in the development of next generation antitubercular drugs.

  4. Mycobacterium tuberculosis Serine/Threonine Protein Kinases

    PubMed Central

    PRISIC, SLADJANA; HUSSON, ROBERT N.

    2014-01-01

    The Mycobacterium tuberculosis genome encodes 11 serine/threonine protein kinases (STPKs). A similar number of two-component systems are also present, indicating that these two signal transduction mechanisms are both important in the adaptation of this bacterial pathogen to its environment. The M. tuberculosis phosphoproteome includes hundreds of Ser- and Thr-phosphorylated proteins that participate in all aspects of M. tuberculosis biology, supporting a critical role for the STPKs in regulating M. tuberculosis physiology. Nine of the STPKs are receptor type kinases, with an extracytoplasmic sensor domain and an intracellular kinase domain, indicating that these kinases transduce external signals. Two other STPKs are cytoplasmic and have regulatory domains that sense changes within the cell. Structural analysis of some of the STPKs has led to advances in our understanding of the mechanisms by which these STPKs are activated and regulated. Functional analysis has provided insights into the effects of phosphorylation on the activity of several proteins, but for most phosphoproteins the role of phosphorylation in regulating function is unknown. Major future challenges include characterizing the functional effects of phosphorylation for this large number of phosphoproteins, identifying the cognate STPKs for these phosphoproteins, and determining the signals that the STPKs sense. Ultimately, combining these STPK-regulated processes into larger, integrated regulatory networks will provide deeper insight into M. tuberculosis adaptive mechanisms that contribute to tuberculosis pathogenesis. Finally, the STPKs offer attractive targets for inhibitor development that may lead to new therapies for drug-susceptible and drug-resistant tuberculosis. PMID:25429354

  5. Genetic engineering of Mycobacterium tuberculosis: a review.

    PubMed

    Lamrabet, Otmane; Drancourt, Michel

    2012-09-01

    Genetic engineering has been used for decades to mutate and delete genes in the Mycobacterium tuberculosis genome with the translational goal of producing attenuated mutants with conserved susceptibility to antituberculous antibiotics. The development of plasmids and mycobacteriophages that can transfer DNA into the M. tuberculosis chromosome has effectively overcome M. tuberculosis slow growth rate and the capsule and mycolic acid wall, which limit DNA uptake. The use of genetic engineering techniques has shed light on many aspects of pathogenesis mechanisms, including cellular growth, mycolic acid biosynthesis, metabolism, drug resistance and virulence. Moreover, such research gave clues to the development of new vaccines or new drugs for routine clinical practice. The use of genetic engineering tools is mainly based on the underlying concept that altering or reducing the M. tuberculosis genome could decrease its virulence. A contrario, recent post-genomic analyses indicated that reduced bacterial genomes are often associated with increased bacterial virulence and that M. tuberculosis acquired genes by lateral genetic exchange during its evolution. Therefore, ancestors utilizing genetic engineering to add genes to the M. tuberculosis genome may lead to new vaccines and the availability of M. tuberculosis isolates with increased susceptibility to antituberculous antibiotics.

  6. Myeloperoxidase Exerts Microbicidal Activity against Mycobacterium tuberculosis

    PubMed Central

    Borelli, Violetta; Banfi, Elena; Perrotta, Maria Giovanna; Zabucchi, Giuliano

    1999-01-01

    We investigated the antimycobacterial role of myeloperoxidase (MPO), one of the most abundant granule proteins in human neutrophils. Our data indicate that purified MPO, in the presence of hydrogen peroxide, exerts a consistent killing activity against Mycobacterium tuberculosis H37Rv and against a clinical isolate. The activity is time and dose dependent and requires the presence of chloride ions in the assay medium. PMID:10417186

  7. Mycobacterium tuberculosis: Manipulator of Protective Immunity

    PubMed Central

    Korb, Vanessa C.; Chuturgoon, Anil A.; Moodley, Devapregasan

    2016-01-01

    Mycobacterium tuberculosis (MTB) is one of the most successful pathogens in human history and remains a global health challenge. MTB has evolved a plethora of strategies to evade the immune response sufficiently to survive within the macrophage in a bacterial-immunological equilibrium, yet causes sufficient immunopathology to facilitate its transmission. This review highlights MTB as the driver of disease pathogenesis and presents evidence of the mechanisms by which MTB manipulates the protective immune response into a pathological productive infection. PMID:26927066

  8. Comparative Mycobacterium tuberculosis spoligotype distribution in Mexico.

    PubMed

    Vera-Cabrera, Lucio; Ramos-Alvarez, Jessica; Molina-Torres, Carmen A; Rivera-Morales, Lydia Guadalupe; Rendón, Adrian; Quiñones-Falconi, Francisco; Ocampo-Candiani, Jorge

    2014-08-01

    In the present work, we studied the genetic diversity of Mycobacterium tuberculosis clinical isolates from patients according to their gender, age, and geographic location in Mexico. We did not observe any statistically significant differences in regard to age or gender. We found that spoligo international type 53 (SIT53) is more frequent in the northern states and that SIT119 predominates in central Mexico. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  9. Comparative Mycobacterium tuberculosis Spoligotype Distribution in Mexico

    PubMed Central

    Ramos-Alvarez, Jessica; Molina-Torres, Carmen A.; Rivera-Morales, Lydia Guadalupe; Rendón, Adrian; Quiñones-Falconi, Francisco; Ocampo-Candiani, Jorge

    2014-01-01

    In the present work, we studied the genetic diversity of Mycobacterium tuberculosis clinical isolates from patients according to their gender, age, and geographic location in Mexico. We did not observe any statistically significant differences in regard to age or gender. We found that spoligo international type 53 (SIT53) is more frequent in the northern states and that SIT119 predominates in central Mexico. PMID:24850349

  10. High Persister Mutants in Mycobacterium tuberculosis

    PubMed Central

    Torrey, Heather L.; Keren, Iris; Via, Laura E.; Lee, Jong Seok; Lewis, Kim

    2016-01-01

    Mycobacterium tuberculosis forms drug-tolerant persister cells that are the probable cause of its recalcitrance to antibiotic therapy. While genetically identical to the rest of the population, persisters are dormant, which protects them from killing by bactericidal antibiotics. The mechanism of persister formation in M. tuberculosis is not well understood. In this study, we selected for high persister (hip) mutants and characterized them by whole genome sequencing and transcriptome analysis. In parallel, we identified and characterized clinical isolates that naturally produce high levels of persisters. We compared the hip mutants obtained in vitro with clinical isolates to identify candidate persister genes. Genes involved in lipid biosynthesis, carbon metabolism, toxin-antitoxin systems, and transcriptional regulators were among those identified. We also found that clinical hip isolates exhibited greater ex vivo survival than the low persister isolates. Our data suggest that M. tuberculosis persister formation involves multiple pathways, and hip mutants may contribute to the recalcitrance of the infection. PMID:27176494

  11. A replication clock for Mycobacterium tuberculosis

    PubMed Central

    Gill, Wendy P; Harik, Nada S; Whiddon, Molly R; Liao, Reiling P; Mittler, John E; Sherman, David R

    2009-01-01

    Few tools exist to assess replication of chronic pathogens during infection. This has been a considerable barrier to understanding latent tuberculosis, and efforts to develop new therapies generally assume that the bacteria are very slowly replicating or nonreplicating during latency1–3. To monitor Mycobacterium tuberculosis replication within hosts, we exploit an unstable plasmid that is lost at a steady, quantifiable rate from dividing cells in the absence of antibiotic selection. By applying a mathematical model, we calculate bacterial growth and death rates during infection of mice. We show that during chronic infection the cumulative bacterial burden—enumerating total live, dead and removed organisms encountered by the mouse lung—is substantially higher than estimates from colony forming units. Our data show that M. tuberculosis replicates throughout the course of chronic infection of mice and is restrained by the host immune system. This approach may also shed light on the replication dynamics of other chronic pathogens. PMID:19182798

  12. Peptide mimotopes of Mycobacterium tuberculosis carbohydrate immunodeterminants

    PubMed Central

    2004-01-01

    Cell-surface saccharides of Mycobacterium tuberculosis appear to be crucial factors in tuberculosis pathogenicity and could be useful antigens in tuberculosis immunodiagnosis. In the present study, we report the successful antigenic and immunogenic mimicry of mannose-containing cell-wall compounds of M. tuberculosis by dodecamer peptides identified by phage-display technology. Using a rabbit antiserum raised against M. tuberculosis cell-surface saccharides as a target for biopanning, peptides with three different consensus sequences were identified. Phage-displayed and chemically synthesized peptides bound to the anticarbohydrate antiserum. Rabbit antibodies elicited against the peptide QEPLMGTVPIRAGGGS recognize the mannosylated M. tuberculosis cell-wall antigens arabinomannan and lipoarabinomannan, and the glycosylated recombinant protein alanine/proline-rich antigen. Furthermore, antibodies were also able to react with mannan from Saccharomyces cerevisiae, but not with phosphatidylinositol dimannosides or arabinogalactan from mycobacteria. These results suggest that the immunogenic peptide mimics oligomannosidic epitopes. Interestingly, this report provides evidence that, in contrast with previously known carbohydrate mimotopes, no aromatic residues are necessary in a peptide sequence for mimicking unusual glycoconjugates synthesized by mycobacteria. The possible usefulness of the identified peptide mimotopes as surrogate reagents for immunodiagnosis and for the study of functional roles of the native non-peptide epitopes is discussed. PMID:15560754

  13. 59 FR- Method Development for Airborne Mycobacterium Tuberculosis; Meeting

    Federal Register 2010, 2011, 2012, 2013, 2014

    1994-03-07

    ... Tuberculosis; Meeting The National Institute for Occupational Safety and Health (NIOSH) of the Centers for... Airborne Mycobacterium Tuberculosis. Time and Date: 1 p.m.-5 p.m., March 29, 1994. Place: Alice Hamilton... peer review of a NIOSH project entitled ``Method Development For Airborne Mycobacterium...

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

    PubMed

    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.

  15. Multispacer Sequence Typing for Mycobacterium tuberculosis Genotyping

    PubMed Central

    Djelouadji, Zoheira; Arnold, Catherine; Gharbia, Saheer; Raoult, Didier; Drancourt, Michel

    2008-01-01

    Background Genotyping methods developed to survey the transmission dynamics of Mycobacterium tuberculosis currently rely on the interpretation of restriction and amplification profiles. Multispacer sequence typing (MST) genotyping is based on the sequencing of several intergenic regions selected after complete genome sequence analysis. It has been applied to various pathogens, but not to M. tuberculosis. Methods and Findings In M. tuberculosis, the MST approach yielded eight variable intergenic spacers which included four previously described variable number tandem repeat loci, one single nucleotide polymorphism locus and three newly evaluated spacers. Spacer sequence stability was evaluated by serial subculture. The eight spacers were sequenced in a collection of 101 M. tuberculosis strains from five phylogeographical lineages, and yielded 29 genetic events including 13 tandem repeat number variations (44.82%), 11 single nucleotide mutations (37.93%) and 5 deletions (17.24%). These 29 genetic events yielded 32 spacer alleles or spacer-types (ST) with an index of discrimination of 0.95. The distribution of M. tuberculosis isolates into ST profiles correlated with their assignment into phylogeographical lineages. Blind comparison of a further 93 M. tuberculosis strains by MST and restriction fragment length polymorphism-IS6110 fingerprinting and mycobacterial interspersed repetitive units typing, yielded an index of discrimination of 0.961 and 0.992, respectively. MST yielded 41 different profiles delineating 16 related groups and proved to be more discriminatory than IS6110-based typing for isolates containing <8 IS6110 copies (P<0.0003). MST was successfully applied to 7/10 clinical specimens exhibiting a Cts ≤ 42 cycles in internal transcribed spacer-real time PCR. Conclusions These results support MST as an alternative, sequencing-based method for genotyping low IS6110 copy-number M. tuberculosis strains. The M. tuberculosis MST database is freely available

  16. Disseminated Mycobacterium tuberculosis Infection in a Dog

    PubMed Central

    Martinho, Anna Paula Vitirito; Franco, Marília Masello Junqueira; Ribeiro, Márcio Garcia; Perrotti, Isabella Belletti Mutt; Mangia, Simone Henriques; Megid, Jane; Vulcano, Luiz Carlos; Lara, Gustavo Henrique Batista; Santos, Adolfo Carlos Barreto; Leite, Clarice Queico Fujimura; de Carvalho Sanches, Osimar; Paes, Antonio Carlos

    2013-01-01

    An uncommon disseminated Mycobacterium tuberculosis infection is described in a 12-year-old female dog presenting with fever, dyspnea, cough, weight loss, lymphadenopathy, melena, epistaxis, and emesis. The dog had a history of close contact with its owner, who died of pulmonary tuberculosis. Radiographic examination revealed diffuse radio-opaque images in both lung lobes, diffuse visible masses in abdominal organs, and hilar and mesenteric lymphadenopathy. Bronchial washing samples and feces were negative for acid-fast organisms. Polymerase chain reaction (PCR)-based species identification of bronchial washing samples, feces, and urine revealed M. tuberculosis using PCR-restriction enzyme pattern analysis-PRA. Because of public health concerns, which were worsened by the physical condition of the dog, euthanasia of the animal was recommended. Rough and tough colonies suggestive of M. tuberculosis were observed after microbiological culture of lung, liver, spleen, heart, and lymph node fragments in Löwenstein-Jensen and Stonebrink media. The PRA analysis enabled diagnosis of M. tuberculosis strains isolated from organs. PMID:23339199

  17. Mycobacterium tuberculosis Infection among Asian Elephants in Captivity

    PubMed Central

    Simpson, Gary; Zimmerman, Ralph; Shashkina, Elena; Chen, Liang; Richard, Michael; Bradford, Carol M.; Dragoo, Gwen A.; Saiers, Rhonda L.; Peloquin, Charles A.; Daley, Charles L.; Planet, Paul; Narachenia, Apurva; Mathema, Barun

    2017-01-01

    Although awareness of tuberculosis among captive elephants is increasing, antituberculosis therapy for these animals is not standardized. We describe Mycobacterium tuberculosis transmission between captive elephants based on whole genome analysis and report a successful combination treatment. Infection control protocols and careful monitoring of treatment of captive elephants with tuberculosis are warranted. PMID:28221115

  18. New Mycobacterium tuberculosis Complex Sublineage, Brazzaville, Congo

    PubMed Central

    Malm, Sven; Linguissi, Laure S. Ghoma; Tekwu, Emmanuel M.; Vouvoungui, Jeannhey C.; Kohl, Thomas A.; Beckert, Patrick; Sidibe, Anissa; Rüsch-Gerdes, Sabine; Madzou-Laboum, Igor K.; Kwedi, Sylvie; Penlap Beng, Véronique; Frank, Matthias; Ntoumi, Francine

    2017-01-01

    Tuberculosis is a leading cause of illness and death in Congo. No data are available about the population structure and transmission dynamics of the Mycobacterium tuberculosis complex strains prevalent in this central Africa country. On the basis of single-nucleotide polymorphisms detected by whole-genome sequencing, we phylogenetically characterized 74 MTBC isolates from Brazzaville, the capital of Congo. The diversity of the study population was high; most strains belonged to the Euro-American lineage, which split into Latin American Mediterranean, Uganda I, Uganda II, Haarlem, X type, and a new dominant sublineage named Congo type (n = 26). Thirty strains were grouped in 5 clusters (each within 12 single-nucleotide polymorphisms), from which 23 belonged to the Congo type. High cluster rates and low genomic diversity indicate recent emergence and transmission of the Congo type, a new Euro-American sublineage of MTBC. PMID:28221129

  19. Diversity and disease pathogenesis in Mycobacterium tuberculosis.

    PubMed

    Warner, Digby F; Koch, Anastasia; Mizrahi, Valerie

    2015-01-01

    The increasing availability of whole-genome sequence (WGS) data for Mycobacterium tuberculosis, the bacterium that causes tuberculosis (TB), suggests that circulating genotypes have been molded by three dominant evolutionary forces: long-term persistence within the human population, which requires a core programme of infection, disease, and transmission; selective pressure on specific genomic loci, which provides evidence of lineage-specific adaptation to host populations; and drug exposure, which has driven the rapid emergence of resistant isolates following the global implementation of anti-TB chemotherapy. Here, we provide an overview of these factors in considering the implications of genotypic diversity for disease pathogenesis, vaccine efficacy, and drug treatment. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Pre-multidrug-resistant Mycobacterium tuberculosis Beijing strain associated with disseminated tuberculosis in a pet dog.

    PubMed

    Botelho, Ana; Perdigão, João; Canto, Ana; Albuquerque, Teresa; Leal, Nuno; Macedo, Rita; Portugal, Isabel; Cunha, Mónica V

    2014-01-01

    Resistance to isoniazid, ethambutol, and streptomycin was detected in a Mycobacterium tuberculosis strain, belonging to the Beijing family lineage, isolated from two nodule exudates of a Yorkshire terrier with generalized tuberculosis. This report alerts medical practitioners to the risk of dissemination of pre-multidrug-resistant tuberculosis (preMDR-TB) through exposure to M. tuberculosis-shedding pets.

  1. Pre-Multidrug-Resistant Mycobacterium tuberculosis Beijing Strain Associated with Disseminated Tuberculosis in a Pet Dog

    PubMed Central

    Perdigão, João; Canto, Ana; Albuquerque, Teresa; Leal, Nuno; Macedo, Rita; Portugal, Isabel; Cunha, Mónica V.

    2014-01-01

    Resistance to isoniazid, ethambutol, and streptomycin was detected in a Mycobacterium tuberculosis strain, belonging to the Beijing family lineage, isolated from two nodule exudates of a Yorkshire terrier with generalized tuberculosis. This report alerts medical practitioners to the risk of dissemination of pre-multidrug-resistant tuberculosis (preMDR-TB) through exposure to M. tuberculosis-shedding pets. PMID:24153119

  2. Triclosan derivatives: Towards potent inhibitors of drug-sensitive and drug-resistant Mycobacterium tuberculosis

    PubMed Central

    Freundlich, Joel S.; Wang, Feng; Vilchèze, Catherine; Gulten, Gulcin; Langley, Robert; Schiehser, Guy A.; Jacobus, David P.; Jacobs, William R.; Sacchettini, James C.

    2012-01-01

    Triclosan has been previously shown to inhibit InhA, an essential enoyl acyl carrier protein reductase of mycolic acid biosynthesis, whose inhibition leads to the lysis of Mycobacterium tuberculosis. Using a structure-based drug design approach, a series of 5-substituted derivatives of triclosan was developed. Two groups of triclosan derivatives with alkyl and aryl substituents, respectively, were identified with dramatically enhanced potency against purified InhA. The most efficacious inhibitor displayed an IC50 value of 21 nM, which was 50-fold more potent than triclosan. X-ray crystal structures of InhA in complex with four triclosan derivatives revealed the structural basis for the inhibitory activity. Six selected triclosan derivatives were tested against isoniazid-sensitive and resistant strains of M. tuberculosis. Among those, the best inhibitor had an MIC value of 4.7 µg/mL (13 µM), which represents a tenfold improvement over the bacteriocidal activity of triclosan. A subset of these triclosan analogs was more potent than isoniazid against two isoniazid-resistant M. tuberculosis strains, demonstrating the significant potential for structure-based design in the development of next generation antitubercular drugs. PMID:19130456

  3. Signature Gene Expression Profiles Discriminate between Isoniazid-, Thiolactomycin-, and Triclosan-Treated Mycobacterium tuberculosis

    PubMed Central

    Betts, Joanna C.; McLaren, Alistair; Lennon, Mark G.; Kelly, Fiona M.; Lukey, Pauline T.; Blakemore, Steve J.; Duncan, Ken

    2003-01-01

    Genomic technologies have the potential to greatly increase the efficiency of the drug development process. As part of our tuberculosis drug discovery program, we used DNA microarray technology to profile drug-induced effects in Mycobacterium tuberculosis. Expression profiles of M. tuberculosis treated with compounds that inhibit key metabolic pathways are required as references for the assessment of novel antimycobacterial agents. We have studied the response of M. tuberculosis to treatment with the mycolic acid biosynthesis inhibitors isoniazid, thiolactomycin, and triclosan. Thiolactomycin targets the β-ketoacyl-acyl carrier protein (ACP) synthases KasA and KasB, while triclosan inhibits the enoyl-ACP reductase InhA. However, controversy surrounds the precise mode of action of isoniazid, with both InhA and KasA having been proposed as the primary target. We have shown that although the global response profiles of isoniazid and thiolactomycin are more closely related to each other than to that of triclosan, there are differences that distinguish the mode of action of these two drugs. In addition, we have identified two groups of genes, possibly forming efflux and detoxification systems, through which M. tuberculosis may limit the effects of triclosan. We have developed a mathematical model, based on the expression of 21 genes, which is able to perfectly discriminate between isoniazid-, thiolactomycin-, or triclosan-treated M. tuberculosis. This model is likely to prove invaluable as a tool to improve the efficiency of our drug development programs by providing a means to rapidly confirm the mode of action of thiolactomycin analogues or novel InhA inhibitors as well as helping to translate enzyme activity into whole-cell activity. PMID:12936993

  4. Disseminated Mycobacterium tuberculosis following renal transplant with alemtuzumab induction.

    PubMed

    Baghban, Adam; Azar, Marwan Mikheal; Bernardo, Raffaele Mario; Malinis, Maricar

    2016-11-16

    Mycobacterium tuberculosis presents unique challenges in the peritransplant period. Here, we describe a case of disseminated tuberculosis following renal transplantation with alemtuzumab induction immunosuppression in a patient with remotely treated pulmonary tuberculosis and ongoing risk factors for re-infection. We also review the available literature regarding the prevalence of tuberculosis infection following solid organ transplant and management of high-risk patients, including the role for isoniazid preventative therapy. 2016 BMJ Publishing Group Ltd.

  5. igr Genes and Mycobacterium tuberculosis cholesterol metabolism.

    PubMed

    Chang, Jennifer C; Miner, Maurine D; Pandey, Amit K; Gill, Wendy P; Harik, Nada S; Sassetti, Christopher M; Sherman, David R

    2009-08-01

    Recently, cholesterol was identified as a physiologically important nutrient for Mycobacterium tuberculosis survival in chronically infected mice. However, it remained unclear precisely when cholesterol is available to the bacterium and what additional bacterial functions are required for its metabolism. Here, we show that the igr locus, which we previously found to be essential for intracellular growth and virulence of M. tuberculosis, is required for cholesterol metabolism. While igr-deficient strains grow identically to the wild type in the presence of short- and long-chain fatty acids, the growth of these bacteria is completely inhibited in the presence of cholesterol. Interestingly, this mutant is still able to respire under cholesterol-dependent growth inhibition, suggesting that the bacteria can metabolize other carbon sources during cholesterol toxicity. Consistent with this hypothesis, we found that the growth-inhibitory effect of cholesterol in vitro depends on cholesterol import, as mutation of the mce4 sterol uptake system partially suppresses this effect. In addition, the Delta igr mutant growth defect during the early phase of disease is completely suppressed by mutating mce4, implicating cholesterol intoxication as the primary mechanism of attenuation. We conclude that M. tuberculosis metabolizes cholesterol throughout infection.

  6. Mycobacterium tuberculosis replicates within necrotic human macrophages

    PubMed Central

    Lerner, Thomas R.; Repnik, Urska; Herbst, Susanne; Collinson, Lucy M.; Griffiths, Gareth

    2017-01-01

    Mycobacterium tuberculosis modulation of macrophage cell death is a well-documented phenomenon, but its role during bacterial replication is less characterized. In this study, we investigate the impact of plasma membrane (PM) integrity on bacterial replication in different functional populations of human primary macrophages. We discovered that IFN-γ enhanced bacterial replication in macrophage colony-stimulating factor–differentiated macrophages more than in granulocyte–macrophage colony-stimulating factor–differentiated macrophages. We show that permissiveness in the different populations of macrophages to bacterial growth is the result of a differential ability to preserve PM integrity. By combining live-cell imaging, correlative light electron microscopy, and single-cell analysis, we found that after infection, a population of macrophages became necrotic, providing a niche for M. tuberculosis replication before escaping into the extracellular milieu. Thus, in addition to bacterial dissemination, necrotic cells provide first a niche for bacterial replication. Our results are relevant to understanding the environment of M. tuberculosis replication in the host. PMID:28242744

  7. Complex multifractal nature in Mycobacterium tuberculosis genome

    NASA Astrophysics Data System (ADS)

    Mandal, Saurav; Roychowdhury, Tanmoy; Chirom, Keilash; Bhattacharya, Alok; Brojen Singh, R. K.

    2017-04-01

    The mutifractal and long range correlation (C(r)) properties of strings, such as nucleotide sequence can be a useful parameter for identification of underlying patterns and variations. In this study C(r) and multifractal singularity function f(α) have been used to study variations in the genomes of a pathogenic bacteria Mycobacterium tuberculosis. Genomic sequences of M. tuberculosis isolates displayed significant variations in C(r) and f(α) reflecting inherent differences in sequences among isolates. M. tuberculosis isolates can be categorised into different subgroups based on sensitivity to drugs, these are DS (drug sensitive isolates), MDR (multi-drug resistant isolates) and XDR (extremely drug resistant isolates). C(r) follows significantly different scaling rules in different subgroups of isolates, but all the isolates follow one parameter scaling law. The richness in complexity of each subgroup can be quantified by the measures of multifractal parameters displaying a pattern in which XDR isolates have highest value and lowest for drug sensitive isolates. Therefore C(r) and multifractal functions can be useful parameters for analysis of genomic sequences.

  8. Complex multifractal nature in Mycobacterium tuberculosis genome

    PubMed Central

    Mandal, Saurav; Roychowdhury, Tanmoy; Chirom, Keilash; Bhattacharya, Alok; Brojen Singh, R. K.

    2017-01-01

    The mutifractal and long range correlation (C(r)) properties of strings, such as nucleotide sequence can be a useful parameter for identification of underlying patterns and variations. In this study C(r) and multifractal singularity function f(α) have been used to study variations in the genomes of a pathogenic bacteria Mycobacterium tuberculosis. Genomic sequences of M. tuberculosis isolates displayed significant variations in C(r) and f(α) reflecting inherent differences in sequences among isolates. M. tuberculosis isolates can be categorised into different subgroups based on sensitivity to drugs, these are DS (drug sensitive isolates), MDR (multi-drug resistant isolates) and XDR (extremely drug resistant isolates). C(r) follows significantly different scaling rules in different subgroups of isolates, but all the isolates follow one parameter scaling law. The richness in complexity of each subgroup can be quantified by the measures of multifractal parameters displaying a pattern in which XDR isolates have highest value and lowest for drug sensitive isolates. Therefore C(r) and multifractal functions can be useful parameters for analysis of genomic sequences. PMID:28440326

  9. Epidemiologic Consequences of Microvariation in Mycobacterium tuberculosis

    PubMed Central

    Mathema, Barun; Kurepina, Natalia; Yang, Guibin; Shashkina, Elena; Manca, Claudia; Mehaffy, Carolina; Bielefeldt-Ohmann, Helle; Ahuja, Shama; Fallows, Dorothy A.; Izzo, Angelo; Bifani, Pablo; Dobos, Karen; Kaplan, Gilla

    2012-01-01

    Background. Evidence from genotype-phenotype studies suggests that genetic diversity in pathogens have clinically relevant manifestations that can impact outcome of infection and epidemiologic success. We studied 5 closely related Mycobacterium tuberculosis strains that collectively caused extensive disease (n = 862), particularly among US-born tuberculosis patients. Methods. Representative isolates were selected using population-based genotyping data from New York City and New Jersey. Growth and cytokine/chemokine response were measured in infected human monocytes. Survival was determined in aerosol-infected guinea pigs. Results. Multiple genotyping methods and phylogenetically informative synonymous single nucleotide polymorphisms showed that all strains were related by descent. In axenic culture, all strains grew similarly. However, infection of monocytes revealed 2 growth phenotypes, slower (doubling ∼55 hours) and faster (∼25 hours). The faster growing strains elicited more tumor necrosis factor α and interleukin 1β than the slower growing strains, even after heat killing, and caused accelerated death of infected guinea pigs (∼9 weeks vs 24 weeks) associated with increased lung inflammation/pathology. Epidemiologically, the faster growing strains were associated with human immunodeficiency virus and more limited in spread, possibly related to their inherent ability to induce a strong protective innate immune response in immune competent hosts. Conclusions. Natural variation, with detectable phenotypic changes, among closely related clinical isolates of M. tuberculosis may alter epidemiologic patterns in human populations. PMID:22315279

  10. Haemophagocytic lymphohistiocytosis associated with Mycobacterium tuberculosis infection

    PubMed Central

    Hui, Yee Man Tracy; Pillinger, Toby; Luqmani, Asad; Cooper, Nichola

    2015-01-01

    Haemophagocytic lymphohistiocytosis (HLH) is a rare, potentially fatal condition that can be primary or secondary. Secondary HLH can occur in association with infections, most commonly viral infections, but has also been reported in association with Mycobacterium tuberculosis (TB). Prompt identification of the underlying cause of HLH is important as it guides treatment decisions. Early initiation of appropriate treatment (eg, anti-TB treatment) reduces morbidity and mortality. We present a case of HLH associated with TB infection. Initial TB investigations were negative and standard combination chemoimmunotherapy for HLH resulted in a limited clinical response. On apparent relapse of HLH, further investigation revealed TB with changes on CT chest, granuloma on bone marrow and eventual positive TB culture on bronchoalveolar lavage. Subsequent treatment with quadruple anti-TB treatment resulted in rapid clinical response and disease remission. We advocate continued monitoring for TB infection in patients with HLH, and prophylaxis or full treatment for those at high risk. PMID:25870214

  11. Rapid radiometric susceptibility testing of Mycobacterium tuberculosis.

    PubMed

    Kertcher, J A; Chen, M F; Charache, P; Hwangbo, C C; Camargo, E E; McIntyre, P A; Wagner, H N

    1978-04-01

    A 48-hour radiometric test for determining the drug susceptibility of Mycobacterium tuberculosis has been developed. The test is based on the measurement of 14CO2 produced by the oxidation of formate labeled with carbon-14. The test system uses 5 X 10(7) organisms in 1 ml of Middlebrook 7H9 medium plus albumin-dextrose-catalase enrichment and 1 muCi of [14C]formate. The 14CO2 produced is measured in an ionization chamber at 24-, 48-, and 72-hour intervals, with and without the addition of antituberculous drugs. Isoniazid, streptomycin, rifampin, and ethambutol were each tested at 3 concentrations by the radiometric method and the reference (agar dilution) method. Six standard strains and 21 patient isolates were compared by both methods. Production of 14CO2 was quantitatively decreased in the presence of drugs that inhibit the organism. The radiometric method requires 2 days; the agar dilution, 14 to 21 days.

  12. A new evolutionary scenario for the Mycobacterium tuberculosis complex

    PubMed Central

    Brosch, R.; Gordon, S. V.; Marmiesse, M.; Brodin, P.; Buchrieser, C.; Eiglmeier, K.; Garnier, T.; Gutierrez, C.; Hewinson, G.; Kremer, K.; Parsons, L. M.; Pym, A. S.; Samper, S.; van Soolingen, D.; Cole, S. T.

    2002-01-01

    The distribution of 20 variable regions resulting from insertion-deletion events in the genomes of the tubercle bacilli has been evaluated in a total of 100 strains of Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium canettii, Mycobacterium microti, and Mycobacterium bovis. This approach showed that the majority of these polymorphisms did not occur independently in the different strains of the M. tuberculosis complex but, rather, resulted from ancient, irreversible genetic events in common progenitor strains. Based on the presence or absence of an M. tuberculosis specific deletion (TbD1), M. tuberculosis strains can be divided into ancestral and “modern” strains, the latter comprising representatives of major epidemics like the Beijing, Haarlem, and African M. tuberculosis clusters. Furthermore, successive loss of DNA, reflected by region of difference 9 and other subsequent deletions, was identified for an evolutionary lineage represented by M. africanum, M. microti, and M. bovis that diverged from the progenitor of the present M. tuberculosis strains before TbD1 occurred. These findings contradict the often-presented hypothesis that M. tuberculosis, the etiological agent of human tuberculosis evolved from M. bovis, the agent of bovine disease. M. canettii and ancestral M. tuberculosis strains lack none of these deleted regions, and, therefore, seem to be direct descendants of tubercle bacilli that existed before the M. africanum→M. bovis lineage separated from the M. tuberculosis lineage. This observation suggests that the common ancestor of the tubercle bacilli resembled M. tuberculosis or M. canettii and could well have been a human pathogen already. PMID:11891304

  13. Mycobacterium tuberculosis Infection in Free-Roaming Wild Asian Elephant

    PubMed Central

    Shivashankar, Beechagondahalli Papanna; Umashankar, Kunigal Srinivasa; Nandini, Poojappa; Giridhar, Papanna; Byregowda, Somenahalli Munivenkatappa; Shrinivasa, Basavegowdanadoddi Marinaik

    2017-01-01

    Postmortem examination of a wild Asian elephant at Rajiv Gandhi National Park, India, revealed nodular lesions, granulomas with central caseation, and acid-fast bacilli in the lungs. PCR and nucleotide sequencing confirmed the presence of Mycobacterium tuberculosis. This study indicates that wild elephants can harbor M. tuberculosis that can become fatal. PMID:28221114

  14. Palaeogenomics of Mycobacterium tuberculosis: epidemic bursts with a degrading genome.

    PubMed

    Djelouadji, Zoheira; Raoult, Didier; Drancourt, Michel

    2011-08-01

    Genome-scale analysis suggests that the last common ancestor of the Mycobacterium tuberculosis complex and Mycobacterium leprae diverged 36 million years ago, and members of the Mycobacterium tuberculosis complex differentiated 40,000 years ago. Analysis of palaeomicrobiological data from a 17,000-year-old sample from a bison and a 9000-year-old sample from a human being suggested that M tuberculosis preceded Mycobacterium bovis and related species. Whole-genome comparisons show that members of the M tuberculosis complex form a unique bacterial species with distinct ecotypes that are transmissible from any infected mammalian species to several others. Genomic deletions identified several M tuberculosis lineages that could be placed on a phylogeographical map, suggesting adaptation to local host populations. The degrees of transmissibility and virulence vary between M tuberculosis clones, with increased virulence mainly linked to gene loss in regulatory pathways. Such data suggest that most M tuberculosis clones have a restricted spreading capacity between the host population, allowing unpredictable bursts of highly transmissible, virulent, and successful clones, such as the east Asian (Beijing) clone. Advances in genomics have helped the development of molecular techniques for accurate identification of species and clones in the M tuberculosis complex, which is essential for tracing the source of infections.

  15. Mycobacterium tuberculosis in Wild Asian Elephants, Southern India

    PubMed Central

    Zachariah, Arun; Pandiyan, Jeganathan; Madhavilatha, G.K.; Mundayoor, Sathish; Chandramohan, Bathrachalam; Sajesh, P.K.; Santhosh, Sam

    2017-01-01

    We tested 3 ild Asian elephants (Elephas maximus) in southern India and confirmed infection in 3 animals with Mycobacterium tuberculosis, an obligate human pathogen, by PCR and genetic sequencing. Our results indicate that tuberculosis may be spilling over from humans (reverse zoonosis) and emerging in wild elephants. PMID:28221104

  16. Mycobacterium tuberculosis in Wild Asian Elephants, Southern India.

    PubMed

    Zachariah, Arun; Pandiyan, Jeganathan; Madhavilatha, G K; Mundayoor, Sathish; Chandramohan, Bathrachalam; Sajesh, P K; Santhosh, Sam; Mikota, Susan K

    2017-03-01

    We tested 3 ild Asian elephants (Elephas maximus) in southern India and confirmed infection in 3 animals with Mycobacterium tuberculosis, an obligate human pathogen, by PCR and genetic sequencing. Our results indicate that tuberculosis may be spilling over from humans (reverse zoonosis) and emerging in wild elephants.

  17. A user-friendly web portal for analyzing conformational changes in structures of Mycobacterium tuberculosis.

    PubMed

    Hassan, Sameer; Thangam, Manonanthini; Vasudevan, Praveen; Kumar, G Ramesh; Unni, Rahul; Devi, P K Gayathri; Hanna, Luke Elizabeth

    2015-10-01

    Initiation of the Tuberculosis Structural Consortium has resulted in the expansion of the Mycobacterium tuberculosis (MTB) protein structural database. Currently, 969 experimentally solved structures are available for 354 MTB proteins. This includes multiple crystal structures for a given protein under different functional conditions, such as the presence of different ligands or mutations. In depth analysis of the multiple structures reveal that subtle differences exist in conformations of a given protein under varied conditions. Therefore, it is immensely important to understand the conformational differences between the multiple structures of a given protein in order to select the most suitable structure for molecular docking and structure-based drug designing. Here, we introduce a web portal ( http://bmi.icmr.org.in/mtbsd/torsion.php ) that we developed to provide comparative data on the ensemble of available structures of MTB proteins, such as Cα root means square deviation (RMSD), sequence identity, presence of mutations and torsion angles. Additionally, torsion angles were used to perform principal component analysis (PCA) to identify the conformational differences between the structures. Additionally, we present a few case studies to demonstrate this database. Graphical Abstract Conformational changes seen in the structures of the enoyl-ACP reductase protein encoded by the Mycobacterial gene inhA.

  18. High Affinity Inha Inhibitors with Activity Against Drug-Resistant Strains of Mycobacterium Tuberculosis

    SciTech Connect

    Sullivan,T.; Truglio, J.; Boyne, M.; Novichenok, P.; Zhang, X.; Stratton, C.; Li, H.; Kaur, T.; Amin, A.; et al.

    2006-01-01

    Novel chemotherapeutics for treating multidrug-resistant (MDR) strains of Mycobacterium tuberculosis (MTB) are required to combat the spread of tuberculosis, a disease that kills more than 2 million people annually. Using structure-based drug design, we have developed a series of alkyl diphenyl ethers that are uncompetitive inhibitors of InhA, the enoyl reductase enzyme in the MTB fatty acid biosynthesis pathway. The most potent compound has a Ki{prime} value of 1 nM for InhA and MIC{sub 99} values of 2-3 {micro}g mL{sup -1} (6-10 {micro}M) for both drug-sensitive and drug-resistant strains of MTB. Overexpression of InhA in MTB results in a 9-12-fold increase in MIC{sub 99}, consistent with the belief that these compounds target InhA within the cell. In addition, transcriptional response studies reveal that the alkyl diphenyl ethers fail to upregulate a putative efflux pump and aromatic dioxygenase, detoxification mechanisms that are triggered by the lead compound triclosan. These diphenyl ether-based InhA inhibitors do not require activation by the mycobacterial KatG enzyme, thereby circumventing the normal mechanism of resistance to the front line drug isoniazid (INH) and thus accounting for their activity against INH-resistant strains of MTB.

  19. Mycobacterium tuberculosis and the macrophage: maintaining a balance.

    PubMed

    Pieters, Jean

    2008-06-12

    Mycobacterium tuberculosis is a highly efficient pathogen, killing millions of infected people annually. The capacity of M. tuberculosis to survive and cause disease is strongly correlated to their ability to escape immune defense mechanisms. In particular, M. tuberculosis has the remarkable capacity to survive within the hostile environment of the macrophage. Understanding M. tuberculosis virulence strategies will not only define novel targets for drug development but will also help to uncover previously unknown signaling pathways related to the host's response to M. tuberculosis infection.

  20. Role of cholesterol in Mycobacterium tuberculosis infection.

    PubMed

    Miner, Maurine D; Chang, Jennifer C; Pandey, Amit K; Sassetti, Christopher M; Sherman, David R

    2009-06-01

    Mycobacterium tuberculosis (MTB) acquisition and utilization of nutrients within the host cell is poorly understood, although it has been hypothesized that host lipids probably play an important role in MTB survival. Cholesterol has recently been identified as an important lipid for mycobacterial infection. The mce4 transport system is required for cholesterol import into bacterial cells, and deletion of mce4 locus resulted in severe attenuation in a chronic mouse model of infection. However, it has remained unclear what additional bacterial functions were required for utilization of this sterol. We have found that the igr locus, which was previously found essential for intracellular growth and virulence of MTB, is required for cholesterol metabolism: igr-deficient bacteria cannot grow using cholesterol as a primary carbon source. The growth-inhibitory effect of cholesterol in vitro depends on cholesterol import, as the delta igr mutant growth defect during the early phase of disease is completely suppressed by mutating mce4, implicating cholesterol intoxication as the primary mechanism of attenuation. We conclude that M. tuberculosis metabolizes cholesterol throughout the course of infection, and that degradation of this sterol is crucial for bacterial persistence.

  1. The Biology of Mycobacterium Tuberculosis Infection

    PubMed Central

    Delogu, Giovanni; Sali, Michela; Fadda, Giovanni

    2013-01-01

    Tuberculosis (TB) still poses a major threat to mankind and during the last thirty years we have seen a recrudescence of the disease even in countries where TB was thought to be conquered. It is common opinion that more effective control tools such as new diagnostics, a new vaccine and new drugs are urgently needed to control the global pandemic, though the so far insufficient understanding of the Mycobacterium tuberculosis (Mtb) mechanism of pathogenesis is a major obstacle for the development of these control tools. In this review, we will summarize the recent advancement in the understanding of Mtb biology and on the pathogenesis of Mtb infection with emphasis on latent infection, with the change in paradigm of the last few years where the dichotomy between latent and active disease has been reconsidered in favor of a dynamic equilibrium between the host and the bacilli, encompassing a continuous spectrum of conditions that has been named TB spectrum. Implications for the diagnosis and control of disease in certain population will also be discussed. PMID:24363885

  2. The cell envelope glycoconjugates of Mycobacterium tuberculosis

    PubMed Central

    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

  3. Human Lung Immunity against Mycobacterium tuberculosis

    PubMed Central

    Schwander, Stephan; Dheda, Keertan

    2011-01-01

    The study of human pulmonary immunity against Mycobacterium tuberculosis (M.tb) provides a unique window into the biological interactions between the human host and M.tb within the broncho-alveolar microenvironment, the site of natural infection. Studies of bronchoalveolar cells (BACs) and lung tissue evaluate innate, adaptive, and regulatory immune mechanisms that collectively contribute to immunological protection or its failure. In aerogenically M.tb–exposed healthy persons lung immune responses reflect early host pathogen interactions that may contribute to sterilization, the development of latent M.tb infection, or progression to active disease. Studies in these persons may allow the identification of biomarkers of protective immunity before the initiation of inflammatory and disease-associated immunopathological changes. In healthy close contacts of patients with tuberculosis (TB) and during active pulmonary TB, immune responses are compartmentalized to the lungs and characterized by an exuberant helper T-cell type 1 response, which as suggested by recent evidence is counteracted by local suppressive immune mechanisms. Here we discuss how exploring human lung immunity may provide insights into disease progression and mechanisms of failure of immunological protection at the site of the initial host–pathogen interaction. These findings may also aid in the identification of new biomarkers of protective immunity that are urgently needed for the development of new and the improvement of current TB vaccines, adjuvant immunotherapies, and diagnostic technologies. To facilitate further work in this area, methodological and procedural approaches for bronchoalveolar lavage studies and their limitations are also discussed. PMID:21075901

  4. The Mycobacterium tuberculosis regulatory network and hypoxia

    PubMed Central

    Galagan, James E.; Minch, Kyle; Peterson, Matthew; Lyubetskaya, Anna; Azizi, Elham; Sweet, Linsday; Gomes, Antonio; Rustad, Tige; Dolganov, Gregory; Glotova, Irina; Abeel, Thomas; Mahwinney, Chris; Kennedy, Adam D.; Allard, René; Brabant, William; Krueger, Andrew; Jaini, Suma; Honda, Brent; Yu, Wen-Han; Hickey, Mark J.; Zucker, Jeremy; Garay, Christopher; Weiner, Brian; Sisk, Peter; Stolte, Christian; Winkler, Jessica K.; Van de Peer, Yves; Iazzetti, Paul; Camacho, Diogo; Dreyfuss, Jonathan; Liu, Yang; Dorhoi, Anca; Mollenkopf, Hans-Joachim; Drogaris, Paul; Lamontagne, Julie; Zhou, Yiyong; Piquenot, Julie; Park, Sang Tae; Raman, Sahadevan; Kaufmann, Stefan H. E.; Mohney, Robert P.; Chelsky, Daniel; Moody, D. Branch; Sherman, David R.; Schoolnik, Gary K.

    2014-01-01

    We have taken the first steps towards a complete reconstruction of the Mycobacterium tuberculosis regulatory network based on ChIP-Seq and combined this reconstruction with system-wide profiling of messenger RNAs, proteins, metabolites and lipids during hypoxia and re-aeration. Adaptations to hypoxia are thought to have a prominent role in M. tuberculosis pathogenesis. Using ChIP-Seq combined with expression data from the induction of the same factors, we have reconstructed a draft regulatory network based on 50 transcription factors. This network model revealed a direct interconnection between the hypoxic response, lipid catabolism, lipid anabolism and the production of cell wall lipids. As a validation of this model, in response to oxygen availability we observe substantial alterations in lipid content and changes in gene expression and metabolites in corresponding metabolic pathways. The regulatory network reveals transcription factors underlying these changes, allows us to computationally predict expression changes, and indicates that Rv0081 is a regulatory hub. PMID:23823726

  5. Mycobacterium tuberculosis Spoligotypes in Monterrey, Mexico▿

    PubMed Central

    Molina-Torres, Carmen A.; Moreno-Torres, Elisa; Ocampo-Candiani, Jorge; Rendon, Adrian; Blackwood, Kym; Kremer, Kristin; Rastogi, Nalin; Welsh, Oliverio; Vera-Cabrera, Lucio

    2010-01-01

    Although tuberculosis is still a public health problem in Mexico, there is little information about the genetic characteristics of the isolates. In the present study, we analyzed by spoligotyping 180 Mycobacterium tuberculosis clinical isolates from the urban area of Monterrey, Mexico, including drug-susceptible and drug-resistant isolates. The spoligotype patterns were compared with those in the international SITVIT2 spoligotyping database. Four isolates presented spoligotype patterns not found in the database (orphan types); the rest were distributed among 44 spoligo international types (SITs). SIT53 (clade T1) and SIT119 (clade X1) were predominant and included 43 (23.8%) and 28 (15.5%) of the isolates, respectively. In order to determine if there was a dominant spoligotype in the group of multidrug-resistant isolates, 37 of them were analyzed by IS6110-based restriction fragment length polymorphism assays, and scarce clustering of strains with more than five bands was observed. Fourteen isolates of this multidrug-resistant group presented four bands or less and were distributed in four SITs: SIT53 (n = 8), SIT92 (n = 3), SIT70 (n = 2), and SIT3038 (n = 1). When the molecular detection of mutations in the katG and rpoB genes were analyzed in these isolates with low copy numbers of IS6110, only two isolates shared the same IS6110, spoligotyping, and mutations patterns. When the distribution of the spoligotypes was analyzed by age cohort, SIT119 was predominantly found in patients 0 to 20 years old, especially in males, accounting for up to 40% of the isolates. In contrast, SIT53 was more prevalent in older females. This analysis demonstrates the variability of M. tuberculosis isolates in Monterrey and the partial dominance of SIT53 and SIT119 in that area of Mexico. PMID:19940048

  6. Insights from the complete genome sequence of Mycobacterium marinum on the evolution of Mycobacterium tuberculosis

    PubMed Central

    Stinear, Timothy P.; Seemann, Torsten; Harrison, Paul F.; Jenkin, Grant A.; Davies, John K.; Johnson, Paul D.R.; Abdellah, Zahra; Arrowsmith, Claire; Chillingworth, Tracey; Churcher, Carol; Clarke, Kay; Cronin, Ann; Davis, Paul; Goodhead, Ian; Holroyd, Nancy; Jagels, Kay; Lord, Angela; Moule, Sharon; Mungall, Karen; Norbertczak, Halina; Quail, Michael A.; Rabbinowitsch, Ester; Walker, Danielle; White, Brian; Whitehead, Sally; Small, Pamela L.C.; Brosch, Roland; Ramakrishnan, Lalita; Fischbach, Michael A.; Parkhill, Julian; Cole, Stewart T.

    2008-01-01

    Mycobacterium marinum, a ubiquitous pathogen of fish and amphibia, is a near relative of Mycobacterium tuberculosis, the etiologic agent of tuberculosis in humans. The genome of the M strain of M. marinum comprises a 6,636,827-bp circular chromosome with 5424 CDS, 10 prophages, and a 23-kb mercury-resistance plasmid. Prominent features are the very large number of genes (57) encoding polyketide synthases (PKSs) and nonribosomal peptide synthases (NRPSs) and the most extensive repertoire yet reported of the mycobacteria-restricted PE and PPE proteins, and related-ESX secretion systems. Some of the NRPS genes comprise a novel family and seem to have been acquired horizontally. M. marinum is used widely as a model organism to study M. tuberculosis pathogenesis, and genome comparisons confirmed the close genetic relationship between these two species, as they share 3000 orthologs with an average amino acid identity of 85%. Comparisons with the more distantly related Mycobacterium avium subspecies paratuberculosis and Mycobacterium smegmatis reveal how an ancestral generalist mycobacterium evolved into M. tuberculosis and M. marinum. M. tuberculosis has undergone genome downsizing and extensive lateral gene transfer to become a specialized pathogen of humans and other primates without retaining an environmental niche. M. marinum has maintained a large genome so as to retain the capacity for environmental survival while becoming a broad host range pathogen that produces disease strikingly similar to M. tuberculosis. The work described herein provides a foundation for using M. marinum to better understand the determinants of pathogenesis of tuberculosis. PMID:18403782

  7. Insertion element IS986 from Mycobacterium tuberculosis: a useful tool for diagnosis and epidemiology of tuberculosis.

    PubMed Central

    Hermans, P W; van Soolingen, D; Dale, J W; Schuitema, A R; McAdam, R A; Catty, D; van Embden, J D

    1990-01-01

    IS986 of Mycobacterium tuberculosis belongs to the IS3-like family of insertion sequences, and it has previously been shown to be present in multiple copies in the chromosome of M. tuberculosis. In this study we investigated the value of a IS986-based DNA probe in the diagnosis and epidemiology of tuberculosis. IS986 was found only in species belonging to the M. tuberculosis complex. Independent isolates of M. tuberculosis complex strains showed a very high degree of polymorphism of restriction fragments which contained IS986 DNA. In contrast, Mycobacterium bovis BCG vaccine strains as well as clinical isolates of M. bovis BCG contained one copy of IS986, which was present at the same location in the chromosome. Different M. tuberculosis isolates from a recent M. tuberculosis outbreak showed an identical banding pattern. We concluded that IS986 is an extremely suitable tool for the diagnosis and epidemiology of tuberculosis. Images PMID:1977765

  8. Mycobacterium tuberculosis volatiles for diagnosis of tuberculosis by Cricetomys rats.

    PubMed

    Mgode, Georgies F; Weetjens, Bart J; Nawrath, Thorben; Lazar, Doris; Cox, Christophe; Jubitana, Maureen; Mahoney, Amanda; Kuipers, Dian; Machang'u, Robert S; Weiner, January; Schulz, Stefan; Kaufmann, Stefan H E

    2012-11-01

    Tuberculosis (TB) diagnosis in regions with limited resources depends on microscopy with insufficient sensitivity. Rapid diagnostic tests of low cost but high sensitivity and specificity are needed for better point-of-care management of TB. Trained African giant pouched rats (Cricetomys sp.) can diagnose pulmonary TB in sputum but the relevant Mycobacterium tuberculosis (Mtb)-specific volatile compounds remain unknown. We investigated the odour volatiles of Mtb detected by rats in reference Mtb, nontuberculous mycobacteria, Nocardia sp., Streptomyces sp., Rhodococcus sp., and other respiratory tract microorganisms spiked into Mtb-negative sputum. Thirteen compounds were specific to Mtb and 13 were shared with other microorganisms. Rats discriminated a blend of Mtb-specific volatiles from individual, and blends of shared, compounds (P = 0.001). The rats' sensitivity for typical TB-positive sputa was 99.15% with 92.23% specificity and 93.14% accuracy. These findings underline the potential of trained Cricetomys rats for rapid TB diagnosis in resource-limited settings, particularly in Africa where Cricetomys rats occur widely and the burden of TB is high. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. Whole chromosomal DNA probes for rapid identification of Mycobacterium tuberculosis and Mycobacterium avium complex.

    PubMed Central

    Roberts, M C; McMillan, C; Coyle, M B

    1987-01-01

    Whole chromosomal DNA probes were used to identify clinical isolates of Mycobacterium tuberculosis, Mycobacterium avium complex, and Mycobacterium gordonae. The probe for M. tuberculosis was prepared from Mycobacterium bovis BCG, which has been shown to be closely related to M. tuberculosis. A probe for the M. avium complex was prepared from three strains representing each of the three DNA homology groups in the M. avium complex. The probes were used in dot blot assays to identify clinical isolates of mycobacteria. The dot blot test correctly identified 57 of the 61 (93%) cultures grown on solid media, and 100% of antibiotic-treated broth-grown cells were correctly identified. Identification by dot blot required a maximum of 48 h. When the probes were tested against 63 positive BACTEC (Johnston Laboratories, Inc., Towson, Md.) cultures of clinical specimens, 59% were correctly identified. However, of the 14 BACTEC cultures that had been treated with antibiotics before being lysed, 13 (93%) were correctly identified. PMID:3112180

  10. CD8 T cells and Mycobacterium tuberculosis infection.

    PubMed

    Lin, Philana Ling; Flynn, JoAnne L

    2015-05-01

    Tuberculosis is primarily a respiratory disease that is caused by Mycobacterium tuberculosis. M. tuberculosis can persist and replicate in macrophages in vivo, usually in organized cellular structures called granulomas. There is substantial evidence for the importance of CD4 T cells in control of tuberculosis, but the evidence for a requirement for CD8 T cells in this infection has not been proven in humans. However, animal model data support a non-redundant role for CD8 T cells in control of M. tuberculosis infection. In humans, infection with this pathogen leads to generation of specific CD8 T cell responses. These responses include classical (MHC Class I restricted) and non-classical CD8 T cells. Here, we discuss the potential roles of CD8 T cells in defense against tuberculosis, and our current understanding of the wide range of CD8 T cell types seen in M. tuberculosis infection.

  11. Surviving the macrophage: tools and tricks employed by Mycobacterium tuberculosis.

    PubMed

    Jayachandran, Rajesh; BoseDasgupta, Somdeb; Pieters, Jean

    2013-01-01

    Mycobacterium tuberculosis has evolved to withstand one of the most inhospitable cells within the human body, namely the macrophage, a cell that is normally geared toward the destruction of any invading microbe. How M. tuberculosis achieves this is still incompletely understood; however, a number of mechanisms are now known that provide advantages to M. tuberculosis for its survival and proliferation inside the macrophage. While some of these mechanisms are mediated by factors released by M. tuberculosis, others rely on host components that are being hijacked to benefit survival of M. tuberculosis within the macrophage as well to avoid the generation of an effective immune response. Here, we describe several of these mechanisms, also pointing out the potential usage of this knowledge toward the development of novel strategies to treat tuberculosis. Furthermore, we attempt to put the 'macrophage niche' into context with other intracellular pathogens and discuss some of the generalities as well as specializations that M. tuberculosis employs to survive.

  12. Role for Mycobacterium tuberculosis Membrane Vesicles in Iron Acquisition

    PubMed Central

    Prados-Rosales, Rafael; Weinrick, Brian C.; Piqué, Daniel G.; Jacobs, William R.; Casadevall, Arturo

    2014-01-01

    Mycobacterium tuberculosis releases membrane vesicles packed with molecules that can modulate the immune response. Because environmental conditions often influence the production and content of bacterial vesicles, this study examined M. tuberculosis microvesicles released under iron limitation, a common condition faced by pathogens inside the host. The findings indicate that M. tuberculosis increases microvesicle production in response to iron restriction and that these microvesicles contain mycobactin, which can serve as an iron donor and supports replication of iron-starved mycobacteria. Consequently, the results revealed a role of microvesicles in iron acquisition in M. tuberculosis, which can be critical for survival in the host. PMID:24415729

  13. Pulmonary disease due to Mycobacterium tuberculosis in a horse: zoonotic concerns and limitations of antemortem testing

    USDA-ARS?s Scientific Manuscript database

    A case of pulmonary tuberculosis caused by Mycobacterium tuberculosis was diagnosed in a horse. Clinical evaluation performed prior to euthanasia did not suggest tuberculosis, but postmortem examination provided pathological and bacteriological evidence of disease. In the lungs, multiple tuberculoid...

  14. Cytokines and Chemokines in Mycobacterium tuberculosis Infection.

    PubMed

    Domingo-Gonzalez, Racquel; Prince, Oliver; Cooper, Andrea; Khader, Shabaana A

    2016-10-01

    Chemokines and cytokines are critical for initiating and coordinating the organized and sequential recruitment and activation of cells into Mycobacterium tuberculosis-infected lungs. Correct mononuclear cellular recruitment and localization are essential to ensure control of bacterial growth without the development of diffuse and damaging granulocytic inflammation. An important block to our understanding of TB pathogenesis lies in dissecting the critical aspects of the cytokine/chemokine interplay in light of the conditional role these molecules play throughout infection and disease development. Much of the data highlighted in this review appears at first glance to be contradictory, but it is the balance between the cytokines and chemokines that is critical, and the "goldilocks" (not too much and not too little) phenomenon is paramount in any discussion of the role of these molecules in TB. Determination of how the key chemokines/cytokines and their receptors are balanced and how the loss of that balance can promote disease is vital to understanding TB pathogenesis and to identifying novel therapies for effective eradication of this disease.

  15. Tuberculosis in seals caused by a novel member of the Mycobacterium tuberculosis complex: Mycobacterium pinnipedii sp. nov.

    PubMed

    Cousins, Debby V; Bastida, Ricardo; Cataldi, Angel; Quse, Viviana; Redrobe, Sharon; Dow, Sue; Duignan, Padraig; Murray, Alan; Dupont, Christine; Ahmed, Niyaz; Collins, Des M; Butler, W Ray; Dawson, David; Rodríguez, Diego; Loureiro, Julio; Romano, Maria Isabel; Alito, A; Zumarraga, M; Bernardelli, Amelia

    2003-09-01

    A comparison of Mycobacterium tuberculosis complex isolates from seals (pinnipeds) in Australia, Argentina, Uruguay, Great Britain and New Zealand was undertaken to determine their relationships to each other and their taxonomic position within the complex. Isolates from 30 cases of tuberculosis in six species of pinniped and seven related isolates were compared to representative and standard strains of the M. tuberculosis complex. The seal isolates could be distinguished from other members of the M. tuberculosis complex, including the recently defined 'Mycobacterium canettii' and 'Mycobacterium caprae', on the basis of host preference and phenotypic and genetic tests. Pinnipeds appear to be the natural host for this 'seal bacillus', although the organism is also pathogenic in guinea pigs, rabbits, humans, Brazilian tapir (Tapirus terrestris) and, possibly, cattle. Infection caused by the seal bacillus is predominantly associated with granulomatous lesions in the peripheral lymph nodes, lungs, pleura, spleen and peritoneum. Cases of disseminated disease have been found. As with other members of the M. tuberculosis complex, aerosols are the most likely route of transmission. The name Mycobacterium pinnipedii sp. nov. is proposed for this novel member of the M. tuberculosis complex (the type strain is 6482(T)=ATCC BAA-688(T)=NCTC 13288(T)).

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

    PubMed Central

    Abdel-Rahman, Zaid; Sengupta, Ruchira; Johnson, Laura

    2016-01-01

    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. PMID:27895946

  17. Radiometric selective inhibition tests for differentiation of Mycobacterium tuberculosis, Mycobacterium bovis, and other mycobacteria.

    PubMed Central

    Gross, W M; Hawkins, J E

    1985-01-01

    In the context of a busy reference laboratory, radiometric selective inhibition tests were evaluated for rapid differentiation of Mycobacterium tuberculosis and Mycobacterium bovis and of the M. tuberculosis complex from other mycobacteria. p-Nitro-alpha-acetylamino-beta-hydroxypropiophenone at 5 micrograms and hydroxylamine hydrochloride at 62.5 and 125 micrograms per ml of 7H12 medium were used to separate the M. tuberculosis complex from other mycobacteria (MOTT bacilli). Since it is important epidemiologically to distinguish M. tuberculosis from M. bovis, susceptibility to 1 microgram of thiophene-2-carboxylic acid per ml was also determined radiometrically. By using these three agents as selective inhibitors, M. tuberculosis, M. bovis, and MOTT bacilli were differentiated with a high degree of specificity by a BACTEC radiometric procedure. Results of tests performed on clinical isolates submitted on solid medium to our reference laboratory were available within 5 days. PMID:3921561

  18. MTBreg: The Database of Conditionally Regulated Proteins in Mycobacterium Tuberculosis

    DOE Data Explorer

    Kaufman, Markus; Pal, Debnath; Eisenberg, David

    Proteins up- and down- regulated in Mycobacterium tuberculosis grown under conditions mimicking infection are included in this database. It also includes information on proteins that are regulated by selected transcription factors or other regulatory proteins. The literature data provided here is complimentary to the databases provided by Michael Strong that include recent TB computational functional linkages and the Prolinks Database by Peter Bowers. The experimental condition, the experimental dataset and a literature reference will be displayed, including links to the computationally linked proteins in the Prolinks Database and the entry in the Mycobacterium tuberculosis Structural Genomics Database.[Copied from information at http://www.doe-mbi.ucla.edu/Services/MTBreg/

  19. Tenosynovitis caused by a novel nontuberculous Mycobacterium species initially misidentified as a member of the Mycobacterium tuberculosis complex.

    PubMed

    Simner, Patricia J; Hyle, Emily P; Buckwalter, Seanne P; Branda, John A; Brown-Elliott, Barbara A; Franklin, Jameelah; Toney, Nadege C; de Man, Tom J B; Wallace, Richard J; Vasireddy, Ravikiran; Gandhi, Rajesh T; Wengenack, Nancy L

    2014-12-01

    We present a case of tenosynovitis caused by a novel, slowly growing, nonchromogenic, nontuberculous mycobacterium (NTM). Originally misidentified as Mycobacterium tuberculosis complex, the NTM cross-reacts with the M. tuberculosis complex nucleic acid hybridization probe, a M. tuberculosis gamma interferon release assay, and is closely related to M. tuberculosis by 16S rRNA gene sequencing. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  20. [Application of recombinant 38000 protein antigen of Mycobacterium tuberculosis in screening Mycobacterium tuberculosis infection].

    PubMed

    He, Xiu-yun; Zhuang, Yu-hui; Zhang, Xiao-gang

    2009-08-01

    To evaluate the potential of recombinant 38000 protein of Mycobacterium tuberculosis (38000 protein) as a tuberculosis-specific tuberculin for screening M. tuberculosis infection. A total of 1342 subjects (706 men and 636 women, age 18-60 years) from several communities in Kazuo County and Xidaziying Town, Chaoyang, Liaoning Province, and Hongdong County, Linfen, Shanxi Province were enrolled from September 2004 to February 2005. The skin tests were performed with double-blinded and the intradermal injections were administered on both forearms with 0.1 ml solution of PPD and 38000 protein at the right side and the left side, respectively. The vertical and transverse diameters of induration or erythema were measured following 24 h for 38000 protein and 48 h for PPD, respectively. The diameters of the the skin test reactions were defined as the means of the vertical and transverse diameters, and positive skin reactions were identified when the diameter was greater than or equal to 5 mm. The comparison of the positive rate was performed via chi(2) test and the consistency of positive skin test reactions between 38000 protein and TB-PPD was analyzed through calculating Kappa coefficients. The positive rate was 55.1% (740/1342) and 28.6% (384/1342) for PPD and 38000 protein, respectively; the difference being significant (chi(2) = 190.6, P < 0.01). The consistency of positive skin test reactions between 38000 protein and PPD was low due to a negative Kappa coefficient. The positive rates induced by PPD and 38000 protein tended to increase with age except for the 33-37 year group. For a given age group, the positive rate of PPD was much higher than that of 38000 protein. The subjects without BCG scar had a lower positive rate for 38000 protein (24.3%, 137/566) than those with BCG scar (31.9%, 247/776) (chi(2) = 4.7, P < 0.05). The subjects with tuberculosis contact history had a higher positive rate for 38000 protein (74.4%, 32/43) than those without tuberculosis contact

  1. Two Cases of Pulmonary Tuberculosis Caused by Mycobacterium tuberculosis subsp. canetti

    PubMed Central

    Morillon, Marc; Koeck, Jean-Louis; Varnerot, Anne; Briant, Jean-François; Nguyen, Gilbert; Verrot, Denis; Bonnet, Daniel; Vincent, Véronique

    2002-01-01

    We identified an unusual strain of mycobacteria from two patients with pulmonary tuberculosis by its smooth, glossy morphotype and, primarily, its genotypic characteristics. Spoligotyping and restriction fragment length polymorphism typing were carried out with the insertion sequence IS6110 patterns. All known cases of tuberculosis caused by Mycobacterium canetti have been contracted in the Horn of Africa. PMID:12453369

  2. Two cases of pulmonary tuberculosis caused by Mycobacterium tuberculosis subsp canetti.

    PubMed

    Miltgen, Jean; Morillon, Marc; Koeck, Jean-Louis; Varnerot, Anne; Briant, Jean-François; Nguyen, Gilbert; Verrot, Denis; Bonnet, Daniel; Vincent, Véronique

    2002-11-01

    We identified an unusual strain of mycobacteria from two patients with pulmonary tuberculosis by its smooth, glossy morphotype and, primarily, its genotypic characteristics. Spoligotyping and restriction fragment length polymorphism typing were carried out with the insertion sequence IS6110 patterns. All known cases of tuberculosis caused by Mycobacterium canetti have been contracted in the Horn of Africa.

  3. Sub-speciation of Mycobacterium tuberculosis complex from tuberculosis patients in Japan.

    PubMed

    Ueyama, Masako; Chikamatsu, Kinuyo; Aono, Akio; Murase, Yoshiro; Kuse, Naoyuki; Morimoto, Kozo; Okumura, Masao; Yoshiyama, Takashi; Ogata, Hideo; Yoshimori, Kozo; Kudoh, Shoji; Azuma, Arata; Gemma, Akihiko; Mitarai, Satoshi

    2014-01-01

    Mycobacterium tuberculosis is the major causative agent of tuberculosis in humans. It is well known that Mycobacterium bovis and other species in the M. tuberculosis complex (MTC) can cause respiratory diseases as zoonosis. We analyzed the MTC isolates collected from tuberculosis patients from Japan in 2002 using a multiplex PCR system that detected cfp32, RD9 and RD12. A total of 970 MTC isolates that were representative of the tuberculosis cases throughout Japan, were examined using this method. As a result, 966 (99.6%) M. tuberculosis, two Mycobacterium africanum and two Mycobacterium canettii were identified using a multiplex PCR system, while no M. bovis was detected. Two isolates that lacked RD9 were initially considered to be M. canettii, but further analysis of the hsp65 sequence revealed them to be M. tuberculosis. Also two M. africanum were identified as M. tuberculosis using the -215 narG nucleotide polymorphism. Though PCR-linked methods have been used for a rapid differentiation of MTC and NTM, from our cases we suggest careful interpretation of RD based identification.

  4. Comparison of two 2,3-diacyl trehalose antigens from Mycobacterium tuberculosis and Mycobacterium fortuitum for serology in tuberculosis patients.

    PubMed Central

    Tórtola, M T; Lanéelle, M A; Martín-Casabona, N

    1996-01-01

    Immunoglobulin G antibodies against two 2,3-diacyl trehalose (DAT) antigens from Mycobacterium tuberculosis (DATT) and Mycobacterium fortuitum (DATF) were studied by enzyme-linked immunosorbent assay of 356 serum samples. The sera were obtained from non-tuberculosis-infected individuals (282 serum samples) and tuberculosis patients (74 serum samples). Non-tuberculosis-infected individuals were healthy people (120 serum samples; positive purified-protein-derivative skin test, 60 patients; negative purified-protein-derivative skin test, 60 patients) patients with nontuberculosis lung disease (59 serum samples), contacts of sputum-smear-positive tuberculosis patients (57 serum samples), and human immunodeficiency virus-infected patients with nontuberculosis lung disease (46 serum samples). Of the 74 patients with tuberculosis, 14 were human immunodeficiency virus infected. The sensitivity of the method using DATT was 44.5%, and that with DATF was 48.6%. The specificities with both antigens were 99.1%. There were no significant differences between the mean values for both antigens (P = 0.2815). We therefore concluded that both antigens were interchangeable. As M. fortuitum, a fast-growing mycobacterium, could be a good source of antigen DAT, these results deserve consideration in the serology of tuberculosis. PMID:8877135

  5. Human Xenobiotic Nuclear Receptor PXR Augments Mycobacterium tuberculosis Survival.

    PubMed

    Bhagyaraj, Ella; Nanduri, Ravikanth; Saini, Ankita; Dkhar, Hedwin Kitdorlang; Ahuja, Nancy; Chandra, Vemika; Mahajan, Sahil; Kalra, Rashi; Tiwari, Drishti; Sharma, Charu; Janmeja, Ashok Kumar; Gupta, Pawan

    2016-07-01

    Mycobacterium tuberculosis can evade host defense processes, thereby ensuring its survival and pathogenesis. In this study, we investigated the role of nuclear receptor, pregnane X receptor (PXR), in M. tuberculosis infection in human monocyte-derived macrophages. In this study, we demonstrate that PXR augments M. tuberculosis survival inside the host macrophages by promoting the foamy macrophage formation and abrogating phagolysosomal fusion, inflammation, and apoptosis. Additionally, M. tuberculosis cell wall lipids, particularly mycolic acids, crosstalk with human PXR (hPXR) by interacting with its promiscuous ligand binding domain. To confirm our in vitro findings and to avoid the reported species barrier in PXR function, we adopted an in vivo mouse model expressing hPXR, wherein expression of hPXR in mice promotes M. tuberculosis survival. Therefore, pharmacological intervention and designing antagonists to hPXR may prove to be a promising adjunct therapy for tuberculosis. Copyright © 2016 by The American Association of Immunologists, Inc.

  6. Radiometric detection of the metabolic activity of Mycobacterium tuberculosis.

    PubMed

    Cummings, D M; Ristroph, D; Camargo, E E; Larson, S M; Wagner, H N

    1975-12-01

    A radiometric test capable of detecting the metabolic rate of M. tuberculosis within 18 hr after inoculation has been developed. The technique is based on the measurement of 14CO2 produced by the bacterial metabolism of 14C-U-glycerol of 14C-U-acetate. The test is an important first step in the development of rapid radiometric techniques for clinical study of Mycobacterium tuberculosis.

  7. Game of 'Somes: Protein Destruction for Mycobacterium tuberculosis Pathogenesis

    PubMed Central

    Samanovic, Marie I.; Darwin, K. Heran

    2015-01-01

    The proteasome system of Mycobacterium tuberculosis is required for causing disease. Proteasomes are multi-subunit chambered proteases and, until recently, were only known to participate in adenosine triphosphate (ATP)-dependent proteolysis in bacteria. In this review, we discuss the latest advances in understanding how both ATP-dependent and ATP-independent proteasome-regulated pathways contribute to M. tuberculosis virulence. PMID:26526503

  8. Efflux inhibition with verapamil potentiates bedaquiline in Mycobacterium tuberculosis.

    PubMed

    Gupta, Shashank; Cohen, Keira A; Winglee, Kathryn; Maiga, Mamoudou; Diarra, Bassirou; Bishai, William R

    2014-01-01

    Drug efflux is an important resistance mechanism in Mycobacterium tuberculosis. We found that verapamil, an efflux inhibitor, profoundly decreases the MIC of bedaquiline and clofazimine to M. tuberculosis by 8- to 16-fold. This exquisite susceptibility was noted among drug-susceptible and drug-resistant clinical isolates. Thus, efflux inhibition is an important sensitizer of bedaquiline and clofazimine, and efflux may emerge as a resistance mechanism to these drugs.

  9. In vitro susceptibilities of Mycobacterium tuberculosis to 10 antimicrobial agents.

    PubMed Central

    Byrne, S K; Crawford, C E; Geddes, G L; Black, W A

    1988-01-01

    After preliminary in vitro screening of 10 antimicrobial agents against Mycobacterium tuberculosis, the MICs of the 6 most promising agents against 27 clinical isolates were determined by agar dilution. The two quinolone compounds tested (difloxacin and A-56620) were the most active, each inhibiting 50% of the strains at concentrations of 4 micrograms/ml. M. tuberculosis strains previously shown to be resistant to isoniazid, streptomycin, rifampin, or ethambutol were as susceptible to these quinolone compounds as susceptible strains. PMID:3143305

  10. Dramatic reduction of culture time of Mycobacterium tuberculosis

    NASA Astrophysics Data System (ADS)

    Ghodbane, Ramzi; Raoult, Didier; Drancourt, Michel

    2014-02-01

    Mycobacterium tuberculosis culture, a critical technique for routine diagnosis of tuberculosis, takes more than two weeks. Here, step-by-step improvements in the protocol including a new medium, microaerophlic atmosphere or ascorbic-acid supplement and autofluorescence detection dramatically shortened this delay. In the best case, primary culture and rifampicin susceptibility testing were achieved in 72 hours when specimens were inoculated directly on the medium supplemented by antibiotic at the beginning of the culture.

  11. Game of 'Somes: Protein Destruction for Mycobacterium tuberculosis Pathogenesis.

    PubMed

    Samanovic, Marie I; Darwin, K Heran

    2016-01-01

    The proteasome system of Mycobacterium tuberculosis is required for causing disease. Proteasomes are multisubunit chambered proteases and, until recently, were only known to participate in adenosine triphosphate (ATP)-dependent proteolysis in bacteria. In this review, we discuss the latest advances in understanding how both ATP-dependent and ATP-independent proteasome-regulated pathways contribute to M. tuberculosis virulence. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Mycobacterium tuberculosis epidemiology in South Dakota.

    PubMed

    Iverson, D A; Hurley, B; Pueringer, R

    1994-03-01

    Tuberculosis incidence in the United States has recently increased from its rate of decline resulting in an excess of cases nationwide. The increase has been attributed largely to the HIV epidemic. Although tuberculosis incidence in South Dakota has increased similar to the national trend, South Dakota has not reported a single HIV-associated case of tuberculosis. Tuberculosis incidence in South Dakota has decreased in younger individuals. As a result, the percentage of tuberculosis cases in the elderly has increased. Though the reported cases of pulmonary tuberculosis have decreased, the reported cases of extrapulmonary tuberculosis have not changed. Furthermore, the percentage of extrapulmonary tuberculosis occurring in the elderly has increased. Tuberculosis incidence in South Dakota is, in part, increasing because of the persistence of extrapulmonary tuberculosis in the elderly.

  13. T cells and adaptive immunity to Mycobacterium tuberculosis in humans.

    PubMed

    Jasenosky, Luke D; Scriba, Thomas J; Hanekom, Willem A; Goldfeld, Anne E

    2015-03-01

    The adaptive immune response mediated by T cells is critical for control of Mycobacterium tuberculosis (M. tuberculosis) infection in humans. However, the M. tuberculosis antigens and host T-cell responses that are required for an effective adaptive immune response to M. tuberculosis infection are yet to be defined. Here, we review recent findings on CD4(+) and CD8(+) T-cell responses to M. tuberculosis infection and examine the roles of distinct M. tuberculosis-specific T-cell subsets in control of de novo and latent M. tuberculosis infection, and in the evolution of T-cell immunity to M. tuberculosis in response to tuberculosis treatment. In addition, we discuss recent studies that elucidate aspects of M. tuberculosis-specific adaptive immunity during human immunodeficiency virus co-infection and summarize recent findings from vaccine trials that provide insight into effective adaptive immune responses to M. tuberculosis infection. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  14. Catabolism of the Last Two Steroid Rings in Mycobacterium tuberculosis and Other Bacteria

    PubMed Central

    Crowe, Adam M.; Casabon, Israël; Brown, Kirstin L.; Liu, Jie; Lian, Jennifer; Rogalski, Jason C.; Hurst, Timothy E.; Snieckus, Victor; Foster, Leonard J.

    2017-01-01

    ABSTRACT Most mycolic acid-containing actinobacteria and some proteobacteria use steroids as growth substrates, but the catabolism of the last two steroid rings has yet to be elucidated. In Mycobacterium tuberculosis, this pathway includes virulence determinants and has been proposed to be encoded by the KstR2-regulated genes, which include a predicted coenzyme A (CoA) transferase gene (ipdAB) and an acyl-CoA reductase gene (ipdC). In the presence of cholesterol, ΔipdC and ΔipdAB mutants of either M. tuberculosis or Rhodococcus jostii strain RHA1 accumulated previously undescribed metabolites: 3aα-H-4α(carboxyl-CoA)-5-hydroxy-7aβ-methylhexahydro-1-indanone (5-OH HIC-CoA) and (R)-2-(2-carboxyethyl)-3-methyl-6-oxocyclohex-1-ene-1-carboxyl-CoA (COCHEA-CoA), respectively. A ΔfadE32 mutant of Mycobacterium smegmatis accumulated 4-methyl-5-oxo-octanedioic acid (MOODA). Incubation of synthetic 5-OH HIC-CoA with purified IpdF, IpdC, and enoyl-CoA hydratase 20 (EchA20), a crotonase superfamily member, yielded COCHEA-CoA and, upon further incubation with IpdAB and a CoA thiolase, yielded MOODA-CoA. Based on these studies, we propose a pathway for the final steps of steroid catabolism in which the 5-member ring is hydrolyzed by EchA20, followed by hydrolysis of the 6-member ring by IpdAB. Metabolites accumulated by ΔipdF and ΔechA20 mutants support the model. The conservation of these genes in known steroid-degrading bacteria suggests that the pathway is shared. This pathway further predicts that cholesterol catabolism yields four propionyl-CoAs, four acetyl-CoAs, one pyruvate, and one succinyl-CoA. Finally, a ΔipdAB M. tuberculosis mutant did not survive in macrophages and displayed severely depleted CoASH levels that correlated with a cholesterol-dependent toxicity. Our results together with the developed tools provide a basis for further elucidating bacterial steroid catabolism and virulence determinants in M. tuberculosis. PMID:28377529

  15. Nicotine Impairs Macrophage Control of Mycobacterium tuberculosis.

    PubMed

    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.

  16. Genomic signal analysis of Mycobacterium tuberculosis

    NASA Astrophysics Data System (ADS)

    Cristea, Paul Dan; Banica, Dorina; Tuduce, Rodica

    2007-02-01

    As previously shown the conversion of nucleotide sequences into digital signals offers the possibility to apply signal processing methods for the analysis of genomic data. Genomic Signal Analysis (GSA) has been used to analyze large scale features of DNA sequences, at the scale of whole chromosomes, including both coding and non-coding regions. The striking regularities of genomic signals reveal restrictions in the way nucleotides and pairs of nucleotides are distributed along nucleotide sequences. Structurally, a chromosome appears to be less of a "plain text", corresponding to certain semantic and grammar rules, but more of a "poem", satisfying additional symmetry restrictions that evoke the "rhythm" and "rhyme". Recurrent patterns in nucleotide sequences are reflected in simple mathematical regularities observed in genomic signals. GSA has also been used to track pathogen variability, especially concerning their resistance to drugs. Previous work has been dedicated to the study of HIV-1, Clade F and Avian Flu. The present paper applies GSA methodology to study Mycobacterium tuberculosis (MT) rpoB gene variability, relevant to its resistance to antibiotics. Isolates from 50 Romanian patients have been studied both by rapid LightCycler PCR and by sequencing of a segment of 190-250 nucleotides covering the region of interest. The variability is caused by SNPs occurring at specific sites along the gene strand, as well as by inclusions. Because of the mentioned symmetry restrictions, the GS variations tend to compensate. An important result is that MT can act as a vector for HIV virus, which is able to retrotranscribe its specific genes both into human and MT genomes.

  17. Novel Cephalosporins Selectively Active on Nonreplicating Mycobacterium tuberculosis

    PubMed Central

    2016-01-01

    We report two series of novel cephalosporins that are bactericidal to Mycobacterium tuberculosis alone of the pathogens tested, which only kill M. tuberculosis when its replication is halted by conditions resembling those believed to pertain in the host, and whose bactericidal activity is not dependent upon or enhanced by clavulanate, a β-lactamase inhibitor. The two classes of cephalosporins bear an ester or alternatively an oxadiazole isostere at C-2 of the cephalosporin ring system, a position that is almost exclusively a carboxylic acid in clinically used agents in the class. Representatives of the series kill M. tuberculosis within macrophages without toxicity to the macrophages or other mammalian cells. PMID:27144688

  18. Novel Cephalosporins Selectively Active on Nonreplicating Mycobacterium tuberculosis.

    PubMed

    Gold, Ben; Smith, Robert; Nguyen, Quyen; Roberts, Julia; Ling, Yan; Lopez Quezada, Landys; Somersan, Selin; Warrier, Thulasi; Little, David; Pingle, Maneesh; Zhang, David; Ballinger, Elaine; Zimmerman, Matthew; Dartois, Véronique; Hanson, Paul; Mitscher, Lester A; Porubsky, Patrick; Rogers, Steven; Schoenen, Frank J; Nathan, Carl; Aubé, Jeffrey

    2016-07-14

    We report two series of novel cephalosporins that are bactericidal to Mycobacterium tuberculosis alone of the pathogens tested, which only kill M. tuberculosis when its replication is halted by conditions resembling those believed to pertain in the host, and whose bactericidal activity is not dependent upon or enhanced by clavulanate, a β-lactamase inhibitor. The two classes of cephalosporins bear an ester or alternatively an oxadiazole isostere at C-2 of the cephalosporin ring system, a position that is almost exclusively a carboxylic acid in clinically used agents in the class. Representatives of the series kill M. tuberculosis within macrophages without toxicity to the macrophages or other mammalian cells.

  19. Oxadiazoles Have Butyrate-Specific Conditional Activity against Mycobacterium tuberculosis

    PubMed Central

    Early, Julie V.; Casey, Allen; Martinez-Grau, Maria Angeles; Gonzalez Valcarcel, Isabel C.; Vieth, Michal; Ollinger, Juliane; Bailey, Mai Ann; Alling, Torey; Files, Megan; Ovechkina, Yulia

    2016-01-01

    Mycobacterium tuberculosis is a global pathogen of huge importance which can adapt to several host niche environments in which carbon source availability is likely to vary. We developed and ran a phenotypic screen using butyrate as the sole carbon source to be more reflective of the host lung environment. We screened a library of ∼87,000 small compounds and identified compounds which demonstrated good antitubercular activity against M. tuberculosis grown with butyrate but not with glucose as the carbon source. Among the hits, we identified an oxadiazole series (six compounds) which had specific activity against M. tuberculosis but which lacked cytotoxicity against mammalian cells. PMID:27044545

  20. Preventing Transmission of Mycobacterium tuberculosis in Health Care Settings.

    PubMed

    Punjabi, Chitra D; Perloff, Sarah R; Zuckerman, Jerry M

    2016-12-01

    Patients with tuberculosis (TB) pose a risk to other patients and health care workers, and outbreaks in health care settings occur when appropriate infection control measures are not used. In this article, we discuss strategies to prevent transmission of Mycobacterium tuberculosis within health care settings. All health care facilities should have an operational TB infection control plan that emphasizes the use of a hierarchy of controls (administrative, environmental, and personal respiratory protection). We also discuss resources available to clinicians who work in the prevention and investigation of nosocomial transmission of M tuberculosis. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Tuberculosis in Alpacas (Lama pacos) Caused by Mycobacterium bovis▿

    PubMed Central

    García-Bocanegra, I.; Barranco, I.; Rodríguez-Gómez, I. M.; Pérez, B.; Gómez-Laguna, J.; Rodríguez, S.; Ruiz-Villamayor, E.; Perea, A.

    2010-01-01

    We report three cases of tuberculosis in alpacas from Spain caused by Mycobacterium bovis. The animals revealed two different lesional patterns. Mycobacterial culture and PCR assay yielded positive results for M. bovis. Molecular typing of the isolates identified spoligotype SB0295 and identical variable-number tandem repeat (VNTR) allele sizes. PMID:20237097

  2. A new antituberculosis drug that selectively kills nonmultiplying Mycobacterium tuberculosis.

    PubMed

    Mitchison, Denis A

    2008-03-13

    An important report by Bryk et al. in this issue of Cell Host & Microbe describes the properties of a rhodanine prodrug active against nonmultiplying Mycobacterium tuberculosis (Mtb). Considering the tolerance of nonreplicating Mtb to most currently available agents, such a drug could be a major addition to our antituberculosis arsenal and would greatly benefit control of the disease.

  3. Tuberculosis in alpacas (Lama pacos) caused by Mycobacterium bovis.

    PubMed

    García-Bocanegra, I; Barranco, I; Rodríguez-Gómez, I M; Pérez, B; Gómez-Laguna, J; Rodríguez, S; Ruiz-Villamayor, E; Perea, A

    2010-05-01

    We report three cases of tuberculosis in alpacas from Spain caused by Mycobacterium bovis. The animals revealed two different lesional patterns. Mycobacterial culture and PCR assay yielded positive results for M. bovis. Molecular typing of the isolates identified spoligotype SB0295 and identical variable-number tandem repeat (VNTR) allele sizes.

  4. Pulmonary Tuberculosis due to Mycobacterium bovis in Captive Siberian Tiger

    PubMed Central

    Lantos, Ákos; Niemann, Stefan; Mezősi, László; Sós, Endre; Erdélyi, Károly; Dávid, Sándor; Parsons, Linda M.; Kubica, Tanja; Rüsch-Gerdes, Sabine

    2003-01-01

    We report the first case of pulmonary tuberculosis caused by Mycobacterium bovis subsp. caprae in a captive Siberian tiger, an endangered feline. The pathogen was isolated from a tracheal aspirate obtained by bronchoscopy. This procedure provided a reliable in vivo diagnostic method in conjunction with conventional and molecular tests for the detection of mycobacteria. PMID:14718093

  5. A strip array for spoligotyping of Mycobacterium tuberculosis complex isolates.

    PubMed

    Tu, Yiling; Zeng, Xiaohong; Li, Hui; Zheng, Rongrong; Xu, Ye; Li, Qingge

    2016-03-01

    A novel strip array was developed for a nine-spacer spoligotyping scheme of Mycobacterium tuberculosis complex (MTBC). The new method was evaluated using 211 MTBC isolates and the results were fully concordant with the traditional spoligotyping approach. The strip array proved to be rapid and convenient for spoligotyping of MTBC.

  6. 21 CFR 866.3370 - Mycobacterium tuberculosis immunofluorescent reagents.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Mycobacterium tuberculosis immunofluorescent reagents. 866.3370 Section 866.3370 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents...

  7. 21 CFR 866.3370 - Mycobacterium tuberculosis immunofluorescent reagents.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Mycobacterium tuberculosis immunofluorescent reagents. 866.3370 Section 866.3370 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents...

  8. [A case of pulmonary multiresistant Mycobacterium bovis tuberculosis in Madagascar].

    PubMed

    Ramarokoto, H; Andrianasolo, D; Rasolonavalona, T; Ramaroson, F; Razafitsiarovana, I; Vincent, V; Ratsimba, L; Rasolofo Razanamparany, V

    2003-01-01

    We report a chronic case of pulmonary tuberculosis in a Malagasy citizen from Antsohihy (West of Madagascar), who was infected with a multi-drug resistant Mycobacterium bovis strain. This is the first case reported of the isolation of such a strain in Madagascar.

  9. Lack of protection afforded by ribonucleic acid preparations from Mycobacterium tuberculosis against Mycobacterium leprae infections in mice.

    PubMed Central

    Shepard, C C; Youmans, A Y; Youmans, G P

    1977-01-01

    Mycobacterial ribonucleic acid preparations from H37Ra, an attenuated strain of Mycobacterium tuberculosis, provide their usual marked protection against M. tuberculosis challenge; however, they provided no protection against Mycobacterium leprae challenge. Suspensions of intact H37Ra were not effective against M. leprae. Suspensions of BCG gave their usual distinct protection against M. leprae challenge. PMID:404242

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

    PubMed

    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.

  11. Co-evolution of Mycobacterium tuberculosis and Homo sapiens

    PubMed Central

    Brites, Daniela; Gagneux, Sebastien

    2015-01-01

    The causative agent of human tuberculosis (TB), Mycobacterium tuberculosis, is an obligate pathogen that evolved to exclusively persist in human populations. For M. tuberculosis to transmit from person to person, it has to cause pulmonary disease. Therefore, M. tuberculosis virulence has likely been a significant determinant of the association between M. tuberculosis and humans. Indeed, the evolutionary success of some M. tuberculosis genotypes seems at least partially attributable to their increased virulence. The latter possibly evolved as a consequence of human demographic expansions. If co-evolution occurred, humans would have counteracted to minimize the deleterious effects of M. tuberculosis virulence. The fact that human resistance to infection has a strong genetic basis is a likely consequence of such a counter-response. The genetic architecture underlying human resistance to M. tuberculosis remains largely elusive. However, interactions between human genetic polymorphisms and M. tuberculosis genotypes have been reported. Such interactions are consistent with local adaptation and allow for a better understanding of protective immunity in TB. Future ‘genome-to-genome’ studies, in which locally associated human and M. tuberculosis genotypes are interrogated in conjunction, will help identify new protective antigens for the development of better TB vaccines. PMID:25703549

  12. Mycobacterium tuberculosis Bacteremia among Acutely Febrile Children in Western Kenya

    PubMed Central

    Pavlinac, Patricia B.; Naulikha, Jaqueline M.; John-Stewart, Grace C.; Onchiri, Frankline M.; Okumu, Albert O.; Sitati, Ruth R.; Cranmer, Lisa M.; Lokken, Erica M.; Singa, Benson O.; Walson, Judd L.

    2015-01-01

    In children, Mycobacterium tuberculosis (M. tuberculosis) frequently disseminates systemically, presenting with nonspecific signs including fever. We determined prevalence of M. tuberculosis bacteremia among febrile children presenting to hospitals in Nyanza, Kenya (a region with high human immunodeficiency virus (HIV) and M. tuberculosis prevalence). Between March 2013 and February 2014, we enrolled children aged 6 months to 5 years presenting with fever (axillary temperature ≥ 37.5°C) and no recent antibiotic use. Blood samples were collected for bacterial and mycobacterial culture using standard methods. Among 148 children enrolled, median age was 3.1 years (interquartile range: 1.8–4.1 years); 10.3% of children were living with a household member diagnosed with M. tuberculosis in the last year. Seventeen percent of children were stunted (height-for-age z-score < −2), 18.6% wasted (weight-for-height z-score < −2), 2.7% were HIV-infected, and 14.2% were HIV-exposed uninfected. Seventeen children (11.5%) had one or more signs of tuberculosis (TB). All children had a Bacille Calmette-Guerin vaccination scar. Among 134 viable blood cultures, none (95% confidence interval: 0–2.7%) had Mycobacterium isolated. Despite exposure to household TB contacts, HIV exposure, and malnutrition, M. tuberculosis bacteremia was not detected in this pediatric febrile cohort, a finding consistent with other pediatric studies. PMID:26324730

  13. Mycobacterium tuberculosis Bacteremia Among Acutely Febrile Children in Western Kenya.

    PubMed

    Pavlinac, Patricia B; Naulikha, Jaqueline M; John-Stewart, Grace C; Onchiri, Frankline M; Okumu, Albert O; Sitati, Ruth R; Cranmer, Lisa M; Lokken, Erica M; Singa, Benson O; Walson, Judd L

    2015-11-01

    In children, Mycobacterium tuberculosis (M. tuberculosis) frequently disseminates systemically, presenting with nonspecific signs including fever. We determined prevalence of M. tuberculosis bacteremia among febrile children presenting to hospitals in Nyanza, Kenya (a region with high human immunodeficiency virus (HIV) and M. tuberculosis prevalence). Between March 2013 and February 2014, we enrolled children aged 6 months to 5 years presenting with fever (axillary temperature ≥ 37.5°C) and no recent antibiotic use. Blood samples were collected for bacterial and mycobacterial culture using standard methods. Among 148 children enrolled, median age was 3.1 years (interquartile range: 1.8-4.1 years); 10.3% of children were living with a household member diagnosed with M. tuberculosis in the last year. Seventeen percent of children were stunted (height-for-age z-score < -2), 18.6% wasted (weight-for-height z-score < -2), 2.7% were HIV-infected, and 14.2% were HIV-exposed uninfected. Seventeen children (11.5%) had one or more signs of tuberculosis (TB). All children had a Bacille Calmette-Guerin vaccination scar. Among 134 viable blood cultures, none (95% confidence interval: 0-2.7%) had Mycobacterium isolated. Despite exposure to household TB contacts, HIV exposure, and malnutrition, M. tuberculosis bacteremia was not detected in this pediatric febrile cohort, a finding consistent with other pediatric studies.

  14. Biosensing Technologies for Mycobacterium tuberculosis Detection: Status and New Developments

    PubMed Central

    Zhou, Lixia; He, Xiaoxiao; He, Dinggeng; Wang, Kemin; Qin, Dilan

    2011-01-01

    Biosensing technologies promise to improve Mycobacterium tuberculosis (M. tuberculosis) detection and management in clinical diagnosis, food analysis, bioprocess, and environmental monitoring. A variety of portable, rapid, and sensitive biosensors with immediate “on-the-spot” interpretation have been developed for M. tuberculosis detection based on different biological elements recognition systems and basic signal transducer principles. Here, we present a synopsis of current developments of biosensing technologies for M. tuberculosis detection, which are classified on the basis of basic signal transducer principles, including piezoelectric quartz crystal biosensors, electrochemical biosensors, and magnetoelastic biosensors. Special attention is paid to the methods for improving the framework and analytical parameters of the biosensors, including sensitivity and analysis time as well as automation of analysis procedures. Challenges and perspectives of biosensing technologies development for M. tuberculosis detection are also discussed in the final part of this paper. PMID:21437177

  15. Biosensing technologies for Mycobacterium tuberculosis detection: status and new developments.

    PubMed

    Zhou, Lixia; He, Xiaoxiao; He, Dinggeng; Wang, Kemin; Qin, Dilan

    2011-01-01

    Biosensing technologies promise to improve Mycobacterium tuberculosis (M. tuberculosis) detection and management in clinical diagnosis, food analysis, bioprocess, and environmental monitoring. A variety of portable, rapid, and sensitive biosensors with immediate "on-the-spot" interpretation have been developed for M. tuberculosis detection based on different biological elements recognition systems and basic signal transducer principles. Here, we present a synopsis of current developments of biosensing technologies for M. tuberculosis detection, which are classified on the basis of basic signal transducer principles, including piezoelectric quartz crystal biosensors, electrochemical biosensors, and magnetoelastic biosensors. Special attention is paid to the methods for improving the framework and analytical parameters of the biosensors, including sensitivity and analysis time as well as automation of analysis procedures. Challenges and perspectives of biosensing technologies development for M. tuberculosis detection are also discussed in the final part of this paper.

  16. Improved understanding of pathogenesis from protein interactions in Mycobacterium tuberculosis.

    PubMed

    Cui, Tao; He, Zheng-Guo

    2014-12-01

    Comprehensive mapping and analysis of protein-protein interactions provide not only systematic approaches for dissecting the infection and survival mechanisms of pathogens but also clues for discovering new antibacterial drug targets. Protein interaction data on Mycobacterium tuberculosis have rapidly accumulated over the past several years. This review summarizes the current progress of protein interaction studies on M. tuberculosis, the causative agent of tuberculosis. These efforts improve our knowledge on the stress response, signaling regulation, protein secretion and drug resistance of the bacteria. M. tuberculosis-host protein interaction studies, although still limited, have recently opened a new door for investigating the pathogenesis of the bacteria. Finally, this review discusses the importance of protein interaction data on identifying and screening new anti-tuberculosis targets and drugs, respectively.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  18. First case of Mycobacterium tuberculosis transmission by heart transplantation from donor to recipient.

    PubMed

    Weile, Jan; Eickmeyer, Holm; Dreier, Jens; Liebke, Michael; Fuchs, Uwe; Wittke, Johann-Wolfgang; Richter, Elvira; Gummert, Jan; Knabbe, Cornelius; Schulz, Uwe

    2013-12-01

    We report the first documented case of a Mycobacterium tuberculosis transmission by an orthotopic heart transplantation from the donor to the recipient. Mycobacterium tuberculosis positive blood culture showed systemic prevalence of the Mycobacteria, however, prophylactic therapy was able to prevent a clinical manifestation of tuberculosis in the recipient.

  19. The methyl-branched fortifications of Mycobacterium tuberculosis.

    PubMed

    Minnikin, David E; Kremer, Laurent; Dover, Lynn G; Besra, Gurdyal S

    2002-05-01

    Mycobacterium tuberculosis continues to be the predominant global infectious agent, annually killing over three million people. Recommended drug regimens have the potential to control tuberculosis, but lack of adherence to such regimens has resulted in the emergence of resistant strains. Mycobacterium tuberculosis has an unusual cell envelope, rich in unique long-chain lipids, that provides a very hydrophobic barrier to antibiotic access. Such lipids, however, can be drug targets, as exemplified by the action of the front-line drug isoniazid on mycolic acid biosynthesis. A number of these lipids are potential key virulence factors and their structures are based on very characteristic methyl-branched long-chain acids and alcohols. This review details the history, structure, and genetic aspects of the biosynthesis of these methyl-branched components, good examples of which are the phthiocerols and the mycocerosic and mycolipenic acids.

  20. Overview on mechanisms of isoniazid action and resistance in Mycobacterium tuberculosis.

    PubMed

    Unissa, Ameeruddin Nusrath; Subbian, Selvakumar; Hanna, Luke Elizabeth; Selvakumar, Nagamiah

    2016-11-01

    Isoniazid (INH) is one of the most active compounds used to treat tuberculosis (TB) worldwide. In addition, INH has been used as a prophylactic drug for individuals with latent Mycobacterium tuberculosis (MTB) infection to prevent reactivation of disease. Importantly, the definition of multidrug resistance (MDR) in TB is based on the resistance of MTB strains to INH and rifampicin (RIF). Despite its simple chemical structure, the mechanism of action of INH is very complex and involves several different concepts. Many pathways pertaining to macromolecular synthesis are affected, notably mycolic acid synthesis. The pro-drug INH is activated by catalase-peroxidase (KatG), and the active INH products are targeted by enzymes namely, enoyl acyl carrier protein (ACP) reductase (InhA) and beta-ketoacyl ACP synthase (KasA). In contrast, INH is inactivated by arylamine N-acetyltransferases (NATs). Consequently, the molecular mechanisms of INH resistance involve several genes in multiple biosynthetic networks and pathways. Mutation in the katG gene is the major cause for INH resistance, followed by inhA, ahpC, kasA, ndh, iniABC,fadE, furA, Rv1592c and Rv1772. The recent association of efflux genes with INH resistance has also gained considerable attention. Interestingly, substitutions have also been observed in nat, fabD, and accD recently in resistant isolates. Understanding the mechanisms operating behind INH action and resistance would enable better detection of INH resistance. This information would aid novel drug design strategies. Herein we review all mechanisms known to potentially contribute to the complexity of INH action and mechanisms of resistance in MTB, with insights into methods for detection of INH resistance as well as their limitations.

  1. A Virtual Screen Discovers Novel, Fragment-Sized Inhibitors of Mycobacterium tuberculosis InhA

    PubMed Central

    Perryman, Alexander L.; Yu, Weixuan; Wang, Xin; Ekins, Sean; Forli, Stefano; Li, Shao-Gang; Freundlich, Joel S.; Tonge, Peter J.; Olson, Arthur J.

    2015-01-01

    Isoniazid (INH) is usually administered to treat latent Mycobacterium tuberculosis (Mtb) infections, and is used in combination therapy to treat active tuberculosis disease (TB). Unfortunately, resistance to this drug is hampering its clinical effectiveness. INH is a prodrug that must be activated by Mtb catalase peroxidase (KatG) before it can inhibit InhA (Mtb enoyl-acyl-carrier-protein reductase). Isoniazid-resistant cases of TB found in clinical settings usually involve mutations in or deletion of katG, which abrogate INH activation. Compounds that inhibit InhA without requiring prior activation by KatG would not be affected by this resistance mechanism and hence would display continued potency against these drug-resistant isolates of Mtb. Virtual screening experiments versus InhA in the GO Fight Against Malaria project (GO FAM) were designed to discover new scaffolds that display base stacking interactions with the NAD cofactor. GO FAM experiments included targets from other pathogens, including Mtb, when they had structural similarity to a malaria target. Eight of the sixteen soluble compounds identified by docking against InhA plus visual inspection were modest inhibitors and did not require prior activation by KatG. The best two inhibitors discovered are both fragment-sized compounds and displayed Ki values of 54 and 59 μM, respectively. Importantly, the novel inhibitors discovered have low structural similarity to known InhA inhibitors and, thus, help expand the number of chemotypes on which future medicinal chemistry efforts can be focused. These new fragment hits could eventually help advance the fight against INH-resistant Mtb strains, which pose a significant global health threat. PMID:25636146

  2. Mycobacterium tuberculosis resistance to antituberculosis drugs in Mozambique*, **

    PubMed Central

    Pires, Germano Manuel; Folgosa, Elena; Nquobile, Ndlovu; Gitta, Sheba; Cadir, Nureisha

    2014-01-01

    OBJECTIVE: To determine the drug resistance profile of Mycobacterium tuberculosis in Mozambique. METHODS: We analyzed secondary data from the National Tuberculosis Referral Laboratory, in the city of Maputo, Mozambique, and from the Beira Regional Tuberculosis Referral Laboratory, in the city of Beira, Mozambique. The data were based on culture-positive samples submitted to first-line drug susceptibility testing (DST) between January and December of 2011. We attempted to determine whether the frequency of DST positivity was associated with patient type or provenance. RESULTS: During the study period, 641 strains were isolated in culture and submitted to DST. We found that 374 (58.3%) were resistant to at least one antituberculosis drug and 280 (43.7%) were resistant to multiple antituberculosis drugs. Of the 280 multidrug-resistant tuberculosis cases, 184 (65.7%) were in previously treated patients, most of whom were from southern Mozambique. Two (0.71%) of the cases of multidrug-resistant tuberculosis were confirmed to be cases of extensively drug-resistant tuberculosis. Multidrug-resistant tuberculosis was most common in males, particularly those in the 21-40 year age bracket. CONCLUSIONS: M. tuberculosis resistance to antituberculosis drugs is high in Mozambique, especially in previously treated patients. The frequency of M. tuberculosis strains that were resistant to isoniazid, rifampin, and streptomycin in combination was found to be high, particularly in samples from previously treated patients. PMID:24831398

  3. Mycobacterium tuberculosis resistance to antituberculosis drugs in Mozambique.

    PubMed

    Pires, Germano Manuel; Folgosa, Elena; Nquobile, Ndlovu; Gitta, Sheba; Cadir, Nureisha

    2014-01-01

    To determine the drug resistance profile of Mycobacterium tuberculosis in Mozambique. We analyzed secondary data from the National Tuberculosis Referral Laboratory, in the city of Maputo, Mozambique, and from the Beira Regional Tuberculosis Referral Laboratory, in the city of Beira, Mozambique. The data were based on culture-positive samples submitted to first-line drug susceptibility testing (DST) between January and December of 2011. We attempted to determine whether the frequency of DST positivity was associated with patient type or provenance. During the study period, 641 strains were isolated in culture and submitted to DST. We found that 374 (58.3%) were resistant to at least one antituberculosis drug and 280 (43.7%) were resistant to multiple antituberculosis drugs. Of the 280 multidrug-resistant tuberculosis cases, 184 (65.7%) were in previously treated patients, most of whom were from southern Mozambique. Two (0.71%) of the cases of multidrug-resistant tuberculosis were confirmed to be cases of extensively drug-resistant tuberculosis. Multidrug-resistant tuberculosis was most common in males, particularly those in the 21-40 year age bracket. M. tuberculosis resistance to antituberculosis drugs is high in Mozambique, especially in previously treated patients. The frequency of M. tuberculosis strains that were resistant to isoniazid, rifampin, and streptomycin in combination was found to be high, particularly in samples from previously treated patients.

  4. Rapid diagnosis of Mycobacterium tuberculosis bacteremia by PCR.

    PubMed

    Folgueira, L; Delgado, R; Palenque, E; Aguado, J M; Noriega, A R

    1996-03-01

    A method based on DNA amplification and hybridization has been used for the rapid detection of Mycobacterium tuberculosis in blood samples from 38 hospitalized patients (15 human immunodeficiency virus [HIV] positive and 23 HIV negative) in whom localized or disseminated forms of tuberculosis were suspected. In 32 of these patients, the diagnosis of tuberculosis was eventually confirmed by conventional bacteriological or histological procedures. M. tuberculosis DNA was detected with the PCR technique in the peripheral blood mononuclear cells from 9 of 11 (82%) HIV-infected patients and in 7 of 21 (33%) HIV-negative patients (P < 0.01), while M. tuberculosis blood cultures were positive in 1 of 8 (12.5%) and 1 of 18 (5.5%) patients, respectively. PCR was positive in all cases with disseminated disease in both HIV-negative and HIV-positive patients and also in the HIV-positive patients with extrapulmonary tuberculosis. Seven samples from patients with documented illness other than tuberculosis and 12 specimens from healthy volunteers, including seven volunteers with a recent positive purified protein derivative test, were used as controls and had a negative PCR. These results suggest that detection of M. tuberculosis DNA in peripheral blood mononuclear cells may be a useful tool for rapid diagnosis of disseminated and extrapulmonary forms of tuberculosis, especially in an HIV-positive population.

  5. Rapid diagnosis of Mycobacterium tuberculosis bacteremia by PCR.

    PubMed Central

    Folgueira, L; Delgado, R; Palenque, E; Aguado, J M; Noriega, A R

    1996-01-01

    A method based on DNA amplification and hybridization has been used for the rapid detection of Mycobacterium tuberculosis in blood samples from 38 hospitalized patients (15 human immunodeficiency virus [HIV] positive and 23 HIV negative) in whom localized or disseminated forms of tuberculosis were suspected. In 32 of these patients, the diagnosis of tuberculosis was eventually confirmed by conventional bacteriological or histological procedures. M. tuberculosis DNA was detected with the PCR technique in the peripheral blood mononuclear cells from 9 of 11 (82%) HIV-infected patients and in 7 of 21 (33%) HIV-negative patients (P < 0.01), while M. tuberculosis blood cultures were positive in 1 of 8 (12.5%) and 1 of 18 (5.5%) patients, respectively. PCR was positive in all cases with disseminated disease in both HIV-negative and HIV-positive patients and also in the HIV-positive patients with extrapulmonary tuberculosis. Seven samples from patients with documented illness other than tuberculosis and 12 specimens from healthy volunteers, including seven volunteers with a recent positive purified protein derivative test, were used as controls and had a negative PCR. These results suggest that detection of M. tuberculosis DNA in peripheral blood mononuclear cells may be a useful tool for rapid diagnosis of disseminated and extrapulmonary forms of tuberculosis, especially in an HIV-positive population. PMID:8904404

  6. Identification of proteins from Mycobacterium tuberculosis missing in attenuated Mycobacterium bovis BCG strains.

    PubMed

    Mattow, J; Jungblut, P R; Schaible, U E; Mollenkopf, H J; Lamer, S; Zimny-Arndt, U; Hagens, K; Müller, E C; Kaufmann, S H

    2001-08-01

    A proteome approach, combining high-resolution two-dimensional electrophoresis (2-DE) with mass spectrometry, was used to compare the cellular protein composition of two virulent strains of Mycobacterium tuberculosis with two attenuated strains of Mycobacterium bovis Bacillus Calmette-Guerin (BCG), in order to identify unique proteins of these strains. Emphasis was given to the identification of M. tuberculosis specific proteins, because we consider these proteins to represent putative virulence factors and interesting candidates for vaccination and diagnosis of tuberculosis. The genome of M. tuberculosis strain H37Rv comprises nearly 4000 predicted open reading frames. In contrast, the separation of proteins from whole mycobacterial cells by 2-DE resulted in silver-stained patterns comprising about 1800 distinct protein spots. Amongst these, 96 spots were exclusively detected either in the virulent (56 spots) or in the attenuated (40 spots) mycobacterial strains. Fifty-three of these spots were analyzed by mass spectrometry, of which 41 were identified, including 32 M. tuberculosis specific spots. Twelve M. tuberculosis specific spots were identified as proteins, encoded by genes previously reported to be deleted in M. bovis BCG. The remaining 20 spots unique for M. tuberculosis were identified as proteins encoded by genes that are not known to be missing in M. bovis BCG.

  7. Asymmetric cell division in Mycobacterium tuberculosis and its unique features.

    PubMed

    Vijay, Srinivasan; Nagaraja, Mukkayyan; Sebastian, Jees; Ajitkumar, Parthasarathi

    2014-03-01

    Recently, several reports showed that about 80 % of mid-log phase Mycobacterium smegmatis, Mycobacterium marinum, and Mycobacterium bovis BCG cells divide symmetrically with 5-10 % deviation in the septum position from the median. However, the mode of cell division of the pathogenic mycobacterial species, Mycobacterium tuberculosis, remained unclear. Therefore, in the present study, using electron microscopy, fluorescence microscopy of septum- and nucleoid-stained live and fixed cells, and live cell time-lapse imaging, we show the occurrence of asymmetric cell division with unusually deviated septum/constriction in 20 % of the 15 % septating M. tuberculosis cells in the mid-log phase population. The remaining 80 % of the 15 % septating cells divided symmetrically but with 2-5 % deviation in the septum/constriction position, as reported for M. smegmatis, M. marinum, and M. bovis BCG cells. Both the long and the short portions of the asymmetrically dividing M. tuberculosis cells with unusually deviated septum contained nucleoids, thereby generating viable short and long cells from each asymmetric division. M. tuberculosis short cells were acid fast positive and, like the long cells, further readily underwent growth and division to generate micro-colony, thereby showing that they were neither mini cells, spores nor dormant forms of mycobacteria. The freshly diagnosed pulmonary tuberculosis patients' sputum samples, which are known for the prevalence of oxidative stress conditions, also contained short cells at the same proportion as that in the mid-log phase population. The probable physiological significance of the generation of the short cells through unusually deviated asymmetric cell division is discussed.

  8. Pyrosequencing assay for rapid identification of Mycobacterium tuberculosis complex species

    PubMed Central

    2011-01-01

    Background Identification of the Mycobacterium tuberculosis complex organisms to the species level is important for diagnostic, therapeutic and epidemiologic perspectives. Indeed, isolates are routinely identified as belonging to the M. tuberculosis complex without further discrimination in agreement with the high genomic similarity of the M. tuberculosis complex members and the resulting complex available identification tools. Findings We herein develop a pyrosequencing assay analyzing polymorphisms within glpK, pykA and gyrB genes to identify members of the M. tuberculosis complex at the species level. The assay was evaluated with 22 M. tuberculosis, 21 M. bovis, 3 M. caprae, 3 M. microti, 2 M. bovis BCG, 2 M. pinnipedii, 1 M. canettii and 1 M. africanum type I isolates. The resulted pyrograms were consistent with conventional DNA sequencing data and successfully identified all isolates. Additionally, 127 clinical M. tuberculosis complex isolates were analyzed and were unambiguously identified as M. tuberculosis. Conclusion We proposed a pyrosequencing-based scheme for the rapid identification of M. tuberculosis complex isolates at the species level. The assay is robust, specific, rapid and can be easily introduced in the routine activity. PMID:22011383

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

    PubMed Central

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

    2013-01-01

    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. PMID:23778657

  10. Proteomic analysis of extracellular vesicles derived from Mycobacterium tuberculosis.

    PubMed

    Lee, Jaewook; Kim, Si-Hyun; Choi, Dong-Sic; Lee, Jong Seok; Kim, Dae-Kyum; Go, Gyeongyun; Park, Seon-Min; Kim, Si Hyun; Shin, Jeong Hwan; Chang, Chulhun L; Gho, Yong Song

    2015-10-01

    The release of extracellular vesicles, also known as outer membrane vesicles, membrane vesicles, exosomes, and microvesicles, is an evolutionarily conserved phenomenon from bacteria to eukaryotes. It has been reported that Mycobacterium tuberculosis releases extracellular vesicles harboring immunologically active molecules, and these extracellular vesicles have been suggested to be applicable in vaccine development and biomarker discovery. However, the comprehensive proteomic analysis has not been performed for M. tuberculosis extracellular vesicles. In this study, we identified a total of 287 vesicular proteins by four LC-MS/MS analyses with high confidence. In addition, we identified several vesicular proteins associated with the virulence of M. tuberculosis. This comprehensive proteome profile will help elucidate the pathogenic mechanism of M. tuberculosis. The data have been deposited to the ProteomeXchange with identifier PXD001160 (http://proteomecentral.proteomexchange.org/dataset/PXD001160). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Dissection of Mycobacterium tuberculosis antigens using recombinant DNA.

    PubMed Central

    Young, R A; Bloom, B R; Grosskinsky, C M; Ivanyi, J; Thomas, D; Davis, R W

    1985-01-01

    A recombinant DNA strategy has been used systematically to survey the Mycobacterium tuberculosis genome for sequences that encode specific antigens detected by monoclonal antibodies. M. tuberculosis genomic DNA fragments with randomly generated endpoints were used to construct a large lambda gt11 recombinant DNA expression library. Sufficient numbers of recombinants were produced to contain inserts whose endpoints occur at nearly every base pair in the pathogen genome. Protein antigens specified by linear segments of pathogen DNA and produced by the recombinant phage of Escherichia coli were screened with monoclonal antibody probes. This approach was coupled with an improved detection method for gene isolation using antibodies to clonally isolate DNA sequences that specify polypeptide components of M. tuberculosis. The methodology described here, which is applicable to other pathogens, offers possibilities for the development of more sensitive and specific immunodiagnostic and seroepidemiological tests for tuberculosis and, ultimately, for the development of more effective vaccines. Images PMID:2581251

  12. Evaluation of the semiautomated Abbott LCx Mycobacterium tuberculosis assay for direct detection of Mycobacterium tuberculosis in respiratory specimens.

    PubMed Central

    Ausina, V; Gamboa, F; Gazapo, E; Manterola, J M; Lonca, J; Matas, L; Manzano, J R; Rodrigo, C; Cardona, P J; Padilla, E

    1997-01-01

    Five hundred twenty processed respiratory specimens from 326 patients received for the diagnosis of tuberculosis or other mycobacterial infections were tested by means of the LCx Mycobacterium tuberculosis Assay from Abbott Laboratories, which uses ligase chain reaction technology for the direct detection of M. tuberculosis complex in respiratory specimens. The results of the LCx M. tuberculosis Assay were compared with the results of culture and staining techniques. After a combination of culture results and the patient's clinical data, a total of 195 specimens were collected from 110 patients who were positively diagnosed as having pulmonary tuberculosis. Twenty-three of these 195 specimens which corresponded to 10 patients with a history of pulmonary tuberculosis (TB) and anti-TB treatment ranging from 1 to 6 months were culture negative. The other 172 specimens were culture positive for M. tuberculosis. With an overall positivity rate of 37.5% (195 of 520 specimens), the sensitivity, specificity, and positive and negative predictive values were 90.8, 100, 100, and 94.7%, respectively, for the LCx M. tuberculosis Assay; 88.2, 100, 100, and 93.4%, respectively, for culture; and 82.6, 92, 72.9, and 97.6%, respectively, for acid-fast staining. For 161 specimens (82.6%) from patients smear positive for the disease and 34 specimens (17.4%) from patients smear negative for the disease, the sensitivity values for the LCx M. tuberculosis Assay were 98.8 and 53%, respectively. There were no statistically significant differences in the sensitivities and specificities between the LCx M. tuberculosis Assay and culture (P > 0.05). Conclusively, the LCx M. tuberculosis Assay has proved to have an acceptable sensitivity and a high specificity in detecting M. tuberculosis and has the potential of reducing the diagnosis time to an 8-h working day. PMID:9230369

  13. Dispersal of Mycobacterium tuberculosis via the Canadian fur trade

    PubMed Central

    Pepperell, Caitlin S.; Granka, Julie M.; Alexander, David C.; Behr, Marcel A.; Chui, Linda; Gordon, Janet; Guthrie, Jennifer L.; Jamieson, Frances B.; Langlois-Klassen, Deanne; Long, Richard; Nguyen, Dao; Wobeser, Wendy; Feldman, Marcus W.

    2011-01-01

    Patterns of gene flow can have marked effects on the evolution of populations. To better understand the migration dynamics of Mycobacterium tuberculosis, we studied genetic data from European M. tuberculosis lineages currently circulating in Aboriginal and French Canadian communities. A single M. tuberculosis lineage, characterized by the DS6Quebec genomic deletion, is at highest frequency among Aboriginal populations in Ontario, Saskatchewan, and Alberta; this bacterial lineage is also dominant among tuberculosis (TB) cases in French Canadians resident in Quebec. Substantial contact between these human populations is limited to a specific historical era (1710–1870), during which individuals from these populations met to barter furs. Statistical analyses of extant M. tuberculosis minisatellite data are consistent with Quebec as a source population for M. tuberculosis gene flow into Aboriginal populations during the fur trade era. Historical and genetic analyses suggest that tiny M. tuberculosis populations persisted for ∼100 y among indigenous populations and subsequently expanded in the late 19th century after environmental changes favoring the pathogen. Our study suggests that spread of TB can occur by two asynchronous processes: (i) dispersal of M. tuberculosis by minimal numbers of human migrants, during which small pathogen populations are sustained by ongoing migration and slow disease dynamics, and (ii) expansion of the M. tuberculosis population facilitated by shifts in host ecology. If generalizable, these migration dynamics can help explain the low DNA sequence diversity observed among isolates of M. tuberculosis and the difficulties in global elimination of tuberculosis, as small, widely dispersed pathogen populations are difficult both to detect and to eradicate. PMID:21464295

  14. Dispersal of Mycobacterium tuberculosis via the Canadian fur trade.

    PubMed

    Pepperell, Caitlin S; Granka, Julie M; Alexander, David C; Behr, Marcel A; Chui, Linda; Gordon, Janet; Guthrie, Jennifer L; Jamieson, Frances B; Langlois-Klassen, Deanne; Long, Richard; Nguyen, Dao; Wobeser, Wendy; Feldman, Marcus W

    2011-04-19

    Patterns of gene flow can have marked effects on the evolution of populations. To better understand the migration dynamics of Mycobacterium tuberculosis, we studied genetic data from European M. tuberculosis lineages currently circulating in Aboriginal and French Canadian communities. A single M. tuberculosis lineage, characterized by the DS6(Quebec) genomic deletion, is at highest frequency among Aboriginal populations in Ontario, Saskatchewan, and Alberta; this bacterial lineage is also dominant among tuberculosis (TB) cases in French Canadians resident in Quebec. Substantial contact between these human populations is limited to a specific historical era (1710-1870), during which individuals from these populations met to barter furs. Statistical analyses of extant M. tuberculosis minisatellite data are consistent with Quebec as a source population for M. tuberculosis gene flow into Aboriginal populations during the fur trade era. Historical and genetic analyses suggest that tiny M. tuberculosis populations persisted for ∼100 y among indigenous populations and subsequently expanded in the late 19th century after environmental changes favoring the pathogen. Our study suggests that spread of TB can occur by two asynchronous processes: (i) dispersal of M. tuberculosis by minimal numbers of human migrants, during which small pathogen populations are sustained by ongoing migration and slow disease dynamics, and (ii) expansion of the M. tuberculosis population facilitated by shifts in host ecology. If generalizable, these migration dynamics can help explain the low DNA sequence diversity observed among isolates of M. tuberculosis and the difficulties in global elimination of tuberculosis, as small, widely dispersed pathogen populations are difficult both to detect and to eradicate.

  15. Antimicrobial resistance in Mycobacterium tuberculosis: mechanistic and evolutionary perspectives.

    PubMed

    Gygli, Sebastian M; Borrell, Sonia; Trauner, Andrej; Gagneux, Sebastien

    2017-03-25

    Antibiotic-resistant Mycobacterium tuberculosis strains are threatening progress in containing the global tuberculosis epidemic. Mycobacterium tuberculosis is intrinsically resistant to many antibiotics, limiting the number of compounds available for treatment. This intrinsic resistance is due to a number of mechanisms including a thick, waxy, hydrophobic cell envelope and the presence of drug degrading and modifying enzymes. Resistance to the drugs which are active against M. tuberculosis is, in the absence of horizontally transferred resistance determinants, conferred by chromosomal mutations. These chromosomal mutations may confer drug resistance via modification or overexpression of the drug target, as well as by prevention of prodrug activation. Drug resistance mutations may have pleiotropic effects leading to a reduction in the bacterium's fitness, quantifiable e.g. by a reduction in the in vitro growth rate. Secondary so-called compensatory mutations, not involved in conferring resistance, can ameliorate the fitness cost by interacting epistatically with the resistance mutation. Although the genetic diversity of M. tuberculosis is low compared to other pathogenic bacteria, the strain genetic background has been demonstrated to influence multiple aspects in the evolution of drug resistance. The rate of resistance evolution and the fitness costs of drug resistance mutations may vary as a function of the genetic background.

  16. Early Events in Mycobacterium tuberculosis Infection in Cynomolgus Macaques†

    PubMed Central

    Lin, Philana Ling; Pawar, Santosh; Myers, Amy; Pegu, Amarenda; Fuhrman, Carl; Reinhart, Todd A.; Capuano, Saverio V.; Klein, Edwin; Flynn, JoAnne L.

    2006-01-01

    Little is known regarding the early events of infection of humans with Mycobacterium tuberculosis. The cynomolgus macaque is a useful model of tuberculosis, with strong similarities to human tuberculosis. In this study, eight cynomolgus macaques were infected bronchoscopically with low-dose M. tuberculosis; clinical, immunologic, microbiologic, and pathologic events were assessed 3 to 6 weeks postinfection. Gross pathological abnormalities were observed as early as 3 weeks, including Ghon complex formation by 5 weeks postinfection. Caseous granulomas were observed in the lung as early as 4 weeks postinfection. Only caseous granulomas were observed in the lungs at these early time points, reflecting a rigorous initial response. T-cell activation (CD29 and CD69) and chemokine receptor (CXCR3 and CCR5) expression appeared localized to different anatomic sites. Activation markers were increased on cells from airways and only at modest levels on cells in peripheral blood. The priming of mycobacterium-specific T cells, characterized by the production of gamma interferon occurred slowly, with responses seen only after 4 weeks of infection. These responses were observed from T lymphocytes in blood, airways, and hilar lymph node, with responses predominantly localized to the site of infection. From these studies, we conclude that immune responses to M. tuberculosis are relatively slow in the local and peripheral compartments and that necrosis occurs surprisingly quickly during granuloma formation. PMID:16790751

  17. Structures of citrate synthase and malate dehydrogenase of Mycobacterium tuberculosis.

    PubMed

    Ferraris, Davide M; Spallek, Ralf; Oehlmann, Wulf; Singh, Mahavir; Rizzi, Menico

    2015-02-01

    The tricarboxylic acid (TCA) cycle is a central metabolic pathway of all aerobic organisms and is responsible for the synthesis of many important precursors and molecules. TCA cycle plays a key role in the metabolism of Mycobacterium tuberculosis and is involved in the adaptation process of the bacteria to the host immune response. We present here the first crystal structures of M. tuberculosis malate dehydrogenase and citrate synthase, two consecutive enzymes of the TCA, at 2.6 Å and 1.5 Å resolution, respectively. General analogies and local differences with the previously reported homologous protein structures are described. © 2014 Wiley Periodicals, Inc.

  18. Zirconia based nucleic acid sensor for Mycobacterium tuberculosis detection

    NASA Astrophysics Data System (ADS)

    Das, Maumita; Sumana, Gajjala; Nagarajan, R.; Malhotra, B. D.

    2010-03-01

    Nanostructured zirconium oxide (ZrO2) film (particle size˜35 nm), electrochemically deposited onto gold(Au) surface, has been used to immobilize 21-mer oligonucleotide probe (ssDNA) specific to Mycobacterium tuberculosis by utilizing affinity between oxygen atom of phosphoric group and zirconium to fabricate DNA biosensor. This DNA-ZrO2/Au bioelectrode, characterized using x-ray diffraction, Fourier transform infrared spectroscopy, cyclic voltammetry, and scanning electron microscopy techniques, can be used for early and rapid diagnosis of M. tuberculosis with detection limit of 0.065 ng/μL within 60s.

  19. Cholesterol catabolism as a therapeutic target in Mycobacterium tuberculosis

    PubMed Central

    Ouellet, Hugues; Johnston, Jonathan B.; Ortiz de Montellano, Paul R.

    2011-01-01

    Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that infects 10 million worldwide and kills 2 million people every year. The uptake and utilization of nutrients by Mtb within the host cell is still poorly understood, although lipids play an important role in Mtb persistence. The recent identification of a large regulon of cholesterol catabolic genes suggests that Mtb can use host sterol for infection and persistence. In this review, we report on recent progress in elucidation of the Mtb cholesterol catabolic reactions and their potential utility as targets for tuberculosis therapeutic agents. PMID:21924910

  20. Evaluation of the inhibitory activity of (aza)isoindolinone-type compounds: toward in vitro InhA action, Mycobacterium tuberculosis growth and mycolic acid biosynthesis.

    PubMed

    Chollet, Aurélien; Stigliani, Jean-Luc; Pasca, Maria Rosalia; Mori, Giorgia; Lherbet, Christian; Constant, Patricia; Quémard, Annaïk; Bernadou, Jean; Pratviel, Geneviève; Bernardes-Génisson, Vania

    2016-11-01

    Inhibitors of the Mycobacterium tuberculosis enoyl-ACP reductase (InhA) are considered as potential promising therapeutics for the treatment of tuberculosis. Previously, we reported that azaisoindolinone-type compounds displayed, in vitro, inhibitory activity toward InhA. Herein, we describe chemical modifications of azaisoindolinone scaffold, the synthesis of 15 new compounds and their evaluations toward the in vitro InhA activity. Based on these results, a structure-InhA inhibitory activity relationship analysis and a molecular docking study, using the conformation of InhA found in the 2H7M crystal structure, were carried out to predict a possible mode of interaction of the best (aza)isoindolinone-type inhibitors with InhA in vitro. Then, the work was extended toward evaluations of these compounds against Mycobacterium tuberculosis (Mtb) growth, and finally, some of them were also investigated in respect of their ability to inhibit mycolic acid biosynthesis inside mycobacteria. Although, some azaisoindolinones were able to inhibit InhA activity and Mtb growth in vitro, they did not inhibit the mycolic acid biosynthesis inside Mtb.

  1. Novel human recombinant antibodies against Mycobacterium tuberculosis antigen 85B.

    PubMed

    Fuchs, Manon; Kämpfer, Susanne; Helmsing, Saskia; Spallek, Ralf; Oehlmann, Wulf; Prilop, Wiebke; Frank, Ronald; Dübel, Stefan; Singh, Mahavir; Hust, Michael

    2014-07-17

    Tuberculosis is the leading cause of death due to bacterial infections worldwide, mainly caused by Mycobacterium tuberculosis. The antigen 85 complex comprises a set of major secreted proteins of M. tuberculosis, which are potential biomarkers for diagnostic. In this work, the first human single chain fragment variable (scFv) antibodies specific for the tuberculosis biomarker 85 B were selected by phage display from naïve antibody gene libraries (HAL7/8). Produced as scFv-Fc in mammalian cells, these antibodies were further characterized and analysed for specificity and applicability in different tuberculosis antigen detection assays. Sandwich detection of recombinant 85 B was successful in enzyme linked immunosorbent assay (ELISA), lateral flow immunoassay and immunoblot. Whereas detection of M. tuberculosis cell extracts and culture filtrates was only possible in direct ELISA and immunoblot assays. It was found that the conformation of 85 B, depending on sample treatment, influenced antigen detection. Recombinant antibodies, selected by phage display, may be applicable for 85 B detection in various assays. These antibodies are candidates for the development of future point of care tuberculosis diagnostic kits. Using 85 B as a biomarker, the antigen conformation influenced by sample treatment is important.

  2. Novel human recombinant antibodies against Mycobacterium tuberculosis antigen 85B

    PubMed Central

    2014-01-01

    Background Tuberculosis is the leading cause of death due to bacterial infections worldwide, mainly caused by Mycobacterium tuberculosis. The antigen 85 complex comprises a set of major secreted proteins of M. tuberculosis, which are potential biomarkers for diagnostic. Results In this work, the first human single chain fragment variable (scFv) antibodies specific for the tuberculosis biomarker 85 B were selected by phage display from naïve antibody gene libraries (HAL7/8). Produced as scFv-Fc in mammalian cells, these antibodies were further characterized and analysed for specificity and applicability in different tuberculosis antigen detection assays. Sandwich detection of recombinant 85 B was successful in enzyme linked immunosorbent assay (ELISA), lateral flow immunoassay and immunoblot. Whereas detection of M. tuberculosis cell extracts and culture filtrates was only possible in direct ELISA and immunoblot assays. It was found that the conformation of 85 B, depending on sample treatment, influenced antigen detection. Conclusions Recombinant antibodies, selected by phage display, may be applicable for 85 B detection in various assays. These antibodies are candidates for the development of future point of care tuberculosis diagnostic kits. Using 85 B as a biomarker, the antigen conformation influenced by sample treatment is important. PMID:25033887

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

    PubMed Central

    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

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

    PubMed

    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

    2016-03-01

    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.

  5. Carbapenems and Rifampin Exhibit Synergy against Mycobacterium tuberculosis and Mycobacterium abscessus.

    PubMed

    Kaushik, Amit; Makkar, Nayani; Pandey, Pooja; Parrish, Nicole; Singh, Urvashi; Lamichhane, Gyanu

    2015-10-01

    An effective regimen for treatment of tuberculosis (TB) is comprised of multiple drugs that inhibit a range of essential cellular activities in Mycobacterium tuberculosis. The effectiveness of a regimen is further enhanced if constituent drugs act with synergy. Here, we report that faropenem (a penem) or biapenem, doripenem, or meropenem (carbapenems), which belong to the β-lactam class of antibiotics, and rifampin, one of the drugs that forms the backbone of TB treatment, act with synergy when combined. One of the reasons (carba)penems are seldom used for treatment of TB is the high dosage levels required, often at the therapeutic limits. The synergistic combination of rifampin and these (carba)penems indicates that (carba)penems can be administered at dosages that are therapeutically relevant. The combination of faropenem and rifampin also limits the frequency of resistant mutants, as we were unable to obtain spontaneous mutants in the presence of these two drugs. The combinations of rifampin and (carba)penems were effective not only against drug-sensitive Mycobacterium tuberculosis but also against drug-resistant clinical isolates that are otherwise resistant to rifampin. A combination of doripenem or biapenem and rifampin also exhibited synergistic activity against Mycobacterium abscessus. Although the MICs of these three drugs alone against M. abscessus are too high to be of clinical relevance, their concentrations in combinations are therapeutically relevant; therefore, they warrant further evaluation for clinical utility to treat Mycobacterium abscessus infection, especially in cystic fibrosis patients.

  6. Genotyping of Mycobacterium tuberculosis: application in epidemiologic studies

    PubMed Central

    Kato-Maeda, Midori; Metcalfe, John Z.; Flores, Laura

    2014-01-01

    Genotyping is used to track specific isolates of Mycobacterium tuberculosis in a community. It has been successfully used in epidemiologic research (termed ‘molecular epidemiology’) to study the transmission dynamics of TB. In this article, we review the genetic markers used in molecular epidemiologic studies including the use of whole-genome sequencing technology. We also review the public health application of molecular epidemiologic tools. PMID:21366420

  7. Cell death paradigms in the pathogenesis of Mycobacterium tuberculosis infection

    PubMed Central

    Parandhaman, Dinesh Kumar; Narayanan, Sujatha

    2014-01-01

    Cell death or senescence is a fundamental event that helps maintain cellular homeostasis, shapes the growth of organism, and provides protective immunity against invading pathogens. Decreased or increased cell death is detrimental both in infectious and non-infectious diseases. Cell death is executed both by regulated enzymic reactions and non-enzymic sudden collapse. In this brief review we have tried to summarize various cell death modalities and their impact on the pathogenesis of Mycobacterium tuberculosis. PMID:24634891

  8. Cell death paradigms in the pathogenesis of Mycobacterium tuberculosis infection.

    PubMed

    Parandhaman, Dinesh Kumar; Narayanan, Sujatha

    2014-01-01

    Cell death or senescence is a fundamental event that helps maintain cellular homeostasis, shapes the growth of organism, and provides protective immunity against invading pathogens. Decreased or increased cell death is detrimental both in infectious and non-infectious diseases. Cell death is executed both by regulated enzymic reactions and non-enzymic sudden collapse. In this brief review we have tried to summarize various cell death modalities and their impact on the pathogenesis of Mycobacterium tuberculosis.

  9. Immune subdominant antigens as vaccine candidates against Mycobacterium tuberculosis.

    PubMed

    Orr, Mark T; Ireton, Gregory C; Beebe, Elyse A; Huang, Po-Wei D; Reese, Valerie A; Argilla, David; Coler, Rhea N; Reed, Steven G

    2014-09-15

    Unlike most pathogens, many of the immunodominant epitopes from Mycobacterium tuberculosis are under purifying selection. This startling finding suggests that M. tuberculosis may gain an evolutionary advantage by focusing the human immune response against selected proteins. Although the implications of this to vaccine development are incompletely understood, it has been suggested that inducing strong Th1 responses against Ags that are only weakly recognized during natural infection may circumvent this evasion strategy and increase vaccine efficacy. To test the hypothesis that subdominant and/or weak M. tuberculosis Ags are viable vaccine candidates and to avoid complications because of differential immunodominance hierarchies in humans and experimental animals, we defined the immunodominance hierarchy of 84 recombinant M. tuberculosis proteins in experimentally infected mice. We then combined a subset of these dominant or subdominant Ags with a Th1 augmenting adjuvant, glucopyranosyl lipid adjuvant in stable emulsion, to assess their immunogenicity in M. tuberculosis-naive animals and protective efficacy as measured by a reduction in lung M. tuberculosis burden of infected animals after prophylactic vaccination. We observed little correlation between immunodominance during primary M. tuberculosis infection and vaccine efficacy, confirming the hypothesis that subdominant and weakly antigenic M. tuberculosis proteins are viable vaccine candidates. Finally, we developed two fusion proteins based on strongly protective subdominant fusion proteins. When paired with the glucopyranosyl lipid adjuvant in stable emulsion, these fusion proteins elicited robust Th1 responses and limited pulmonary M. tuberculosis for at least 6 wk postinfection with a single immunization. These findings expand the potential pool of M. tuberculosis proteins that can be considered as vaccine Ag candidates. Copyright © 2014 by The American Association of Immunologists, Inc.

  10. Mycobacterium tuberculosis: implications for district nurses.

    PubMed

    James, Stephanie; Watson, Michael

    2010-10-01

    Despite the belief of many health professionals, tuberculosis is not a disease of the past but is on the increase (Department of Health (DH), 2004) and the UK has seen a year on year increase in the number of new cases (Health Protection Agency, 2008). The DH have made a number of recommendations to combat this increase and one of those recommendations is to raise awareness among health staff (2004). This review has set out to examine district nurses' knowledge about tuberculosis and the consequences of poor knowledge. Five themes emerged from the literature search with the most prominent being the subject of adherence and how this could be addressed. The review has identified that district nurses should have a greater knowledge of tuberculosis and patient treatment could be improved by the nurse having a better understanding about tuberculosis care.

  11. EmbA is an essential arabinosyltransferase in Mycobacterium tuberculosis

    PubMed Central

    Amin, Anita G.; Goude, Renan; Shi, Libin; Zhang, Jian; Chatterjee, Delphi; Parish, Tanya

    2008-01-01

    The Emb proteins (EmbA, EmbB, EmbC) are mycobacterial arabinosyltransferases involved in the biogenesis of the mycobacterial cell wall. EmbA and EmbB are predicted to work in unison as a heterodimer. EmbA and EmbB are involved in the formation of the crucial terminal hexaarabinoside motif [Araβ(1→2)Araα(1→5)] [Araβ(1→2)Araα(1→3)]Araα(1→5)Araα1→(Ara6) in the cell wall polysaccharide arabinogalactan. Studies conducted in Mycobacterium smegmatis revealed that mutants with disruptions in embA or embB are viable, although the growth rate was affected. In contrast, we demonstrate here that embA is an essential gene in Mycobacterium tuberculosis, since a deletion of the chromosomal gene could only be achieved when a second functional copy was provided on an integrated vector. Complementation of an embA mutant of M. smegmatis by M. tuberculosis embA confirmed that it encodes a functional arabinosyltransferase. We identified a promoter for M. tuberculosis embA located immediately upstream of the gene, indicating that it is expressed independently from the upstream gene, embC. Promoter activity from PembA(Mtb) was sevenfold lower when assayed in M. smegmatis compared to M. tuberculosis, indicating that the latter is not a good host for genetic analysis of M. tuberculosis embA expression. PembA(Mtb) activity remained constant throughout growth phases and after stress treatment, although it was reduced during hypoxia-induced non-replicating persistence. Ethambutol exposure had no effect on PembA(Mtb) activity. These data demonstrate that M. tuberculosis embA encodes a functional arabinosyltransferase which is constitutively expressed and plays a critical role in M. tuberculosis. PMID:18174142

  12. EmbA is an essential arabinosyltransferase in Mycobacterium tuberculosis.

    PubMed

    Amin, Anita G; Goude, Renan; Shi, Libin; Zhang, Jian; Chatterjee, Delphi; Parish, Tanya

    2008-01-01

    The Emb proteins (EmbA, EmbB, EmbC) are mycobacterial arabinosyltransferases involved in the biogenesis of the mycobacterial cell wall. EmbA and EmbB are predicted to work in unison as a heterodimer. EmbA and EmbB are involved in the formation of the crucial terminal hexaarabinoside motif [Arabeta(1-->2)Araalpha(1-->5)] [Arabeta(1-->2)Araalpha(1-->3)]Araalpha(1-->5)Araalpha1-->(Ara(6)) in the cell wall polysaccharide arabinogalactan. Studies conducted in Mycobacterium smegmatis revealed that mutants with disruptions in embA or embB are viable, although the growth rate was affected. In contrast, we demonstrate here that embA is an essential gene in Mycobacterium tuberculosis, since a deletion of the chromosomal gene could only be achieved when a second functional copy was provided on an integrated vector. Complementation of an embA mutant of M. smegmatis by M. tuberculosis embA confirmed that it encodes a functional arabinosyltransferase. We identified a promoter for M. tuberculosis embA located immediately upstream of the gene, indicating that it is expressed independently from the upstream gene, embC. Promoter activity from P(embA)((Mtb)) was sevenfold lower when assayed in M. smegmatis compared to M. tuberculosis, indicating that the latter is not a good host for genetic analysis of M. tuberculosis embA expression. P(embA)((Mtb)) activity remained constant throughout growth phases and after stress treatment, although it was reduced during hypoxia-induced non-replicating persistence. Ethambutol exposure had no effect on P(embA)((Mtb)) activity. These data demonstrate that M. tuberculosis embA encodes a functional arabinosyltransferase which is constitutively expressed and plays a critical role in M. tuberculosis.

  13. The rpoB gene of Mycobacterium tuberculosis.

    PubMed Central

    Miller, L P; Crawford, J T; Shinnick, T M

    1994-01-01

    A portion of the Mycobacterium tuberculosis gene encoding the beta subunit of RNA polymerase (rpoB) was amplified by PCR using degenerate oligonucleotides and used as a hybridization probe to isolate plasmid clones carrying the entire rpoB gene of M. tuberculosis H37Rv, a virulent, rifampin-susceptible strain. Sequence analysis of a 5,084-bp SacI genomic DNA fragment revealed a 3,534-bp open reading frame encoding an 1,178-amino-acid protein with 57% identity with the Escherichia coli beta subunit. This SacI fragment also carried a portion of the rpoC gene located 43 bp downstream from the 3' end of the rpoB open reading frame; this organization is similar to that of the rpoBC operon of E. coli. The M. tuberculosis rpoB gene was cloned into the shuttle plasmid pMV261 and electroporated into the LR223 strain of Mycobacterium smegmatis, which is highly resistant to rifampin (MIC > 200 micrograms/ml). The resulting transformants were relatively rifampin susceptible (MIC = 50 micrograms/ml). Using PCR mutagenesis techniques, we introduced a specific rpoB point mutation (associated with clinical strains of rifampin-resistant M. tuberculosis) into the cloned M. tuberculosis rpoB gene and expressed this altered gene in the LR222 strain of M. smegmatis, which is susceptible to rifampin (MIC = 25 micrograms/ml). The resulting transformants were rifampin resistant (MIC = 200 micrograms/ml). The mutagenesis and expression strategy of the cloned M. tuberculosis rpoB gene that we have employed in this study will allow us to determine the rpoB mutations that are responsible for rifampin resistance in M. tuberculosis. PMID:8031050

  14. Mixed-Strain Mycobacterium tuberculosis Infections and the Implications for Tuberculosis Treatment and Control

    PubMed Central

    van Helden, Paul D.; Wilson, Douglas; Colijn, Caroline; McLaughlin, Megan M.; Abubakar, Ibrahim; Warren, Robin M.

    2012-01-01

    Summary: Numerous studies have reported that individuals can simultaneously harbor multiple distinct strains of Mycobacterium tuberculosis. To date, there has been limited discussion of the consequences for the individual or the epidemiological importance of mixed infections. Here, we review studies that documented mixed infections, highlight challenges associated with the detection of mixed infections, and discuss possible implications of mixed infections for the diagnosis and treatment of patients and for the community impact of tuberculosis control strategies. We conclude by highlighting questions that should be resolved in order to improve our understanding of the importance of mixed-strain M. tuberculosis infections. PMID:23034327

  15. Mycobacterium tuberculosis pili (MTP), a putative biomarker for a tuberculosis diagnostic test.

    PubMed

    Naidoo, Natasha; Ramsugit, Saiyur; Pillay, Manormoney

    2014-05-01

    Novel biomarkers are urgently needed for point of care TB diagnostics. In this study, we investigated the potential of the pilin subunit protein encoded by the mtp gene as a diagnostic biomarker. BLAST analysis of the mtp gene on published genome databases, and amplicon sequencing were performed in Mycobacterium tuberculosis Complex (MTBC) strains and other organisms. The protein secondary structure of the amino acid sequences of non-tuberculous Mycobacteria that partially aligned with the mtp sequence was analysed with PredictProtein software. The mtp gene and corresponding amino acid sequence of MTBC were 100% homologous with H37Rv, in contrast to the partial alignment of the non-tuberculous Mycobacteria. The mtp gene was present in all 91 clinical isolates of MTBC. Except for 2 strains with point mutations, the sequence was 100% conserved among the clinical strains. The mtp gene could not be amplified in all non-tuberculous Mycobacteria and respiratory organisms. The predicted MTP protein structure of Mycobacterium avium, Mycobacterium ulcerans and Mycobacterium abscessus differed significantly from that of the M. tuberculosis, which was similar to Mycobacterium marinum. The absence of the mtp gene in non-tuberculous Mycobacteria and other respiratory bacteria suggests that its encoded product, the pilin subunit protein of M. tuberculosis may be a suitable marker for a point of care TB test.

  16. Predictive factors of Mycobacterium tuberculosis infection and pulmonary tuberculosis in prisoners.

    PubMed

    Martín Sánchez, V; Alvarez-Guisasola, F; Caylá, J A; Alvarez, J L

    1995-06-01

    Tuberculosis currently represents a serious problem in prison populations. With the aim of studying the predictive factors for, and the prevalence of, Mycobacterium tuberculosis infection and pulmonary tuberculosis in a Spanish prison, all those admitted during 1991 and 1992 were included (N = 1314). The tuberculin skin test, HIV serology, chest X-ray and bacteriological examination of sputum were carried out. Statistical analysis was done by univariant tests, stratified analysis and logistic regression. The prevalence of M. tuberculosis infection was 55.5% (95% confidence interval [CI] 52.5-58.5). An association was found with sex, imprisonment more than once, HIV infection and age. The co-infection rate (tuberculosis plus HIV) was 9.2%. Logistic regression showed a greater risk with age (4.4% per year), time spent in prison and for males. The prevalence of pulmonary tuberculosis was 1.26% and an association was found with M. tuberculosis infection, HIV infection (odds ratio [OR] = 13.7), intravenous drug users (OR = 17.2) and imprisonment more than once (OR = 7.3). Logistic regression showed an association with HIV co-infection (OR = 20.2). The prevalence of M. tuberculosis infection and pulmonary tuberculosis is high when compared with similar studies. The influence of age, time spent in prison and co-infection with HIV is relevant to recommendations for specific tuberculosis prevention programmes in correctional facilities.

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

    NASA Astrophysics Data System (ADS)

    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.

  18. Effect of decalcifying agents on the staining of Mycobacterium tuberculosis.

    PubMed Central

    Anderson, G; Coup, A J

    1975-01-01

    Lymph nodes from guinea pigs inoculated with Mycobacterium tuberculosis were fixed in buffered formalin, then treated for the recommended times in Gooding and Stewart's fluid, EDTA, aqueous nitric acid, von Ebner's fluid, and rapid decalcifier (RDC). The blocks were processed to paraffin wax and sections were stained by the Ziehl-Neelsen technique. Only in sections of the blocks treated with RDC were no acid alcohol fast bacilli demonstrable. Hydrochloric acid is a known constituent of RDC and it was found that Myco. tuberculosis is altered by treatment with 2-5M solutions of hydrochloric acid and above and cannot subseuqently be demonstrated by the Ziehl-Neelsen stain. From these results it is recommended that calcified tissue from patients in whom there is a suspicion of tuberculosis should be decalcified with an agent other than RDC. PMID:51859

  19. Shared characteristics between Mycobacterium tuberculosis and fungi contribute to virulence.

    PubMed

    Willcocks, Sam; Wren, Brendan W

    2014-01-01

    Mycobacterium tuberculosis, an etiologic agent of tuberculosis, exacts a heavy toll in terms of human morbidity and mortality. Although an ancient disease, new strains are emerging as human population density increases. The emergent virulent strains appear adept at steering the host immune response from a protective Th1 type response towards a Th2 bias, a feature shared with some pathogenic fungi. Other common characteristics include infection site, metabolic features, the composition and display of cell surface molecules, the range of innate immune receptors engaged during infection, and the ability to form granulomas. Literature from these two distinct fields of research are reviewed to propose that the emergent virulent strains of M. tuberculosis are in the process of convergent evolution with pathogenic fungi, and are increasing the prominence of conserved traits from environmental phylogenetic ancestors that facilitate their evasion of host defenses and dissemination.

  20. A Focused Screen Identifies Antifolates with Activity on Mycobacterium tuberculosis

    PubMed Central

    Kumar, Anuradha; Colmenarejo, Gonzalo; Pérez, Esther; Gonzalez, Ruben R.; Torres, Pedro; Calvo, David; Gómez, Ruben M.; Ortega, Fátima; Jiménez, Elena; Gabarro, Raquel C.; Rullás, Joaquín; Ballell, Lluis

    2015-01-01

    Antifolates are widely used to treat several diseases but are not currently used in the first-line treatment of tuberculosis, despite evidence that some of these molecules can target Mycobacterium tuberculosis (Mtb) bacilli in vitro. To identify new antifolate candidates for animal-model efficacy studies of tuberculosis, we paired knowledge and tools developed in academia with the infrastructure and chemistry resources of a large pharmaceutical company. Together we curated a focused library of 2508 potential antifolates, which were then tested for activity against live Mtb. We identified 210 primary hits, confirmed the on-target activity of potent compounds, and now report the identification and characterization of 5 hit compounds, representative of 5 different chemical scaffolds. These antifolates have potent activity against Mtb and represent good starting points for improvement that could lead to in vivo efficacy studies. PMID:26771003

  1. Genomic deletions suggest a phylogeny for the Mycobacterium tuberculosis complex.

    PubMed

    Mostowy, Serge; Cousins, Debby; Brinkman, Jacqui; Aranaz, Alicia; Behr, Marcel A

    2002-07-01

    To better understand the evolution of the Mycobacterium tuberculosis complex, subspecies were tested for large sequence polymorphisms. Samples with greater numbers of deletions, without exception, were missing all the same regions that were deleted from samples with lesser numbers of deletions. Principal genetic groups based on single-nucleotide polymorphisms were restricted to one of the deletion-based groups, and isolates that shared genotypes based on molecular epidemiological markers were assigned almost exclusively to the same deletion type. The data provide compelling evidence that human tuberculosis did not originate from the present-day bovine form. Genomic deletions present themselves as an attractive modality to study the evolution of the M. tuberculosis complex.

  2. Manipulation of the Mononuclear Phagocyte System by Mycobacterium tuberculosis

    PubMed Central

    Lugo-Villarino, Geanncarlo; Neyrolles, Olivier

    2014-01-01

    Over the past 20 years, there has been an emerging appreciation about the role of the mononuclear phagocyte system (MPS) to control and eradicate pathogens. Likewise, there have been significant advances in dissecting the mechanisms involved in the microbial subversion of MPS cells, mainly affecting their differentiation and effector functions. Mycobacterium tuberculosis is a chronic bacterial pathogen that represents an enigma to the field because of its remarkable ability to thrive in humans. One reason is that M. tuberculosis renders a defective MPS compartment, which is perhaps the most ingenious strategy for survival in the host given the prominence of these cells to modulate microenvironments, their function as sentinels and orchestrators of the immune response, and their pathogenic role as reservoirs for microbial persistence. In this article, the principal strategies used by M. tuberculosis to subvert the MPS compartment are presented along with emerging concepts. PMID:25147188

  3. Novel Mycobacterium tuberculosis Complex Isolate from a Wild Chimpanzee

    PubMed Central

    Coscolla, Mireia; Lewin, Astrid; Metzger, Sonja; Maetz-Rennsing, Kerstin; Calvignac-Spencer, Sébastien; Nitsche, Andreas; Dabrowski, Pjotr Wojtek; Radonic, Aleksandar; Niemann, Stefan; Parkhill, Julian; Couacy-Hymann, Emmanuel; Feldman, Julia; Comas, Iñaki; Boesch, Christophe; Gagneux, Sebastien

    2013-01-01

    Tuberculosis (TB) is caused by gram-positive bacteria known as the Mycobacterium tuberculosis complex (MTBC). MTBC include several human-associated lineages and several variants adapted to domestic and, more rarely, wild animal species. We report an M. tuberculosis strain isolated from a wild chimpanzee in Côte d’Ivoire that was shown by comparative genomic and phylogenomic analyses to belong to a new lineage of MTBC, closer to the human-associated lineage 6 (also known as M. africanum West Africa 2) than to the other classical animal-associated MTBC strains. These results show that the general view of the genetic diversity of MTBC is limited and support the possibility that other MTBC variants exist, particularly in wild mammals in Africa. Exploring this diversity is crucial to the understanding of the biology and evolutionary history of this widespread infectious disease. PMID:23735084

  4. Mycolic Acid Index Susceptibility Method for Mycobacterium tuberculosis

    PubMed Central

    Viader-Salvadó, José M.; Garza-González, Elvira; Valdez-Leal, Ramón; de los Angeles del Bosque-Moncayo, M.; Tijerina-Menchaca, Rolando; Guerrero-Olazarán, Martha

    2001-01-01

    A rapid drug susceptibility test to measure the susceptibility of Mycobacterium tuberculosis to isoniazid (INH) and rifampin (RIF) using clinical isolates and a newly defined mycolic acid index (MAI) was evaluated. A total of 200 clinical isolates of M. tuberculosis were tested for susceptibility or resistance to INH and RIF by the MAI susceptibility and indirect-proportion methods. Overall, there was agreement between the two methods for 398 (99.5%) of the 400 total tests. Specifically, the sensitivity of the MAI susceptibility method for INH and RIF was 97.6 and 100%, respectively. The specificity and positive predictive value were 100% for both drugs, and the negative predictive value for INH and RIF was 98.3 and 100%, respectively. In conclusion, the MAI susceptibility method described here can be used for rapid drug susceptibility testing of M. tuberculosis clinical isolates within 5 days after clinical isolates are incubated in the presence or absence of an antituberculosis drug. PMID:11427584

  5. Development of Rifapentine Susceptibility Tests for Mycobacterium tuberculosis

    PubMed Central

    Heifets, L.; Sanchez, T.; Vanderkolk, J.; Pham, V.

    1999-01-01

    Two methods for testing the susceptibility of Mycobacterium tuberculosis to rifapentine have been developed: the agar proportion method and the radiometric BACTEC technique. A critical concentration of 0.5 μg of rifapentine per ml is proposed for both methods since it provides a reliable means of distinguishing between susceptible and resistant M. tuberculosis isolates. It is recommended that two quality control M. tuberculosis strains be used at the introduction of these tests in a clinical laboratory: one that is pansusceptible (H37Rv) and one that is resistant to rifapentine. The resistant strain can be obtained from the American Type Culture Collection, where it is deposited under the number ATCC 700457. PMID:9869560

  6. The efficacy of the heat killing of Mycobacterium tuberculosis

    PubMed Central

    Doig, C; Seagar, A L; Watt, B; Forbes, K J

    2002-01-01

    There is concern that current procedures for the heat inactivation of Mycobacterium tuberculosis may not be adequate. This raises serious safety issues for laboratory staff performing molecular investigations such as IS6110 restriction fragment length polymorphism typing. This paper confirms that the protocol of van Embden et al, as performed routinely in this laboratory, is safe and effective for the heat inactivation of M tuberculosis. This procedure involves complete immersion of a tube containing a suspension of one loopfull of growth in a water bath at 80°C for 20 minutes. Seventy four isolates were included in this investigation. Despite prolonged incubation for 20 weeks, none of the heat killed M tuberculosis suspensions produced visible colonies or gave a positive growth signal from liquid culture. This method did not affect the integrity of the DNA for subsequent molecular investigations. PMID:12354807

  7. Novel Mycobacterium tuberculosis complex isolate from a wild chimpanzee.

    PubMed

    Coscolla, Mireia; Lewin, Astrid; Metzger, Sonja; Maetz-Rennsing, Kerstin; Calvignac-Spencer, Sébastien; Nitsche, Andreas; Dabrowski, Pjotr Wojtek; Radonic, Aleksandar; Niemann, Stefan; Parkhill, Julian; Couacy-Hymann, Emmanuel; Feldman, Julia; Comas, Iñaki; Boesch, Christophe; Gagneux, Sebastien; Leendertz, Fabian H

    2013-06-01

    Tuberculosis (TB) is caused by gram-positive bacteria known as the Mycobacterium tuberculosis complex (MTBC). MTBC include several human-associated lineages and several variants adapted to domestic and, more rarely, wild animal species. We report an M. tuberculosis strain isolated from a wild chimpanzee in Côte d'Ivoire that was shown by comparative genomic and phylogenomic analyses to belong to a new lineage of MTBC, closer to the human-associated lineage 6 (also known as M. africanum West Africa 2) than to the other classical animal-associated MTBC strains. These results show that the general view of the genetic diversity of MTBC is limited and support the possibility that other MTBC variants exist, particularly in wild mammals in Africa. Exploring this diversity is crucial to the understanding of the biology and evolutionary history of this widespread infectious disease.

  8. A new approach for pyrazinamide susceptibility testing in Mycobacterium tuberculosis.

    PubMed

    Zimic, Mirko; Loli, Sebastian; Gilman, Robert H; Gutierrez, Andrés; Fuentes, Patricia; Cotrina, Milagros; Kirwan, Daniela; Sheen, Patricia

    2012-08-01

    Pyrazinamide (PZA) is an important drug in the treatment of tuberculosis. Microbiological methods of PZA susceptibility testing are controversial and have low reproducibility. After conversion of PZA into pyrazinoic acid (POA) by the bacterial pyrazinamidase enzyme, the drug is expelled from the bacteria by an efflux pump. To evaluate the rate of POA extrusion from Mycobacterium tuberculosis as a parameter to detect PZA resistance. The rate of POA extrusion and PZA susceptibility determined by BACTEC 460 were measured for 34 strains in a previous study. PZA resistance was modeled in a logistic regression with the pyrazinoic efflux rate. POA efflux rate predicted PZA resistance with 70.83%-92.85% sensitivity and 100% specificity compared with BACTEC 460. POA efflux rate could be a useful tool for predicting PZA resistance in M. tuberculosis. Further exploration of this approach may lead to the development of new tools for diagnosing PZA resistance, which may be of public health importance.

  9. 5-Arylaminouracil Derivatives: New Inhibitors of Mycobacterium tuberculosis.

    PubMed

    Matyugina, Elena; Novikov, Mikhail; Babkov, Denis; Ozerov, Alexander; Chernousova, Larisa; Andreevskaya, Sofia; Smirnova, Tatiana; Karpenko, Inna; Chizhov, Alexander; Murthu, Pravin; Lutz, Stefan; Kochetkov, Sergei; Seley-Radtke, Katherine L; Khandazhinskaya, Anastasia L

    2015-12-01

    Three series of 5-arylaminouracil derivatives, including 5-(phenylamino)uracils, 1-(4'-hydroxy-2'-cyclopenten-1'-yl)-5-(phenylamino)uracils, and 1,3-di-(4'-hydroxy-2'-cyclopenten-1'-yl)-5-(phenylamino)uracils, were synthesized and screened for potential antimicrobial activity. Most of compounds had a negative effect on the growth of the Mycobacterium tuberculosis H37Rv strain, with 100% inhibition observed at concentrations between 5 and 40 μg/mL. Of those, 1-(4'-hydroxy-2'-cyclopenten-1'-yl)-3-(4‴-hydroxy-2‴-cyclopenten-1‴-yl)-5-(4″-butyloxyphenylamino)uracil proved to be the most active among tested compounds against the M. tuberculosis multidrug-resistant strain MS-115 (MIC90 5 μg/mL). In addition, the thymidylate kinase of M. tuberculosis was evaluated as a possible enzymatic target.

  10. Advances in Mycobacterium tuberculosis therapeutics discovery utlizing structural biology

    PubMed Central

    Chim, Nicholas; Owens, Cedric P.; Contreras, Heidi; Goulding, Celia W.

    2013-01-01

    In 2012, tuberculosis (TB) remains a global health threat and is exacerbated both by the emergence of drug resistant Mycobacterium tuberculosis strains and its synergy with HIV infection. The waning effectiveness of current treatment regimens necessitates the development of new or repurposed anti-TB therapeutics for improved combination therapies against the disease. Exploiting atomic resolution structural information of proteins in complex with their substrates and/or inhibitors can facilitate structure-based rational drug design. Since our last review in 2009, there has been a wealth of new M. tuberculosis protein structural information. Once again, we have compiled the most promising structures with regards to potential anti-TB drug development and present them in this updated review. PMID:23167715

  11. Prevalence of latent Mycobacterium tuberculosis infection in prisoners

    PubMed Central

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

    2016-01-01

    ABSTRACT Objective: To determine the prevalence of and the factors associated with latent Mycobacterium tuberculosis infection (LTBI) in prisoners in the state of Minas Gerais, Brazil. Methods: 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. Results: 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. Conclusions: 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. PMID:27812634

  12. Soluble TNFRp75 regulates host protective immunity against Mycobacterium tuberculosis.

    PubMed

    Keeton, Roanne; Allie, Nasiema; Dambuza, Ivy; Abel, Brian; Hsu, Nai-Jen; Sebesho, Boipelo; Randall, Philippa; Burger, Patricia; Fick, Elizabeth; Quesniaux, Valerie F J; Ryffel, Bernhard; Jacobs, Muazzam

    2014-04-01

    Development of host protective immunity against Mycobacterium tuberculosis infection is critically dependent on the inflammatory cytokine TNF. TNF signals through 2 receptors, TNFRp55 and TNFRp75; however, the role of TNFRp75-dependent signaling in immune regulation is poorly defined. Here we found that mice lacking TNFRp75 exhibit greater control of M. tuberculosis infection compared with WT mice. TNFRp75-/- mice developed effective bactericidal granulomas and demonstrated increased pulmonary recruitment of activated DCs. Moreover, IL-12p40-dependent migration of DCs to lung draining LNs of infected TNFRp75-/- mice was substantially higher than that observed in WT M. tuberculosis-infected animals and was associated with enhanced frequencies of activated M. tuberculosis-specific IFN-γ-expressing CD4+ T cells. In WT mice, TNFRp75 shedding correlated with markedly reduced bioactive TNF levels and IL-12p40 expression. Neutralization of TNFRp75 in M. tuberculosis-infected WT BM-derived DCs (BMDCs) increased production of bioactive TNF and IL-12p40 to a level equivalent to that produced by TNFRp75-/- BMDCs. Addition of exogenous TNFRp75 to TNFRp75-/- BMDCs infected with M. tuberculosis decreased IL-12p40 synthesis, demonstrating that TNFRp75 shedding regulates DC activation. These data indicate that TNFRp75 shedding downmodulates protective immune function and reduces host resistance and survival; therefore, targeting TNFRp75 may be beneficial for improving disease outcome.

  13. Indoleamides are active against drug-resistant Mycobacterium tuberculosis

    PubMed Central

    Lun, Shichun; Guo, Haidan; Onajole, Oluseye K.; Pieroni, Marco; Gunosewoyo, Hendra; Chen, Gang; Tipparaju, Suresh K.; Ammerman, Nicole C.; Kozikowski, Alan P.; Bishai, William R.

    2014-01-01

    Responsible for nearly two million deaths each year, the infectious disease tuberculosis remains a serious global health challenge. The emergence of multidrug- and extensively drug-resistant strains of Mycobacterium tuberculosis confounds control efforts, and new drugs with novel molecular targets are desperately needed. Here we describe lead compounds, the indoleamides, with potent activity against both drug-susceptible and drug-resistant strains of M. tuberculosis by targeting the mycolic acid transporter MmpL3. We identify a single mutation in mmpL3 which confers high resistance to the indoleamide class while remaining susceptible to currently used first- and second-line tuberculosis drugs, indicating a lack of cross-resistance. Importantly, an indoleamide derivative exhibits dose-dependent anti-mycobacterial activity when orally administered to M. tuberculosis-infected mice. The bioavailability of the indoleamides, combined with their ability to kill tubercle bacilli, indicates great potential for translational developments of this structure class for the treatment of drug-resistant tuberculosis. PMID:24352433

  14. Mycobacterium tuberculosis Lineage Distribution in Xinjiang and Gansu Provinces, China.

    PubMed

    Chen, Haixia; He, Li; Huang, Hairong; Shi, Chengmin; Ni, Xumin; Dai, Guangming; Ma, Liang; Li, Weimin

    2017-04-21

    Mycobacterium tuberculosis (M. tuberculosis) genotyping has dramatically improved the understanding of the epidemiology of tuberculosis (TB). In this study, 187 M. tuberculosis isolates from Xinjiang Uygur Autonomous Region (Xinjiang) and Gansu province in China were genotyped using large sequence polymorphisms (LSPs) and variable number tandem repeats (VNTR). Ten isolates, which represent major nodes of VNTR-based minimum spanning tree, were selected and subsequently subjected to multi-locus sequence analyses (MLSA) that include 82 genes. Based on a robust lineage assignment, we tested the association between lineages and clinical characteristics by logistic regression. There are three major lineages of M. tuberculosis prevalent in Xinjiang, viz. the East Asian Lineage 2 (42.1%; 56/133), the Euro-American Lineage 4 (33.1%; 44/133), and the Indian and East African Lineage 3 (24.8%; 33/133); two lineages prevalent in Gansu province, which are the Lineage 2 (87%; 47/54) and the Lineage 4 (13%; 7/54). The topological structures of the MLSA-based phylogeny support the LSP-based identification of M. tuberculosis lineages. The statistical results suggest an association between the Lineage 2 and the hemoptysis/bloody sputum symptom, fever in Uygur patients. The pathogenicity of the Lineage 2 remains to be further investigated.

  15. A vitamin B12 transporter in Mycobacterium tuberculosis

    PubMed Central

    Gopinath, Krishnamoorthy; Venclovas, Česlovas; Ioerger, Thomas R.; Sacchettini, James C.; McKinney, John D.; Mizrahi, Valerie; Warner, Digby F.

    2013-01-01

    Vitamin B12-dependent enzymes function in core biochemical pathways in Mycobacterium tuberculosis, an obligate pathogen whose metabolism in vivo is poorly understood. Although M. tuberculosis can access vitamin B12 in vitro, it is uncertain whether the organism is able to scavenge B12 during host infection. This question is crucial to predictions of metabolic function, but its resolution is complicated by the absence in the M. tuberculosis genome of a direct homologue of BtuFCD, the only bacterial B12 transport system described to date. We applied genome-wide transposon mutagenesis to identify M. tuberculosis mutants defective in their ability to use exogenous B12. A small proportion of these mapped to Rv1314c, identifying the putative PduO-type ATP : co(I)rrinoid adenosyltransferase as essential for B12 assimilation. Most notably, however, insertions in Rv1819c dominated the mutant pool, revealing an unexpected function in B12 acquisition for an ATP-binding cassette (ABC)-type protein previously investigated as the mycobacterial BacA homologue. Moreover, targeted deletion of Rv1819c eliminated the ability of M. tuberculosis to transport B12 and related corrinoids in vitro. Our results establish an alternative to the canonical BtuCD-type system for B12 uptake in M. tuberculosis, and elucidate a role in B12 metabolism for an ABC protein implicated in chronic mycobacterial infection. PMID:23407640

  16. Mycobacterium tuberculosis causing tuberculous lymphadenitis in Maputo, Mozambique.

    PubMed

    Viegas, Sofia Omar; Ghebremichael, Solomon; Massawo, Leguesse; Alberto, Matos; Fernandes, Fabíola Couto; Monteiro, Eliane; Couvin, David; Matavele, José Maiane; Rastogi, Nalin; Correia-Neves, Margarida; Machado, Adelina; Carrilho, Carla; Groenheit, Ramona; Källenius, Gunilla; Koivula, Tuija

    2015-11-21

    The zoonosis bovine tuberculosis (TB) is known to be responsible for a considerable proportion of extrapulmonary TB. In Mozambique, bovine TB is a recognised problem in cattle, but little has been done to evaluate how Mycobacterium bovis has contributed to human TB. We here explore the public health risk for bovine TB in Maputo, by characterizing the isolates from tuberculous lymphadenitis (TBLN) cases, a common manifestation of bovine TB in humans, in the Pathology Service of Maputo Central Hospital, in Mozambique, during one year. Among 110 patients suspected of having TBLN, 49 had a positive culture result. Of those, 48 (98%) were positive for Mycobacterium tuberculosis complex and one for nontuberculous mycobacteria. Of the 45 isolates analysed by spoligotyping and Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat (MIRU-VNTR), all were M. tuberculosis. No M. bovis was found. Cervical TBLN, corresponding to 39 (86.7%) cases, was the main cause of TBLN and 66.7% of those where from HIV positive patients. We found that TBLN in Maputo was caused by a variety of M. tuberculosis strains. The most prevalent lineage was the EAI (n = 19; 43.2%). Particular common spoligotypes were SIT 48 (EAI1_SOM sublineage), SIT 42 (LAM 9), SIT 1 (Beijing) and SIT53 (T1), similar to findings among pulmonary cases. M. tuberculosis was the main etiological agent of TBLN in Maputo. M. tuberculosis genotypes were similar to the ones causing pulmonary TB, suggesting that in Maputo, cases of TBLN arise from the same source as pulmonary TB, rather than from an external zoonotic source. Further research is needed on other forms of extrapulmonary TB and in rural areas where there is high prevalence of bovine TB in cattle, to evaluate the risk of transmission of M. bovis from cattle to humans.

  17. Mixed Infection of Mycobacterium abscessus subsp. abscessus and Mycobacterium tuberculosis in the Lung

    PubMed Central

    Sohn, Sungmin; Wang, Sungho; Shi, Hyejin; Park, Sungrock; Lee, Sangki; Park, Kyoung Taek

    2017-01-01

    A mixed infection of Mycobacterium abscessus subsp. abscessus (Mab) and Mycobacterium tuberculosis (MTB) in the lung is an unusual clinical manifestation and has not yet been reported. A 61-year-old woman had been treated for Mab lung disease and concomitant pneumonia, and was diagnosed with pulmonary tuberculosis (PTB). Despite both anti-PTB and anti-Mab therapy, her entire left lung was destroyed and collapsed. She underwent left pneumonectomy and received medical therapy. We were able to successfully treat her mixed infection by pneumonectomy followed by inhaled amikacin therapy. To the best of our knowledge, thus far, this is the first description of a mixed Mab and MTB lung infection. PMID:28180105

  18. Host-pathogen interactions in latent Mycobacterium tuberculosis infection: identification of new targets for tuberculosis intervention.

    PubMed

    Lin, May Young; Ottenhoff, Tom H M

    2008-03-01

    Mycobacterium tuberculosis (M. tuberculosis) is one of the worlds' most successful and sophisticated pathogens. It is estimated that over 2 billion people today harbour latent M. tuberculosis infection without any clinical symptoms. Since most new cases of active tuberculosis (TB) arise from this (growing) number of latently infected individuals, urgent measures to control TB reactivation are required, including more effective drugs and new TB vaccines. The currently widely used BCG vaccines, as well as most new generation TB-vaccines that are being developed are designed as prophylactic or as BCG-booster vaccines. Unfortunately, many of these vaccines are unlikely to be effective in individuals already latently infected with M. tuberculosis. Here we argue that detailed analysis of M. tuberculosis genes that are switched on predominantly during the latent stage of infection may lead to the identification of new M. tuberculosis targets for drug and vaccine development. First, we will describe essential host-pathogen interactions in TB with particular emphasis on TB latency and persistent infection. Subsequently, we will focus on a novel group of late-stage specific genes, encoded by the M. tuberculosis dormancy (dosR) regulon, and summarize recent studies describing human T-cell recognition of these dormancy antigens in relation to (latent) M. tuberculosis infection. We will discuss the possible relevance of these new classes of antigens for new TB intervention strategies.

  19. Role of Mycobacterium tuberculosis pknD in the pathogenesis of central nervous system tuberculosis.

    PubMed

    Be, Nicholas A; Bishai, William R; Jain, Sanjay K

    2012-01-13

    Central nervous system disease is the most serious form of tuberculosis, and is associated with high mortality and severe neurological sequelae. Though recent clinical reports suggest an association of distinct Mycobacterium tuberculosis strains with central nervous system disease, the microbial virulence factors required have not been described previously. We screened 398 unique M. tuberculosis mutants in guinea pigs to identify genes required for central nervous system tuberculosis. We found M. tuberculosis pknD (Rv0931c) to be required for central nervous system disease. These findings were central nervous system tissue-specific and were not observed in lung tissues. We demonstrated that pknD is required for invasion of brain endothelia (primary components of the blood-brain barrier protecting the central nervous system), but not macrophages, lung epithelia, or other endothelia. M. tuberculosis pknD encodes a "eukaryotic-like" serine-threonine protein kinase, with a predicted intracellular kinase and an extracellular (sensor) domain. Using confocal microscopy and flow cytometry we demonstrated that the M. tuberculosis PknD sensor is sufficient to trigger invasion of brain endothelia, a process which was neutralized by specific antiserum. Our findings demonstrate a novel in vivo role for M. tuberculosis pknD and represent an important mechanism for bacterial invasion and virulence in central nervous system tuberculosis, a devastating and understudied disease primarily affecting young children.

  20. Role of Mycobacterium tuberculosis pknD in the Pathogenesis of central nervous system tuberculosis

    PubMed Central

    2012-01-01

    Background Central nervous system disease is the most serious form of tuberculosis, and is associated with high mortality and severe neurological sequelae. Though recent clinical reports suggest an association of distinct Mycobacterium tuberculosis strains with central nervous system disease, the microbial virulence factors required have not been described previously. Results We screened 398 unique M. tuberculosis mutants in guinea pigs to identify genes required for central nervous system tuberculosis. We found M. tuberculosis pknD (Rv0931c) to be required for central nervous system disease. These findings were central nervous system tissue-specific and were not observed in lung tissues. We demonstrated that pknD is required for invasion of brain endothelia (primary components of the blood-brain barrier protecting the central nervous system), but not macrophages, lung epithelia, or other endothelia. M. tuberculosis pknD encodes a "eukaryotic-like" serine-threonine protein kinase, with a predicted intracellular kinase and an extracellular (sensor) domain. Using confocal microscopy and flow cytometry we demonstrated that the M. tuberculosis PknD sensor is sufficient to trigger invasion of brain endothelia, a process which was neutralized by specific antiserum. Conclusions Our findings demonstrate a novel in vivo role for M. tuberculosis pknD and represent an important mechanism for bacterial invasion and virulence in central nervous system tuberculosis, a devastating and understudied disease primarily affecting young children. PMID:22243650

  1. Multiplex-PCR for differentiation of Mycobacterium bovis from Mycobacterium tuberculosis complex.

    PubMed

    Spositto, F L E; Campanerut, P A Z; Ghiraldi, L D; Leite, C Q F; Hirata, M H; Hirata, R D C; Siqueira, V L D; Cardoso, R Fressatti

    2014-01-01

    We evaluated a multiplex-PCR to differentiate Mycobacterium bovis from M. tuberculosis Complex (MTC) by one step amplification based on simultaneous detection of pncA 169 C > G change in M. bovis and the IS6110 present in MTC species. Our findings showed the proposed multiplex-PCR is a very useful tool for complementation in differentiating M. bovis from other cultured MTC species.

  2. Proteogenomic analysis of Mycobacterium tuberculosis by high resolution mass spectrometry.

    PubMed

    Kelkar, Dhanashree S; Kumar, Dhirendra; Kumar, Praveen; Balakrishnan, Lavanya; Muthusamy, Babylakshmi; Yadav, Amit Kumar; Shrivastava, Priyanka; Marimuthu, Arivusudar; Anand, Sridhar; Sundaram, Hema; Kingsbury, Reena; Harsha, H C; Nair, Bipin; Prasad, T S Keshava; Chauhan, Devendra Singh; Katoch, Kiran; Katoch, Vishwa Mohan; Kumar, Prahlad; Chaerkady, Raghothama; Ramachandran, Srinivasan; Dash, Debasis; Pandey, Akhilesh

    2011-12-01

    The genome sequencing of H37Rv strain of Mycobacterium tuberculosis was completed in 1998 followed by the whole genome sequencing of a clinical isolate, CDC1551 in 2002. Since then, the genomic sequences of a number of other strains have become available making it one of the better studied pathogenic bacterial species at the genomic level. However, annotation of its genome remains challenging because of high GC content and dissimilarity to other model prokaryotes. To this end, we carried out an in-depth proteogenomic analysis of the M. tuberculosis H37Rv strain using Fourier transform mass spectrometry with high resolution at both MS and tandem MS levels. In all, we identified 3176 proteins from Mycobacterium tuberculosis representing ~80% of its total predicted gene count. In addition to protein database search, we carried out a genome database search, which led to identification of ~250 novel peptides. Based on these novel genome search-specific peptides, we discovered 41 novel protein coding genes in the H37Rv genome. Using peptide evidence and alternative gene prediction tools, we also corrected 79 gene models. Finally, mass spectrometric data from N terminus-derived peptides confirmed 727 existing annotations for translational start sites while correcting those for 33 proteins. We report creation of a high confidence set of protein coding regions in Mycobacterium tuberculosis genome obtained by high resolution tandem mass-spectrometry at both precursor and fragment detection steps for the first time. This proteogenomic approach should be generally applicable to other organisms whose genomes have already been sequenced for obtaining a more accurate catalogue of protein-coding genes.

  3. Culture and molecular method for detection of Mycobacterium tuberculosis complex and Mycobacterium avium subsp. paratuberculosis in milk and dairy products.

    PubMed

    Messelhäusser, U; Kämpf, P; Hörmansdorfer, S; Wagner, B; Schalch, B; Busch, U; Höller, C; Wallner, P; Barth, G; Rampp, A

    2012-01-01

    A combined molecular and cultural method for the detection of the Mycobacterium tuberculosis complex (MTBC) and Mycobacterium avium subsp. paratuberculosis was developed and tested with artificially contaminated milk and dairy products. Results indicate that the method can be used for a reliable detection as a basis for first risk assessments.

  4. Murine Mycobacterium marinum Infection as a Model for Tuberculosis.

    PubMed

    Lienard, Julia; Carlsson, Fredric

    2017-01-01

    Mycobacteria are a major human health problem globally. Regarding tuberculosis the situation is worsened by the poor efficacy of current vaccine regimens and by emergence of drug-resistant strains (Manjelievskaia J et al, Trans R Soc Trop Med Hyg 110: 110, 2016; Pereira et al., Lancet Infect Dis 12:300-306, 2012; http://www.who.int/tb/publications/global_report/en/) undermining both disease-prevention and available treatments. Thus, increased basic understanding of mycobacterial-and particularly Mycobacterium tuberculosis-virulence strategies and pathogenesis is of great importance. To this end several in vivo infection models are available (Guirado and Schlesinger, Front Immunol 4:98, 2013; Leung et al., Eur J Immunol 43:2246-2254, 2013; Patel et al., J Lab Physicians 3:75-79, 2011; van Leeuwen et al., Cold Spring Harb Perspect Med 5:a018580, 2015). While these models all have their merits they also exhibit limitations, and none perfectly mimics all aspects of human tuberculosis. Thus, there is a need for multiple models that may complement each other, ultimately allowing us to gain true insight into the pathogenesis of mycobacterial infections.Here, we describe a recently developed mouse model of Mycobacterium marinum infection that allows kinetic and quantitative studies of disease progression in live animals [8]. Notably, this model exhibits features of human tuberculosis not replicated in M. tuberculosis infected mice, and may provide an important complement to the field. For example, granulomas in the M. marinum model develop central caseating necrosis (Carlsson et al., PLoS Pathog 6:e1000895, 2010), a hallmark of granulomas in human tuberculosis normally not replicated in murine M. tuberculosis infection. Moreover, while tuberculosis is heterogeneous and presents with a continuum of active and latent disease, M. tuberculosis infected mice essentially lack this dynamic range and do not replicate latency (Guirado and Schlesinger, Front Immunol 4:98, 2013

  5. The Human Antibody Response to the Surface of Mycobacterium tuberculosis

    PubMed Central

    Perley, Casey C.; Frahm, Marc; Click, Eva M.; Dobos, Karen M.; Ferrari, Guido; Stout, Jason E.; Frothingham, Richard

    2014-01-01

    Background Vaccine-induced human antibodies to surface components of Haemophilus influenzae and Streptococcus pneumonia are correlated with protection. Monoclonal antibodies to surface components of Mycobacterium tuberculosis are also protective in animal models. We have characterized human antibodies that bind to the surface of live M. tuberculosis. Methods Plasma from humans with latent tuberculosis (TB) infection (n = 23), active TB disease (n = 40), and uninfected controls (n = 9) were assayed by ELISA for reactivity to the live M. tuberculosis surface and to inactivated M. tuberculosis fractions (whole cell lysate, lipoarabinomannan, cell wall, and secreted proteins). Results When compared to uninfected controls, patients with active TB disease had higher antibody titers to the surface of live M. tuberculosis (Δ = 0.72 log10), whole cell lysate (Δ = 0.82 log10), and secreted proteins (Δ = 0.62 log10), though there was substantial overlap between the two groups. Individuals with active disease had higher relative IgG avidity (Δ = 1.4 to 2.6) to all inactivated fractions. Surprisingly, the relative IgG avidity to the live M. tuberculosis surface was lower in the active disease group than in uninfected controls (Δ = –1.53, p = 0.004). Patients with active disease had higher IgG than IgM titers for all inactivated fractions (ratios, 2.8 to 10.1), but equal IgG and IgM titers to the live M. tuberculosis surface (ratio, 1.1). Higher antibody titers to the M. tuberculosis surface were observed in active disease patients who were BCG-vaccinated (Δ = 0.55 log10, p = 0.008), foreign-born (Δ = 0.61 log10, p = 0.004), or HIV-seronegative (Δ = 0.60 log10, p = 0.04). Higher relative IgG avidity scores to the M. tuberculosis surface were also observed in active disease patients who were BCG-vaccinated (Δ = 1.12, p<0.001) and foreign-born (Δ = 0.87, p = 0.01). Conclusions/Significance Humans

  6. Synthetic Long Peptide Derived from Mycobacterium tuberculosis Latency Antigen Rv1733c Protects against Tuberculosis

    PubMed Central

    Coppola, Mariateresa; van den Eeden, Susan J. F.; Wilson, Louis; Franken, Kees L. M. C.; Ottenhoff, Tom H. M.

    2015-01-01

    Responsible for 9 million new cases of active disease and nearly 2 million deaths each year, tuberculosis (TB) remains a global health threat of overwhelming dimensions. Mycobacterium bovis BCG, the only licensed vaccine available, fails to confer lifelong protection and to prevent reactivation of latent infection. Although 15 new vaccine candidates are now in clinical trials, an effective vaccine against TB remains elusive, and new strategies for vaccination are vital. BCG vaccination fails to induce immunity against Mycobacterium tuberculosis latency antigens. Synthetic long peptides (SLPs) combined with adjuvants have been studied mostly for therapeutic cancer vaccines, yet not for TB, and proved to induce efficient antitumor immunity. This study investigated an SLP derived from Rv1733c, a major M. tuberculosis latency antigen which is highly expressed by “dormant” M. tuberculosis and well recognized by T cells from latently M. tuberculosis-infected individuals. In order to assess its in vivo immunogenicity and protective capacity, Rv1733c SLP in CpG was administered to HLA-DR3 transgenic mice. Immunization with Rv1733c SLP elicited gamma interferon-positive/tumor necrosis factor-positive (IFN-γ+/TNF+) and IFN-γ+ CD4+ T cells and Rv1733c-specific antibodies and led to a significant reduction in the bacterial load in the lungs of M. tuberculosis-challenged mice. This was observed both in a pre- and in a post-M. tuberculosis challenge setting. Moreover, Rv1733c SLP immunization significantly boosted the protective efficacy of BCG, demonstrating the potential of M. tuberculosis latency antigens to improve BCG efficacy. These data suggest a promising role for M. tuberculosis latency antigen Rv1733c-derived SLPs as a novel TB vaccine approach, both in a prophylactic and in a postinfection setting. PMID:26202436

  7. How B cells shape the immune response against Mycobacterium tuberculosis.

    PubMed

    Maglione, Paul J; Chan, John

    2009-03-01

    Extensive work illustrating the importance of cellular immune mechanisms for protection against Mycobacterium tuberculosis has largely relegated B-cell biology to an afterthought within the tuberculosis (TB) field. However, recent studies have illustrated that B lymphocytes, through a variety of interactions with the cellular immune response, play previously underappreciated roles in shaping host defense against non-viral intracellular pathogens, including M. tuberculosis. Work in our laboratory has recently shown that, by considering these lymphocytes more broadly within their variety of interactions with cellular immunity, B cells have a significant impact on the outcome of airborne challenge with M. tuberculosis as well as the resultant inflammatory response. In this review, we advocate for a revised view of TB immunology in which roles of cellular and humoral immunity are not mutually exclusive. In the context of our current understanding of host defense against non-viral intracellular infections, we review recent data supporting a more significant role of B cells during M. tuberculosis infection than previously thought.

  8. The progress made in determining the Mycobacterium tuberculosis structural proteome

    PubMed Central

    Hecker, Michael

    2011-01-01

    Mycobacterium tuberculosis is a highly infectious pathogen that is still responsible for millions of deaths annually. Effectively treating this disease typically requires a course of antibiotics, most of which were developed decades ago. These drugs are, however, not effective against persistent tubercle bacilli and the emergence of drug-resistant stains threatens to make many of them obsolete. The identification of new drug targets, allowing the development of new potential drugs, is therefore imperative. Both proteomics and structural biology have important roles to play in this process, the former as a means of identifying promising drug targets and the latter allowing understanding of protein function and protein–drug interactions at atomic resolution. The determination of M. tuberculosis protein structures has been a goal of the scientific community for the last decade, who have aimed to supply a large amount of structural data that can be used in structure-based approaches for drug discovery and design. Only since the genome sequence of M. tuberculosis has been available has the determination of large numbers of tuberculosis protein structures been possible. Currently, the molecular structures of 8.5% of all the pathogen's protein-encoding ORFs have been determined. In this review, we look at the progress made in determining the M. tuberculosis structural proteome and the impact this has had on the development of potential new drugs, as well as the discovery of the function of crucial mycobaterial proteins. PMID:21674801

  9. TIM3 Mediates T Cell Exhaustion during Mycobacterium tuberculosis Infection.

    PubMed

    Jayaraman, Pushpa; Jacques, Miye K; Zhu, Chen; Steblenko, Katherine M; Stowell, Britni L; Madi, Asaf; Anderson, Ana C; Kuchroo, Vijay K; Behar, Samuel M

    2016-03-01

    While T cell immunity initially limits Mycobacterium tuberculosis infection, why T cell immunity fails to sterilize the infection and allows recrudescence is not clear. One hypothesis is that T cell exhaustion impairs immunity and is detrimental to the outcome of M. tuberculosis infection. Here we provide functional evidence for the development T cell exhaustion during chronic TB. Second, we evaluate the role of the inhibitory receptor T cell immunoglobulin and mucin domain-containing-3 (TIM3) during chronic M. tuberculosis infection. We find that TIM3 expressing T cells accumulate during chronic infection, co-express other inhibitory receptors including PD1, produce less IL-2 and TNF but more IL-10, and are functionally exhausted. Finally, we show that TIM3 blockade restores T cell function and improves bacterial control, particularly in chronically infected susceptible mice. These data show that T cell immunity is suboptimal during chronic M. tuberculosis infection due to T cell exhaustion. Moreover, in chronically infected mice, treatment with anti-TIM3 mAb is an effective therapeutic strategy against tuberculosis.

  10. Diversity of Mycobacterium tuberculosis lineages in French Polynesia.

    PubMed

    Osman, Djaltou Aboubaker; Phelippeau, Michael; Drancourt, Michel; Musso, Didier

    2017-04-01

    French Polynesia is an overseas territory located in the South Pacific. The incidence of tuberculosis in French Polynesia has been stable since 2000 with an average of 20 cases/y/100,000 inhabitants. Molecular epidemiology of Mycobacterium tuberculosis in French Polynesia is unknown because M. tuberculosis isolates have not been routinely genotyped. From 2009 to 2012, 34 isolates collected from 32 French Polynesian patients were identified as M. tuberculosis by probe hybridization. These isolates were genotyped using spoligotyping and 24-loci mycobacterial interspersed repetitive units (MIRUs)-variable number of tandem repeat (VNTR). Spoligotype patterns obtained using commercial kits were compared with the online international database SITVIT. MIRU-VNTR genotyping was performed using an in-house protocol based on capillary electrophoresis sizing for 24-loci MIRU-VNTR genotyping. The results of the spoligotyping method revealed that 25 isolates grouped into six previously described spoligotypes [H1, H3, U likely (S), T1, Manu, and Beijing] and nine isolates grouped into six new spoligotypes. Comparison with the international database MIRU-VNTRplus distributed 30 isolates into five lineages (Haarlem, Latin American Mediterranean, S, X, and Beijing) and four as unassigned isolates. Genotyping identified four phylogenetic lineages belonging to the modern Euro-American subgroup, one Beijing genotype responsible for worldwide pandemics, including remote islands in the South Pacific, and one Manu genotype of the ancestral lineage of M. tuberculosis. Copyright © 2015. Published by Elsevier B.V.

  11. Structural and functional characterization of Mycobacterium tuberculosis triosephosphate isomerase

    SciTech Connect

    Connor, Sean E.; Capodagli, Glenn C.; Deaton, Michelle K.; Pegan, Scott D.

    2012-04-18

    Tuberculosis (TB) is a major infectious disease that accounts for over 1.7 million deaths every year. Mycobacterium tuberculosis, the causative agent of tuberculosis, enters the human host by the inhalation of infectious aerosols. Additionally, one third of the world's population is likely to be infected with latent TB. The incidence of TB is on the rise owing in part to the emergence of multidrug-resistant strains. As a result, there is a growing need to focus on novel M. tuberculosis enzyme targets. M. tuberculosis triosephosphate isomerase (MtTPI) is an essential enzyme for gluconeogenetic pathways, making it a potential target for future therapeutics. In order to determine its structure, the X-ray crystal structure of MtTPI has been determined, as well as that of MtTPI bound with a reaction-intermediate analog. As a result, two forms of the active site were revealed. In conjunction with the kinetic parameters obtained for the MtTPI-facilitated conversion of dihydroxyacetone phosphate (DHAP) to D-glyceraldehyde-3-phosphate (D-GAP), this provides a greater structural and biochemical understanding of this enzyme. Additionally, isothermal titration calorimetry was used to determine the binding constant for a reaction-intermediate analog bound to the active site of MtTPI.

  12. Bystander Macrophage Apoptosis after Mycobacterium tuberculosis H37Ra Infection▿

    PubMed Central

    Kelly, Deirdre M.; ten Bokum, Annemieke M. C.; O'Leary, Seonadh M.; O'Sullivan, Mary P.; Keane, Joseph

    2008-01-01

    Human macrophages infected with Mycobacterium tuberculosis may undergo apoptosis. Macrophage apoptosis contributes to the innate immune response against M. tuberculosis by containing and limiting the growth of mycobacteria and also by depriving the bacillus of its niche cell. Apoptosis of infected macrophages is well documented; however, bystander apoptosis of uninfected macrophages has not been described in the setting of M. tuberculosis. We observed that uninfected human macrophages underwent significant bystander apoptosis 48 and 96 h after they came into contact with macrophages infected with avirulent M. tuberculosis. The bystander apoptosis was significantly greater than the background apoptosis observed in uninfected control cells cultured for the same length of time. There was no evidence of the involvement of tumor necrosis factor alpha, Fas, tumor necrosis factor-related apoptosis-inducing ligand, transforming growth factor β, Toll-like receptor 2, or MyD88 in contact-mediated bystander apoptosis. This newly described phenomenon may further limit the spread of M. tuberculosis by eliminating the niche cells on which the bacillus relies. PMID:17954721

  13. Gene Transfer in Mycobacterium tuberculosis: Shuttle Phasmids to Enlightenment

    PubMed Central

    JACOBS, WILLIAM R.

    2016-01-01

    Infectious diseases have plagued humankind throughout history and have posed serious public health problems. Yet vaccines have eradicated smallpox and antibiotics have drastically decreased the mortality rate of many infectious agents. These remarkable successes in the control of infections came from knowing the causative agents of the diseases, followed by serendipitous discoveries of attenuated viruses and antibiotics. The discovery of DNA as genetic material and the understanding of how this information translates into specific phenotypes have changed the paradigm for developing new vaccines, drugs, and diagnostic tests. Knowledge of the mechanisms of immunity and mechanisms of action of drugs has led to new vaccines and new antimicrobial agents. The key to the acquisition of the knowledge of these mechanisms has been identifying the elemental causes (i.e., genes and their products) that mediate immunity and drug resistance. The identification of these genes is made possible by being able to transfer the genes or mutated forms of the genes into causative agents or surrogate hosts. Such an approach was limited in Mycobacterium tuberculosis by the difficulty of transferring genes or alleles into M. tuberculosis or a suitable surrogate mycobacterial host. The construction of shuttle phasmids—chimeric molecules that replicate in Escherichia coli as plasmids and in mycobacteria as mycobacteriophages—was instrumental in developing gene transfer systems for M. tuberculosis. This review will discuss M. tuberculosis genetic systems and their impact on tuberculosis research. “I had to know my enemy in order to prevail against him.”Nelson Mandela PMID:26105819

  14. Early clearance of Mycobacterium tuberculosis: a new frontier in prevention

    PubMed Central

    Verrall, Ayesha J; Netea, Mihai G; Alisjahbana, Bachti; Hill, Philip C; van Crevel, Reinout

    2014-01-01

    Early clearance (EC) is the successful eradication of inhaled Mycobacterium tuberculosis before an adaptive immune response develops. Evidence for EC comes from case contact studies that consistently show that a proportion of heavily exposed individuals do not develop M. tuberculosis infection. Further support for the existence of this phenotype comes from genetic loci associated with tuberculin reactivity. In this review we discuss aspects of the innate response that may underpin EC and hypotheses that can be tested through field laboratory link studies in M. tuberculosis case contacts. Specifically, we consider mechanisms whereby alveolar macrophages recognize and kill intracellular M. tuberculosis, and how other cell types, such as neutrophils, natural killer T cells, mucosa-associated invariant T cells and γδ T cells may assist. How EC may be impaired by HIV infection or vitamin D deficiency is also explored. As EC is a form of protective immunity, further study may advance the development of vaccines and immunotherapies to prevent M. tuberculosis infection. PMID:24754048

  15. Evasion of Innate and Adaptive Immunity by Mycobacterium tuberculosis.

    PubMed

    Goldberg, Michael F; Saini, Neeraj K; Porcelli, Steven A

    2014-10-01

    Through thousands of years of reciprocal coevolution, Mycobacterium tuberculosis has become one of humanity's most successful pathogens, acquiring the ability to establish latent or progressive infection and persist even in the presence of a fully functioning immune system. The ability of M. tuberculosis to avoid immune-mediated clearance is likely to reflect a highly evolved and coordinated program of immune evasion strategies that interfere with both innate and adaptive immunity. These include the manipulation of their phagosomal environment within host macrophages, the selective avoidance or engagement of pattern recognition receptors, modulation of host cytokine production, and the manipulation of antigen presentation to prevent or alter the quality of T-cell responses. In this article we review an extensive array of published studies that have begun to unravel the sophisticated program of specific mechanisms that enable M. tuberculosis and other pathogenic mycobacteria to persist and replicate in the face of considerable immunological pressure from their hosts. Unraveling the mechanisms by which M. tuberculosis evades or modulates host immune function is likely to be of major importance for the development of more effective new vaccines and targeted immunotherapy against tuberculosis.

  16. Mycobacterium tuberculosis Pathogenesis and Molecular Determinants of Virulence

    PubMed Central

    Smith, Issar

    2003-01-01

    Tuberculosis (TB), one of the oldest known human diseases. is still is one of the major causes of mortality, since two million people die each year from this malady. TB has many manifestations, affecting bone, the central nervous system, and many other organ systems, but it is primarily a pulmonary disease that is initiated by the deposition of Mycobacterium tuberculosis, contained in aerosol droplets, onto lung alveolar surfaces. From this point, the progression of the disease can have several outcomes, determined largely by the response of the host immune system. The efficacy of this response is affected by intrinsic factors such as the genetics of the immune system as well as extrinsic factors, e.g., insults to the immune system and the nutritional and physiological state of the host. In addition, the pathogen may play a role in disease progression since some M. tuberculosis strains are reportedly more virulent than others, as defined by increased transmissibility as well as being associated with higher morbidity and mortality in infected individuals. Despite the widespread use of an attenuated live vaccine and several antibiotics, there is more TB than ever before, requiring new vaccines and drugs and more specific and rapid diagnostics. Researchers are utilizing information obtained from the complete sequence of the M. tuberculosis genome and from new genetic and physiological methods to identify targets in M. tuberculosis that will aid in the development of these sorely needed antitubercular agents. PMID:12857778

  17. Building a better bacillus: the emergence of Mycobacterium tuberculosis

    PubMed Central

    Wang, Joyce; Behr, Marcel A.

    2014-01-01

    The genus Mycobacterium is comprised of more than 150 species that reside in a wide variety of habitats. Most mycobacteria are environmental organisms that are either not associated with disease or are opportunistic pathogens that cause non-transmissible disease in immunocompromised individuals. In contrast, a small number of species, such as the tubercle bacillus, Mycobacterium tuberculosis, are host-adapted pathogens for which there is no known environmental reservoir. In recent years, gene disruption studies using the host-adapted pathogen have uncovered a number of “virulence factors,” yet genomic data indicate that many of these elements are present in non-pathogenic mycobacteria. This suggests that much of the genetic make-up that enables virulence in the host-adapted pathogen is already present in environmental members of the genus. In addition to these generic factors, we hypothesize that molecules elaborated exclusively by professional pathogens may be particularly implicated in the ability of M. tuberculosis to infect, persist, and cause transmissible pathology in its host species, Homo sapiens. One approach to identify these molecules is to employ comparative analysis of mycobacterial genomes, to define evolutionary events such as horizontal gene transfer (HGT) that contributed M. tuberculosis-specific genetic elements. Independent studies have now revealed the presence of HGT genes in the M. tuberculosis genome and their role in the pathogenesis of disease is the subject of ongoing investigations. Here we review these studies, focusing on the hypothesized role played by HGT loci in the emergence of M. tuberculosis from a related environmental species into a highly specialized human-adapted pathogen. PMID:24765091

  18. Allelic exchange in Mycobacterium tuberculosis with long linear recombination substrates.

    PubMed Central

    Balasubramanian, V; Pavelka, M S; Bardarov, S S; Martin, J; Weisbrod, T R; McAdam, R A; Bloom, B R; Jacobs, W R

    1996-01-01

    Genetic studies of Mycobacterium tuberculosis have been greatly hampered by the inability to introduce specific chromosomal mutations. Whereas the ability to perform allelic exchanges has provided a useful method of gene disruption in other organisms, in the clinically important species of mycobacteria, such as M. tuberculosis and Mycobacterium bovis, similar approaches have thus far been unsuccessful. In this communication, we report the development of a shuttle mutagenesis strategy that involves the use of long linear recombination substrates to reproducibly obtain recombinants by allelic exchange in M. tuberculosis. Long linear recombination substrates, approximately 40 to 50 kb in length, were generated by constructing libraries in the excisable cosmid vector pYUB328. The cosmid vector could be readily excised from the recombinant cosmids by digestion with PacI, a restriction endonuclease for which there exist few, if any, sites in mycobacterial genomes. A cosmid containing the mycobacterial leuD gene was isolated, and a selectable marker conferring resistance to kanamycin was inserted into the leuD gene in the recombinant cosmid by interplasmid recombination in Escherichia coli. A long linear recombination substrate containing the insertionally mutated leuD gene was generated by PacI digestion. Electroporation of this recombination substrate containing the insertionally mutated leuD allele resulted in the generation of leucine auxotrophic mutants by homologous recombination in 6% of the kanamycin-resistant transformants for both the Erdman and H37Rv strains of M. tuberculosis. The ability to perform allelic exchanges provides an important approach for investigating the biology of this pathogen as well as developing new live-cell M. tuberculosis-based vaccines. PMID:8550428

  19. A human challenge model for Mycobacterium tuberculosis using Mycobacterium bovis bacille Calmette-Guerin.

    PubMed

    Minassian, Angela M; Satti, Iman; Poulton, Ian D; Meyer, Joel; Hill, Adrian V S; McShane, Helen

    2012-04-01

    There is currently no safe human challenge model of Mycobacterium tuberculosis infection to enable proof-of-concept efficacy evaluation of candidate vaccines against tuberculosis. In vivo antimycobacterial immunity could be assessed using intradermal Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccination as a surrogate for M. tuberculosis infection. Healthy BCG-naive and BCG-vaccinated volunteers were challenged with intradermal BCG. BCG load was quantified from skin biopsy specimens by polymerase chain reaction (PCR) and culture colony-forming units. Cellular infiltrate was isolated by suction blisters and examined by flow cytometry. Prechallenge immune readouts were correlated with BCG load after challenge. In BCG-naive volunteers, live BCG was detected at the challenge site for up to 4 weeks and peaked at 2 weeks. Infiltration of mainly CD15(+) neutrophils was observed in blister fluid. In previously BCG-vaccinated individuals, PCR analysis of skin biopsy specimens reflected a degree of mycobacterial immunity. There was no significant correlation between BCG load after challenge and mycobacterial-specific memory T cells measured before challenge by cultured enzyme-linked immunospot assay. This novel experimental human challenge model provides a platform for the identification of correlates of antimycobacterial immunity and will greatly facilitate the rational down-selection of candidate tuberculosis vaccines. Further evaluation of this model with BCG and new vaccine candidates is warranted.

  20. A genomic view of sugar transport in Mycobacterium smegmatis and Mycobacterium tuberculosis.

    PubMed

    Titgemeyer, Fritz; Amon, Johannes; Parche, Stephan; Mahfoud, Maysa; Bail, Johannes; Schlicht, Maximilian; Rehm, Nadine; Hillmann, Dietmar; Stephan, Joachim; Walter, Britta; Burkovski, Andreas; Niederweis, Michael

    2007-08-01

    We present a comprehensive analysis of carbohydrate uptake systems of the soil bacterium Mycobacterium smegmatis and the human pathogen Mycobacterium tuberculosis. Our results show that M. smegmatis has 28 putative carbohydrate transporters. The majority of sugar transport systems (19/28) in M. smegmatis belong to the ATP-binding cassette (ABC) transporter family. In contrast to previous reports, we identified genes encoding all components of the phosphotransferase system (PTS), including permeases for fructose, glucose, and dihydroxyacetone, in M. smegmatis. It is anticipated that the PTS of M. smegmatis plays an important role in the global control of carbon metabolism similar to those of other bacteria. M. smegmatis further possesses one putative glycerol facilitator of the major intrinsic protein family, four sugar permeases of the major facilitator superfamily, one of which was assigned as a glucose transporter, and one galactose permease of the sodium solute superfamily. Our predictions were validated by gene expression, growth, and sugar transport analyses. Strikingly, we detected only five sugar permeases in the slow-growing species M. tuberculosis, two of which occur in M. smegmatis. Genes for a PTS are missing in M. tuberculosis. Our analysis thus brings the diversity of carbohydrate uptake systems of fast- and a slow-growing mycobacteria to light, which reflects the lifestyles of M. smegmatis and M. tuberculosis in their natural habitats, the soil and the human body, respectively.

  1. Mycobacterium tuberculosis: ecology and evolution of a human bacterium.

    PubMed

    Bañuls, Anne-Laure; Sanou, Adama; Anh, Nguyen Thi Van; Godreuil, Sylvain

    2015-11-01

    Some species of the Mycobacterium tuberculosis complex (MTBC), particularly Mycobacterium tuberculosis, which causes human tuberculosis (TB), are the first cause of death linked to a single pathogen worldwide. In the last decades, evolutionary studies have much improved our knowledge on MTBC history and have highlighted its long co-evolution with humans. Its ability to remain latent in humans, the extraordinary proportion of asymptomatic carriers (one-third of the entire human population), the deadly epidemics and the observed increasing level of resistance to antibiotics are proof of its evolutionary success. Many MTBC molecular signatures show not only that these bacteria are a model of adaptation to humans but also that they have influenced human evolution. Owing to the unbalance between the number of asymptomatic carriers and the number of patients with active TB, some authors suggest that infection by MTBC could have a protective role against active TB disease and also against other pathologies. However, it would be inappropriate to consider these infectious pathogens as commensals or symbionts, given the level of morbidity and mortality caused by TB.

  2. Targeting Mycobacterium tuberculosis Topoisomerase I by Small-Molecule Inhibitors

    PubMed Central

    Godbole, Adwait Anand; Ahmed, Wareed; Bhat, Rajeshwari Subray; Bradley, Erin K.; Ekins, Sean

    2014-01-01

    We describe inhibition of Mycobacterium tuberculosis topoisomerase I (MttopoI), an essential mycobacterial enzyme, by two related compounds, imipramine and norclomipramine, of which imipramine is clinically used as an antidepressant. These molecules showed growth inhibition of both Mycobacterium smegmatis and M. tuberculosis cells. The mechanism of action of these two molecules was investigated by analyzing the individual steps of the topoisomerase I (topoI) reaction cycle. The compounds stimulated cleavage, thereby perturbing the cleavage-religation equilibrium. Consequently, these molecules inhibited the growth of the cells overexpressing topoI at a low MIC. Docking of the molecules on the MttopoI model suggested that they bind near the metal binding site of the enzyme. The DNA relaxation activity of the metal binding mutants harboring mutations in the DxDxE motif was differentially affected by the molecules, suggesting that the metal coordinating residues contribute to the interaction of the enzyme with the drug. Taken together, the results highlight the potential of these small molecules, which poison the M. tuberculosis and M. smegmatis topoisomerase I, as leads for the development of improved molecules to combat mycobacterial infections. Moreover, targeting metal coordination in topoisomerases might be a general strategy to develop new lead molecules. PMID:25534741

  3. Dielectrophoretic characterization of antibiotic-treated Mycobacterium tuberculosis complex cells.

    PubMed

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

    2015-10-01

    Multi-drug resistant tuberculosis (MDR-TB) has become a serious concern for proper treatment of patients. As a phenotypic method, dielectrophoresis can be useful but is yet to be attempted to evaluate Mycobacterium tuberculosis complex cells. This paper investigates the dielectrophoretic behavior of Mycobacterium bovis (Bacillus Calmette-Guérin, BCG) cells that are treated with heat or antibiotics rifampin (RIF) or isoniazid (INH). The experimental parameters are designed on the basis of our sensitivity analysis. The medium conductivity (σ(m)) and the frequency (f) for a crossover frequency (f(xo1)) test are decided to detect the change of σ(m)-f(xo1) in conjunction with the drug mechanism. Statistical modeling is conducted to estimate the distributions of viable and nonviable cells from the discrete measurement of f (xo1). Finally, the parameters of the electrophysiology of BCG cells, C(envelope) and σ(cyto), are extracted through a sampling algorithm. This is the first evaluation of the dielectrophoresis (DEP) approach as a means to assess the effects of antimicrobial drugs on M. tuberculosis complex cells.

  4. Targeting Mycobacterium tuberculosis topoisomerase I by small-molecule inhibitors.

    PubMed

    Godbole, Adwait Anand; Ahmed, Wareed; Bhat, Rajeshwari Subray; Bradley, Erin K; Ekins, Sean; Nagaraja, Valakunja

    2015-03-01

    We describe inhibition of Mycobacterium tuberculosis topoisomerase I (MttopoI), an essential mycobacterial enzyme, by two related compounds, imipramine and norclomipramine, of which imipramine is clinically used as an antidepressant. These molecules showed growth inhibition of both Mycobacterium smegmatis and M. tuberculosis cells. The mechanism of action of these two molecules was investigated by analyzing the individual steps of the topoisomerase I (topoI) reaction cycle. The compounds stimulated cleavage, thereby perturbing the cleavage-religation equilibrium. Consequently, these molecules inhibited the growth of the cells overexpressing topoI at a low MIC. Docking of the molecules on the MttopoI model suggested that they bind near the metal binding site of the enzyme. The DNA relaxation activity of the metal binding mutants harboring mutations in the DxDxE motif was differentially affected by the molecules, suggesting that the metal coordinating residues contribute to the interaction of the enzyme with the drug. Taken together, the results highlight the potential of these small molecules, which poison the M. tuberculosis and M. smegmatis topoisomerase I, as leads for the development of improved molecules to combat mycobacterial infections. Moreover, targeting metal coordination in topoisomerases might be a general strategy to develop new lead molecules.

  5. Mycobacterium tuberculosis infection in women with unexplained infertility

    PubMed Central

    Eftekhar, Maryam; Pourmasumi, Soheila; Sabeti, Parvin; Aflatoonian, Abbas; Sheikhha, Mohammad Hasan

    2015-01-01

    Background: Genital tuberculosis (GTB) is an important cause of female infertility, especially in developing countries. The positive results of polymerase chain reaction (PCR) in endometrial GTB in the absence of tubal damage raise the possibility of the detection of sub-clinical or latent disease, with doubtful benefits of treatment. Objective: To evaluate the mycobacterium tuberculosis infection in endometrial biopsy samples collected from unexplained infertile women attending Yazd Research and Clinical Center for Infertility by using PCR techniques. Materials and Methods: In this cross sectional study, 144 infertile women with unexplained infertility aged 20-35 years old and normal Histro-saplango graphy findings were enrolled. Endometrial biopsy samples from each participant were tested for mycobacterium tuberculosis detecting by PCR. In 93 patients, peritoneal fluid was also taken for culture and PCR. Results: The PCR results of endometrial specimens were negative in all cases, demonstrating that there was no GTB infection among our patients. Conclusion: Our results showed that GTB could not be considered as a major problem in women with unexplained infertility. Although, studies have indicated that PCR is a useful method in diagnosing early GTB disease in infertile women with no demonstrable evidence of tubal or endometrial involvement. PMID:27141534

  6. Mycobacterium tuberculosis adhesins: potential biomarkers as anti-tuberculosis therapeutic and diagnostic targets.

    PubMed

    Govender, Viveshree S; Ramsugit, Saiyur; Pillay, Manormoney

    2014-09-01

    Adhesion to host cells is a precursor to host colonization and evasion of the host immune response. Conversely, it triggers the induction of the immune response, a process vital to the host's defence against infection. Adhesins are microbial cell surface molecules or structures that mediate the attachment of the microbe to host cells and thus the host-pathogen interaction. They also play a crucial role in bacterial aggregation and biofilm formation. In this review, we discuss the role of adhesins in the pathogenesis of the aetiological agent of tuberculosis, Mycobacterium tuberculosis. We also provide insight into the structure and characteristics of some of the characterized and putative M. tuberculosis adhesins. Finally, we examine the potential of adhesins as targets for the development of tuberculosis control strategies.

  7. Overview and phylogeny of Mycobacterium tuberculosis complex organisms: implications for diagnostics and legislation of bovine tuberculosis.

    PubMed

    Rodriguez-Campos, Sabrina; Smith, Noel H; Boniotti, Maria B; Aranaz, Alicia

    2014-10-01

    Members of the Mycobacterium tuberculosis complex (MTBC) cause a serious disease with similar pathology, tuberculosis; in this review, bovine tuberculosis will be considered as disease caused by any member of the MTBC in bovids. Bovine tuberculosis is responsible for significant economic loss due to costly eradication programs and trade limitations and poses a threat to both endangered and protected species as well as to public health. We here give an overview on all members of the MTBC, focusing on their isolation from different animal hosts. We also review the recent advances made in elucidating the evolutionary and phylogenetic relationships of members of the MTBC. Because the nomenclature of the MTBC is controversial, its members have been considered species, subspecies or ecotypes, this review discusses the possible implications for diagnostics and the legal consequences of naming of new species. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Genetic Lineages of Mycobacterium tuberculosis Isolates in Isfahan, Iran.

    PubMed

    Riyahi Zaniani, Fatemeh; Moghim, Sharareh; Mirhendi, Hossein; Ghasemian Safaei, Hajieh; Fazeli, Hossein; Salehi, Mahshid; Nasr Esfahani, Bahram

    2017-01-01

    In this study, we aimed to identify the genetic lineages of Mycobacterium tuberculosis isolates in Isfahan via the mycobacterial interspersed repetitive-unit-variable number tandem repeat typing method based on 15 loci. Forty-nine M. tuberculosis isolates were collected between 2013 and 2015 from Tuberculosis patients in Mollahadi Sabzevari Tuberculosis Center in Isfahan. All isolates were typed by 15-locus MIRU-VNTR typing. The highest percentage of isolates, 44.89 % (22/49), belonged to the Euro-American lineage, while the frequencies of the East-African-Indian, East-Asian, and Indo-Oceanic lineages were 28.57 % (14/49), 24.4 % (12/49), and 2.04 % (1/49), respectively. Among the 22 isolates of the Euro-American lineage, those belonging to the NEW-1 sub-lineage were most prevalent (24.4 %). Approximately, the same proportion of isolates belonging to the Delhi/CAS, Beijing, and NEW-1 sub-lineages were identified in Iranian and Afghan immigrant patients. The Delhi/CAS and Beijing sub-lineage isolates were prevalent among patients who had been previously treated for TB. Results showed that all of the 49 MIRU-VNTR patterns were unique and the clustering rate of the 15-locus MIRU-VNTR was 0.0 (minimum recent transmission). The results of this study show that the lineages of M. tuberculosis isolates in Isfahan are similar to those reported in the Eastern Mediterranean region (indicative of the epidemiological relationship between the countries in the region). The low clustering rate in our results reveals that transmission of tuberculosis in Isfahan is, in most cases, a reactivation of previous tuberculosis infection and the role of recently transmitted disease is minor.

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

    PubMed

    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

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

    PubMed Central

    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

  11. Tuberculosis Caused by Mycobacterium bovis in a Capybara (Hydrochoerus hydrochaeris).

    PubMed

    Mol, J P S; Carvalho, T F; Fonseca, A A; Sales, E B; Issa, M A; Rezende, L C; Hodon, M A; Tinoco, H P; Malta, M C C; Pessanha, A T; Pierezan, F; Mota, P M P C; Paixão, T A; Santos, R L

    2016-01-01

    Tuberculosis, associated with Mycobacterium bovis, was diagnosed post mortem in an adult female capybara (Hydrochoerus hydrochaeris), kept at the Pampulha Ecological Park, Belo Horizonte, Brazil, in a large metropolitan area. On post-mortem examination, there were numerous firm white nodules scattered throughout all lobes of both lungs. Tissue samples were collected for histological and microbiological examination. Microscopically, the pulmonary nodules were multifocal to coalescing granulomas and intralesional acid-fast bacilli were evident in Ziehl-Neelsen-stained sections of the lung and spleen. Colonies with morphological features of Mycobacterium spp. were isolated from lung samples and conventional polymerase chain reaction (PCR) with genomic DNA from the isolates was positive for M. bovis; sequencing indicated 100% identity with the region of difference 4 (RD4) of M. bovis. In addition, M. bovis DNA was detected in the lung by quantitative PCR. The finding of M. bovis in a capybara indicates a potential public health risk in a zoological collection.

  12. Encoded library technology as a source of hits for the discovery and lead optimization of a potent and selective class of bactericidal direct inhibitors of Mycobacterium tuberculosis InhA.

    PubMed

    Encinas, Lourdes; O'Keefe, Heather; Neu, Margarete; Remuiñán, Modesto J; Patel, Amish M; Guardia, Ana; Davie, Christopher P; Pérez-Macías, Natalia; Yang, Hongfang; Convery, Maire A; Messer, Jeff A; Pérez-Herrán, Esther; Centrella, Paolo A; Alvarez-Gómez, Daniel; Clark, Matthew A; Huss, Sophie; O'Donovan, Gary K; Ortega-Muro, Fátima; McDowell, William; Castañeda, Pablo; Arico-Muendel, Christopher C; Pajk, Stane; Rullás, Joaquín; Angulo-Barturen, Iñigo; Alvarez-Ruíz, Emilio; Mendoza-Losana, Alfonso; Ballell Pages, Lluís; Castro-Pichel, Julia; Evindar, Ghotas

    2014-02-27

    Tuberculosis (TB) is one of the world's oldest and deadliest diseases, killing a person every 20 s. InhA, the enoyl-ACP reductase from Mycobacterium tuberculosis, is the target of the frontline antitubercular drug isoniazid (INH). Compounds that directly target InhA and do not require activation by mycobacterial catalase peroxidase KatG are promising candidates for treating infections caused by INH resistant strains. The application of the encoded library technology (ELT) to the discovery of direct InhA inhibitors yielded compound 7 endowed with good enzymatic potency but with low antitubercular potency. This work reports the hit identification, the selected strategy for potency optimization, the structure-activity relationships of a hundred analogues synthesized, and the results of the in vivo efficacy studies performed with the lead compound 65.

  13. Extended culture enhances sensitivity of a gamma interferon assay for latent Mycobacterium tuberculosis infection.

    PubMed

    Cehovin, Ana; Cliff, Jacqueline M; Hill, Philip C; Brookes, Roger H; Dockrell, Hazel M

    2007-06-01

    To test the hypothesis that prolonged culture would enhance the sensitivity of latent tuberculosis detection by a gamma interferon release assay, blood samples from 33 household contacts of Gambian tuberculosis patients were stimulated with Mycobacterium tuberculosis-specific antigens. After 24 h of culture, 66% were positive, compared to 93% after 6 days of culture.

  14. Detection of Mycobacterium tuberculosis in latently infected lungs by immunohistochemistry and confocal microscopy

    PubMed Central

    Eugenin, Eliseo; Kaplan, Gilla

    2014-01-01

    Detection of latent Mycobacterium tuberculosis is a challenge in the diagnosis of asymptomatic, subclinical tuberculosis. We report the development of an immunofluorescence technique to visualize and enumerate M. tuberculosis in latently infected rabbit lungs where no acid-fast–stained organisms were seen and no cultivable bacilli were obtained by the agar-plating method. PMID:25161200

  15. Mechanisms of cell recruitment in the immune response to Mycobacterium tuberculosis.

    PubMed

    Peters, Wendy; Ernst, Joel D

    2003-02-01

    Recent advances in understanding cell traffic, especially the roles of adhesion proteins, chemokines, and chemokine receptors, provide the opportunity for understanding mechanisms involved in the immune response to tuberculosis. This review concentrates on the roles of these molecules and the immune response in tuberculosis, based on studies of humans and mice infected with Mycobacterium tuberculosis.

  16. Prenatal passive transfer of Mycobacterium tuberculosis antibodies in Asian elephant (Elephas maximus) calves.

    PubMed

    McGee, Jennifer L; Wiedner, Ellen; Isaza, Ramiro

    2014-12-01

    Asian elephant (Elephas maximus) dams and their newborn calves were tested for Mycobacterium tuberculosis antibodies in serum. Blood was drawn from dams prior to calving and from calves on their day of birth. All six calves born to tuberculosis-reactive dams were also tuberculosis reactive, suggesting prenatal passive placental transfer of tuberculosis antibodies. In contrast, all three calves born to tuberculosis-nonreactive dams lacked detectable tuberculosis antibodies in pre-suckling or day-of-birth blood samples. Of the living tuberculosis-reactive calves observed from 1 to 11 yr of age, none exhibited clinical signs of tuberculosis infection or became tuberculosis culture positive. This is the first report of prenatal passive placental transfer of tuberculosis antibodies in elephants and demonstrates that detectible tuberculosis antibodies in newborn elephant calves should not be assumed to correlate with clinical tuberculosis.

  17. Tuberculosis part 4: Control of Mycobacterium tuberculosis transmission in dental care facilities.

    PubMed

    Feller, L; Wood, N H; Chikte, U M E; Khammissa, R A G; Meyerov, R; Lemmer, J

    2009-10-01

    The risk of transmission of Mycobacterium tuberculosis (Mtb) in oral healthcare facilities is probably low, but the consequences if it occurs, are grave. The greatest risk of exposure to Mtb transmission is associated with treating dental patients from communities with a high prevalence of tuberculosis (TB) and HIV disease because these patients may have active TB but be unaware of their status. The risk of Mtb transmission in the dental surgery is heightened by dental treatment with ultrasonic and air operated high speed instruments that generate aerosols. This article offers recommendations for the necessary components of an effective Mtb infection control programme.

  18. Mycobacterium tuberculosis infection in an orangutan (Pongo pygmaeus).

    PubMed

    Shin, N S; Kwon, S W; Han, D H; Bai, G H; Yoon, J; Cheon, D S; Son, Y S; Ahn, K; Chae, C; Lee, Y S

    1995-10-01

    A respiratory disorder was noted in a 5-year-old female orangutan kept in the Yongin Farmland. Radiographically, multiple radiodense foci ranging from 2 to 6 mm diameter were seen throughout the lung lobes. Grossly, the thoracic cavity revealed a firm texture and grayish-pink discoloration of the left apical lung lobe. Histopathologically, multifocal areas of granulomatous pneumonia present the right and left apical lung lobes. Both primers from IS1081 and IS6110 targeting 196 bp and 245 bp respectively were used in polymerase chain reaction, Mycobacterium tuberculosis was isolated from liver and confirmed by polymerase chain reaction.

  19. Anti-Mycobacterium tuberculosis activity of fungus Phomopsis stipata

    PubMed Central

    de Prince, Karina Andrade; Sordi, Renata; Pavan, Fernando Rogério; Barreto Santos, Adolfo Carlos; Araujo, Angela R.; Leite, Sergio R.A.; Leite, Clarice Q. F.

    2012-01-01

    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. PMID:24031821

  20. Zoonotic tuberculosis due to Mycobacterium bovis in developing countries.

    PubMed Central

    Cosivi, O.; Grange, J. M.; Daborn, C. J.; Raviglione, M. C.; Fujikura, T.; Cousins, D.; Robinson, R. A.; Huchzermeyer, H. F.; de Kantor, I.; Meslin, F. X.

    1998-01-01

    The World Health Organization (WHO) estimates that human tuberculosis (TB) incidence and deaths for 1990 to 1999 will be 88 million and 30 million, respectively, with most cases in developing countries. Zoonotic TB (caused by Mycobacterium bovis) is present in animals in most developing countries where surveillance and control activities are often inadequate or unavailable; therefore, many epidemiologic and public health aspects of infection remain largely unknown. We review available information on zoonotic TB in developing countries, analyze risk factors that may play a role in the disease, review recent WHO activities, and recommend actions to assess the magnitude of the problem and control the disease in humans and animals. PMID:9452399

  1. Chlorhexidine decontamination of sputum for culturing Mycobacterium tuberculosis.

    PubMed

    Asmar, Shady; Drancourt, Michel

    2015-08-05

    Culture of Mycobacterium tuberculosis is the gold standard method for the laboratory diagnosis of pulmonary tuberculosis, after effective decontamination. We evaluated squalamine and chlorhexidine to decontaminate sputum specimens for the culture of mycobacteria. Eight sputum specimens were artificially infected with 10(5) colony-forming units (cfu)/mL Mycobacterium tuberculosis and Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans as contaminants. In the second step, we tested chlorhexidine-based decontamination on 191 clinical specimens, (Chlorhexidine, 0.1, 0.5 and 0.7 %). In a last step, growth of contaminants and mycobacteria was measured in 75 consecutive sputum specimens using the routine NALC-NaOH decontamination protocol or with 0.7 % chlorhexidine decontamination and an inoculation on Coletsos medium. In the artificially model, contaminants grew in 100 % of the artificially infected sputum specimens decontaminated using 100 mg/mL squalamine, in 62.5 % of specimens decontaminated using N-Acetyl-L-Cysteine-Sodium Hydroxide (NALC-NaOH), and in 0 % of specimens decontaminated using 0.1 %, 0.35 %, or 1 % chlorhexidine (P < 0.05). These specimens yielded <10(2) cfu M. tuberculosis using NALC-NaOH and > 1.4.10(2) cfu M. tuberculosis when any concentration of chlorhexidine was used (P < 0.05). In the second step we found that 0.7 %-chlorhexidine yielded 0 % contamination rate, 3.2 % for 0.5 %-chlorhexidine and 28.3 % for 0.1 %-chlorhexidine. As for the 75 specimens treated in parallel by both methods we found that when using the standard NALC-NaOH decontamination method, 8/75 (10.7 %) specimens yielded M. tuberculosis colonies with a time to detection of 17.5 ± 3 days and an 8 % contamination rate. Additionally, 14 specimens yielded mycobacteria colonies (12 M. tuberculosis, and 2 Mycobacterium bolletii) (18.7 %) (P = 0.25), which has yielded a 100 % sensitivity for the chlorhexidine protocol. Time

  2. Selective Mycobacterium tuberculosis Shikimate Kinase Inhibitors as Potential Antibacterials

    PubMed Central

    Gordon, Sara; Simithy, Johayra; Goodwin, Douglas C; Calderón, Angela I

    2015-01-01

    Owing to the persistence of tuberculosis (TB) as well as the emergence of multidrug-resistant and extensively drug-resistant (XDR) forms of the disease, the development of new antitubercular drugs is crucial. Developing inhibitors of shikimate kinase (SK) in the shikimate pathway will provide a selective target for antitubercular agents. Many studies have used in silico technology to identify compounds that are anticipated to interact with and inhibit SK. To a much more limited extent, SK inhibition has been evaluated by in vitro methods with purified enzyme. Currently, there are no data on in vivo activity of Mycobacterium tuberculosis shikimate kinase (MtSK) inhibitors available in the literature. In this review, we present a summary of the progress of SK inhibitor discovery and evaluation with particular attention toward development of new antitubercular agents. PMID:25861218

  3. Immunoinformatics study on highly expressed Mycobacterium tuberculosis genes during infection.

    PubMed

    Nguyen Thi, Le Thuy; Sarmiento, Maria Elena; Calero, Romel; Camacho, Frank; Reyes, Fatima; Hossain, Md Murad; Gonzalez, Gustavo Sierra; Norazmi, Mohd Nor; Acosta, Armando

    2014-09-01

    The most important targets for vaccine development are the proteins that are highly expressed by the microorganisms during infection in-vivo. A number of Mycobacterium tuberculosis (Mtb) proteins are also reported to be expressed in-vivo at different phases of infection. In the present study, we analyzed multiple published databases of gene expression profiles of Mtb in-vivo at different phases of infection in animals and humans and selected 38 proteins that are highly expressed in the active, latent and reactivation phases. We predicted T- and B-cell epitopes from the selected proteins using HLAPred for T-cell epitope prediction and BCEPred combined with ABCPred for B-cell epitope prediction. For each selected proteins, regions containing both T- and B-cell epitopes were identified which might be considered as important candidates for vaccine design against tuberculosis.

  4. Clonal complexity in Mycobacterium tuberculosis can hamper diagnostic procedures.

    PubMed

    Pérez-Lago, Laura; Herranz, Marta; Navarro, Yurena; Ruiz Serrano, María Jesús; Miralles, Pilar; Bouza, Emilio; García-de-Viedma, Darío

    2017-02-15

    Clonal complexity is increasingly accepted in Mycobacterium tuberculosis infection, including mixed infections by ≥2 strains, which usually occur in settings with a high burden of tuberculosis and/or a high risk of overexposure to infected patients. Mixed infections can hamper diagnostic procedures: obtaining an accurate antibiogram is difficult when the susceptibility patterns of the strains differ. Here, we show how mixed infections can also prove challenging for other diagnostic procedures, even outside settings where mixed infections are traditionally expected. We show how an unnoticed mixed infection in an HIV-positive patient diagnosed in Madrid, Spain, with differences in the representativeness of the coinfecting strains in different sputum samples, markedly complicated the resolution of a laboratory cross-contamination false-positivity alert.

  5. Structural enzymology of sulphur metabolism in Mycobacterium tuberculosis.

    PubMed

    Schnell, Robert; Schneider, Gunter

    2010-05-21

    The emergence of multidrug-resistant strains of Mycobacterium tuberculosis poses a serious threat to human health and has led to world-wide efforts focusing on the development of novel vaccines and antibiotics against this pathogen. Sulphur metabolism in this organism has been linked to essential processes such as virulence and redox defence. The cysteine biosynthetic pathway is up-regulated in models of persistent M. tuberculosis infections and provides potential targets for novel anti-mycobacterial agents, directed specifically toward the pathogen in its persistent phase. Functional and structural characterization of enzymes from sulfur metabolism establishes a necessary framework for the design of strong binding inhibitors that might be developed into new drugs. This review summarizes recent progress in the elucidation of the structural enzymology of the sulphate reduction and cysteine biosynthesis pathways.

  6. Streptomyces as host for recombinant production of Mycobacterium tuberculosis proteins.

    PubMed

    Vallin, Carlos; Ramos, Astrid; Pimienta, Elsa; Rodríguez, Caridad; Hernández, Tairí; Hernández, Ivones; Del Sol, Ricardo; Rosabal, Grisel; Van Mellaert, Lieve; Anné, Jozef

    2006-01-01

    The 45/47 kDa APA protein (Rv1860) of Mycobacterium tuberculosis was produced by Streptomyces lividans. The recombinant protein could be recovered from the culture medium of an S. lividans clone containing the apa gene under control of the promoter and signal sequence of the Streptomyces coelicolor agarase gene. The recombinant protein production was further scaled-up using fermentation conditions. The APA protein was subsequently purified from the culture supernatant by means of immunochromatography. About 80 mg of recombinant protein were obtained per liter of culture media. In vivo tests with the APA protein purified from S. lividans TK24/pRGAPA1 revealed that the recombinant protein was antigenic and could induce high titers of specific antibodies in the mouse biological model. Results obtained concerning heterologous production of APA, its immunogenic and antigenic capacity, demonstrated the potential of S. lividans as a valuable host for the production of recombinant proteins from M. tuberculosis.

  7. The transmission of Mycobacterium tuberculosis in high burden settings.

    PubMed

    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

    2016-02-01

    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 transmission in populations and trials and describe the Wells-Riley equation, which is used to estimate transmission risk in indoor spaces. Present research priorities include the identification of effective strategies for tuberculosis infection control, improved understanding of where transmission occurs and the transmissibility of drug-resistant strains, and estimates of the effect of HIV and antiretroviral therapy on transmission dynamics. When research is planned and interventions are designed to interrupt transmission, resource constraints that are common in high burden settings-including shortages of health-care workers-must be considered.

  8. Antimycobacterial Metabolism: Illuminating Mycobacterium tuberculosis Biology and Drug Discovery.

    PubMed

    Awasthi, Divya; Freundlich, Joel S

    2017-09-01

    Bacteria are capable of performing a number of biotransformations that may activate or deactivate xenobiotics. Recent efforts have utilized metabolomics techniques to study the fate of small-molecule antibacterials within the targeted organism. Examples involving Mycobacterium tuberculosis are reviewed and analyzed with regard to the insights they provide as to both activation and deactivation of the antibacterial. The studies, in particular, shed light on biosynthetic transformations performed by M. tuberculosis while suggesting avenues for the evolution of chemical tools, highlighting potential areas for drug discovery, and mechanisms of approved drugs. A two-pronged approach investigating the metabolism of antibacterials within both the host and bacterium is outlined and will be of value to both the chemical biology and drug discovery fields. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Disinfecting endoscopes: how not to transmit Mycobacterium tuberculosis by bronchoscopy.

    PubMed Central

    Leers, W D

    1980-01-01

    Mycobacterium tuberculosis was cultured from the bronchial washings of two patients who underwent bronchoscopy consecutively with the same bronchoscope. Active pulmonary tuberculosis was later confirmed in the first patient, whereas the second patient had clinical and serologic evidence of infection with respiratory syncytial virus. The bronchoscope had been cleaned with an iodophor disinfectant, which had not destroyed the tubercle bacilli. The agent recommended for chemical disinfection of fibreoptic bronchoscopes is 2% glutaraldehyde solution; the instrument should be immersed in it for 10 to 30 minutes. Five hours' exposure to ethylene oxide is recommended for sterilization of instruments. These procedures must be preceded by adequate mechanical cleaning. Then transmission of pathogenic organisms during endoscopy, which can result in nosocomial disease, misdiagnosis or inappropriate treatment, will be avoided. Images FIG. 1 FIG. 2 FIG. 3 PMID:6790150

  10. Eosinophilic cystitis and cholangitis - systemic disease triggered by mycobacterium tuberculosis?

    PubMed

    Buda, Piotr; Grenda, Ryszard; Wieteska-Klimczak, Anna; Gietka, Piotr; Skobejko-Włodarska, Lidia; Felberg, Karina; Książyk, Janusz

    Eosinophilic cystitis (EC) is a rare inflammatory disorder of the urinary tract characterized by infiltration of bladder with eosinophils. The cause remains unclear, immunological mechanisms have been implicated in pathogenesis. Potential etiological factors include: tumors, allergy, parasitic infections, trauma. The disease may have a variable course, from a mild self-limiting, through common symptoms like: dysuria, hematuria, abdominal pain, tumor, to severe renal failure, with eosinophilic infiltration of the other organs and systemic complications. Treatment depending on disease severity and etiology is pharmacological and/or surgical. Here we report a case of a previously healthy 16-year old girl with inflammatory tumor in the liver hilum infiltrating extrahepatic biliary tract who developed three months later haematuria with acute dysuric signs and renal failure. Based on histopathological findings diagnosis of eosinophilic cystitis was established. Tests for Mycobacterium tuberculosis were positive. To our knowledge, EC association with cholangitis and tuberculosis have never been reported before.

  11. Mycobacterium tuberculosis Infection following Kidney Transplantation

    PubMed Central

    Boubaker, Karima; Gargah, Tahar; Abderrahim, Ezzedine; Ben Abdallah, Taieb; Kheder, Adel

    2013-01-01

    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. PMID:24222903

  12. Molecular Biology of Drug Resistance in Mycobacterium tuberculosis

    PubMed Central

    Smith, Tasha; Wolff, Kerstin A.; Nguyen, Liem

    2014-01-01

    Tuberculosis (TB) has become a curable disease thanks to the discovery of antibiotics. However, it has remained one of the most difficult infections to treat. Most current TB regimens consist of six to nine months of daily doses of four drugs that are highly toxic to patients. The purpose of these lengthy treatments is to completely eradicate Mycobacterium tuberculosis, notorious for its ability to resist most antibacterial agents, thereby preventing the formation of drug resistant mutants. On the contrary, the prolonged therapies have led to poor patient adherence. This, together with a severe limit of drug choices, has resulted in the emergence of strains that are increasingly resistant to the few available antibiotics. Here we review our current understanding of molecular mechanisms underlying the profound drug resistance of M. tuberculosis. This knowledge is essential for the development of more effective antibiotics that not only are potent against drug resistant M. tuberculosis strains but also help shorten the current treatment courses required for drug susceptible TB. PMID:23179675

  13. Rv3168 phosphotransferase activity mediates kanamycin resistance in Mycobacterium tuberculosis.

    PubMed

    Ahn, Jae-Woo; Kim, Kyung-Jin

    2013-11-28

    Tuberculosis is a worldwide epidemic disease caused by Mycobacterium tuberculosis, with an estimated one-third of the human population currently affected. Treatment of this disease with aminoglycoside antibiotics has become less effective owing to antibiotic resistance. Recent determination of the crystal structure of the M. tuberculosis Rv3168 protein suggests a structure similar to that of Enterococcus faecalis APH(3')-IIIa, and that this protein may be an aminoglycoside phosphotransferase. To determine whether Rv3168 confers antibiotic resistance against kanamycin, we performed dose-response antibiotic resistance experiments using kanamycin. Expression of the Rv3168 protein in Escherichia coli conferred antibiotic resistance against 100 μM kanamycin, a concentration that effected cell growth arrest in the parental E. coli strain and an E. coli strain expressing the Rv3168(D249A) mutant, in which the catalytic Asp249 residue was mutated to alanine. Furthermore, we detected phosphotransferase activity of Rv3168 against kanamycin as a substrate. Moreover, docking simulation of kanamycin into the Rv3168 structure suggests that kanamycin fits well into the substrate binding pocket of the protein, and that the phosphorylation-hydroxyl-group of kanamycin was located at a position similar to that in E. faecalis APH(3')-IIIa. On the basis of these results, we suggest that the Rv3168 mediates kanamycin resistance in M. tuberculosis, likely through phosphotransferase targeting of kanamycin.

  14. Latent Mycobacterium tuberculosis Infection and Interferon-Gamma Release Assays.

    PubMed

    Pai, Madhukar; Behr, Marcel

    2016-10-01

    The identification of individuals with latent tuberculosis infection (LTBI) is useful for both fundamental understanding of the pathogenesis of disease and for clinical and public health interventions (i.e., to prevent progression to disease). Basic research suggests there is a pathogenetic continuum from exposure to infection to disease, and individuals may advance or reverse positions within the spectrum, depending on changes in the host immunity. Unfortunately, there is no diagnostic test that resolves the various stages within the spectrum of Mycobacterium tuberculosis infection. Two main immune-based approaches are currently used for identification of LTBI: the tuberculin skin test (TST) and the interferon-gamma release assay (IGRA). TST can use either the conventional purified protein derivative or more specific antigens. Extensive research suggests that both TST and IGRA represent indirect markers of M. tuberculosis exposure and indicates a cellular immune response to M. tuberculosis. The imperfect concordance between these two tests suggests that neither test is perfect, presumably due to both technical and biological reasons. Neither test can accurately differentiate between LTBI and active TB. Both IGRA and TST have low sensitivity in a variety of immunocompromised populations. Cohort studies have shown that both TST and IGRA have low predictive value for progression from infection to active TB. For fundamental applications, basic research is necessary to identify those at highest risk of disease with a positive TST and/or IGRA. For clinical applications, the identification of such biomarkers can help prioritize efforts to interrupt progression to disease through preventive therapy.

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

    PubMed Central

    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

  16. Inhibition studies of Mycobacterium tuberculosis salicylate synthase (MbtI).

    PubMed

    Manos-Turvey, Alexandra; Bulloch, Esther M M; Rutledge, Peter J; Baker, Edward N; Lott, J Shaun; Payne, Richard J

    2010-07-05

    Mycobacterium tuberculosis salicylate synthase (MbtI), a member of the chorismate-utilizing enzyme family, catalyses the first committed step in the biosynthesis of the siderophore mycobactin T. This complex secondary metabolite is essential for both virulence and survival of M. tuberculosis, the etiological agent of tuberculosis (TB). It is therefore anticipated that inhibitors of this enzyme may serve as TB therapies with a novel mode of action. Herein we describe the first inhibition study of M. tuberculosis MbtI using a library of functionalized benzoate-based inhibitors designed to mimic the substrate (chorismate) and intermediate (isochorismate) of the MbtI-catalyzed reaction. The most potent inhibitors prepared were those designed to mimic the enzyme intermediate, isochorismate. These compounds, based on a 2,3-dihydroxybenzoate scaffold, proved to be low-micromolar inhibitors of MbtI. The most potent inhibitors in this series possessed hydrophobic enol ether side chains at C3 in place of the enol-pyruvyl side chain found in chorismate and isochorismate.

  17. Enhanced Specialized Transduction Using Recombineering in Mycobacterium tuberculosis

    PubMed Central

    Tufariello, JoAnn M.; Malek, Adel A.; Vilchèze, Catherine; Cole, Laura E.; Ratner, Hannah K.; González, Pablo A.; Jain, Paras; Hatfull, Graham F.; Larsen, Michelle H.

    2014-01-01

    ABSTRACT Genetic engineering has contributed greatly to our understanding of Mycobacterium tuberculosis biology and has facilitated antimycobacterial and vaccine development. However, methods to generate M. tuberculosis deletion mutants remain labor-intensive and relatively inefficient. Here, methods are described that significantly enhance the efficiency (greater than 100-fold) of recovering deletion mutants by the expression of mycobacteriophage recombineering functions during the course of infection with specialized transducing phages delivering allelic exchange substrates. This system has been successfully applied to the CDC1551 strain of M. tuberculosis, as well as to a ΔrecD mutant generated in the CDC1551 parental strain. The latter studies were undertaken as there were precedents in both the Escherichia coli literature and mycobacterial literature for enhancement of homologous recombination in strains lacking RecD. In combination, these measures yielded a dramatic increase in the recovery of deletion mutants and are expected to facilitate construction of a comprehensive library of mutants with every nonessential gene of M. tuberculosis deleted. The findings also open up the potential for sophisticated genetic screens, such as synthetic lethal analyses, which have so far not been feasible for the slow-growing mycobacteria. PMID:24865558

  18. Native New Zealand plants with inhibitory activity towards Mycobacterium tuberculosis

    PubMed Central

    2010-01-01

    Background Plants have long been investigated as a source of antibiotics and other bioactives for the treatment of human disease. New Zealand contains a diverse and unique flora, however, few of its endemic plants have been used to treat tuberculosis. One plant, Laurelia novae-zelandiae, was reportedly used by indigenous Maori for the treatment of tubercular lesions. Methods Laurelia novae-zelandiae and 44 other native plants were tested for direct anti-bacterial activity. Plants were extracted with different solvents and extracts screened for inhibition of the surrogate species, Mycobacterium smegmatis. Active plant samples were then tested for bacteriostatic activity towards M. tuberculosis and other clinically-important species. Results Extracts of six native plants were active against M. smegmatis. Many of these were also inhibitory towards M. tuberculosis including Laurelia novae-zelandiae (Pukatea). M. excelsa (Pohutukawa) was the only plant extract tested that was active against Staphylococcus aureus. Conclusions Our data provide support for the traditional use of Pukatea in treating tuberculosis. In addition, our analyses indicate that other native plant species possess antibiotic activity. PMID:20537175

  19. An acidic sphingomyelinase Type C activity from Mycobacterium tuberculosis.

    PubMed

    Castro-Garza, Jorge; González-Salazar, Francisco; Quinn, Frederick D; Karls, Russell K; De La Garza-Salinas, Laura Hermila; Guzmán-de la Garza, Francisco J; Vargas-Villarreal, Javier

    2016-01-01

    Sphingomyelinases (SMases) catalyze the hydrolysis of sphingomyelin to ceramide and phosphorylcholine. Sphingolipids are recognized as diverse and dynamic regulators of a multitude of cellular processes mediating cell cycle control, differentiation, stress response, cell migration, adhesion, and apoptosis. Bacterial SMases are virulence factors for several species of pathogens. Whole cell extracts of Mycobacterium tuberculosis strains H37Rv and CDC1551 were assayed using [N-methyl-(14)C]-sphingomyelin as substrate. Acidic Zn(2+)-dependent SMase activity was identified in both strains. Peak SMase activity was observed at pH 5.5. Interestingly, overall SMase activity levels from CDC1551 extracts are approximately 1/3 of those of H37Rv. The presence of exogenous SMase produced by M. tuberculosis during infection may interfere with the normal host inflammatory response thus allowing the establishment of infection and disease development. This Type C activity is different from previously identified M. tuberculosis SMases. Defining the biochemical characteristics of M. tuberculosis SMases helps to elucidate the roles that these enzymes play during infection and disease. Copyright © 2016 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  20. Prospective genotyping of Mycobacterium tuberculosis from fresh clinical samples.

    PubMed

    Bidovec-Stojkovič, Urška; Seme, Katja; Žolnir-Dovč, Manca; Supply, Philip

    2014-01-01

    Shorter time-to-result is key for improving molecular-guided epidemiological investigation of tuberculosis (TB) cases. We performed a prospective study to evaluate the use of standardized MIRU-VNTR (mycobacterial interspersed repetitive-unit-variable-number tandem-repeat) typing of Mycobacterium tuberculosis directly on 79 fresh clinical samples from 26 TB patients consecutively enrolled over a 17-month period. Overall, complete 24-locus types were obtained for 18 out of the 26 (69.2%) patients and 14 of the 16 grade 3+ and grade 2+ samples (87.5%). The degree of completion of the genotypes obtained significantly correlated with smear microscopy grade both for 26 first samples (p = 0.0003) and for 53 follow-up samples (p = 0.002). For 20 of the 26 patients for whom complete or even incomplete M. tuberculosis isolate genotypes were obtained, typing applied to the clinical samples allowed the same unambiguous conclusions regarding case clustering or uniqueness as those that could have been drawn based on the corresponding cultured isolates. Standard 24 locus MIRU-VNTR typing of M. tuberculosis can be applied directly to fresh clinical samples, with typeability depending on the bacterial load in the sample.

  1. LAG3 expression in active Mycobacterium tuberculosis infections.

    PubMed

    Phillips, Bonnie L; Mehra, Smriti; Ahsan, Muhammad H; Selman, Moises; Khader, Shabaana A; Kaushal, Deepak

    2015-03-01

    Mycobacterium tuberculosis (MTB) is a highly successful pathogen because of its ability to persist in human lungs for long periods of time. MTB modulates several aspects of the host immune response. Lymphocyte-activation gene 3 (LAG3) is a protein with a high affinity for the CD4 receptor and is expressed mainly by regulatory T cells with immunomodulatory functions. To understand the function of LAG3 during MTB infection, a nonhuman primate model of tuberculosis, which recapitulates key aspects of natural human infection in rhesus macaques (Macaca mulatta), was used. We show that the expression of LAG3 is highly induced in the lungs and particularly in the granulomatous lesions of macaques experimentally infected with MTB. Furthermore, we show that LAG3 expression is not induced in the lungs and lung granulomas of animals exhibiting latent tuberculosis infection. However, simian immunodeficiency virus-induced reactivation of latent tuberculosis infection results in an increased expression of LAG3 in the lungs. This response is not observed in nonhuman primates infected with non-MTB bacterial pathogens, nor with simian immunodeficiency virus alone. Our data show that LAG3 was expressed primarily on CD4(+) T cells, presumably by regulatory T cells but also by natural killer cells. The expression of LAG3 coincides with high bacterial burdens and changes in the host type 1 helper T-cell response.

  2. The T cell response to secreted antigens of Mycobacterium tuberculosis.

    PubMed

    Andersen, P

    1994-10-01

    Recent information from several laboratories points to proteins secreted from live Mycobacterium tuberculosis as being involved in protective immunity. We have studied protein release from M. tuberculosis during growth and have defined 3 different groups of proteins: excreted proteins, secreted proteins of the outer cell wall and cytoplasmic proteins released at late culture timepoints. These findings have lead to the definition of a short-term culture filtrate (ST-CF) enriched in excreted/secreted proteins and with a minimal content of autolytic products. ST-CF was tested as antigen in experimental vaccines against tuberculosis. A vaccine based on the adjuvant dimethyldioctadecylammonium chloride (DDA) was constructed and demonstrated to induce a potent cell mediated immune response of the Th-1 type. The vaccine was tested in parallel with a BCG standard vaccine and both vaccines induced a highly significant protection of the same magnitude. Molecules within the Ag85 complex and a 6-kDA secreted protein were mapped as the major antigenic targets for long-lived T cells involved in protective immunity against M. tuberculosis.

  3. Rapid diagnosis of Mycobacterium tuberculosis infection and drug susceptibility testing.

    PubMed

    Wilson, Michael L

    2013-06-01

    The global control of tuberculosis remains a challenge from the standpoint of diagnosis, detection of drug resistance, and treatment. This is an area of special concern to the health of women and children, particularly in regions of the world with high infant mortality rates and where women have limited access to health care. Because treatment can only be initiated when infection is detected, and is guided by the results of antimicrobial susceptibility testing, there recently has been a marked increase in the development and testing of novel assays designed to detect Mycobacterium tuberculosis complex, with or without simultaneous detection of resistance to isoniazid and/or rifampin. Both nonmolecular and molecular assays have been developed. This review will summarize the current knowledge about the use of rapid tests to detect M tuberculosis and drug resistance. Review of the most recent World Health Organization Global Tuberculosis Report, as well as selected publications in the primary research literature, meta-analyses, and review articles. To a large extent, nonmolecular methods are refinements or modifications of conventional methods, with the primary goal of providing more rapid test results. In contrast, molecular methods use novel technologies to detect the presence of M tuberculosis complex and genes conferring drug resistance. Evaluations of molecular assays have generally shown that these assays are of variable sensitivity for detecting the presence of M tuberculosis complex, and in particular are insensitive when used with smear-negative specimens. As a group, molecular assays have been shown to be of high sensitivity for detecting resistance to rifampin, but of variable sensitivity for detecting resistance to isoniazid.

  4. Response of Mycobacterium tuberculosis to reactive oxygen and nitrogen intermediates.

    PubMed Central

    Garbe, T. R.; Hibler, N. S.; Deretic, V.

    1996-01-01

    BACKGROUND: Mycobacterium tuberculosis is a significant human pathogen capable of replicating in mononuclear phagocytic cells. Exposure to reactive oxygen and nitrogen intermediates is likely to represent an important aspect of the life cycle of this organism. The response of M. tuberculosis to these agents may be of significance for its survival in the host. MATERIALS AND METHODS: Patterns of de novo proteins synthesized in M. tuberculosis H37Rv exposed to compounds that generate reactive oxygen and nitrogen intermediates were studied by metabolic labeling and two-dimensional electrophoresis. RESULTS: Menadione, a redox cycling compound which increases intracellular superoxide levels, caused enhanced synthesis of seven polypeptides, six of which appeared to be heat shock proteins. Chemical release of nitric oxide induced eight polypeptides of which only one could be identified as a heat shock protein. Nitric oxide also exhibited a mild inhibitory action on general protein synthesis in the concentration range tested. Hydrogen peroxide did not cause differential gene expression and exerted a generalized inhibition in a dose-dependent manner. Cumene hydroperoxide caused mostly inhibition but induction of two heat shock proteins was detectable. CONCLUSIONS: The presented findings indicate major differences between M. tuberculosis and the paradigms of oxidative stress response in enteric bacteria, and are consistent with the multiple lesions found in oxyR of this organism. The effect of hydrogen peroxide, which in Escherichia coli induces eight polypeptides known to be controlled by the central regulator oxyR, appears to be absent in M. tuberculosis. Superoxide and nitric oxide responses, which in E. coli overlap and are controlled by the same regulatory system soxRS, represent discrete and independent phenomena in M. tuberculosis. Images FIG. 1 FIG. 2 FIG. 3 FIG. 4 FIG. 5 FIG. 6 PMID:8900541

  5. Essential Metabolites of Mycobacterium tuberculosis and Their Mimics

    PubMed Central

    Lamichhane, Gyanu; Freundlich, Joel S.; Ekins, Sean; Wickramaratne, Niluka; Nolan, Scott T.; Bishai, William R.

    2011-01-01

    An organism requires a range of biomolecules for its growth. By definition, these are essential molecules which constitute the basic metabolic requirements of an organism. A small organic molecule with chemical similarity to that of an essential metabolite may bind to the enzyme that catalyzes its production and inhibit it, likely resulting in the stasis or death of the organism. Here, we report a high-throughput approach for identifying essential metabolites of an organism using genetic and biochemical approaches and then implement computational approaches to identify metabolite mimics. We generated and genotyped 5,126 Mycobacterium tuberculosis mutants and performed a statistical analysis to determine putative essential genes. The essential molecules of M. tuberculosis were classified as products of enzymes that are encoded by genes in this list. Although incomplete, as many enzymes of M. tuberculosis have yet to be identified and characterized, this is the first report of a large number of essential molecules of the organism. We identified essential metabolites of three distinct metabolic pathways in M. tuberculosis and selected molecules with chemical similarity using cheminformatics strategies that illustrate a variety of different pharmacophores. Our approach is aimed at systematic identification of essential molecules and their mimics as a blueprint for development of effective chemical probes of M. tuberculosis metabolism, with the ultimate goal of seeking drugs that can kill this pathogen. As an illustration of this approach, we report that compounds JFD01307SC and l-methionine-S-sulfoximine, which share chemical similarity with an essential molecule of M. tuberculosis, inhibited the growth of this organism at micromolar concentrations. PMID:21285434

  6. Resistance mechanisms of Mycobacterium tuberculosis against phagosomal copper overload

    PubMed Central

    Rowland, Jennifer L.; Niederweis, Michael

    2012-01-01

    SUMMARY Mycobacterium tuberculosis is an important bacterial pathogen with an extremely slow growth rate, an unusual outer membrane of very low permeability and a cunning ability to survive inside the human host despite a potent immune response. A key trait of M. tuberculosis is to acquire essential nutrients while still preserving its natural resistance to toxic compounds. In this regard, copper homeostasis mechanisms are particularly interesting, because copper is an important element for bacterial growth, but copper overload is toxic. In M. tuberculosis at least two enzymes require copper as a cofactor: the Cu/Zn-superoxide dismutase SodC and the cytochrome c oxidase which is essential for growth in vitro. Mutants of M. tuberculosis lacking the copper metallothionein MymT, the efflux pump CtpV and the membrane protein MctB are more susceptible to copper indicating that these proteins are part of a multipronged system to balance intracellular copper levels. Recent evidence showed that part of copper toxicity is a reversible damage of accessible Fe-S clusters of dehydratases and the displacement of other divalent cations such as zinc and manganese as cofactors in proteins. There is accumulating evidence that macrophages use copper to poison bacteria trapped inside phagosomes. Here, we review the rapidly increasing knowledge about copper homeostasis mechanisms in M. tuberculosis and contrast those with similar mechanisms in E. coli. These findings reveal an intricate interplay between the host which aims to overload the phagosome with copper and M. tuberculosis which utilizes several mechanisms to reduce the toxic effects of excess copper. PMID:22361385

  7. Energy Metabolism and Drug Efflux in Mycobacterium tuberculosis

    PubMed Central

    Black, Philippa A.; Warren, Robin M.; Louw, Gail E.; van Helden, Paul D.; Victor, Thomas C.

    2014-01-01

    The inherent drug susceptibility of microorganisms is determined by multiple factors, including growth state, the rate of drug diffusion into and out of the cell, and the intrinsic vulnerability of drug targets with regard to the corresponding antimicrobial agent. Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), remains a significant source of global morbidity and mortality, further exacerbated by its ability to readily evolve drug resistance. It is well accepted that drug resistance in M. tuberculosis is driven by the acquisition of chromosomal mutations in genes encoding drug targets/promoter regions; however, a comprehensive description of the molecular mechanisms that fuel drug resistance in the clinical setting is currently lacking. In this context, there is a growing body of evidence suggesting that active extrusion of drugs from the cell is critical for drug tolerance. M. tuberculosis encodes representatives of a diverse range of multidrug transporters, many of which are dependent on the proton motive force (PMF) or the availability of ATP. This suggests that energy metabolism and ATP production through the PMF, which is established by the electron transport chain (ETC), are critical in determining the drug susceptibility of M. tuberculosis. In this review, we detail advances in the study of the mycobacterial ETC and highlight drugs that target various components of the ETC. We provide an overview of some of the efflux pumps present in M. tuberculosis and their association, if any, with drug transport and concomitant effects on drug resistance. The implications of inhibiting drug extrusion, through the use of efflux pump inhibitors, are also discussed. PMID:24614376

  8. Molecular Epidemiology of Mycobacterium tuberculosis among South African Gold Miners

    PubMed Central

    Lewis, James J.; Connors, Jeremy; Chihota, Violet N.; Shashkina, Elena; van der Meulen, Minty; Graviss, Edward A.; Ha, Ngan P.; Kreiswirth, Barry N.; Grant, Alison D.; Fielding, Katherine L.; Dorman, Susan E.; Churchyard, Gavin J.

    2015-01-01

    Rationale: HIV-associated tuberculosis remains a major health problem among the gold-mining workforce in South Africa. We postulate that high levels of recent transmission, indicated by strain clustering, are fueling the tuberculosis epidemic among gold miners. Objectives: To combine molecular and epidemiologic data to describe Mycobacterium tuberculosis genetic diversity, estimate levels of transmission, and examine risk factors for clustering. Methods: We conducted a cross-sectional study of culture-positive M. tuberculosis isolates in 15 gold mine shafts across three provinces in South Africa. All isolates were subject IS6110-based restriction fragment length polymorphisms, and we performed spoligotyping analysis and combined it with basic demographic and clinical information. Measurements and Main Results: Of the 1,602 M. tuberculosis patient isolates, 1,240 (78%) had genotyping data available for analysis. A highly diverse bacillary population was identified, comprising a total of 730 discrete genotypes. Four genotypic families (Latin American Mediterranean spoligotype family; W-Beijing; AH or X; and T1–T4) accounted for over 50% of all strains. Overall, 45% (560/1,240) of strains were genotypically clustered. The minimum estimate for recent transmission (n − 1 method) was 32% (range, 27–34%). There were no individual-level risk factors for clustering, apart from borderline evidence for being non–South African and having self-reported HIV infection. Conclusions: The high M. tuberculosis genetic diversity and lack of risk factors for clustering are indicative of a universal risk for disease among gold miners and likely mixing with nonmining populations. Our results underscore the urgent need to intensify interventions to interrupt transmission across the entire gold-mining workforce in South Africa. PMID:25419914

  9. Genetic Diversity and Dynamic Distribution of Mycobacterium tuberculosis Isolates Causing Pulmonary and Extrapulmonary Tuberculosis in Thailand

    PubMed Central

    Srilohasin, Prapaporn; Tokunaga, Katsushi; Nishida, Nao; Prammananan, Therdsak; Smittipat, Nat; Mahasirimongkol, Surakameth; Chaiyasirinroje, Boonchai; Yanai, Hideki; Palittapongarnpim, Prasit

    2014-01-01

    This study examined the genetic diversity and dynamicity of circulating Mycobacterium tuberculosis strains in Thailand using nearly neutral molecular markers. The single nucleotide polymorphism (SNP)-based genotypes of 1,414 culture-positive M. tuberculosis isolates from 1,282 pulmonary tuberculosis (PTB) and 132 extrapulmonary TB (EPTB) patients collected from 1995 to 2011 were characterized. Among the eight SNP cluster groups (SCG), SCG2 (44.1%), which included the Beijing (BJ) genotype, and SCG1 (39.4%), an East African Indian genotype, were dominant. Comparisons between the genotypes of M. tuberculosis isolates causing PTB and EPTB in HIV-negative cases revealed similar prevalence trends although genetic diversity was higher in the PTB patients. The identification of 10 reported sequence types (STs) and three novel STs was hypothesized to indicate preferential expansion of the SCG2 genotype, especially the modern BJ ST10 (15.6%) and ancestral BJ ST19 (13.1%). An association between SCG2 and SCG1 genotypes and particular patient age groups implies the existence of different genetic advantages among the bacterial populations. The results revealed that increasing numbers of young patients were infected with M. tuberculosis SCGs 2 and 5, which contrasts with the reduction of the SCG1 genotype. Our results indicate the selection and dissemination of potent M. tuberculosis genotypes in this population. The determination of heterogeneity and dynamic population changes of circulating M. tuberculosis strains in countries using the Mycobacterium bovis BCG (bacillus Calmette-Guérin) vaccine are beneficial for vaccine development and control strategies. PMID:25297330

  10. Genetic diversity and dynamic distribution of Mycobacterium tuberculosis isolates causing pulmonary and extrapulmonary tuberculosis in Thailand.

    PubMed

    Srilohasin, Prapaporn; Chaiprasert, Angkana; Tokunaga, Katsushi; Nishida, Nao; Prammananan, Therdsak; Smittipat, Nat; Mahasirimongkol, Surakameth; Chaiyasirinroje, Boonchai; Yanai, Hideki; Palittapongarnpim, Prasit

    2014-12-01

    This study examined the genetic diversity and dynamicity of circulating Mycobacterium tuberculosis strains in Thailand using nearly neutral molecular markers. The single nucleotide polymorphism (SNP)-based genotypes of 1,414 culture-positive M. tuberculosis isolates from 1,282 pulmonary tuberculosis (PTB) and 132 extrapulmonary TB (EPTB) patients collected from 1995 to 2011 were characterized. Among the eight SNP cluster groups (SCG), SCG2 (44.1%), which included the Beijing (BJ) genotype, and SCG1 (39.4%), an East African Indian genotype, were dominant. Comparisons between the genotypes of M. tuberculosis isolates causing PTB and EPTB in HIV-negative cases revealed similar prevalence trends although genetic diversity was higher in the PTB patients. The identification of 10 reported sequence types (STs) and three novel STs was hypothesized to indicate preferential expansion of the SCG2 genotype, especially the modern BJ ST10 (15.6%) and ancestral BJ ST19 (13.1%). An association between SCG2 and SCG1 genotypes and particular patient age groups implies the existence of different genetic advantages among the bacterial populations. The results revealed that increasing numbers of young patients were infected with M. tuberculosis SCGs 2 and 5, which contrasts with the reduction of the SCG1 genotype. Our results indicate the selection and dissemination of potent M. tuberculosis genotypes in this population. The determination of heterogeneity and dynamic population changes of circulating M. tuberculosis strains in countries using the Mycobacterium bovis BCG (bacillus Calmette-Guérin) vaccine are beneficial for vaccine development and control strategies.

  11. Uptake of sulfate but not phosphate by Mycobacterium tuberculosis is slower than that for Mycobacterium smegmatis.

    PubMed

    Song, Houhui; Niederweis, Michael

    2012-03-01

    Knowledge of the metabolic pathways used by Mycobacterium tuberculosis during infection is important for understanding its nutrient requirements and host adaptation. However, uptake, the first step in the utilization of nutrients, is poorly understood for many essential nutrients, such as inorganic anions. Here, we show that M. tuberculosis utilizes nitrate as the sole nitrogen source, albeit at lower efficiency than asparagine, glutamate, and arginine. The growth of the porin triple mutant M. smegmatis ML16 in media with limiting amounts of nitrate and sulfate as sole nitrogen and sulfur sources, respectively, was delayed compared to that of the wild-type strain. The uptake of sulfate was 40-fold slower than that of the wild-type strain, indicating that the efficient uptake of these anions is dependent on porins. The uptake by M. tuberculosis of sulfate and phosphate was approximately 40- and 10-fold slower than that of M. smegmatis, respectively, which is consistent with the slower growth of M. tuberculosis. However, the uptake of these anions by M. tuberculosis is orders of magnitude faster than diffusion through lipid membranes, indicating that unknown outer membrane proteins are required to facilitate this process.

  12. High throughput phenotypic analysis of Mycobacterium tuberculosis and Mycobacterium bovis strains' metabolism using biolog phenotype microarrays.

    PubMed

    Khatri, Bhagwati; Fielder, Mark; Jones, Gareth; Newell, William; Abu-Oun, Manal; Wheeler, Paul R

    2013-01-01

    Tuberculosis is a major human and animal disease of major importance worldwide. Genetically, the closely related strains within the Mycobacterium tuberculosis complex which cause disease are well-characterized but there is an urgent need better to understand their phenotypes. To search rapidly for metabolic differences, a working method using Biolog Phenotype MicroArray analysis was developed. Of 380 substrates surveyed, 71 permitted tetrazolium dye reduction, the readout over 7 days in the method. By looking for ≥5-fold differences in dye reduction, 12 substrates differentiated M. tuberculosis H37Rv and Mycobacterium bovis AF2122/97. H37Rv and a Beijing strain of M. tuberculosis could also be distinguished in this way, as could field strains of M. bovis; even pairs of strains within one spoligotype could be distinguished by 2 to 3 substrates. Cluster analysis gave three clear groups: H37Rv, Beijing, and all the M. bovis strains. The substrates used agreed well with prior knowledge, though an unexpected finding that AF2122/97 gave greater dye reduction than H37Rv with hexoses was investigated further, in culture flasks, revealing that hexoses and Tween 80 were synergistic for growth and used simultaneously rather than in a diauxic fashion. Potential new substrates for growth media were revealed, too, most promisingly N-acetyl glucosamine. Osmotic and pH arrays divided the mycobacteria into two groups with different salt tolerance, though in contrast to the substrate arrays the groups did not entirely correlate with taxonomic differences. More interestingly, these arrays suggested differences between the amines used by the M. tuberculosis complex and enteric bacteria in acid tolerance, with some hydrophobic amino acids being highly effective. In contrast, γ-aminobutyrate, used in the enteric bacteria, had no effect in the mycobacteria. This study proved principle that Phenotype MicroArrays can be used with slow-growing pathogenic mycobacteria and already has

  13. High Throughput Phenotypic Analysis of Mycobacterium tuberculosis and Mycobacterium bovis Strains' Metabolism Using Biolog Phenotype Microarrays

    PubMed Central

    Khatri, Bhagwati; Fielder, Mark; Jones, Gareth; Newell, William; Abu-Oun, Manal; Wheeler, Paul R.

    2013-01-01

    Tuberculosis is a major human and animal disease of major importance worldwide. Genetically, the closely related strains within the Mycobacterium tuberculosis complex which cause disease are well-characterized but there is an urgent need better to understand their phenotypes. To search rapidly for metabolic differences, a working method using Biolog Phenotype MicroArray analysis was developed. Of 380 substrates surveyed, 71 permitted tetrazolium dye reduction, the readout over 7 days in the method. By looking for ≥5-fold differences in dye reduction, 12 substrates differentiated M. tuberculosis H37Rv and Mycobacterium bovis AF2122/97. H37Rv and a Beijing strain of M. tuberculosis could also be distinguished in this way, as could field strains of M. bovis; even pairs of strains within one spoligotype could be distinguished by 2 to 3 substrates. Cluster analysis gave three clear groups: H37Rv, Beijing, and all the M. bovis strains. The substrates used agreed well with prior knowledge, though an unexpected finding that AF2122/97 gave greater dye reduction than H37Rv with hexoses was investigated further, in culture flasks, revealing that hexoses and Tween 80 were synergistic for growth and used simultaneously rather than in a diauxic fashion. Potential new substrates for growth media were revealed, too, most promisingly N-acetyl glucosamine. Osmotic and pH arrays divided the mycobacteria into two groups with different salt tolerance, though in contrast to the substrate arrays the groups did not entirely correlate with taxonomic differences. More interestingly, these arrays suggested differences between the amines used by the M. tuberculosis complex and enteric bacteria in acid tolerance, with some hydrophobic amino acids being highly effective. In contrast, γ-aminobutyrate, used in the enteric bacteria, had no effect in the mycobacteria. This study proved principle that Phenotype MicroArrays can be used with slow-growing pathogenic mycobacteria and already has

  14. Mean Platelet Volume in Mycobacterium tuberculosis Infection

    PubMed Central

    Lee, Min Young; Kim, Young Jin; Lee, Hee Joo; Park, Tae Sung

    2016-01-01

    Introduction. Mean platelet volume (MPV) has been thought as a useful index of platelet activation. It is supposed that MPV is also associated with several inflammatory and infectious diseases. Korea still has a high incidence of tuberculosis (TB). The aim of this study was to investigate MPV as an inflammatory marker in TB patients. Materials and Methods. MPV were determined in 221 patients with TB and 143 individuals for control group. MPV was estimated by an Advia 2120 (Siemens Healthcare Diagnostics, Tarrytown, NY, USA). Results. In the TB patient group, a positive correlation was found between CRP and MPV. Age and MPV had a positive correlation in TB patient group. Conclusions. We conclude that there is a significant relation between MPV and inflammatory conditions. MPV can be an inflammatory marker to determine the disease activity in TB patients. PMID:27419136

  15. Acquired resistance of Mycobacterium tuberculosis to bedaquiline.

    PubMed

    Andries, Koen; Villellas, Cristina; Coeck, Nele; Thys, Kim; Gevers, Tom; Vranckx, Luc; Lounis, Nacer; de Jong, Bouke C; Koul, Anil

    2014-01-01

    Bedaquiline (BDQ), an ATP synthase inhibitor, is the first drug to be approved for treatment of multi-drug resistant tuberculosis in decades. In vitro resistance to BDQ was previously shown to be due to target-based mutations. Here we report that non-target based resistance to BDQ, and cross-resistance to clofazimine (CFZ), is due to mutations in Rv0678, a transcriptional repressor of the genes encoding the MmpS5-MmpL5 efflux pump. Efflux-based resistance was identified in paired isolates from patients treated with BDQ, as well as in mice, in which it was confirmed to decrease bactericidal efficacy. The efflux inhibitors verapamil and reserpine decreased the minimum inhibitory concentrations of BDQ and CFZ in vitro, but verapamil failed to increase the bactericidal effect of BDQ in mice and was unable to reverse efflux-based resistance in vivo. Cross-resistance between BDQ and CFZ may have important clinical implications.

  16. Disseminated Mycobacterium tuberculosis Infection Masquerading as Metastasis after Heavy Ion Radiotherapy for Prostate Cancer

    PubMed Central

    Ando, Masaru; Mukai, Yutaka; Ushijima, Ryo-ichi; Shioyama, Yoshiyuki; Umeki, Kenji; Okada, Fumito; Nureki, Shin-ichi; Mimata, Hiromitsu; Kadota, Jun-ichi

    2016-01-01

    Fluorodeoxyglucose (FDG)-positron emission tomography with computed tomography (FDG-PET/CT) is useful in disease monitoring of malignancies after therapy, while an FDG uptake may also be present in benign diseases. We herein demonstrate a case of disseminated Mycobacterium tuberculosis mimicking systemic metastasis of prostate cancer. This case highlights that clinicians should consider Mycobacterium tuberculosis in patients with prostate cancer who demonstrate multifocal FDG uptakes masquerading as metastasis, even when the chest photographs reveal a normal appearance and a sputum examination demonstrates negative results. An invasive surgical biopsy may be required and a pathological analysis would be critical in the diagnosis of Mycobacterium tuberculosis. PMID:27853089

  17. Tuberculosis

    MedlinePlus

    Tuberculosis (TB) is a disease caused by bacteria called Mycobacterium tuberculosis. The bacteria usually attack the lungs, but they can also damage other parts of the body. TB spreads through the air when a person with ...

  18. Impaired Cytokine but Enhanced Cytotoxic Marker Expression in Mycobacterium tuberculosis-Induced CD8+ T Cells in Individuals With Type 2 Diabetes and Latent Mycobacterium tuberculosis Infection.

    PubMed

    Kumar, Nathella Pavan; Moideen, Kadar; George, Parakkal Jovvian; Dolla, Chandrakumar; Kumaran, Paul; Babu, Subash

    2016-03-01

    Type 2 diabetes mellitus (DM) is a risk factor for tuberculosis among individuals with latent Mycobacterium tuberculosis infection. To explore the influence of DM on CD8(+) T-cell responses during latent M. tuberculosis infection, we estimated the cytokine and cytotoxic marker expression pattern in individuals with latent M. tuberculosis infection with DM and those with latent M. tuberculosis infection without DM. Among individuals with latent M. tuberculosis infection, those with DM had diminished frequencies of CD8(+) T-helper type 1 (Th1), Th2, and Th17 cells following stimulation by M. tuberculosis antigen and enhanced frequencies of CD8(+) T cells expressing cytotoxic markers, compared with those without DM. Thus, our results suggest that coincident DM modulates CD8(+) T-cell function during latent M. tuberculosis infection.

  19. [Advances in the research of an animal model of wound due to Mycobacterium tuberculosis infection].

    PubMed

    Chen, Ling; Jia, Chiyu

    2015-12-01

    Tuberculosis ranks as the second deadly infectious disease worldwide. The incidence of tuberculosis is high in China. Refractory wound caused by Mycobacterium tuberculosis infection ranks high in misdiagnosis, and it is accompanied by a protracted course, and its pathogenic mechanism is still not so clear. In order to study its pathogenic mechanism, it is necessary to reproduce an appropriate animal model. Up to now the study of the refractory wound caused by Mycobacterium tuberculosis infection is just beginning, and there is still no unimpeachable model for study. This review describes two models which may reproduce a wound similar to the wound caused by Mycobacterium tuberculosis infection, so that they could be used to study the pathogenesis and characteristics of a tuberculosis wound in an animal.

  20. A High-Throughput Cidality Screen for Mycobacterium Tuberculosis

    PubMed Central

    Kaur, Parvinder; Ghosh, Anirban; Krishnamurthy, Ramya Vadageri; Bhattacharjee, Deepa Gagwani; Achar, Vijayashree; Datta, Santanu; Narayanan, Shridhar; Anbarasu, Anand; Ramaiah, Sudha

    2015-01-01

    Exposure to Mycobacterium tuberculosis (Mtb) aerosols is a major threat to tuberculosis (TB) researchers, even in bio-safety level-3 (BSL-3) facilities. Automation and high-throughput screens (HTS) in BSL3 facilities are essential for minimizing manual aerosol-generating interventions and facilitating TB research. In the present study, we report the development and validation of a high-throughput, 24-well ‘spot-assay’ for selecting bactericidal compounds against Mtb. The bactericidal screen concept was first validated in the fast-growing surrogate Mycobacterium smegmatis (Msm) and subsequently confirmed in Mtb using the following reference anti-tubercular drugs: rifampicin, isoniazid, ofloxacin and ethambutol (RIOE, acting on different targets). The potential use of the spot-assay to select bactericidal compounds from a large library was confirmed by screening on Mtb, with parallel plating by the conventional gold standard method (correlation, r2 = 0.808). An automated spot-assay further enabled an MBC90 determination on resistant and sensitive Mtb clinical isolates. The implementation of the spot-assay in kinetic screens to enumerate residual Mtb after either genetic silencing (anti-sense RNA, AS-RNA) or chemical inhibition corroborated its ability to detect cidality. This relatively simple, economical and quantitative HTS considerably minimized the bio-hazard risk and enabled the selection of novel vulnerable Mtb targets and mycobactericidal compounds. Thus, spot-assays have great potential to impact the TB drug discovery process. PMID:25693161

  1. [In vitro interferon gamma production in mice vaccinated with 'Mycobacterium habana' against Mycobacterium tuberculosis antigens].

    PubMed

    Valdés Hernández, Iliana; Montoro Cardoso, C Ernesto; Hernández-Pando, Rogelio

    2012-01-01

    development of new antituberculosis vaccines requires the characterization of the cell-mediated immune responses induced by mycobacterial antigens. to determine the immunogenic potential of 'Mycobacterium habana' TMC-5135 when using subcutaneous vaccine in Balb/c mice. in this study, Balb/c mice were inoculated subcutaneously with live 'Mycobacterium habana' TMC-5135. The production of IFN gamma in cell suspensions obtained from the lungs, the spleen and the lymph nodes after stimulation with mycobacterial antigens Ag85b or culture filtrate antigens (CFA) was recorded. the production of IFN gamma after stimulation with CFA and Ag85b was higher in mice vaccinated with 'M. habana' than in animals immunized with BCG. these results encourage new research on 'M. habana' as vaccinal candidate against tuberculosis.

  2. Mycobacterium tuberculosis and Copper: A Newly Appreciated Defense against an Old Foe?*

    PubMed Central

    Darwin, K. Heran

    2015-01-01

    Several independent studies have recently converged upon the conclusion that the human bacterial pathogen Mycobacterium tuberculosis encounters copper during infections. At least three independently regulated pathways respond to excess copper and are required for the full virulence of M. tuberculosis in animals. In this review, I will discuss the functions of the best-characterized copper-responsive proteins in M. tuberculosis, the potential sources of copper during an infection, and remaining questions about the interface between copper and tuberculosis. PMID:26055711

  3. The RD1 virulence locus of Mycobacterium tuberculosis regulates DNA transfer in Mycobacterium smegmatis

    PubMed Central

    Flint, Jessica L.; Kowalski, Joseph C.; Karnati, Pavan K.; Derbyshire, Keith M.

    2004-01-01

    Conjugal DNA transfer occurs by an atypical mechanism in Mycobacterium smegmatis. The transfer system is chromosomally encoded and requires recipient recombination functions for both chromosome and plasmid transfer. Cis-acting sequences have been identified that confer mobility on nontransferable plasmids, but these are larger and have different properties to canonical oriT sites found in bacterial plasmids. To identify trans-acting factors required for mediating DNA transfer, a library of transposon insertion mutants was generated in the donor strain, and individual mutants were screened for their effect on transfer. From this screen, a collection of insertion mutants was isolated that increased conjugation frequencies relative to wild type. Remarkably, the mutations map to a 25-kb region of the M. smegmatis chromosome that is syntenous with the RD1 region of Mycobacterium tuberculosis, which is considered to be the primary attenuating deletion in the related vaccine strain Mycobacterium bovis bacillus Calmette–Guérin. The genes of the RD1 region encode a secretory apparatus responsible for exporting Cfp10- and Esat-6, both potent antigens and virulence factors. In crosses using two M. smegmatis donors, we show that wild-type cells can suppress the elevated transfer phenotype of mutant donors, which is consistent with the secretion of a factor that suppresses conjugation. Most importantly, the RD1 region of M. tuberculosis complements the conjugation phenotype of the RD1 mutants in M. smegmatis. Our results indicate that the M. tuberculosis and M. smegmatis RD1 regions are functionally equivalent and provide a unique perspective on the role of this critical secretion apparatus. PMID:15314236

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

    PubMed

    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.

  5. Effect of common and experimental anti-tuberculosis treatments on Mycobacterium tuberculosis growing as biofilms

    PubMed Central

    Dalton, James P.; Uy, Benedict; Phummarin, Narisa; Copp, Brent R.; Denny, William A.; Swift, Simon

    2016-01-01

    Much is known regarding the antibiotic susceptibility of planktonic cultures of Mycobacterium tuberculosis, the bacterium responsible for the lung disease tuberculosis (TB). As planktonically-grown M. tuberculosis are unlikely to be entirely representative of the bacterium during infection, we set out to determine how effective a range of anti-mycobacterial treatments were against M. tuberculosis growing as a biofilm, a bacterial phenotype known to be more resistant to antibiotic treatment. Light levels from bioluminescently-labelled M. tuberculosis H37Rv (strain BSG001) were used as a surrogate for bacterial viability, and were monitored before and after one week of treatment. After treatment, biofilms were disrupted, washed and inoculated into fresh broth and plated onto solid media to rescue any surviving bacteria. We found that in this phenotypic state M. tuberculosis was resistant to the majority of the compounds tested. Minimum inhibitory concentrations (MICs) increased by 20-fold to greater than 1,000-fold, underlying the potential of this phenotype to cause significant problems during treatment. PMID:27904808

  6. Genotypic characteristics of Mycobacterium tuberculosis isolated from household contacts of tuberculosis patients in the Philippines

    PubMed Central

    2013-01-01

    Background The Philippines has an extremely high rate of tuberculosis but little is known about M. tuberculosis genotypes and transmission dynamics in this country. The aim of this study was to determine the proportion of household contacts who develop active TB due to direct transmission from an index case in that household. Methods Mycobacterium tuberculosis isolates from household contacts of tuberculosis patients in the Philippines were characterized using restriction-fragment-length polymorphism analysis, spoligotyping, and mycobacterial interspersed repetitive units – variable number tandem repeats typing (12-loci) to determine their utility in elucidating transmission in an area of high tuberculosis prevalence. Drug susceptibility patterns for these isolates were also determined. Results Spoligotyping and MIRU-VNTR typing results matched in 10 (62.5%) of 16 index patient-household contact pairs while IS6110 fingerprints matched in only six (37.5%) pairs. Only 3/16 (18.8%) index patient-household contact pairs had identical drug susceptibility results. Conclusions Strain typing of M. tuberculosis isolates from household contacts in the Philippines indicates that transmission of strains does not necessarily occur directly from the index patient living in close proximity in the same household but rather that community-based transmission also frequently occurs. Accurate susceptibility testing of all isolates is necessary to insure optimal care of both the index patients and any culture-positive household contacts. PMID:24308751

  7. [A case of pulmonary Mycobacterium kansasii infection with pleural effusion, distinguished from pulmonary tuberculosis].

    PubMed

    Kimura, Yosuke; Kurosawa, Takayuki; Hosaka, Kiminori

    2014-09-01

    A case of pulmonary Mycobacterium kansasii infection with pleural effusion is very rare. We report a case of pulmonary Mycobacterium kansasii infection with pleural effusion, distinguished from pulmonary tuberculosis. A 44-year-old man presented to a clinic with a productive cough, sputum, and loss of appetite for several months. Chest X-ray and chest computed tomography (CT) showed right pleural effusion, centrilobular nodules and infiltrative shadows with cavities in the bilateral lung fields. The direct smear examination showed positive acid-fast bacilli (Gaffky 5). He was referred to our hospital for suspected recurrent pulmonary tuberculosis. We started anti-tuberculosis drugs because pulmonary tuberculosis complicated with pleurisy was first suspected from the findings of high ADA level (78.6 IU/l) of the effusion and positive result of interferon-gamma release assay (QuantiFERON TB-2G). But Mycobacterium tuberculosis and M. avium complex was not identified by the polymerase chain reaction method and the culture of the sputum was negative. At a later date, Mycobacterium kansasii was detected by sputum culture. The patient was diagnosed as pulmonary Mycobacterium kansasii infection and treatment with anti-tuberculosis drugs including RFP resulted in a good clinical response. This case was a rare case of pulmonary Mycobacterium kansasii infection with pleural effusion, distinguished from pulmonary tuberculosis.

  8. Comparing Galactan Biosynthesis in Mycobacterium tuberculosis and Corynebacterium diphtheriae.

    PubMed

    Wesener, Darryl A; Levengood, Matthew R; Kiessling, Laura L

    2017-02-17

    The suborder Corynebacterineae encompasses species like Corynebacterium glutamicum, which has been harnessed for industrial production of amino acids, as well as Corynebacterium diphtheriae and Mycobacterium tuberculosis, which cause devastating human diseases. A distinctive component of the Corynebacterineae cell envelope is the mycolyl-arabinogalactan (mAG) complex. The mAG is composed of lipid mycolic acids, and arabinofuranose (Araf) and galactofuranose (Galf) carbohydrate residues. Elucidating microbe-specific differences in mAG composition could advance biotechnological applications and lead to new antimicrobial targets. To this end, we compare and contrast galactan biosynthesis in C. diphtheriae and M. tuberculosis In each species, the galactan is constructed from uridine 5'-diphosphate-α-d-galactofuranose (UDP-Galf), which is generated by the enzyme UDP-galactopyranose mutase (UGM or Glf). UGM and the galactan are essential in M. tuberculosis, but their importance in Corynebacterium species was not known. We show that small molecule inhibitors of UGM impede C. glutamicum growth, suggesting that the galactan is critical in corynebacteria. Previous cell wall analysis data suggest the galactan polymer is longer in mycobacterial species than corynebacterial species. To explore the source of galactan length variation, a C. diphtheriae ortholog of the M. tuberculosis carbohydrate polymerase responsible for the bulk of galactan polymerization, GlfT2, was produced, and its catalytic activity was evaluated. The C. diphtheriae GlfT2 gave rise to shorter polysaccharides than those obtained with the M. tuberculosis GlfT2. These data suggest that GlfT2 alone can influence galactan length. Our results provide tools, both small molecule and genetic, for probing and perturbing the assembly of the Corynebacterineae cell envelope.

  9. Transcriptional Profiling of Mycobacterium Tuberculosis During Infection: Lessons Learned

    PubMed Central

    Ward, Sarah K.; Abomoelak, Bassam; Marcus, Sarah A.; Talaat, Adel M.

    2010-01-01

    Infection with Mycobacterium tuberculosis, the causative agent of tuberculosis, is considered one of the biggest infectious disease killers worldwide. A significant amount of attention has been directed toward revealing genes involved in the virulence and pathogenesis of this air-born pathogen. With the advances in technologies for transcriptional profiling, several groups, including ours, took advantage of DNA microarrays to identify transcriptional units differentially regulated by M. tuberculosis within a host. The main idea behind this approach is that pathogens tend to regulate their gene expression levels depending on the host microenvironment, and preferentially express those needed for survival. Identifying this class of genes will improve our understanding of pathogenesis. In our case, we identified an in vivo expressed genomic island that was preferentially active in murine lungs during early infection, as well as groups of genes active during chronic tuberculosis. Other studies have identified additional gene groups that are active during macrophage infection and even in human lungs. Despite all of these findings, one of the lingering questions remaining was whether in vivo expressed transcripts are relevant to the virulence, pathogenesis, and persistence of the organism. The work of our group and others addressed this question by examining the contribution of in vivo expressed genes using a strategy based on gene deletions followed by animal infections. Overall, the analysis of most of the in vivo expressed genes supported a role of these genes in M. tuberculosis pathogenesis. Further, these data suggest that in vivo transcriptional profiling is a valid approach to identify genes required for bacterial pathogenesis. PMID:21738523

  10. Is Adipose Tissue a Place for Mycobacterium tuberculosis Persistence?

    PubMed Central

    Neyrolles, Olivier; Hernández-Pando, Rogelio; Pietri-Rouxel, France; Fornès, Paul; Tailleux, Ludovic; Payán, Jorge Alberto Barrios; Pivert, Elisabeth; Bordat, Yann; Aguilar, Diane; Prévost, Marie-Christine; Petit, Caroline; Gicquel, Brigitte

    2006-01-01

    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 persistent

  11. Mycobacterium tuberculosis Malate Synthase- and MPT51-Based Serodiagnostic Assay as an Adjunct to Rapid Identification of Pulmonary Tuberculosis

    PubMed Central

    Achkar, Jacqueline M.; Dong, Yuxin; Holzman, Robert S.; Belisle, John; Kourbeti, Irene S.; Sherpa, Tsering; Condos, Rany; Rom, William N.; Laal, Suman

    2006-01-01

    The 81-kDa malate synthase (MS; Rv 1837c) and the 27-kDa MPT51 (Rv 3803c) of Mycobacterium tuberculosis are immunodominant antigens recognized by serum antibodies from ∼80% of human immunodeficiency virus-negative smear-positive tuberculosis patients from India. We now provide evidence that the use of the MS/MPT51-based serodiagnostic assay can serve as an adjunct to sputum microscopy in the rapid diagnosis of pulmonary tuberculosis. PMID:17090645

  12. Rapid susceptibility testing of Mycobacterium avium complex and Mycobacterium tuberculosis isolated from AIDS patients

    NASA Technical Reports Server (NTRS)

    Dhople, Arvind M.

    1994-01-01

    In ominous projections issued by both U.S. Public Health Service and the World Health Organization, the epidemic of HIV infection will continue to rise more rapidly worldwide than predicted earlier. The AIDS patients are susceptible to diseases called opportunistic infections of which tuberculosis and Mycobacterium avium complex (MAC) infection are most common. This has created an urgent need to uncover new drugs for the treatment of these infections. In the seventies, NASA scientists at Goddard Space Flight Center, Greenbelt, MD, had adopted a biochemical indicator, adenosine triphosphate (ATP), to detect presence of life in extraterrestrial space. We proposed to develop ATP assay technique to determine sensitivity of antibacterial compounds against MAC and M. tuberculosis.

  13. Origin, Spread and Demography of the Mycobacterium tuberculosis Complex

    PubMed Central

    Wirth, Thierry; Hildebrand, Falk; Allix-Béguec, Caroline; Wölbeling, Florian; Kubica, Tanja; Kremer, Kristin; van Soolingen, Dick; Rüsch-Gerdes, Sabine; Locht, Camille; Brisse, Sylvain; Meyer, Axel

    2008-01-01

    The evolutionary timing and spread of the Mycobacterium tuberculosis complex (MTBC), one of the most successful groups of bacterial pathogens, remains largely unknown. Here, using mycobacterial tandem repeat sequences as genetic markers, we show that the MTBC consists of two independent clades, one composed exclusively of M. tuberculosis lineages from humans and the other composed of both animal and human isolates. The latter also likely derived from a human pathogenic lineage, supporting the hypothesis of an original human host. Using Bayesian statistics and experimental data on the variability of the mycobacterial markers in infected patients, we estimated the age of the MTBC at 40,000 years, coinciding with the expansion of “modern” human populations out of Africa. Furthermore, coalescence analysis revealed a strong and recent demographic expansion in almost all M. tuberculosis lineages, which coincides with the human population explosion over the last two centuries. These findings thus unveil the dynamic dimension of the association between human host and pathogen populations. PMID:18802459

  14. Molecular epidemiology and genotyping of Mycobacterium tuberculosis isolated in Baghdad.

    PubMed

    Mustafa Ali, Ruqaya; Trovato, Alberto; Couvin, David; Al-Thwani, Amina N; Borroni, Emanuele; Dhaer, Fahim H; Rastogi, Nalin; Cirillo, Daniela M

    2014-01-01

    Tuberculosis (TB) remains a major health problem in Iraq but the strains responsible for the epidemic have been poorly characterized. Our aim was to characterize the TB strains circulating in Bagdad (Iraq). A total of 270 Mycobacterium tuberculosis complex (MTBC) strains isolated between 2010 and 2011 from TB patients attending the Center of Chest and Respiratory diseases in Baghdad were analyzed by Spoligotyping. The analysis indicated that 94.1% of the isolates belong to known genotype clades: CAS 39.6%, ill-defined T clade 29.6%, Manu 7.4%, Haarlem 7%, Ural 4.1%, LAM 3.3%, X 0.7%, LAM7-TUR 0.7%, EAI 0.7%, S 0.7%, and unknown 5.9%. Comparison with the international multimarker database SITVIT2 showed that SIT 309 (CAS1-Delhi) and SIT1144 (T1) were the most common types. In addition, 44 strains were included in SITVIT2 database under 16 new Spoligotype International Types (SITs); of these, 6 SITs (SIT3346, SIT3497, SIT3708, SIT3790, SIT3791, and SIT3800) (n = 32 strains) were created within the present study and 10 were created after a match with an orphan in the database. By using 24-loci MIRU-VNTR-typing on a subset of 110 samples we found a high recent transmission index (RTI) of 33.6%. In conclusion, we present the first unifying framework for both epidemiology and evolutionary analysis of M. tuberculosis in Iraq.

  15. Molecular Epidemiology and Genotyping of Mycobacterium tuberculosis Isolated in Baghdad

    PubMed Central

    Mustafa Ali, Ruqaya; Trovato, Alberto; Al-Thwani, Amina N.; Dhaer, Fahim H.; Cirillo, Daniela M.

    2014-01-01

    Tuberculosis (TB) remains a major health problem in Iraq but the strains responsible for the epidemic have been poorly characterized. Our aim was to characterize the TB strains circulating in Bagdad (Iraq). A total of 270 Mycobacterium tuberculosis complex (MTBC) strains isolated between 2010 and 2011 from TB patients attending the Center of Chest and Respiratory diseases in Baghdad were analyzed by Spoligotyping. The analysis indicated that 94.1% of the isolates belong to known genotype clades: CAS 39.6%, ill-defined T clade 29.6%, Manu 7.4%, Haarlem 7%, Ural 4.1%, LAM 3.3%, X 0.7%, LAM7-TUR 0.7%, EAI 0.7%, S 0.7%, and unknown 5.9%. Comparison with the international multimarker database SITVIT2 showed that SIT 309 (CAS1-Delhi) and SIT1144 (T1) were the most common types. In addition, 44 strains were included in SITVIT2 database under 16 new Spoligotype International Types (SITs); of these, 6 SITs (SIT3346, SIT3497, SIT3708, SIT3790, SIT3791, and SIT3800) (n = 32 strains) were created within the present study and 10 were created after a match with an orphan in the database. By using 24-loci MIRU-VNTR-typing on a subset of 110 samples we found a high recent transmission index (RTI) of 33.6%. In conclusion, we present the first unifying framework for both epidemiology and evolutionary analysis of M. tuberculosis in Iraq. PMID:24719873

  16. Pyrazinamide inhibits trans-translation in Mycobacterium tuberculosis.

    PubMed

    Shi, Wanliang; Zhang, Xuelian; Jiang, Xin; Yuan, Haiming; Lee, Jong Seok; Barry, Clifton E; Wang, Honghai; Zhang, Wenhong; Zhang, Ying

    2011-09-16

    Pyrazinamide (PZA) is a first-line tuberculosis drug that plays a unique role in shortening the duration of tuberculosis chemotherapy. PZA is hydrolyzed intracellularly to pyrazinoic acid (POA) by pyrazinamidase (PZase, encoded by pncA), an enzyme frequently lost in PZA-resistant strains, but the target of POA in Mycobacterium tuberculosis has remained elusive. Here, we identify a previously unknown target of POA as the ribosomal protein S1 (RpsA), a vital protein involved in protein translation and the ribosome-sparing process of trans-translation. Three PZA-resistant clinical isolates without pncA mutation harbored RpsA mutations. RpsA overexpression conferred increased PZA resistance, and we confirmed that POA bound to RpsA (but not a clinically identified ΔAla mutant) and subsequently inhibited trans-translation rather than canonical translation. Trans-translation is essential for freeing scarce ribosomes in nonreplicating organisms, and its inhibition may explain the ability of PZA to eradicate persisting organisms.

  17. Immunological consequences of strain variation within the Mycobacterium tuberculosis complex

    PubMed Central

    Tientcheu, Leopold D.; Ndengane, Mthawelenga; Andoseh, Genevieve; Kampmann, Beate; Wilkinson, Robert J

    2017-01-01

    In 2015, there were an estimated 10.4 million new cases of tuberculosis (TB) globally, making it one of the leading causes of death due to an infectious disease. TB is caused by members of the Mycobacterium tuberculosis complex (MTBC), with human disease resulting from infection by M. tuberculosis sensu stricto and M. africanum. Recent progress in genotyping techniques, in particular the increasing availability of whole genome sequence data, has revealed previously under appreciated levels of genetic diversity within the MTBC. Several studies have shown that this genetic diversity may translate into differences in TB transmission, clinical manifestations of disease, and host immune responses. This suggests the existence of MTBC genotype‐dependent host–pathogen interactions which may influence the outcome of infection and progression of disease. In this review, we highlight the studies demonstrating differences in innate and adaptive immunological outcomes consequent on MTBC genetic diversity, and discuss how these differences in immune response might influence the development of TB vaccines, diagnostics and new therapies. PMID:28150302

  18. Structure-activity relationships of macrolides against Mycobacterium tuberculosis.

    PubMed

    Zhu, Zhaohai J; Krasnykh, Olga; Pan, Dahua; Petukhova, Valentina; Yu, Gengli; Liu, Yinghui; Liu, Huiwen; Hong, Saweon; Wang, Yuehong; Wan, Baojie; Liang, Wenzhong; Franzblau, Scott G

    2008-08-01

    Existing 14, 15 and 16-membered macrolide antibiotics, while effective for other bacterial infections, including some mycobacteria, have not demonstrated significant efficacy in tuberculosis. Therefore an attempt was made to optimize this class for activity against Mycobacterium tuberculosis through semisyntheses and bioassay. Approximately 300 macrolides were synthesized and screened for anti-TB activity. Structural modifications on erythromycin were carried out at positions 3, 6, 9, 11, and 12 of the 14-membered lactone ring; as well as at position 4'' of cladinose and position 2' of desosamine. In general, the synthesized macrolides belong to four subclasses: 9-oxime, 11,12-carbamate, 11,12-carbazate, and 6-O-substituted derivatives. Selected compounds were assessed for mammalian cell toxicity and in some cases were further assessed for CYP3A4 inhibition, microsome stability, in vivo tolerance and efficacy. The activity of 11,12-carbamates and carbazates as well as 9-oximes is highly influenced by the nature of the substitution at these positions. For hydrophilic macrolides, lipophilic substitution may result in enhanced potency, presumably by enhanced passive permeation through the cell envelope. This strategy, however, has limitations. Removal of the C-3 cladinose generally reduces the activity. Acetylation at C-2' or 4'' maintains potency of C-9 oximes but dramatically decreases that of 11,12-substituted compounds. Further significant increases in the potency of macrolides for M. tuberculosis may require a strategy for the concurrent reduction of ribosome methylation.

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

    PubMed Central

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

    2014-01-01

    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. PMID:24573854

  20. Macrophage polarization drives granuloma outcome during Mycobacterium tuberculosis infection.

    PubMed

    Marino, Simeone; Cilfone, Nicholas A; Mattila, Joshua T; Linderman, Jennifer J; Flynn, JoAnne L; Kirschner, Denise E

    2015-01-01

    Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), induces formation of granulomas, structures in which immune cells and bacteria colocalize. Macrophages are among the most abundant cell types in granulomas and have been shown to serve as both critical bactericidal cells and targets for M. tuberculosis infection and proliferation throughout the course of infection. Very little is known about how these processes are regulated, what controls macrophage microenvironment-specific polarization and plasticity, or why some granulomas control bacteria and others permit bacterial dissemination. We take a computational-biology approach to investigate mechanisms that drive macrophage polarization, function, and bacterial control in granulomas. We define a "macrophage polarization ratio" as a metric to understand how cytokine signaling translates into polarization of single macrophages in a granuloma, which in turn modulates cellular functions, including antimicrobial activity and cytokine production. Ultimately, we extend this macrophage ratio to the tissue scale and define a "granuloma polarization ratio" describing mean polarization measures for entire granulomas. Here we coupled experimental data from nonhuman primate TB granulomas to our computational model, and we predict two novel and testable hypotheses regarding macrophage profiles in TB outcomes. First, the temporal dynamics of granuloma polarization ratios are predictive of granuloma outcome. Second, stable necrotic granulomas with low CFU counts and limited inflammation are characterized by short NF-κB signal activation intervals. These results suggest that the dynamics of NF-κB signaling is a viable therapeutic target to promote M1 polarization early during infection and to improve outcome.

  1. Immunological consequences of strain variation within the Mycobacterium tuberculosis complex.

    PubMed

    Tientcheu, Leopold D; Koch, Anastasia; Ndengane, Mthawelenga; Andoseh, Genevieve; Kampmann, Beate; Wilkinson, Robert J

    2017-03-01

    In 2015, there were an estimated 10.4 million new cases of tuberculosis (TB) globally, making it one of the leading causes of death due to an infectious disease. TB is caused by members of the Mycobacterium tuberculosis complex (MTBC), with human disease resulting from infection by M. tuberculosis sensu stricto and M. africanum. Recent progress in genotyping techniques, in particular the increasing availability of whole genome sequence data, has revealed previously under appreciated levels of genetic diversity within the MTBC. Several studies have shown that this genetic diversity may translate into differences in TB transmission, clinical manifestations of disease, and host immune responses. This suggests the existence of MTBC genotype-dependent host-pathogen interactions which may influence the outcome of infection and progression of disease. In this review, we highlight the studies demonstrating differences in innate and adaptive immunological outcomes consequent on MTBC genetic diversity, and discuss how these differences in immune response might influence the development of TB vaccines, diagnostics and new therapies. © 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. [Pyrazinamide monoresistant Mycobacterium tuberculosis in Manisa region, Turkey].

    PubMed

    Ozkütük, Nuri; Ecemiş, Talat; Sürücüoğlu, Süheyla

    2008-10-01

    Pyrazinamide (PZA) is a primary antituberculous drug. BACTEC 460TB is the recommended reference method for the detection of PZA resistance in Mycobacterium tuberculosis. This method is more expensive than the conventional susceptibility methods and therefore, it is recommended that each laboratory should establish their own protocol for the inclusion of PZA in the panel of primary drugs tested. One of the most important factors that help this decision is the prevalence of PZA resistance, particularly PZA monoresistance in the related community. The aim of the present study was to determine the extent of PZA monoresistance in M. tuberculosis complex (MTBC) isolates in our region. In this study, PZA susceptibility testing of 109 MTBC strains (susceptible to isoniazid, rifampicin, ethambutol and streptomycin) isolated from Manisa province in the Aegean region of Turkey was performed by using the BACTEC 460TB radiometric system (Becton Dickinson, MD). Two (1.8%) of the 109 isolates which were susceptible to all primary drugs revealed monoresistance against PZA. One of the PZA-monoresistant isolates has been identified as M. bovis and the other as M. tuberculosis by molecular method (Genotype MTBC, Hain Lifescience, Germany). The results of our study indicated that since the rate of PZA monoresistance was low, susceptibility testing of a panel of primary drugs without PZA may be an economical alternative in our region.

  3. A New Approach for Pyrazinamide Susceptibility Testing in Mycobacterium tuberculosis

    PubMed Central

    Loli, Sebastian; Gilman, Robert H.; Gutierrez, Andrés; Fuentes, Patricia; Cotrina, Milagros; Kirwan, Daniela; Sheen, Patricia

    2012-01-01

    Background: Pyrazinamide (PZA) is an important drug in the treatment of tuberculosis. Microbiological methods of PZA susceptibility testing are controversial and have low reproducibility. After conversion of PZA into pyrazinoic acid (POA) by the bacterial pyrazinamidase enzyme, the drug is expelled from the bacteria by an efflux pump. Objective: To evaluate the rate of POA extrusion from Mycobacterium tuberculosis as a parameter to detect PZA resistance. Methods: The rate of POA extrusion and PZA susceptibility determined by BACTEC 460 were measured for 34 strains in a previous study. PZA resistance was modeled in a logistic regression with the pyrazinoic efflux rate. Result: POA efflux rate predicted PZA resistance with 70.83%–92.85% sensitivity and 100% specificity compared with BACTEC 460. Conclusion: POA efflux rate could be a useful tool for predicting PZA resistance in M. tuberculosis. Further exploration of this approach may lead to the development of new tools for diagnosing PZA resistance, which may be of public health importance. PMID:22372927

  4. Molecular Characterization of Mycobacterium tuberculosis Strains with TB-SPRINT.

    PubMed

    Molina-Moya, Barbara; Gomgnimbou, Michel Kiréopori; Lafoz, Carmen; Lacoma, Alicia; Prat, Cristina; Refrégier, Guislaine; Samper, Sofia; Dominguez, Jose; Sola, Christophe

    2017-07-10

    We evaluated Tuberculosis-Spoligo-Rifampicin-Isoniazid Typing (TB-SPRINT), a microbead-based method for spoligotyping and detection of rifampicin and isoniazid resistance in Mycobacterium tuberculosis. For that, 67 M. tuberculosis complex strains were retrospectively selected. Membrane-based spoligotyping, restriction fragment length polymorphism, DNA sequencing/pyrosequencing of rpoB, katG, and inhA promoter, TB-SPRINT, and SNP typing were performed. Concordance between spoligotyping methods was 99.6% (2,785/2,795 spoligotype data points). For most of the discordant cases, the same lineage was assigned with both methods. Concordance between phenotypic drug susceptibility testing and TB-SPRINT for detecting rifampicin and isoniazid resistance was 98.4% (63/64) and 93.8% (60/64), respectively. Concordance between DNA sequencing/pyrosequencing and TB-SPRINT for detecting mutations in rpoB, katG, and inhA were 98.4% (60/61), 100% (64/64), and 96.9% (62/64), respectively. In conclusion, TB-SPRINT is a rapid and easy-to-perform assay for genotyping and detecting drug resistance in a single tube; therefore, it may be a useful tool to improve epidemiological surveillance.

  5. Molecular typing of Mycobacterium tuberculosis circulated in Moscow, Russian Federation.

    PubMed

    Afanas'ev, M V; Ikryannikova, L N; Il'ina, E N; Kuz'min, A V; Larionova, E E; Smirnova, T G; Chernousova, L N; Govorun, V M

    2011-02-01

    The present study investigates epidemiological diversity and multidrug resistance spreading among Mycobacterium tuberculosis strains circulating in Moscow, Russian Federation. Among 115 M. tuberculosis strains selected randomly from the sputum of epidemiologically unrelated tuberculosis (TB) patients, multidrug-resistant (MDR) strains predominated. Mutations in the RRDR of the rpoB gene were detected in 64 (83.1%) of 77 rifampicin (RIF)-resistant strains. The Ser531→Leu substitution was prevalent among them (76.5%). Aberrations in the Ser315 codon of katG and/or in the inhA promoter region were found in 79 (84.0%) of 94 isoniazid (INH)-resistant strains. Strains belonging to the Beijing family prevailed. Seventy-one different patterns were identified using the 24-VNTR loci typing scheme. Three main 24-loci VNTR clusters included 34 strains which belonged to the Beijing family. The spoligotyping and 24-loci VNTR typing combination demonstrated maximal discriminatory power. Among the Beijing strains, the MDR phenotype was revealed more frequently than among the others. High genetic heterogeneity of the studied population was shown by the assessment of VNTR loci variability in the analyzed group and in the strains from other parts of Russia. Comparison of the 24-VNTR locus typing and spoligotyping data with revealed resistance-associated mutation allows us to make a suggestion that the active transmission of MDR strains and the independent appearance of drug resistance during chemotherapy occurred in the studied population simultaneously.

  6. Isolation of Mycobacterium tuberculosis from captive Ateles paniscus.

    PubMed

    Rocha, Vivianne Cambuí Mesquita; Ikuta, Cássia Yumi; Gomes, Marcelo S; Quaglia, Fausto; Matushima, Eliana R; Ferreira Neto, José Soares

    2011-05-01

    An adult female red-faced black spider monkey (Ateles paniscus), housed for 2 years in the Parque Estoril Zoo in São Paulo, Brazil, showed apathy. Clinical examination revealed discrete emaciation, swelling and induration of lymph nodes, and presence of a mass in the abdominal cavity. Therapies with enrofloxacin, azithromycin, and ceftiofur were ineffective. The animal died after 6 months. Necropsy and histopathology confirmed granulommas in lymph nodes, parietal and visceral pleura, lungs, liver, spleen, and kidneys. Acid-fast bacilli were isolated and identified as Mycobacterium tuberculosis by polymerase chain reaction restriction analysis and Spoligotyping techniques. The zoo personnel and other animals that had had contact with the infected primate were negative to tuberculosis diagnostic procedures, such as sputum exam (baciloscopy) and thorax radiography. It was impossible to determine whether the infection occurred before or after the arrival of the animal to the Parque Estoril Zoo. This is the first report of M. tuberculosis infection in Ateles paniscus, a neotropical primate.

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

    PubMed Central

    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

  8. The Role of ESX-1 in Mycobacterium tuberculosis Pathogenesis.

    PubMed

    Wong, Ka-Wing

    2017-05-01

    In this article, we have described several cellular pathological effects caused by the Mycobacterium tuberculosis ESX-1. The effects include induction of necrosis, NOD2 signaling, type I interferon production, and autophagy. We then attempted to suggest that these pathological effects are mediated by the cytosolic access of M. tuberculosis-derived materials as a result of the phagosome-disrupting activity of the major ESX-1 substrate ESAT-6. Such activity of ESAT-6 is most likely due to its pore-forming activity at the membrane. The amyloidogenic characteristic of ESAT-6 is reviewed here as a potential mechanism of membrane pore formation. In addition to ESAT-6, the ESX-1 substrate EspB interferes with membrane-mediated innate immune mechanisms such as efferocytosis and autophagy, most likely through its ability to bind phospholipids. Overall, the M. tuberculosis ESX-1 secretion system appears to be a specialized system for the deployment of host membrane-targeting proteins, whose primary function is to interrupt key steps in innate immune mechanisms against pathogens. Inhibitors that block the ESX-1 system or block host factors critical for ESX-1 toxicity have been identified and should represent attractive potential new antituberculosis drugs.

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

    PubMed

    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 SO2 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 SO2. 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 SO2 vaccinated goats had the lowest lesion and culture scores. These results suggest that the SO2 vaccine provides some protection against TB infection acquired from natural exposure. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Systems-Based Approaches to Probing Metabolic Variation within the Mycobacterium tuberculosis Complex

    PubMed Central

    Lofthouse, Emma K.; Wheeler, Paul R.; Beste, Dany J. V.; Khatri, Bhagwati L.; Wu, Huihai; Mendum, Tom A.; Kierzek, Andrzej M.; McFadden, Johnjoe

    2013-01-01

    The Mycobacterium tuberculosis complex includes bovine and human strains of the tuberculosis bacillus, including Mycobacterium tuberculosis, Mycobacterium bovis and the Mycobacterium bovis BCG vaccine strain. M. bovis has evolved from a M. tuberculosis-like ancestor and is the ancestor of the BCG vaccine. The pathogens demonstrate distinct differences in virulence, host range and metabolism, but the role of metabolic differences in pathogenicity is poorly understood. Systems biology approaches have been used to investigate the metabolism of M. tuberculosis, but not to probe differences between tuberculosis strains. In this study genome scale metabolic networks of M. bovis and M. bovis BCG were constructed and interrogated, along with a M. tuberculosis network, to predict substrate utilisation, gene essentiality and growth rates. The models correctly predicted 87-88% of high-throughput phenotype data, 75-76% of gene essentiality data and in silico-predicted growth rates matched measured rates. However, analysis of the metabolic networks identified discrepancies between in silico predictions and in vitro data, highlighting areas of incomplete metabolic knowledge. Additional experimental studies carried out to probe these inconsistencies revealed novel insights into the metabolism of these strains. For instance, that the reduction in metabolic capability observed in bovine tuberculosis strains, as compared to M. tuberculosis, is not reflected by current genetic or enzymatic knowledge. Hence, the in silico networks not only successfully simulate many aspects of the growth and physiology of these mycobacteria, but also provide an invaluable tool for future metabolic studies. PMID:24098743

  11. Comparative analyses of transport proteins encoded within the genomes of Mycobacterium tuberculosis and Mycobacterium leprae.

    PubMed

    Youm, Jiwon; Saier, Milton H

    2012-03-01

    The co-emergence of multidrug resistant pathogenic bacterial strains and the Human Immunodeficiency Virus pandemic has made tuberculosis a leading public health threat. The causative agent is Mycobacterium tuberculosis (Mtu), a facultative intracellular parasite. Mycobacterium leprae (Mle), a related organism that causes leprosy, is an obligate intracellular parasite. Given that different transporters are required for bacterial growth and persistence under a variety of growth conditions, we conducted comparative analyses of transport proteins encoded within the genomes of these two organisms. A minimal set of genes required for intracellular and extracellular life was identified. Drug efflux systems utilizing primary active transport mechanisms have been preferentially retained in Mle and still others preferentially lost. Transporters associated with environmental adaptation found in Mtu were mostly lost in Mle. These findings provide starting points for experimental studies that may elucidate the dependencies of pathogenesis on transport for these two pathogenic mycobacteria. They also lead to suggestions regarding transporters that function in intra- versus extra-cellular growth.

  12. Comparative analyses of transport proteins encoded within the genomes of Mycobacterium tuberculosis and Mycobacterium leprae

    PubMed Central

    Youm, Jiwon; Saier, Milton H.

    2012-01-01

    The co-emergence of multidrug resistant pathogenic bacterial strains and the HIV pandemic has made tuberculosis a leading public health threat. The causative agent is Mycobacterium tuberculosis (Mtu), a facultative intracellular parasite. Mycobacterium leprae (Mle), a related organism that causes leprosy, is an obligate intracellular parasite. Given that different transporters are required for bacterial growth and persistence under a variety of growth conditions, we conducted comparative analyses of transport proteins encoded within the genomes of these two organisms. A minimal set of genes required for intracellular and extracellular life were identified. Drug efflux systems utilizing primary active transport mechanisms have been preferentially retained in Mle and still others preferentially lost. Transporters associated with environmental adaptation found in Mtu were mostly lost in Mle. These findings provide starting points for experimental studies that may elucidate the dependencies of pathogenesis on transport for these two pathogenic mycobacteria. They also lead to suggestions regarding transporters that function in intra- versus extra-cellular growth. PMID:22179038

  13. Differences in T-cell responses between Mycobacterium tuberculosis and Mycobacterium africanum-infected patients.

    PubMed

    Tientcheu, Leopold D; Sutherland, Jayne S; de Jong, Bouke C; Kampmann, Beate; Jafali, James; Adetifa, Ifedayo M; Antonio, Martin; Dockrell, Hazel M; Ota, Martin O

    2014-05-01

    In The Gambia, Mycobacterium tuberculosis (Mtb) and Mycobacterium africanum (Maf) are major causes of tuberculosis (TB). Maf is more likely to cause TB in immune suppressed individuals, implying differences in virulence. Despite this, few studies have assessed the underlying immunity to the two pathogens in human. In this study, we analyzed T-cell responses from 19 Maf- and 29 Mtb-infected HIV-negative patients before and after TB chemotherapy following overnight stimulation of whole blood with TB-specific antigens. Before treatment, percentages of early secreted antigenic target-6(ESAT-6)/culture filtrate protein-10(CFP-10) and purified protein derivative-specific single-TNF-α-producing CD4(+) and CD8(+) T cells were significantly higher while single-IL-2-producing T cells were significantly lower in Maf- compared with Mtb-infected patients. Purified protein derivative-specific polyfunctional CD4(+) T cells frequencies were significantly higher before than after treatment, but there was no difference between the groups at both time points. Furthermore, the proportion of CD3(+) CD11b(+) T cells was similar in both groups pretreatment, but was significantly lower with higher TNF-α, IL-2, and IFN-γ production in Mtb- compared with that of Maf-infected patients posttreatment. Our data provide evidence of differences in T-cell responses to two mycobacterial strains with differing virulence, providing some insight into TB pathogenesis with different Mtb strains that could be prospectively explored as biomarkers for TB protection or susceptibility.

  14. Prevalence of latent Mycobacterium tuberculosis infection in prisoners.

    PubMed

    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

  15. Digitally Barcoding Mycobacterium tuberculosis Reveals In Vivo Infection Dynamics in the Macaque Model of Tuberculosis

    PubMed Central

    Martin, Constance J.; Cadena, Anthony M.; Leung, Vivian W.; Lin, Philana Ling; Maiello, Pauline; Hicks, Nathan; Chase, Michael R.

    2017-01-01

    ABSTRACT Infection with Mycobacterium tuberculosis causes a spectrum of outcomes; the majority of individuals contain but do not eliminate the infection, while a small subset present with primary active tuberculosis (TB) disease. This variability in infection outcomes is recapitulated at the granuloma level within each host, such that some sites of infection can be fully cleared while others progress. Understanding the spectrum of TB outcomes requires new tools to deconstruct the mechanisms underlying differences in granuloma fate. Here, we use novel genome-encoded barcodes to uniquely tag individual M. tuberculosis bacilli, enabling us to quantitatively track the trajectory of each infecting bacterium in a macaque model of TB. We also introduce a robust bioinformatics pipeline capable of identifying and counting barcode sequences within complex mixtures and at various read depths. By coupling this tagging strategy with serial positron emission tomography coregistered with computed tomography (PET/CT) imaging of lung pathology in macaques, we define a lesional map of M. tuberculosis infection dynamics. We find that there is no significant infection bottleneck, but there are significant constraints on productive bacterial trafficking out of primary granulomas. Our findings validate our barcoding approach and demonstrate its utility in probing lesion-specific biology and dissemination. This novel technology has the potential to greatly enhance our understanding of local dynamics in tuberculosis. PMID:28487426

  16. Roles of PE_PGRS family in Mycobacterium tuberculosis pathogenesis and novel measures against tuberculosis.

    PubMed

    Tian, Chen; Jian-Ping, Xie

    2010-12-01

    Tuberculosis remains a serious threat to global public health. As one of the most successful pathogens, Mycobacterium tuberculosis is an adept in evasion the host immune response. M. tuberculosis PE_PGRS family has been widely proposed as molecular mantra to deflect host immunity. The feature of this family is the conserved N-terminal and variable C-terminal. PE_PGRS proteins, with functions largely unknown, are only found among mycobacteria and located in the mycobacterial cell wall and cell membrane. Most PE_PGRS proteins have antigenicity. Polymorphic PGRS domain might play a role in neutralize immune response. Several members of PE_PGRS family have been heterologously expressed for further function characterization. PE_PGRS gene expressing stage-specifically, especially during M. tuberculosis persistence, is promising intervention target to prevent reactivation. The origin, physiological role and spatiotemporal regulation feature of this family remain elusive. The understanding of these questions will shed light on the pathogenesis of M. tuberculosis and facilitate the discovery of new effective antibacterial intervention. Copyright © 2010 Elsevier Ltd. All rights reserved.

  17. 78 FR 36698 - Microbiology Devices; Reclassification of Nucleic Acid-Based Systems for Mycobacterium tuberculosis

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-19

    ... Nucleic Acid-Based Systems for Mycobacterium tuberculosis Complex in Respiratory Specimens AGENCY: Food...) is proposing to reclassify nucleic acid-based in vitro diagnostic devices for the detection of... Controls Guideline: Nucleic Acid-Based In Vitro Diagnostic Devices for the Detection of Mycobacterium...

  18. mmr, a Mycobacterium tuberculosis Gene Conferring Resistance to Small Cationic Dyes and Inhibitors

    PubMed Central

    De Rossi, Edda; Branzoni, Manuela; Cantoni, Rita; Milano, Anna; Riccardi, Giovanna; Ciferri, Orio

    1998-01-01

    The mmr gene, cloned from Mycobacterium tuberculosis, was shown to confer to Mycobacterium smegmatis resistance to tetraphenylphosphonium (TPP), erythromycin, ethidium bromide, acriflavine, safranin O, and pyronin Y. The gene appears to code for a protein containing four transmembrane domains. Studies of [3H]TPP intracellular accumulation strongly suggest that the resistance mediated by the Mmr protein involves active extrusion of TPP. PMID:9811672

  19. Diagnosis of extrapulmonary tuberculosis by Gen-Probe amplified Mycobacterium tuberculosis direct test.

    PubMed Central

    Ehlers, S; Ignatius, R; Regnath, T; Hahn, H

    1996-01-01

    A total of 294 specimens collected from nonrespiratory sites of 268 patients were tested for direct detection of Mycobacterium tuberculosis complex by the Gen-Probe Amplified Mycobacterium tuberculosis Direct Test (AMTD). The specimens included ascitic, pleural, pericardial, and synovial fluids, abscess aspirates, and tissue and lymph node biopsy samples, as well as gastric aspirates and cerebrospinal fluid samples. All samples were processed by the N-acetyl-L-cysteine-sodium hydroxide decontamination procedure prior to testing. Twenty samples showed acid-fast bacilli on auramine staining, and 48 samples were positive by AMTD, 9 of which were negative for M. tuberculosis complex by culture. After reviewing the patients clinical charts to resolve discrepancies, the test result of one cerebrospinal fluid sample was considered to be false positive by AMTD. The overall sensitivity, specificity, positive predictive value, and negative predictive value were 83.9, 99.6, 97.9, and 96.3%, respectively. No significant differences were found when AMTD results obtained with specimens of nonrespiratory origin were compared with assay results obtained with samples of respiratory origin (P > 0.05). In conclusion, our results demonstrate that AMTD performs equally well with all types of specimens. PMID:8862598

  20. Characterisation of methionine adenosyltransferase from Mycobacterium smegmatis and M. tuberculosis.

    PubMed

    Berger, Bradley J; Knodel, Marvin H

    2003-06-16

    Tuberculosis remains a serious world-wide health threat which requires the characterisation of novel drug targets for the development of future antimycobacterials. One of the key obstacles in the definition of new targets is the large variety of metabolic alterations that occur between cells in the active growth and chronic/dormant phases of tuberculosis. The ideal biochemical target should be active in both growth phases. Methionine adenosyltransferase, which catalyses the formation of S-adenosylmethionine from methionine and ATP, is involved in polyamine biosynthesis during active growth and is also required for the methylation and cyclopropylation of mycolipids necessary for survival in the chronic phase. The gene encoding methionine adenosyltransferase has been cloned from Mycobacterium tuberculosis and the model organism M. smegmatis. Both enzymes retained all amino acids known to be involved in catalysing the reaction. While the M. smegmatis enzyme could be functionally expressed, the M. tuberculosis homologue was insoluble and inactive under a large variety of expression conditions. For the M. smegmatis enzyme, the Vmax for S-adenosylmethionine formation was 1.30 micromol/min/mg protein and the Km for methionine and ATP was 288 microM and 76 microM respectively. In addition, the enzyme was competitively inhibited by 8-azaguanine and azathioprine with a Ki of 4.7 mM and 3.7 mM respectively. Azathioprine inhibited the in vitro growth of M. smegmatis with a minimal inhibitory concentration (MIC) of 500 microM, while the MIC for 8-azaguanine was >1.0 mM. The methionine adenosyltransferase from both organisms had a primary structure very similar those previously characterised in other prokaryotic and eukaryotic organisms. The kinetic properties of the M. smegmatis enzyme were also similar to known prokaryotic methionine adenosyltransferases. Inhibition of the enzyme by 8-azaguanine and azathioprine provides a starting point for the synthesis of higher affinity

  1. Intracardiac tuberculomas caused by Mycobacterium tuberculosis in a dog.

    PubMed

    Szaluś-Jordanow, Olga; Augustynowicz-Kopeć, Ewa; Czopowicz, Michał; Olkowski, Arkadiusz; Łobaczewski, Andrzej; Rzewuska, Magdalena; Sapierzyński, Rafał; Wiatr, Elżbieta; Garncarz, Magdalena; Frymus, Tadeusz

    2016-06-14

    This paper presents an unusual form of disseminated Mycobacterium tuberculosis infection in a dog. The infection lasted at least one year and its main gross lesions were massive cardiac tuberculomas. To the best of our knowledge, this is the first report of heart tuberculomas in a dog. A 9-year-old mixed-breed male dog weighing 10 kg was referred to the clinic for cardiological evaluation before general anesthesia. The echocardiography revealed a lump of about 20 mm in diameter in the area of the left atrium. Almost one year later the same dog was presented again in severe clinical state (fever, anorexia, weight loss, depression, cough, dyspnea, lymphadenomegaly, vomiting, recent episodes of fainting). Due to progression of the disease and poor effects of treatment the owner decided to euthanize the dog. Most prominent lesions observed during autopsy were diffuse pneumonia, fibrinous pericarditis and epicarditis as well as large, yellow, semisolid masses of caseous necrosis in the left and right atrium (30 mm and 15 mm in diameter, respectively). From both pulmonary and cardiac lesions M. tuberculosis was isolated on Lowenstein-Jensen slants and in Bactec Mycobacteria Growth Indicator Tube 960 liquid media, and confirmed by BD ProbeTec ET Direct Detection Assay and spoligotyping. Companion animals may occasionally suffer from tuberculosis but majority of cases probably remain misdiagnosed or undetected. Typically tuberculosis in dogs affects lungs and their regional lymph nodes. Even in humans tuberculomas are rare manifestation of mycobacterial infection, mostly seen in the central nervous system. Atypical location of main tuberculous lesions may account for lack of correct ante mortem diagnosis in this case.

  2. Efficacy of Microencapsulated Rifampin in Mycobacterium tuberculosis-Infected Mice

    PubMed Central

    Quenelle, Debra C.; Staas, Jay K.; Winchester, Gary A.; Barrow, Esther L. W.; Barrow, William W.

    1999-01-01

    Rifampin is a first-line drug useful in the treatment of tuberculosis. By using biocompatible polymeric excipients of lactide and glycolide copolymers, two microsphere formulations were developed for targeted and sustained delivery of rifampin, with minimal dosing. A small-microsphere formulation, with demonstrated ability to inhibit intracellularly replicating Mycobacterium tuberculosis H37Rv, was tested along with a large-microsphere formulation in an infected mouse model. Results revealed that by using a single treatment of the large-microsphere formulation, it was possible to achieve a significant reduction in M. tuberculosis H37Rv CFUs in the lungs of mice by 26 days postinfection. A combination of small (given as two injections on day 0 and day 7) and large (given as one injection at day 0) rifampin-loaded microsphere formulations resulted in significant reductions in CFUs in the lungs by 26 days, achieving a 1.23 log10 reduction in CFUs. By comparison, oral treatment with 5, 10, or 20 mg of rifampin/kg of body weight, administered every day, resulted in a reduction of 0.42, 1.7, or 1.8 log10 units, respectively. Thus the microsphere formulations, administered in one or two doses, were able to achieve results in mice similar to those obtained with a daily drug regimen within the range of the highest clinically tolerated dosage in humans. These results demonstrate that microsphere formulations of antimycobacterial drugs such as rifampin can be used for therapy of tuberculosis with minimal dosing. PMID:10223927

  3. Rifampin induces hydroxyl radical formation in Mycobacterium tuberculosis.

    PubMed

    Piccaro, Giovanni; Pietraforte, Donatella; Giannoni, Federico; Mustazzolu, Alessandro; Fattorini, Lanfranco

    2014-12-01

    The antituberculosis (anti-TB) drug rifampin (RIF) binds to the beta subunit of the RNA polymerase (RpoB) of Mycobacterium tuberculosis, but the bactericidal responses triggered after target interaction are not known. To evaluate whether RIF induced an oxidative burst, lysates of RIF-treated M. tuberculosis were tested for determination of reactive oxygen species (ROS) by the electron paramagnetic resonance (EPR) technique using 1-hydroxy-3-carboxy-pyrrolidine (CPH) and 5,5-dimethyl-1-pyrrolidine-N-oxide (DMPO) as spin traps. M. tuberculosis killing by RIF stimulated an increase in the rate of formation of the CPH radical (CP·). Lysate pretreatment with the O2·(-) and ·OH scavengers superoxide dismutase (SOD) and thiourea (THIO), respectively, or with the metal chelator diethylene triamine pentaacetic acid (DTPA) inhibited CP· formation, arguing in favor of a metal-catalyzed ROS response. Formation of CP· did not increase following treatment of RIF-resistant strains with RIF, indicating that the ROS were induced after RpoB binding. To identify the ROS formed, lysates of RIF-treated bacilli were incubated with DMPO, a spin trap specific for ·OH and O2·(-), with or without pretreatment with SOD, catalase, THIO, or DTPA. Superoxide dismutase, catalase, and THIO decreased formation of the DMPO-OH adduct, and SOD plus DTPA completely suppressed it, suggesting that RIF activated metal-dependent O2·(-)-mediated mechanisms producing ·OH inside tubercle bacilli. The finding that the metal chelator DTPA reduced the bactericidal activity of RIF supported the possibility that ·OH was generated through these mechanisms and that it participated at least in part in M. tuberculosis killing by the drug.

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

    NASA Astrophysics Data System (ADS)

    Badr, Hesham M.

    2011-11-01

    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 °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.

  5. [Susceptibilities of Mycobacterium tuberculosis strains collected from regional tuberculosis laboratories to major antituberculous drugs].

    PubMed

    Sayğan, M Bakir; Ocak, Fatih; Cesur, Salih; Tarhan, Gülnur; Ceyhan, Ismail; Gümüişlü, Feyzullah; Beker, Gülşan; Güner, Uğur; Coşkun, Erol

    2007-07-01

    The aim of this study was to investigate the susceptibility rates of Mycobacterium tuberculosis strains sent to Refik Saydam Hygiene Center, Tuberculosis Reference and Research Laboratory, Ankara, from seven different regional tuberculosis laboratories between the 1999-2002 period against major antituberculous drugs. The sensitivities of a total 505 M. tuberculosis strains to isoniazid (INAH), rifampicin (RIF), streptomycin (SM) and ethambutol (EMB) were determined by using proportion method in Lowenstein-Jensen medium. Of the strains, 385 (76.2%) were found sensitive to all of the tested drugs, while 120 strains were resistant to at least one of the antituberculous drugs. The resistant strains showed 14 different resistance patterns. The resistance rates were detected as 13.3% for INAH and RIF (67 strains of each), 9.1% for SM (46 strains), and 3.4% (17 strains) for EMB. Multidrug resistant (INAH+RIF) M. tuberculosis was 7.9% (40 strains). The highest resistance rate to INAH, RIF and EMB (21.2%, 21.2% and 10.6%, respectively) was detected in the isolates which were sent from Bursa province (located in northwestern Turkey); the highest SM (18.8%) and multidrug resistance (INAH+RIF) rates (18.8% and 10.6%, respectively) were detected in the strains sent from Elazig and Van provinces (both located in eastern Turkey). Since the inappropriate use of the first and second line antituberculous drugs leads to the development and spread of the resistant strains, "Directly Observed Therapy Shortcourse (DOTS)" is a very important practice. Therefore regional tuberculosis laboratories should be worth considering as the chains of a well-organized national laboratory network, in order to detect the antituberculous drug resistance patterns of the M. tuberculosis strains over the country.

  6. Rapid Diagnosis of Tuberculosis by Real-Time High-Resolution Imaging of Mycobacterium tuberculosis Colonies.

    PubMed

    Ghodbane, Ramzi; Asmar, Shady; Betzner, Marlena; Linet, Marie; Pierquin, Joseph; Raoult, Didier; Drancourt, Michel

    2015-08-01

    Culture remains the cornerstone of diagnosis for pulmonary tuberculosis, but the fastidiousness of Mycobacterium tuberculosis may delay culture-based diagnosis for weeks. We evaluated the performance of real-time high-resolution imaging for the rapid detection of M. tuberculosis colonies growing on a solid medium. A total of 50 clinical specimens, including 42 sputum specimens, 4 stool specimens, 2 bronchoalveolar lavage fluid specimens, and 2 bronchial aspirate fluid specimens were prospectively inoculated into (i) a commercially available Middlebrook broth and evaluated for mycobacterial growth indirectly detected by measuring oxygen consumption (standard protocol) and (ii) a home-made solid medium incubated in an incubator featuring real-time high-resolution imaging of colonies (real-time protocol). Isolates were identified by Ziehl-Neelsen staining and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Use of the standard protocol yielded 14/50 (28%) M. tuberculosis isolates, which is not significantly different from the 13/50 (26%) M. tuberculosis isolates found using the real-time protocol (P = 1.00 by Fisher's exact test), and the contamination rate of 1/50 (2%) was not significantly different from the contamination rate of 2/50 (4%) using the real-time protocol (P = 1.00). The real-time imaging protocol showed a 4.4-fold reduction in time to detection, 82 ± 54 h versus 360 ± 142 h (P < 0.05). These preliminary data give the proof of concept that real-time high-resolution imaging of M. tuberculosis colonies is a new technology that shortens the time to growth detection and the laboratory diagnosis of pulmonary tuberculosis.

  7. Innate and Adaptive Cellular Immune Responses to Mycobacterium tuberculosis Infection.

    PubMed

    Mayer-Barber, Katrin D; Barber, Daniel L

    2015-07-17

    Host resistance to Mycobacterium tuberculosis (Mtb) infection requires the coordinated efforts of innate and adaptive immune cells. Diverse pulmonary myeloid cell populations respond to Mtb with unique contributions to both host-protective and potentially detrimental inflammation. Although multiple cell types of the adaptive immune system respond to Mtb infection, CD4 T cells are the principal antigen-specific cells responsible for containment of Mtb infection, but they can also be major contributors to disease during Mtb infection in several different settings. Here, we will discuss the role of different myeloid populations as well as the dual nature of CD4 T cells in Mtb infection with a primary focus on data generated using in vivo cellular immunological studies in experimental animal models and in humans when available. Copyright © 2015 Cold Spring Harbor Laboratory Press; all rights reserved.

  8. An elucidation of neutrophil functions against Mycobacterium tuberculosis infection.

    PubMed

    Morris, Devin; Nguyen, Thien; Kim, John; Kassissa, Christine; Khurasany, Melissa; Luong, Jennifer; Kasko, Sarah; Pandya, Shalin; Chu, Michael; Chi, Po-Ting; Ly, Judy; Lagman, Minette; Venketaraman, Vishwanath

    2013-01-01

    We characterized the functions of neutrophils in response to Mycobacterium tuberculosis (M. tb) infection, with particular reference to glutathione (GSH). We examined the effects of GSH in improving the ability of neutrophils to control intracellular M. tb infection. Our findings indicate that increasing the intracellular levels of GSH with a liposomal formulation of GSH (L-GSH) resulted in reduction in the levels of free radicals and increased acidification of M. tb containing phagosomes leading to the inhibition in the growth of M. tb. This inhibitory mechanism is dependent on the presence of TNF-α and IL-6. Our studies demonstrate a novel regulatory mechanism adapted by the neutrophils to control M. tb infection.

  9. An Elucidation of Neutrophil Functions against Mycobacterium tuberculosis Infection

    PubMed Central

    Morris, Devin; Nguyen, Thien; Kim, John; Kassissa, Christine; Khurasany, Melissa; Luong, Jennifer; Kasko, Sarah; Pandya, Shalin; Chu, Michael; Chi, Po-Ting; Lagman, Minette; Venketaraman, Vishwanath

    2013-01-01

    We characterized the functions of neutrophils in response to Mycobacterium tuberculosis (M. tb) infection, with particular reference to glutathione (GSH). We examined the effects of GSH in improving the ability of neutrophils to control intracellular M. tb infection. Our findings indicate that increasing the intracellular levels of GSH with a liposomal formulation of GSH (L-GSH) resulted in reduction in the levels of free radicals and increased acidification of M. tb containing phagosomes leading to the inhibition in the growth of M. tb. This inhibitory mechanism is dependent on the presence of TNF-α and IL-6. Our studies demonstrate a novel regulatory mechanism adapted by the neutrophils to control M. tb infection. PMID:24312131

  10. Cholesterol catabolism by Mycobacterium tuberculosis requires transcriptional and metabolic adaptations

    PubMed Central

    Griffin, Jennifer E.; Pandey, Amit K.; Gilmore, Sarah A.; Mizrahi, Valerie; Mckinney, John D.; Bertozzi, Carolyn R.; Sassetti, Christopher M.

    2012-01-01

    SUMMARY To understand the adaptation of Mycobacterium tuberculosis to the intracellular environment, we used comprehensive metabolite profiling to identify the biochemical pathways utilized during growth on cholesterol, a critical carbon source during chronic infection. Metabolic alterations observed during cholesterol catabolism centered on propionyl-CoA and pyruvate pools. Consequently, growth on this substrate required the transcriptional induction of the propionyl-CoA assimilating methylcitrate cycle (MCC) enzymes, via the Rv1129c regulatory protein. We show that both Rv1129c and the MCC enzymes are required for intracellular growth in macrophages, and the growth defect of MCC mutants is largely attributable to the degradation of host-derived cholesterol. Together, these observations define a coordinated transcriptional and metabolic adaptation that is required for scavenging carbon during intracellular growth. PMID:22365605

  11. Turning the respiratory flexibility of Mycobacterium tuberculosis against itself

    PubMed Central

    Lamprecht, Dirk A.; Finin, Peter M.; Rahman, Md. Aejazur; Cumming, Bridgette M.; Russell, Shannon L.; Jonnala, Surendranadha R.; Adamson, John H.; Steyn, Adrie J. C.

    2016-01-01

    The Mycobacterium tuberculosis (Mtb) electron transport chain (ETC) has received significant attention as a drug target, however its vulnerability may be affected by its flexibility in response to disruption. Here we determine the effect of the ETC inhibitors bedaquiline, Q203 and clofazimine on the Mtb ETC, and the value of the ETC as a drug target, by measuring Mtb's respiration using extracellular flux technology. We find that Mtb's ETC rapidly reroutes around inhibition by these drugs and increases total respiration to maintain ATP levels. Rerouting is possible because Mtb rapidly switches between terminal oxidases, and, unlike eukaryotes, is not susceptible to back pressure. Increased ETC activity potentiates clofazimine's production of reactive oxygen species, causing rapid killing in vitro and in a macrophage model. Our results indicate that combination therapy targeting the ETC can be exploited to enhance killing of Mtb. PMID:27506290

  12. In vitro susceptibility testing of Mycobacterium tuberculosis complex strains isolated from seals to antituberculosis drugs.

    PubMed

    Bernardelli, Amelia; Morcillo, Nora; Loureiro, Julio; Quse, Viviana; Davenport, Silvana

    2004-06-01

    Mycobacteria strains belonging to the Mycobacterium tuberculosis complex were isolated from seals found in the South Atlantic. The animals were received in Mundo Marino installations and treated for Mycobacterium tuberculosis complex by conventional therapy of intensive care and enriched food supply; however, in all cases treatment failed. Necropsies of all animals revealed extensive lesions compatible with tuberculosis involving lungs, liver, spleen and lymphatic nodes. Classical biochemical methods as well as molecular techniques using the IS6110 probes were performed for mycobacterial identification. Furthermore, the LCx M. tuberculosis assay (Abbott Laboratories) identified all strains as Mycobacterium tuberculosis complex members. The in vitro susceptibility pattern was examined in mycobacterial strains isolated from seven seals and in 3 reference strains--BCG, H37Rv (M. tuberculosis) and AN5 (Mycobacterium bovis)--to 4 medications--isoniazid, rifampin, streptomycin and ethambutol. Minimal inhibitory drug concentrations were determined by the Mycobacterial Growth Indicator Tube (BD Argentina) method and a microdilution and colorimetric assay using 3-(4-5 dimethyltiazol-2)-2,5 diphenyltetrazolium bromide. All the isolates and the reference strains BCG and AN5 were inhibited by MIC values similar to those of H37Rv with good agreement obtained by both techniques. These findings suggest that a therapeutic regimen aimed to seals diagnosed with tuberculosis play an important role in the prevention of tuberculosis transmission from infected animals to humans that are in routine contact with them.

  13. Emergence of Potential Superbug Mycobacterium Tuberculosis, Lessons from New Delhi Mutant-1 Bacterial Strains

    PubMed Central

    Nazir, Taha; Abraham, Suraj; Islam, Azharul

    2012-01-01

    Recent reports have shown that certain bacterial strains attain the New Delhi Metallo-beta-lactamase-1 (NDM-1) enzyme and become resistant to a broad range of antibiotics. Similarly, more dangerous “superbugs” of multi-drug resistant (MDR) and extensive drug resistant (XDR) Mycobacterium tuberculosis strains are gradually emerging through rapid genetic mutation caused by prescription non-compliance or unsupervised indiscriminate use of anti-tubercular drugs or other antibiotics. Mycobacterium tuberculosis cases have been reported in highly susceptible population groups including the aboriginal communities of US and Canada. In Canada alone, the total number of reported tuberculosis cases has decreased over the past decade. However, there is a steady increase in HIV cases in certain communities including the aboriginal communities. Reintroduction of MDR/XDR strains of tuberculosis is possible in these susceptible communities, which in turn may pose serious public health situation. MDR/XDR strains of tuberculosis are virtually untreatable using current anti-tubercular medication protocols. Thus, MDR/XDR tuberculosis presents a grave global public health threat. The unpredictable genetic mechanism involved in generating MDR/XDR resistant strains of Mycobacterium tuberculosis may pose greater challenges in developing appropriate treatment strategies. In this article, we briefly review potential genetic mechanism of emerging NDM-1 bacterial strains and draw a rationale parallel to the underlying genetic mechanism of MDR/XDR Mycobacterium tuberculosis strain development. PMID:23267308

  14. Primary Multidrug-Resistant Mycobacterium tuberculosis in 2 Regions, Eastern Siberia, Russian Federation

    PubMed Central

    Zhdanova, Svetlana; Ogarkov, Oleg; Boyarinova, Galina; Alexeeva, Galina; Pholwat, Suporn; Zorkaltseva, Elena; Houpt, Eric R.; Savilov, Eugeniy

    2013-01-01

    Of 235 Mycobacterium tuberculosis isolates from patients who had not received tuberculosis treatment in the Irkutsk oblast and the Sakha Republic (Yakutia), eastern Siberia, 61 (26%) were multidrug resistant. A novel strain, S 256, clustered among these isolates and carried eis-related kanamycin resistance, indicating a need for locally informed diagnosis and treatment strategies. PMID:24047678

  15. Primary multidrug-resistant Mycobacterium tuberculosis in 2 regions, Eastern Siberia, Russian Federation.

    PubMed

    Zhdanova, Svetlana; Heysell, Scott K; Ogarkov, Oleg; Boyarinova, Galina; Alexeeva, Galina; Pholwat, Suporn; Zorkaltseva, Elena; Houpt, Eric R; Savilov, Eugeniy

    2013-10-01

    Of 235 Mycobacterium tuberculosis isolates from patients who had not received tuberculosis treatment in the Irkutsk oblast and the Sakha Republic (Yakutia), eastern Siberia, 61 (26%) were multidrug resistant. A novel strain, S 256, clustered among these isolates and carried eis-related kanamycin resistance, indicating a need for locally informed diagnosis and treatment strategies.

  16. Mycobacterium tuberculosis Meets the Cytosol: The Role of cGAS in Anti-mycobacterial Immunity.

    PubMed

    Majlessi, Laleh; Brosch, Roland

    2015-06-10

    The intracellular fate of Mycobacterium tuberculosis is a subject of long debate. In this issue of Cell Host & Microbe, three independent studies reveal the detection of cytosolic mycobacterial DNA by the nucleotidyltransferase cGAS, emphasizing the concept of cytosolic access by M. tuberculosis and its role in balancing immune-protection and immune-pathogenesis.

  17. Beijing Lineage of MDR Mycobacterium tuberculosis in Bulgaria, 2007–2011

    PubMed Central

    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 Beijing genotype among these patients was 3.2%. PMID:25340498

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

    PubMed

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

    2014-11-01

    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 Beijing genotype among these patients was 3.2%.

  19. Rapid Accumulation of Eosinophils in Lung Lesions in Guinea Pigs Infected with Mycobacterium tuberculosis

    PubMed Central

    Lasco, Todd M.; Turner, Oliver C.; Cassone, Lynne; Sugawara, Isamu; Yamada, Hiroyuki; McMurray, David N.; Orme, Ian M.

    2004-01-01

    Guinea pig eosinophils were positively identified in bronchoalveolar lavage populations and in the lung granulomas of Mycobacterium tuberculosis-infected guinea pigs. It is possible that the rapid influx of these cells, and their subsequent degranulation during acute pulmonary tuberculosis, may play a key role in the susceptibility of this animal model. PMID:14742563

  20. Draft Genome Sequence of Mycobacterium tuberculosis Clinical Strain G-12-005

    PubMed Central

    de Carvalho, Fabíola Marques; de Almeida, Luiz Gonzaga Paula; Bablishvili, Nino; Gauthier, Marie; Paranhos-Baccalà, Glaucia; de Vasconcelos, Ana Tereza Ribeiro

    2014-01-01

    Infection caused by drug-resistant Mycobacterium tuberculosis is a growing concern, especially in eastern Europe. We report an annotated draft genome sequence of M. tuberculosis strain G-12-005 obtained from a patient in Georgia. PMID:24812221

  1. Emergence of potential superbug mycobacterium tuberculosis, lessons from new delhi mutant-1 bacterial strains.

    PubMed

    Nazir, Taha; Abraham, Suraj; Islam, Azharul

    2012-01-01

    Recent reports have shown that certain bacterial strains attain the New Delhi Metallo-beta-lactamase-1 (NDM-1) enzyme and become resistant to a broad range of antibiotics. Similarly, more dangerous "superbugs" of multi-drug resistant (MDR) and extensive drug resistant (XDR) Mycobacterium tuberculosis strains are gradually emerging through rapid genetic mutation caused by prescription non-compliance or unsupervised indiscriminate use of anti-tubercular drugs or other antibiotics. Mycobacterium tuberculosis cases have been reported in highly susceptible population groups including the aboriginal communities of US and Canada. In Canada alone, the total number of reported tuberculosis cases has decreased over the past decade. However, there is a steady increase in HIV cases in certain communities including the aboriginal communities. Reintroduction of MDR/XDR strains of tuberculosis is possible in these susceptible communities, which in turn may pose serious public health situation. MDR/XDR strains of tuberculosis are virtually untreatable using current anti-tubercular medication protocols. Thus, MDR/XDR tuberculosis presents a grave global public health threat. The unpredictable genetic mechanism involved in generating MDR/XDR resistant strains of Mycobacterium tuberculosis may pose greater challenges in developing appropriate treatment strategies. In this article, we briefly review potential genetic mechanism of emerging NDM-1 bacterial strains and draw a rationale parallel to the underlying genetic mechanism of MDR/XDR Mycobacterium tuberculosis strain development.

  2. Discordant resistance to kanamycin and amikacin in drug-resistant Mycobacterium tuberculosis.

    PubMed

    Krüüner, Annika; Jureen, Pontus; Levina, Klavdia; Ghebremichael, Solomon; Hoffner, Sven

    2003-09-01

    It is generally thought that there is full cross-resistance in Mycobacterium tuberculosis between the aminoglycoside drugs kanamycin and amikacin. However, kanamycin resistance and amikacin susceptibility were seen in 43 of 79 (54%) multidrug-resistant Estonian isolates, indicating that there might be a need to test the resistance of M. tuberculosis isolates to both drugs.

  3. Mycobacterium tuberculosis Rv1987 induces Th2 immune responses and enhances Mycobacterium smegmatis survival in mice.

    PubMed

    Sha, Shanshan; Shi, Xiaoxia; Deng, Guoying; Chen, Lina; Xin, Yi; Ma, Yufang

    2017-04-01

    Mycobacterium tuberculosis can interfere with host immune response and escape clearance through its specific antigens. M. tuberculosis Rv1987 encoded by region of difference (RD)-2 gene is a secretory protein with immunogenic potency. Here, we investigated the impact of Rv1987 on host cytokine responses and T cell polarization in mouse aerosol model. A recombinant M. smegmatis mc(2)155 strain that overexpressed Rv1987 protein (named MS1987) was constructed and used to infect C57BL/6 mice. The mc(2)155 harbored the empty vector (named MSVec) was as a control. The results showed that MS1987 challenged mice promoted Th2-biased cytokine responses with lower secretion of IFN-γ but higher production of IL-4 and Rv1987-specific IgG antibody compared to MSVec infected mice. Neutrophilic inflammation and high bacterial burden were observed in the lung tissues of MS1987 infected mice probably own to the failed Th1 cell immunity. Besides, subcutaneous injection of Rv1987 protein could mediate the Th1 cytokine responses caused by M. bovis BCG in mice. These results indicated that M. tuberculosis Rv1987 protein could modulate host immune response towards Th2 profile, which probably contributed to the immune evasion of bacteria from host elimination. Copyright © 2017 Elsevier GmbH. All rights reserved.

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

    PubMed Central

    Ihama, Yasushi; Hokama, Akira; Hibiya, Kenji; Kishimoto, Kazuto; Nakamoto, Manabu; Hirata, Tetsuo; Kinjo, Nagisa; Cash, Haley L; Higa, Futoshi; Tateyama, Masao; Kinjo, Fukunori; Fujita, Jiro

    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

  5. In Vitro Activity of a New Isothiazoloquinolone, ACH-702, against Mycobacterium tuberculosis and Other Mycobacteria▿

    PubMed Central

    Molina-Torres, Carmen A.; Ocampo-Candiani, Jorge; Rendón, Adrian; Pucci, Michael J.; Vera-Cabrera, Lucio

    2010-01-01

    In this work, we describe the activity of ACH-702 against clinical isolates of Mycobacterium tuberculosis and six different nontuberculous mycobacteria. The MIC50 and MIC90 of both susceptible and drug-resistant M. tuberculosis strains tested were 0.0625 and 0.125 μg/ml, respectively. The MIC50 and MIC90 values for Mycobacterium fortuitum isolates were 0.0625 μg/ml in both cases; Mycobacterium avium complex isolates showed MIC50 and MIC90 values of 0.25 and 4 μg/ml, respectively. PMID:20231398

  6. Reactogenicity to major tuberculosis antigens absent in BCG is linked to improved protection against Mycobacterium tuberculosis.

    PubMed

    Aguilo, Nacho; Gonzalo-Asensio, Jesus; Alvarez-Arguedas, Samuel; Marinova, Dessislava; Gomez, Ana Belen; Uranga, Santiago; Spallek, Ralf; Singh, Mahavir; Audran, Regine; Spertini, François; Martin, Carlos

    2017-07-14

    MTBVAC is a live-attenuated Mycobacterium tuberculosis vaccine, currently under clinical development, that contains the major antigens ESAT6 and CFP10. These antigens are absent from the current tuberculosis vaccine, BCG. Here we compare the protection induced by BCG and MTBVAC in several mouse strains that naturally express different MHC haplotypes differentially recognizing ESAT6 and CFP10. MTBVAC induces improved protection in C3H mice, the only of the three tested strains reactive to both ESAT6 and CFP10. Deletion of both antigens in MTBVAC reduces its efficacy to BCG levels, supporting a link between greater efficacy and CFP10- and ESAT6-specific reactogenicity. In addition, MTBVAC (but not BCG) triggers a specific response in human vaccinees against ESAT6 and CFP10. Our results warrant further exploration of this response as potential biomarker of protection in MTBVAC clinical trials.

  7. Global Efforts in the Development of Vaccines for Tuberculosis: Requirements for Improved Vaccines Against Mycobacterium tuberculosis.

    PubMed

    Méndez-Samperio, P

    2016-10-01

    Currently, more than 9.0 million people develop acute pulmonary tuberculosis (TB) each year and about 1.5 million people worldwide die from this infection. Thus, developing vaccines to prevent active TB disease remains a priority. This article discusses recent progress in the development of new vaccines against TB and focusses on the main requirements for development of improved vaccines against Mycobacterium tuberculosis (M. tb). Over the last two decades, significant progress has been made in TB vaccine development, and some TB vaccine candidates have currently completed a phase III clinical trial. The potential public health benefits of these vaccines are possible, but it will need much more effort, including new global governance investment on this research. This investment would certainly be less than the annual global financial toll of TB treatment.

  8. Zoonotic tuberculosis in human beings caused by Mycobacterium bovis-a call for action.

    PubMed

    Olea-Popelka, Francisco; Muwonge, Adrian; Perera, Alejandro; Dean, Anna S; Mumford, Elizabeth; Erlacher-Vindel, Elisabeth; Forcella, Simona; Silk, Benjamin J; Ditiu, Lucica; El Idrissi, Ahmed; Raviglione, Mario; Cosivi, Ottorino; LoBue, Philip; Fujiwara, Paula I

    2017-01-01

    Mycobacterium tuberculosis is recognised as the primary cause of human tuberculosis worldwide. However, substantial evidence suggests that the burden of Mycobacterium bovis, the cause of bovine tuberculosis, might be underestimated in human beings as the cause of zoonotic tuberculosis. In 2013, results from a systematic review and meta-analysis of global zoonotic tuberculosis showed that the same challenges and concerns expressed 15 years ago remain valid. These challenges faced by people with zoonotic tuberculosis might not be proportional to the scientific attention and resources allocated in recent years to other diseases. The burden of zoonotic tuberculosis in people needs important reassessment, especially in areas where bovine tuberculosis is endemic and where people live in conditions that favour direct contact with infected animals or animal products. As countries move towards detecting the 3 million tuberculosis cases estimated to be missed annually, and in view of WHO's end TB strategy endorsed by the health authorities of WHO Member States in 2014 to achieve a world free of tuberculosis by 2035, we call on all tuberculosis stakeholders to act to accurately diagnose and treat tuberculosis caused by M bovis in human beings. Copyright © 2017 World Health Organization. Published by Elsevier Ltd/Inc/BV. All rights reserved. Published by Elsevier Ltd.. All rights reserved.

  9. Genetic diversity of Mycobacterium tuberculosis isolated from tuberculosis patients in the Serengeti ecosystem in Tanzania.

    PubMed

    Mbugi, Erasto V; Katale, Bugwesa Z; Siame, Keith K; Keyyu, Julius D; Kendall, Sharon L; Dockrell, Hazel M; Streicher, Elizabeth M; Michel, Anita L; Rweyemamu, Mark M; Warren, Robin M; Matee, Mecky I; van Helden, Paul D

    2015-03-01

    This study was part of a larger cross-sectional survey that was evaluating tuberculosis (TB) infection in humans, livestock and wildlife in the Serengeti ecosystem in Tanzania. The study aimed at evaluating the genetic diversity of Mycobacterium tuberculosis isolates from TB patients attending health facilities in the Serengeti ecosystem. DNA was extracted from 214 sputum cultures obtained from consecutively enrolled newly diagnosed untreated TB patients aged ≥18 years. Spacer oligonucleotide typing (spoligotyping) and Mycobacterium Interspersed Repetitive Units and Variable Number Tandem Repeat (MIRU-VNTR) were used to genotype M. tuberculosis to establish the circulating lineages. Of the214 M. tuberculosis isolates genotyped, 55 (25.7%) belonged to the Central Asian (CAS) family, 52 (24.3%) were T family (an ill-defined family), 38 (17.8%) belonged to the Latin American Mediterranean (LAM) family, 25 (11.7%) to the East-African Indian (EAI) family, 25 (11.7%) comprised of different unassigned ('Serengeti') strain families, while 8 (3.7%) belonged to the Beijing family. A minority group that included Haarlem, X, U and S altogether accounted for 11 (5.2%) of all genotypes. MIRU-VNTR typing produced diverse patterns within and between families indicative of unlinked transmission chains. We conclude that, in the Serengeti ecosystem only a few successful families predominate namely CAS, T, LAM and EAI families. Other types found in lower prevalence are Beijing, Haarlem, X, S and MANU. The Haarlem, EAI_Somalia, LAM3 and S/convergent and X2 subfamilies found in this study were not reported in previous studies in Tanzania.

  10. Genetic diversity of Mycobacterium tuberculosis isolated from tuberculosis patients in the Serengeti ecosystem in Tanzania

    PubMed Central

    Mbugi, Erasto V.; Katale, Bugwesa Z.; Siame, Keith K.; Keyyu, Julius D.; Kendall, Sharon L.; Dockrell, Hazel M.; Streicher, Elizabeth M.; Michel, Anita L.; Rweyemamu, Mark M.; Warren, Robin M.; Matee, Mecky I.; van Helden, Paul D.

    2015-01-01

    Summary This study was part of a larger cross-sectional survey that was evaluating tuberculosis (TB) infection in humans, livestock and wildlife in the Serengeti ecosystem in Tanzania. The study aimed at evaluating the genetic diversity of Mycobacterium tuberculosis isolates from TB patients attending health facilities in the Serengeti ecosystem. DNA was extracted from 214 sputum cultures obtained from consecutively enrolled newly diagnosed untreated TB patients aged ≥18 years. Spacer oligonucleotide typing (spoligotyping) and Mycobacterium Interspersed Repetitive Units and Variable Number Tandem Repeat (MIRU-VNTR) were used to genotype M. tuberculosis to establish the circulating lineages. Of the214 M. tuberculosis isolates genotyped, 55 (25.7%) belonged to the Central Asian (CAS) family, 52 (24.3%) were T family (an ill-defined family), 38 (17.8%) belonged to the Latin American Mediterranean (LAM) family, 25 (11.7%) to the East-African Indian (EAI) family, 25 (11.7%) comprised of different unassigned (‘Serengeti’) strain families, while 8 (3.7%) belonged to the Beijing family. A minority group that included Haarlem, X, U and S altogether accounted for 11 (5.2%) of all genotypes. MIRU-VNTR typing produced diverse patterns within and between families indicative of unlinked transmission chains. We conclude that, in the Serengeti ecosystem only a few successful families predominate namely CAS, T, LAM and EAI families. Other types found in lower prevalence are Beijing, Haarlem, X, S and MANU. The Haarlem, EAI_Somalia, LAM3 and S/convergent and X2 subfamilies found in this study were not reported in previous studies in Tanzania. PMID:25522841

  11. Genetics-directed drug discovery for combating Mycobacterium tuberculosis infection.

    PubMed

    Quan, Yuan; Xiong, Le; Chen, Jing; Zhang, Hong-Yu

    2017-02-01

    Mycobacterium tuberculosis (Mtb), the pathogen of tuberculosis (TB), is one of the most infectious bacteria in the world. The traditional strategy to combat TB involves targeting the pathogen directly; however, the rapid evolution of drug resistance lessens the efficiency of this anti-TB method. Therefore, in recent years, some researchers have turned to an alternative anti-TB strategy, which hinders Mtb infection through targeting host genes. In this work, using a theoretical genetic analysis, we identified 170 Mtb infection-associated genes from human genetic variations related to Mtb infection. Then, the agents targeting these genes were identified to have high potential as anti-TB drugs. In particular, the agents that can target multiple Mtb infection-associated genes are more druggable than the single-target counterparts. These potential anti-TB agents were further screened by gene expression data derived from connectivity map. As a result, some agents were revealed to have high interest for experimental evaluation. This study not only has important implications for anti-TB drug discovery, but also provides inspirations for streamlining the pipeline of modern drug discovery.

  12. Biochemical Characterization of Uracil Phosphoribosyltransferase from Mycobacterium tuberculosis

    PubMed Central

    Villela, Anne Drumond; Ducati, Rodrigo Gay; Rosado, Leonardo Astolfi; Bloch, Carlos Junior; Prates, Maura Vianna; Gonçalves, Danieli Cristina; Ramos, Carlos Henrique Inacio; Basso, Luiz Augusto; Santos, Diogenes Santiago

    2013-01-01

    Uracil phosphoribosyltransferase (UPRT) catalyzes the conversion of uracil and 5-phosphoribosyl-α-1-pyrophosphate (PRPP) to uridine 5′-monophosphate (UMP) and pyrophosphate (PPi). 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 PPi 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. PMID:23424660

  13. Oxygen Availability and Metabolic Dynamics During Mycobacterium tuberculosis Latency.

    PubMed

    May, Elebeoba E; Sershen, Cheryl L

    2016-10-01

    In vitro models of Mycobacterium tuberculosis (Mtb) nonreplicating persistence (NRP) suggest the rate of oxygen ( O2 ) depletion is a significant determinant in persistence. However, few studies have characterized the metabolic association between slow and rapid O2 depletion rates and successful versus failed persistence. We developed a theoretical model of Mtb metabolic adaptation that includes O2 driven genetic modulation of enzymes in the tricarboxylic acid cycle, energy, and redox recycling pathways. We conducted an in silico study of Mtb adaptation, and investigated the metabolic dynamics that enable persistence during slow versus rapid anaerobiosis. Consistent with in vitro studies, during rapid anaerobiosis the most significant enzymatic changes occurred during the active growth period while the majority of the slow anaerobic system's adaptive response occurred during the first phase of aerobic shiftdown (NRP1). The characteristic response of the two conditions differed, with the slow anaerobic system exhibiting positive adaptation response, upregulating six enzymes during NRP1, while the rapid system exhibited a negative adaptation response, decreasing the levels of seven enzymes during active growth. Results of our study illustrate the intricate metabolic balance Mtb achieves during adaptation to anaerobic conditions, and how failure in redox recycling correlates to failure in persistence. We have provided the first theoretical description of the genetic and metabolic death profile for Mtb during anaerobic growth. Insight into Mtbs dynamic adaptation response provides a unique systems framework for the development of targeted therapies to reduce the likelihood of mycobacterial persistence and the related incidence of latent tuberculosis infection.

  14. Biochemical characterization of the Mycobacterium tuberculosis phosphoribosyl-1-pyrophosphate synthetase

    PubMed Central

    Alderwick, Luke J; Lloyd, Georgina S; Lloyd, Adrian J; Lovering, Andrew L; Eggeling, Lothar; Besra, Gurdyal S

    2011-01-01

    Mycobacterium tuberculosis arabinogalactan (AG) is an essential cell wall component. It provides a molecular framework serving to connect peptidoglycan to the outer mycolic acid layer. The biosynthesis of the arabinan domains of AG and lipoarabinomannan (LAM) occurs via a combination of membrane bound arabinofuranosyltransferases, all of which utilize decaprenol-1-monophosphorabinose as a substrate. The source of arabinose ultimately destined for deposition into cell wall AG or LAM originates exclusively from phosphoribosyl-1-pyrophosphate (pRpp), a central metabolite which is also required for other essential metabolic processes, such as de novo purine and pyrimidine biosyntheses. In M. tuberculosis, a single pRpp synthetase enzyme (Mt-PrsA) is solely responsible for the generation of pRpp, by catalyzing the transfer of pyrophosphate from ATP to the C1 hydroxyl position of ribose-5-phosphate. Here, we report a detailed biochemical and biophysical study of Mt-PrsA, which exhibits the most rapid enzyme kinetics reported for a pRpp synthetase. PMID:21045009

  15. Anaerobic incubation conditions enhance pyrazinamide activity against Mycobacterium tuberculosis.

    PubMed

    Wade, Mary Margaret; Zhang, Ying

    2004-08-01

    Pyrazinamide (PZA) is an unconventional front line tuberculosis drug characterized by high in vivo sterilizing activity, but poor in vitro activity. This disparity in PZA activity may reflect differences between the in vivo tissue environment and in vitro culture conditions. This study examined the effect of anaerobic conditions, which exist in granulomatous lesions in vivo, on PZA activity in vitro. Low oxygen enhanced the activity of PZA against Mycobacterium tuberculosis, with anaerobic conditions resulting in greater enhancement than microaerobic conditions. ATPase and respiratory chain enzyme inhibitors enhanced PZA activity under normal atmospheric conditions, but not under anaerobic conditions. Furthermore, the inhibitors did not enhance isoniazid or rifampicin activity. Nitrate as an alternative electron acceptor antagonized PZA activity under anaerobic conditions. These findings provide further support for a proposed mechanism of action of PZA in which the active form of PZA (pyrazinoic acid) depletes the membrane energy reserve. They also provide another explanation for the higher sterilizing activity of PZA within in vivo lesions with low oxygen than under in vitro drug susceptibility testing conditions with ambient oxygen.

  16. Macrophage polarization: convergence point targeted by mycobacterium tuberculosis and HIV.

    PubMed

    Lugo-Villarino, Geanncarlo; Vérollet, Christel; Maridonneau-Parini, Isabelle; Neyrolles, Olivier

    2011-01-01

    In the arms race of host-microbe co-evolution, macrophages (Mɸs) have been endowed with strategies to neutralize pathogenic challenge while preserving host integrity. During steady-states conditions, Mɸs perform multiple house-keeping functions governed by their differentiation state, tissue distribution, and signals from the microenvironment. In response to pathogenic challenge and host mediators, however, Mɸs undergo different programs of activation rendering them either pro-inflammatory and microbicidal (M1), or immunosuppressants and tissue repairers (M2). An excessive or prolonged polarization of either program may be detrimental to the host due to potential tissue injury or contribution to pathogenesis. Conversely, intracellular microbes that cause chronic diseases such as tuberculosis and acquired immunodeficiency syndrome exemplify strategies for survival in the host. Indeed, both Mycobacterium tuberculosis (Mtb) and human immunodeficiency virus (HIV-1) are successful intracellular microbes that thrive in Mɸs. Given these microbes not only co-circulate throughout the developing world but each has contributed to prevalence and mortality caused by the other, substantial insights into microbe physiology and host defenses then rest in the attempt to fully understand their influence on Mɸ polarization. This review addresses the role of Mɸ polarization in the immune response to, and pathogenesis of, Mtb and HIV.

  17. Chemokine response in mice infected with Mycobacterium tuberculosis.

    PubMed Central

    Rhoades, E R; Cooper, A M; Orme, I M

    1995-01-01

    We show here that infection of murine macrophages with various strains of Mycobacterium tuberculosis induces the rapid in vitro expression of genes encoding chemokines macrophage inflammatory protein 1 alpha and macrophage inflammatory protein 2, which recruit neutrophils to sites of infection, and macrophage-recruiting chemokines 10-kDa, interferon-inducible protein (IP-10) and macrophage chemotactic protein 1. Three strains of M. tuberculosis, Erdman and the clinical isolates CSU 22 and CSU 46, induced similar levels of secretion of macrophage chemotactic protein 1 from infected macrophage monolayers; however, the Erdman strain failed to induce levels of secretion of tumor necrosis factor alpha similar to those induced by either CSU 22 or CSU 46. Using a low-dose aerosol infection model, we also found that while the Erdman strain induced negligible increases in chemokine mRNA levels in the lungs, infection with either CSU 22 or CSU 46 resulted in greater levels of mRNA production for all four chemokines tested. The growth of these strains in the lungs was, however, equally well contained by acquired host immunity. These data allow us to hypothesize that the chemokine response in the lungs probably does not control the protective granulomatous response and that perhaps other T-cell- or macrophage-associated cytokines such as tumor necrosis factor alpha or interleukin 12 may be involved in this process. PMID:7558294

  18. SInCRe—structural interactome computational resource for Mycobacterium tuberculosis

    PubMed Central

    Metri, Rahul; Hariharaputran, Sridhar; Ramakrishnan, Gayatri; Anand, Praveen; Raghavender, Upadhyayula S.; Ochoa-Montaño, Bernardo; Higueruelo, Alicia P.; Sowdhamini, Ramanathan; Chandra, Nagasuma R.; Blundell, Tom L.; Srinivasan, Narayanaswamy

    2015-01-01

    We have developed an integrated database for Mycobacterium tuberculosis H37Rv (Mtb) that collates information on protein sequences, domain assignments, functional annotation and 3D structural information along with protein–protein and protein–small molecule interactions. SInCRe (Structural Interactome Computational Resource) is developed out of CamBan (Cambridge and Bangalore) collaboration. The motivation for development of this database is to provide an integrated platform to allow easily access and interpretation of data and results obtained by all the groups in CamBan in the field of Mtb informatics. In-house algorithms and databases developed independently by various academic groups in CamBan are used to generate Mtb-specific datasets and are integrated in this database to provide a structural dimension to studies on tuberculosis. The SInCRe database readily provides information on identification of functional domains, genome-scale modelling of structures of Mtb proteins and characterization of the small-molecule binding sites within Mtb. The resource also provides structure-based function annotation, information on small-molecule binders including FDA (Food and Drug Administration)-approved drugs, protein–protein interactions (PPIs) and natural compounds that bind to pathogen proteins potentially and result in weakening or elimination of host–pathogen protein–protein interactions. Together they provide prerequisites for identification of off-target binding. Database URL: http://proline.biochem.iisc.ernet.in/sincre PMID:26130660

  19. Characterization and Transcriptome Analysis of Mycobacterium tuberculosis Persisters

    PubMed Central

    Keren, Iris; Minami, Shoko; Rubin, Eric; Lewis, Kim

    2011-01-01

    ABSTRACT Tuberculosis continues to be a major public health problem in many parts of the world. Significant obstacles in controlling the epidemic are the length of treatment and the large reservoir of latently infected people. Bacteria form dormant, drug-tolerant persister cells, which may be responsible for the difficulty in treating both acute and latent infections. We find that in Mycobacterium  tuberculosis, low numbers of drug-tolerant persisters are present in lag and early exponential phases, increasing sharply at late exponential and stationary phases to make up ~1% of the population. This suggests that persister formation is governed by both stochastic and deterministic mechanisms. In order to isolate persisters, an exponentially growing population was treated with d-cycloserine, and cells surviving lysis were collected by centrifugation. A transcriptome of persisters was obtained by using hybridization to an Affymetrix array. The transcriptome shows downregulation of metabolic and biosynthetic pathways, consistent with a certain degree of dormancy. A set of genes was upregulated in persisters, and these are likely involved in persister formation and maintenance. A comparison of the persister transcriptome with transcriptomes obtained for several in vitro dormancy models identified a small number of genes upregulated in all cases, which may represent a core dormancy response. PMID:21673191

  20. Mycobacterium microti Infection (Vole Tuberculosis) in Wild Rodent Populations

    PubMed Central

    Cavanagh, Rachel; Begon, Michael; Bennett, Malcolm; Ergon, Torbjørn; Graham, Isla M.; de Haas, Petra E. W.; Hart, C. A.; Koedam, Marianne; Kremer, Kristin; Lambin, Xavier; Roholl, Paul; Soolingen, Dick van

    2002-01-01

    Mycobacterium microti (vole tuberculosis) infections in small wild mammals were first described more than 60 years ago in several populations in Great Britain. Few studies of vole tuberculosis have been undertaken since then, and little is known about the relationship between M. microti isolates originating from different populations or at different times or of the prevalence of this infection in wild rodent populations, despite human cases of M. microti infections being increasingly reported. In this study, field voles (Microtus agrestis), bank voles (Clethrionomys glareolus), and wood mice (Apodemus sylvaticus) were found to be infected, with up to 8% having external tuberculous signs, in wild populations in Northumberland and Cheshire, England. Spoligotyping applied directly to the clinical material simultaneously detected and typed M. microti bacteria in skin lesions, lymph glands, and internal abcesses. IS6110 restriction fragment length polymorphism typing of cultured bacteria was used to compare these isolates with previously isolated strains from both animals and humans. This demonstrated that although the current rodent isolates were distinct from those isolated from voles in the 1930s in Great Britain, they had a high degree of similarity to these strains and were distinct from the M. microti isolates from humans, a pig, and a ferret from The Netherlands. Thus, M. microti infection seems to be widespread in wild rodent populations, but more studies are needed to understand how M. microti might be transmitted from animals to humans and to determine better the zoonotic risk posed. PMID:12202566

  1. Mycobacterium tuberculosis chorismate mutase: A potential target for TB.

    PubMed

    Khanapur, Manjulatha; Alvala, Mallika; Prabhakar, Maddela; Shiva Kumar, K; Edwin, R K; Sri Saranya, P S V K; Patel, Raj Kumar; Bulusu, Gopalakrishnan; Misra, P; Pal, Manojit

    2017-03-15

    Mycobacterium tuberculosis chorismate mutase (MtbCM) catalyzes the rearrangement of chorismate to prephenate in the shikimate biosynthetic pathway to form the essential amino acids, phenylalanine and tyrosine. Two genes encoding chorismate mutase have been identified in Mtb. The secretory form,∗MtbCM (encoded by Rv1885c) is assumed to play a key role in pathogenesis of tuberculosis. Also, the inhibition of MtbCM may hinder the supply of nutrients to the organism. Indeed, the existence of chorismate mutase (CM) in bacteria, fungi and higher plants but not in human and low sequence homology among known CM makes it an interesting target for the discovery of anti-tubercular agents. The present article mainly focuses on the recent developments in the structure, function and inhibition of MtbCM. The understanding of various aspects of MtbCM as presented in the current article may facilitate the design and subsequent chemical synthesis of new inhibitors against ∗MtbCM, that could lead to the discovery and development of novel and potent anti-tubercular agents in future.

  2. Mycobacterium tuberculosis Infection Interferes with HIV Vaccination in Mice

    PubMed Central

    Ignatowicz, Lech; Mazurek, Jolanta; Leepiyasakulchai, Chaniya; Sköld, Markus; Hinkula, Jorma; Källenius, Gunilla; Pawlowski, Andrzej

    2012-01-01

    Tuberculosis (TB) has emerged as the most prominent bacterial disease found in human immunodeficiency virus (HIV)-positive individuals worldwide. Due to high prevalence of asymptomatic Mycobacterium tuberculosis (Mtb) infections, the future HIV vaccine in areas highly endemic for TB will often be administrated to individuals with an ongoing Mtb infection. The impact of concurrent Mtb infection on the immunogenicity of a HIV vaccine candidate, MultiHIV DNA/protein, was investigated in mice. We found that, depending on the vaccination route, mice infected with Mtb before the administration of the HIV vaccine showed impairment in both the magnitude and the quality of antibody and T cell responses to the vaccine components p24Gag and gp160Env. Mice infected with Mtb prior to intranasal HIV vaccination exhibited reduced p24Gag-specific serum IgG and IgA, and suppressed gp160Env-specific serum IgG as compared to respective titers in uninfected HIV-vaccinated controls. Importantly, in Mtb-infected mice that were HIV-vaccinated by the intramuscular route the virus neutralizing activity in serum was significantly decreased, relative to uninfected counterparts. In addition mice concurrently infected with Mtb had fewer p24Gag-specific IFN-γ-expressing T cells and multifunctional T cells in their spleens. These results suggest that Mtb infection might interfere with the outcome of prospective HIV vaccination in humans. PMID:22848444

  3. Mycobacterium tuberculosis-induced neutrophil ectosomes decrease macrophage activation.

    PubMed

    Duarte, Tonya Azevedo; Noronha-Dutra, Alberto Augusto; Nery, Joilda Silva; Ribeiro, Samantha Brum; Pitanga, Thassila Nogueira; Lapa E Silva, José R; Arruda, Sérgio; Boéchat, Neio

    2012-05-01

    The existence of ectosome-like microvesicles released by neutrophils was proposed a few decades ago. Other studies revealed that the innate immune response during mycobacterial infection is accompanied by an intense migration of neutrophils to the site of infection, which may be important during the acute phase of tuberculosis. We found that the ectosomes derived from infected neutrophils are biologically active and can influence the survival of Mycobacterium tuberculosis within macrophages. Mycobacteria were cultured on supplemented Middlebrook-7H9 broth. All strains were grown to the exponential phase and quantitated by serial dilution. Human neutrophils and macrophages were infected with mycobacteria. Ectosomes from neutrophils were isolated post-infection and characterized by transmission electron microscopy and flow cytometry. To determine whether these microvesicles influenced mycobactericidal activity, mycobacteria-infected macrophages were treated with isolated ectosomes. Ectosomes were released from neutrophils infected with mycobacteria. These ectosomes were derived from neutrophil plasma membrane and a small proportion stained with PKH26. These microvesicles, when incubated with infected macrophages, influenced antimycobacterial activity. This is the first study to demonstrate that ectosomes that are shed from infected neutrophils influence mycobactericidal activity in macrophages in vitro, suggesting that these microvesicles have biological significance. Nevertheless, major gaps in our knowledge of microvesicle biology remain. Copyright © 2012 Elsevier Ltd. All rights reserved.

  4. Structural Basis of Mycobacterium tuberculosis Transcription and Transcription Inhibition.

    PubMed

    Lin, Wei; Mandal, Soma; Degen, David; Liu, Yu; Ebright, Yon W; Li, Shengjian; Feng, Yu; Zhang, Yu; Mandal, Sukhendu; Jiang, Yi; Liu, Shuang; Gigliotti, Matthew; Talaue, Meliza; Connell, Nancy; Das, Kalyan; Arnold, Eddy; Ebright, Richard H

    2017-04-20

    Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, which kills 1.8 million annually. Mtb RNA polymerase (RNAP) is the target of the first-line antituberculosis drug rifampin (Rif). We report crystal structures of Mtb RNAP, alone and in complex with Rif, at 3.8-4.4 Å resolution. The results identify an Mtb-specific structural module of Mtb RNAP and establish that Rif functions by a steric-occlusion mechanism that prevents extension of RNA. We also report non-Rif-related compounds-Nα-aroyl-N-aryl-phenylalaninamides (AAPs)-that potently and selectively inhibit Mtb RNAP and Mtb growth, and we report crystal structures of Mtb RNAP in complex with AAPs. AAPs bind to a different site on Mtb RNAP than Rif, exhibit no cross-resistance with Rif, function additively when co-administered with Rif, and suppress resistance emergence when co-administered with Rif. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Amperometric immunosensor for rapid detection of Mycobacterium tuberculosis

    NASA Astrophysics Data System (ADS)

    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.

  6. Tuberculosis Caused by Mycobacterium africanum, United States, 2004-2013.

    PubMed

    Sharma, Aditya; Bloss, Emily; Heilig, Charles M; Click, Eleanor S

    2016-03-01

    Mycobacterium africanum is endemic to West Africa and causes tuberculosis (TB). We reviewed reported cases of TB in the United States during 2004-2013 that had lineage assigned by genotype (spoligotype and mycobacterial interspersed repetitive unit variable number tandem repeats). M. africanum caused 315 (0.4%) of 73,290 TB cases with lineage assigned by genotype. TB caused by M. africanum was associated more with persons from West Africa (adjusted odds ratio [aOR] 253.8, 95% CI 59.9-1,076.1) and US-born black persons (aOR 5.7, 95% CI 1.2-25.9) than with US-born white persons. TB caused by M. africanum did not show differences in clinical characteristics when compared with TB caused by M. tuberculosis. Clustered cases defined as >2 cases in a county with identical 24-locus mycobacterial interspersed repetitive unit genotypes, were less likely for M. africanum (aOR 0.1, 95% CI 0.1-0.4), which suggests that M. africanum is not commonly transmitted in the United States.

  7. Nonweekend schedule for BACTEC drug susceptibility testing of Mycobacterium tuberculosis.

    PubMed Central

    Hawkins, J E

    1986-01-01

    Determination of the drug susceptibility of Mycobacterium tuberculosis by conventional methods using an agar-based medium may take 3 weeks or more to complete. On the other hand, results on positive cultures are generally available in 4 to 7 days with the radiometric (BACTEC, Johnston Laboratories, Towson, Md.) procedure. One disadvantage to the latter is the requirement to determine the quantity of 14CO2 in each test vial on a daily basis from the day of inoculation. Growth index readings often must be made over weekends, adding to the work load of clinical laboratories during periods of reduced staff or necessitating compensatory pay or time. Susceptibility tests with streptomycin, isoniazid, ethambutol, and rifampin against 104 M. tuberculosis strains were performed by the submerged disk method, the recommended BACTEC method with daily growth index readings, and the radiometric procedure with readings delayed for 2 days after inoculation. Criteria for interpretation of "delayed" tests were established. Drug concentrations tested included some modifications of those available commercially. Overall agreement for the four drugs by the three methods was greater than 90%. We conclude that under our test conditions a schedule of inoculation of radiometric test vials on Friday with growth index readings commencing on Monday gives susceptibility results that correlate well with the daily BACTEC method and with a conventional 7H10 agar method. PMID:3086371

  8. Drug susceptibility testing of Mycobacterium tuberculosis with nitrate reductase assay.

    PubMed

    Coban, Ahmet Yilmaz; Birinci, Asuman; Ekinci, Bora; Durupinar, Belma

    2004-09-01

    The nitrate reductase assay (NRA) was evaluated for susceptibility testing of Mycobacterium tuberculosis using 80 clinical isolates of M. tuberculosis and H37Rv as a control strain. All isolates were tested by the proportion method and the NRA for isoniazid (INH), rifampicin (RIF), streptomycin (STR) and ethambutol (ETM). The proportion method was carried out according to NCCLS on Löwenstein-Jensen (LJ) medium and the NRA on LJ medium containing 1000 microg/ml potassium nitrate (KNO(3)). After incubation for 7, 10, 14 and 21 days, Griess reagent was added to each LJ medium and nitrate reduction was determined by a colour change. Comparing the NRA with the proportion method, sensitivities were 100, 100, 82.1 and 92.2% for INH, RIF, STR and ETM, respectively. Specificities were 100, 100, 92.3 and 100% for INH, RIF, STR and ETM, respectively. The results of 2, 22 and 56 isolates were obtained after 7, 10 and 14 days, respectively. The proportion method result were read at 21-28 days. The NRA is rapid, inexpensive and easy to perform. Our results indicated that the NRA is suitable for the early determination of INH and RIF resistance in countries where sophisticated procedures are not always available.

  9. Inhibitors of Mycobacterium tuberculosis DosRST signaling and persistence.

    PubMed

    Zheng, Huiqing; Colvin, Christopher J; Johnson, Benjamin K; Kirchhoff, Paul D; Wilson, Michael; Jorgensen-Muga, Katriana; Larsen, Scott D; Abramovitch, Robert B

    2017-02-01

    The Mycobacterium tuberculosis (Mtb) DosRST two-component regulatory system promotes the survival of Mtb during non-replicating persistence (NRP). NRP bacteria help drive the long course of tuberculosis therapy; therefore, chemical inhibition of DosRST may inhibit the ability of Mtb to establish persistence and thus shorten treatment. Using a DosRST-dependent fluorescent Mtb reporter strain, a whole-cell phenotypic high-throughput screen of a ∼540,000 compound small-molecule library was conducted. The screen discovered novel inhibitors of the DosRST regulon, including three compounds that were subject to follow-up studies: artemisinin, HC102A and HC103A. Under hypoxia, all three compounds inhibit Mtb-persistence-associated physiological processes, including triacylglycerol synthesis, survival and antibiotic tolerance. Artemisinin functions by disabling the heme-based DosS and DosT sensor kinases by oxidizing ferrous heme and generating heme-artemisinin adducts. In contrast, HC103A inhibits DosS and DosT autophosphorylation activity without targeting the sensor kinase heme.

  10. Use of immunochromatographic assay for rapid identification of Mycobacterium tuberculosis complex from liquid culture

    PubMed Central

    Považan, Anika; Vukelić, Anka; Savković, Tijana; Kurucin, Tatjana

    2012-01-01

    A new, simple immunochromatographic assay for rapid identification of Mycobacterium tuberculosis complex in liquid cultures has been developed. The principle of the assay is binding of the Mycobacterium tuberculosis complex specific antigen to the monoclonal antibody conjugated on the test strip. The aim of this study is evaluation of the performance of immunochromatographic assay in identification of Mycobacterium tuberculosis complex in primary positive liquid cultures of BacT/Alert automated system. A total of 159 primary positive liquid cultures were tested using the immunochromatographic assay (BD MGIT TBc ID) and the conventional subculture, followed by identification using biochemical tests. Of 159 positive liquid cultures, using the conventional method, Mycobacterium tuberculos is was identified in 119 (74.8%), nontuberculous mycobacteria were found in 4 (2.5%), 14 (8.8%) cultures were contaminated and 22 (13.8%) cultures were found to be negative. Using the immunochromatographic assay, Mycobacterium tuberculosis complex was detected in 118 (74.2%) liquid cultures, and 41 (25.8%) tests were negative. Sensitivity, specificity, positive and negative predictive values of the test were 98.3%; 97.5%; 99.15%; 95.12%, respectively. The value of kappa test was 0.950, and McNemar test was 1.00. The immunochromatographic assay is a simple and rapid test which represents a suitable alternative to the conventional subculture method for the primary identification of Mycobacterium tuberculosis complex in liquid cultures of BacT/Alert automated system. PMID:22364301

  11. Fast and efficient detection of tuberculosis antigens using liposome encapsulated secretory proteins of Mycobacterium tuberculosis.

    PubMed

    Tiwari, Dileep; Haque, Shafiul; Tiwari, Ram P; Jawed, Arshad; Govender, Thavendran; Kruger, Hendrik G

    2017-04-01

    A rapid and efficient diagnostic test was developed for the detection of Mycobacterium tuberculosis antigens in serum samples of active tuberculosis (TB) and extrapulmonary TB patients via a liposomal agglutination-based method. A rapid card test has been developed to facilitate the recognition of high-affinity binding rabbit raised purified culture filtrate protein antibodies coupled on the surface of activated liposomal preparation. In the presence of TB antigens, the polyclonal antibodies bound to the liposomal particles demonstrate a visible agglutination reaction. The developed assay was simple, rapid, reliable, sensitive, and specific as a diagnostic test for the detection of antigens in serum samples of clinically confirmed cases of TB within 4-5 minutes' duration. The test was evaluated at different hospitals, medical colleges, and pathology centers, and involved 1483 participants. This investigation was conducted to detect the presence of these antigens during the period of active growth of the microorganism in serum samples for pulmonary TB and processed tissue biopsy for other extrapulmonary TB. Results obtained using this test were compared with acid-fast bacilli smear and culture results. Our study demonstrated that the newly developed liposome tuberculosis antigen card test detected antigens in our study population with approximately 97.48% sensitivity and 95.79% specificity. This is the first study to report the liposomal encapsulation of culture filtrate proteins from M. tuberculosis for diagnostic application. Copyright © 2015. Published by Elsevier B.V.

  12. Drug resistance of Mycobacterium tuberculosis isolates from tuberculosis lymphadenitis patients in Ethiopia.

    PubMed

    Biadglegne, Fantahun; Tessema, Belay; Sack, Ulrich; Rodloff, Arne C

    2014-07-01

    The emergence of drug resistance tuberculosis (TB) is a significant challenge for TB control and prevention programmes, and the major problem is multidrug resistant tuberculosis (MDR-TB). The present study was carried out to determine the frequency of drug resistant Mycobacterium tuberculosis isolates among newly and retreated TB lymphadenitis patients and risk factors for acquiring this infection. Two hundred twenty five M. tuberculosis isolates from TB lymphadenitis patients who were diagnosed as new and retreated tuberculosis cases between April 2012 and May 2012 were included in this study. Isolates were tested for susceptibility to isoniazed (INH), rifampicin (RMP), streptomycin (SM), ethambutol (EMB) and pyrazinamide (PZA) using the BacT/AlerT 3D system protocol. Among 225 isolates, 15 (6.7%) were resistant to at least one first line anti-TB drug. Three (1.3%) were MDR-TB. Resistance to INH, RMP, SM, and EMB was found in 8 (3.6%), 4 (1.8%), 10 (4.4%), and 4 (1.8%) isolates, respectively. Of the 212 new TB lymphadenitis cases three (1.4%) were MDR-TB. A rifampicin resistant M. tuberculosis isolate was diagnosed from smear and culture negative newly treated cases. All isolates were susceptible to PZA. Matted cervical lymph nodes were the prominent sites involved. Newly treated TB lymphadenitis patients had a greater risk for presenting resistance to anti-TB drugs ( p =0.046). Our study showed that TB lymphadenitis patients harboured drug resistant TB and MDR-TB, although at a low rate. Resistance was not associated with age, sex, patients' education and contact history. Further research is required to determine transmission dynamics of drug resistant strains.

  13. Structural and functional studies of phosphoenolpyruvate carboxykinase from Mycobacterium tuberculosis.

    PubMed

    Machová, Iva; Snášel, Jan; Dostál, Jiří; Brynda, Jiří; Fanfrlík, Jindřich; Singh, Mahavir; Tarábek, Ján; Vaněk, Ondřej; Bednárová, Lucie; Pichová, Iva

    2015-01-01

    Tuberculosis, the second leading infectious disease killer after HIV, remains a top public health priority. The causative agent of tuberculosis, Mycobacterium tuberculosis (Mtb), which can cause both acute and clinically latent infections, reprograms metabolism in response to the host niche. Phosphoenolpyruvate carboxykinase (Pck) is the enzyme at the center of the phosphoenolpyruvate-pyruvate-oxaloacetate node, which is involved in regulating the carbon flow distribution to catabolism, anabolism, or respiration in different states of Mtb infection. Under standard growth conditions, Mtb Pck is associated with gluconeogenesis and catalyzes the metal-dependent formation of phosphoenolpyruvate. In non-replicating Mtb, Pck can catalyze anaplerotic biosynthesis of oxaloacetate. Here, we present insights into the regulation of Mtb Pck activity by divalent cations. Through analysis of the X-ray structure of Pck-GDP and Pck-GDP-Mn2+ complexes, mutational analysis of the GDP binding site, and quantum mechanical (QM)-based analysis, we explored the structural determinants of efficient Mtb Pck catalysis. We demonstrate that Mtb Pck requires presence of Mn2+ and Mg2+ cations for efficient catalysis of gluconeogenic and anaplerotic reactions. The anaplerotic reaction, which preferably functions in reducing conditions that are characteristic for slowed or stopped Mtb replication, is also effectively activated by Fe2+ in the presence of Mn2+ or Mg2+ cations. In contrast, simultaneous presence of Fe2+ and Mn2+ or Mg2+ inhibits the gluconeogenic reaction. These results suggest that inorganic ions can contribute to regulation of central carbon metabolism by influencing the activity of Pck. Furthermore, the X-ray structure determination, biochemical characterization, and QM analysis of Pck mutants confirmed the important role of the Phe triad for proper binding of the GDP-Mn2+ complex in the nucleotide binding site and efficient catalysis of the anaplerotic reaction.

  14. Interaction of Pattern Recognition Receptors with Mycobacterium Tuberculosis.

    PubMed

    Mortaz, Esmaeil; Adcock, Ian M; Tabarsi, Payam; Masjedi, Mohammad Reza; Mansouri, Davood; Velayati, Ali Akbar; Casanova, Jean-Laurent; Barnes, Peter J

    2015-01-01

    Tuberculosis (TB) is considered a major worldwide health problem with 10 million new cases diagnosed each year. Our understanding of TB immunology has become greater and more refined since the identification of Mycobacterium tuberculosis (MTB) as an etiologic agent and the recognition of new signaling pathways modulating infection. Understanding the mechanisms through which the cells of the immune system recognize MTB can be an important step in designing novel therapeutic approaches, as well as improving the limited success of current vaccination strategies. A great challenge in chronic disease is to understand the complexities, mechanisms, and consequences of host interactions with pathogens. Innate immune responses along with the involvement of distinct inflammatory mediators and cells play an important role in the host defense against the MTB. Several classes of pattern recognition receptors (PRRs) are involved in the recognition of MTB including Toll-Like Receptors (TLRs), C-type lectin receptors (CLRs) and Nod-like receptors (NLRs) linked to inflammasome activation. Among the TLR family, TLR1, TLR2, TLR4, and TLR9 and their down-stream signaling proteins play critical roles in the initiation of the immune response in the pathogenesis of TB. The inflammasome pathway is associated with the coordinated release of cytokines such as IL-1β and IL-18 which also play a role in the pathogenesis of TB. Understanding the cross-talk between these signaling pathways will impact on the design of novel therapeutic strategies and in the development of vaccines and immunotherapy regimes. Abnormalities in PRR signaling pathways regulated by TB will affect disease pathogenesis and need to be elucidated. In this review we provide an update on PRR signaling during M. tuberculosis infection and indicate how greater knowledge of these pathways may lead to new therapeutic opportunities.

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

    PubMed Central

    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

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

    PubMed

    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

    2015-06-05

    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. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. A Defined Tuberculosis Vaccine Candidate Boosts BCG and Protects Against Multidrug Resistant Mycobacterium tuberculosis

    PubMed Central

    Bertholet, Sylvie; Ireton, Gregory C.; Ordway, Diane J.; Windish, Hillarie Plessner; Pine, Samuel O.; Kahn, Maria; Phan, Tony; Orme, Ian M.; Vedvick, Thomas S.; Baldwin, Susan L.; Coler, Rhea N.; Reed, Steven G.

    2011-01-01

    Despite the widespread use of Mycobacterium bovis bacillus Calmette-Guerin (BCG) childhood vaccine, tuberculosis (TB) remains a serious global health problem. A successful vaccine against TB that replaces or boosts BCG will include antigens that induce or recall appropriate T cell responses. Four Mycobacterium tuberculosis (Mtb) antigens, including members of the virulence factor families PE/PPE and EsX, or antigens associated with latency were produced as a single recombinant fusion protein. When administered with the adjuvant GLA-SE, a stable oil-in-water nanoemulsion, the fusion protein ID93 was immunogenic in mice, guinea pigs, and cynomolgus monkeys. In mice, ID93/GLA-SE combination induced polyfunctional CD4 TH1-cell responses characterized by antigen-specific IFN-gamma, tumor necrosis factor and interleukin-2, as well as a reduction in the number of bacteria in the lungs of animals subsequently infected with virulent or multidrug resistant Mtb strains. Furthermore, boosting BCG-vaccinated guinea pigs with ID93/GLA-SE resulted in reduced pathology and fewer bacilli, and prevented the death of animals challenged with virulent Mtb. Finally, ID93 elicited polyfunctional effector CD4 and CD8 T-cell responses in BCG-vaccinated or Mtb-exposed human peripheral blood mononuclear cells. This study establishes that the protein subunit vaccine ID93/GLA-SE protects against TB and MDR-TB in animals, and is a candidate for boosting the protective efficacy of the childhood BCG vaccine. PMID:20944089

  18. The Bioinformatics Analysis of Comparative Genomics of Mycobacterium tuberculosis Complex (MTBC) Provides Insight into Dissimilarities between Intraspecific Groups Differing in Host Association, Virulence, and Epitope Diversity

    PubMed Central

    Jia, Xinmiao; Yang, Li; Dong, Mengxing; Chen, Suting; Lv, Lingna; Cao, Dandan; Fu, Jing; Yang, Tingting; Zhang, Ju; Zhang, Xiangli; Shang, Yuanyuan; Wang, Guirong; Sheng, Yongjie; Huang, Hairong; Chen, Fei

    2017-01-01

    Tuberculosis now exceeds HIV as the top infectious disease cause of mortality, and is caused by the Mycobacterium tuberculosis complex (MTBC). MTBC strains have highly conserved genome sequences (similarity >99%) but dramatically different phenotypes. To analyze the relationship between genotype and phenotype, we conducted the comparative genomic analysis on 12 MTBC strains representing different lineages (i.e., Mycobacterium bovis; M. bovis BCG; M. microti; M. africanum; M. tuberculosis H37Rv; M. tuberculosis H37Ra, and six M. tuberculosis clinical isolates). The analysis focused on the three aspects of pathogenicity: host association, virulence, and epitope variations. Host association analysis indicated that eight mce3 genes, two enoyl-CoA hydratases, and five PE/PPE family genes were present only in human isolates; these may have roles in host-pathogen interactions. There were 15 SNPs found on virulence factors (including five SNPs in three ESX secretion proteins) only in the Beijing strains, which might be related to their more virulent phenotype. A comparison between the virulent H37Rv and non-virulent H37Ra strains revealed three SNPs that were likely associated with the virulence attenuation of H37Ra: S219L (PhoP), A219E (MazG) and a newly identified I228M (EspK). Additionally, a comparison of animal-associated MTBC strains showed that the deletion of the first four genes (i.e., pe35, ppe68, esxB, esxA), rather than all eight genes of RD1, might play a central role in the virulence attenuation of animal isolates. Finally, by comparing epitopes among MTBC strains, we found that four epitopes were lost only in the Beijing strains; this may render them better capable of evading the human immune system, leading to enhanced virulence. Overall, our comparative genomic analysis of MTBC strains reveals the relationship between the highly conserved genotypes and the diverse phenotypes of MTBC, provides insight into pathogenic mechanisms, and facilitates the

  19. Characterization of Novel Mycobacterium tuberculosis and Mycobacterium smegmatis Mutants Hypersusceptible to β-Lactam Antibiotics

    PubMed Central

    Flores, Anthony R.; Parsons, Linda M.; Pavelka, Martin S.

    2005-01-01

    Our laboratory previously constructed mutants of Mycobacterium tuberculosis and Mycobacterium smegmatis with deletions in the genes for their major β-lactamases, BlaC and BlaS, respectively, and showed that the mutants have increased susceptibilities to most β-lactam antibiotics, particularly the penicillins. However, there is still a basal level of resistance in the mutants to certain penicillins, and the susceptibilities of the mutants to some cephalosporin-based β-lactams are essentially the same as those of the wild types. We hypothesized that characterizing additional mutants (derived from β-lactamase deletion mutants) that are hypersusceptible to β-lactam antibiotics might reveal novel genes involved with other mechanisms of β-lactam resistance, peptidoglycan assembly, and cell envelope physiology. We report here the isolation and characterization of nine β-lactam antibiotic-hypersusceptible transposon mutants, two of which have insertions in genes known to be involved with peptidoglycan biosynthesis (ponA2 and dapB); the other seven mutants have insertions which affect novel genes. These genes can be classified into three groups: those involved with peptidoglycan biosynthesis, cell division, and other cell envelope processes. Two of the peptidoglycan-biosynthetic genes (ponA2 and pbpX) may encode β-lactam antibiotic-resistant enzymes proposed to be involved with the synthesis of the unusual diaminopimelyl linkages within the mycobacterial peptidoglycan. PMID:15743935

  20. Molecular typing of Mycobacterium tuberculosis complex isolated from pulmonary tuberculosis patients in central Ethiopia.

    PubMed

    Bedewi, Zufan; Worku, Adane; Mekonnen, Yalemtsehay; Yimer, Getnet; Medhin, Girmay; Mamo, Gezahegne; Pieper, Rembert; Ameni, Gobena

    2017-03-01

    Identification of the types of strains of Mycobacterium tuberculosis (M. tuberculosis) complex causing tuberculosis (TB) could contribute to TB control program of specific geographic region as well as it could add knowledge onto the existing literature on TB worldwide. The objective of the present study was to identify the species and strains of M. tuberculosis complex causing pulmonary tuberculosis in central Ethiopia. A health institution- based cross-sectional study was conducted on 338 smear positive TB cases visiting three hospitals between October 2012 and September 2013. Morning and spot sputum samples were collected before the starting of treatment regimens. Thus, a total of 338 pooled sputum samples collected from these cases. Samples were cultured on Löwenstein Jensen media and the isolates were identified by the region of difference (RD) 9 based polymerase chain reaction (PCR) and spoligotyping. Of the total isolates 98.6% of the isolates were identified to be M. tuberculosis while the remaining 1.4% were identified as M. africanum. Further, typing of M. tuberculosis using spoligotyping lead to the identification of 90 different strains of M. tuberculosis. Of these strains, 32 were clustered consisting of more than one isolate while the remaining 58 strains were unique consisting of single isolate. Thus, 79.3% (223/281) of the isolates were found in the clustered while only 20.6% (58/281) of the strains were unique. Forty-five of the spolgotyping patterns were registeredin the SITVIT2 or SpolDB4 database in while the remaining 45 were notfound in the database and hence were orphan strains. The dominant strains were SIT53, SIT149, and SIT54, consisting of 43, 37 and 34 isolates, respectively. Classification of the spoligotype patterns using TB-insight RUN TB-Lineage showed that 86.8, 6.4, 5, 1.4% ofthe isolatesbelonged to the Euro-American lineage, East-African-Indian, Indo-oceanic and M. africanum, respectively. The identification of clustered and new

  1. Detection of Mycobacterium tuberculosis and Mycobacterium avium Complexes by Real-Time PCR in Bovine Milk from Brazilian Dairy Farms.

    PubMed

    Bezerra, André Vinícius Andrade; Dos Reis, Emily Marques; Rodrigues, Rogério Oliveira; Cenci, Alexander; Cerva, Cristine; Mayer, Fabiana Quoos

    2015-05-01

    Foodborne diseases are a public health problem worldwide. The consumption of contaminated raw milk has been recognized as a major cause of transmission of bovine tuberculosis to humans. Other mycobacteria that may be present in raw milk and may cause diseases are those belonging to the Mycobacterium avium complex. In this study, molecular biology tools were applied to investigate raw milk contamination with Mycobacterium spp. in family dairy farms from Rio Grande do Sul, southern Brazil. Furthermore, different variables related to the source of the milk, herd characteristics, and management were evaluated for their effect on milk contamination. Five hundred and two samples were analyzed, of which 354 were from the Northwest region (102 farms with samples from 93 bulk tanks and 261 animals) and 148 from the South region of the state (22 farms with samples from 23 bulk tanks and 125 animals). Among them, 10 (1.99%) and 7 (1.39%) were positive for Mycobacterium tuberculosis (9 confirmed as Mycobacterium bovis) and M. avium complexes, respectively. There was no difference in the frequencies of positive samples between the regions or the sample sources. Of the positive samples, 4 were collected from a bulk tank (1 positive for M. avium and 3 for M. tuberculosis). Moreover, 1 sample was positive concomitantly for M. tuberculosis and M. avium complexes. On risk analysis, no variable was associated with raw milk contamination by M. tuberculosis complex species. However, washing the udders of all animals and drying them with paper towels were weakly classified as risk factors for M. avium contamination. Positive samples were obtained from both animals and bulk tanks, which emphasizes the importance of tuberculosis control programs and provides evidence that milk monitoring can be used as a control practice. Moreover, the findings of this study reinforce the need for awareness of the problems of raw milk consumption among the general population.

  2. Added epidemiologic value to tuberculosis prevention and control of the investigation of clustered genotypes of Mycobacterium tuberculosis isolates.

    PubMed

    McNabb, Scott J N; Kammerer, J Steve; Hickey, Andrew C; Braden, Christopher R; Shang, Nong; Rosenblum, Lisa S; Navin, Thomas R

    2004-09-15

    The Centers for Disease Control and Prevention established the US National Tuberculosis Genotyping and Surveillance Network to study the utility of genotyping Mycobacterium tuberculosis isolates for prevention and control. From 1998 to 2000, four sites performed conventional contact investigations and epidemiologic investigations of cases with genotypically matched M. tuberculosis isolates, called cluster investigations. The authors compared cluster pairs (two cases with M. tuberculosis isolates having identical genotypes) whose epidemiologic linkages were discovered only during cluster investigation with those whose epidemiologic linkages were discovered during conventional contact investigation. Among the 2,141 reported culture-positive tuberculosis cases, 2,055 (96%) M. tuberculosis isolates were genotyped. By itself and at a minimum, cluster investigation added 43 (38%) of the 113 total epidemiologic linkages discovered. Of the epidemiologic linkages discovered during conventional contact investigation, 29% of tuberculosis case pairs were not supported by genotyping data. The linkages discovered only during cluster investigation were more likely discovered in nontraditional settings and relationships and among larger clusters (cluster size of >5: adjusted odds ratio = 57.6, 95% confidence interval: 31.8, 104.6). Information gained from genotyping M. tuberculosis isolates should initiate cluster investigations of tuberculosis cases not previously discovered as being epidemiologically linked during conventional contact investigation. Cluster investigations will play a crucial role in predicting recent tuberculosis transmission more accurately, as we move toward tuberculosis elimination in the United States.

  3. Implication of the RD(Rio) Mycobacterium tuberculosis sublineage in multidrug resistant tuberculosis in Portugal.

    PubMed

    David, Susana; Duarte, Elsa L; Leite, Clarice Queico Fugimura; Ribeiro, João-Nuno; Maio, José-Nuno; Paixão, Eleonora; Portugal, Clara; Sancho, Luísa; Germano de Sousa, José

    2012-10-01

    Multidrug and extensively drug resistant Mycobacterium tuberculosis are a threat to tuberculosis control programs. Genotyping methods, such as spoligotyping and MIRU-VNTR typing (Mycobacterial Interspersed Repetitive Units), are useful in monitoring potentially epidemic strains and estimating strain phylogenetic lineages and/or genotypic families. M. tuberculosis Latin American Mediterranean (LAM) family is a major worldwide contributor to tuberculosis (TB). LAM specific molecular markers, Ag85C(103) single nucleotide polymorphism (SNP) and RD(Rio) long-sequence polymorphism (LSP), were used to characterize spoligotype signatures from 859 patient isolates from Portugal. LAM strains were found responsible for 57.7% of all tuberculosis cases. Strains with the RD(Rio) deletion (referred to as RD(Rio)) were estimated to represent 1/3 of all the strains and over 60% of the multidrug resistant (MDR) strains. The major spoligotype signature SIT20 belonging to the LAM1 RD(Rio) sublineage, represented close to 1/5th of all the strains, over 20% of which were MDR. Analysis of published datasets according to stipulated 12loci MIRU-VNTR RD(Rio) signatures revealed that 96.3% (129/134) of MDR and extensively drug resistant (XDR) clusters were RD(Rio). This is the first report associating the LAM RD(Rio) sublineage with MDR. These results are an important contribution to the monitoring of these strains with heightened transmission for future endeavors to arrest MDR-TB and XDR-TB. Copyright © 2012 Elsevier B.V. All rights reserved.

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

    PubMed Central

    Moraes, Eloise Brasil; Slompo, Letícia; Finardi, Amanda Juliane; da Silveira, Heloisa Paro Pedro; Ruiz, Luciana; Gomes, Harrison Magdinier; Richini, Virginia Bodelão; Suffys, Philip; Fortaleza, Carlos Magno Castelo Branco; Cavalcanti, Ricardo; Baptista, Ida Maria Foschiani Dias

    2017-01-01

    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 RDRio Mycobacterium 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. PMID:28225901

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

    PubMed Central

    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

  6. Whole genome analysis of Mycobacterium tuberculosis isolates from recurrent episodes of tuberculosis, Finland, 1995-2013.

    PubMed

    Korhonen, V; Smit, P W; Haanperä, M; Casali, N; Ruutu, P; Vasankari, T; Soini, H

    2016-06-01

    Recurrent tuberculosis (TB) is caused by an endogenous re-activation of the same strain of Mycobacterium tuberculosis (relapse) or exogenous infection with a new strain (re-infection). Recurrence of TB in Finland was analysed in a population-based, 19-year study, and genotyping was used to define relapse and re-infection. The M. tuberculosis isolates from patients with suspected relapse were further analysed by whole genome sequencing (WGS) to determine the number and type of mutations occurring in the bacterial genome between the first and second disease episodes. In addition, publicly available tools (PhyResSE and SpolPred) were used to predict drug resistance and spoligotype profile from the WGS data. Of the 8299 notified TB cases, 48 (0.6%) patients had episodes classified as recurrent. Forty-two patients had more than one culture-confirmed TB episode, and isolates from two episodes in 21 patients were available for genotyping. In 18 patients, the M. tuberculosis isolates obtained from the first and second TB episodes had identical spoligotypes. The WGS analysis of the 36 M. tuberculosis isolates from the 18 suspected relapse patients (average time between isolates 2.8 years) revealed 0 to 38 single nucleotide polymorphisms (median 1, mean 3.78) between the first and second isolate. There seemed to be no direct relation between the number of years between the two isolates, or treatment outcome, and the number of single nucleotide polymorphisms. The results suggest that the mutation rate may depend on multiple host-, strain- and treatment-related factors. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  7. Cloning and sequence analysis of a class A beta-lactamase from Mycobacterium tuberculosis H37Ra.

    PubMed Central

    Hackbarth, C J; Unsal, I; Chambers, H F

    1997-01-01

    A cosmid library from Mycobacterium tuberculosis H37Ra was introduced into Mycobacterium smegmatis, and eight recombinant clones with increased resistance to cefoxitin were identified. Isoelectric focusing detected an M. tuberculosis-derived beta-lactamase in one of these recombinant clones. A sequence analysis identified it as a class A beta-lactamase whose expression correlated with the increased resistance phenotype. PMID:9145897

  8. Mycobacterium bovis infection of cattle and white-tailed deer: Translational research of relevance to human tuberculosis

    USDA-ARS?s Scientific Manuscript database

    Tuberculosis (TB) is a premier example of a disease complex with pathogens primarily affecting humans (i.e., Mycobacterium tuberculosis) or livestock and wildlife (i.e., Mycobacterium bovis) and with a long history of inclusive collaborations between physicians and veterinarians. Advances with the s...

  9. Molecular cloning and immunologic reactivity of a novel low molecular mass antigen of Mycobacterium tuberculosis.

    PubMed

    Coler, R N; Skeiky, Y A; Vedvick, T; Bement, T; Ovendale, P; Campos-Neto, A; Alderson, M R; Reed, S G

    1998-09-01

    Polypeptide Ags present in the culture filtrate of Mycobacterium tuberculosis were purified and evaluated for their ability to stimulate PBMC from purified protein derivative (PPD)-positive healthy donors. One such Ag, which elicited strong proliferation and IFN-gamma production, was further characterized. The N-terminal amino acid sequence of this polypeptide was determined and used to design oligonucleotides for screening a recombinant M. tuberculosis genomic DNA library. The gene (Mtb 8.4) corresponding to the identified polypeptide was cloned, sequenced, and expressed in Escherichia coli. The predicted m.w. of the recombinant protein without its signal peptide was 8.4 kDa. By Southern analysis, the DNA encoding this mycobacterial protein was found in the M. tuberculosis substrains H37Rv, H37Ra, Erdman, and "C" strain, as well as in certain other mycobacterial species, including Mycobacterium avium and Mycobacterium bovis BCG (bacillus Calmette-Guerin, Pasteur). The Mtb 8.4 gene appears to be absent from the environmental mycobacterial species examined thus far, including Mycobacterium smegmatis, Mycobacterium gordonae, Mycobacterium chelonae, Mycobacterium fortuitum, and Mycobacterium scrofulaceum. Recombinant Mtb 8.4 Ag induced significant proliferation as well as production of IFN-gamma, IL-10, and TNF-alpha, but not IL-5, from human PBMC isolated from PPD-positive healthy donors. Mtb 8.4 did not stimulate PBMC from PPD-negative donors. Furthermore, immunogenicity studies in mice indicate that Mtb 8.4 elicits a Th1 cytokine profile, which is considered important for protective immunity to tuberculosis. Collectively, these results demonstrate that Mtb 8.4 is an immunodominant T cell Ag of M. tuberculosis.

  10. Characterization of activity and expression of isocitrate lyase in Mycobacterium avium and Mycobacterium tuberculosis.

    PubMed

    Höner Zu Bentrup, K; Miczak, A; Swenson, D L; Russell, D G

    1999-12-01

    Analysis by two-dimensional gel electrophoresis revealed that Mycobacterium avium expresses several proteins unique to an intracellular infection. One abundant protein with an apparent molecular mass of 50 kDa was isolated, and the N-terminal sequence was determined. It matches a sequence in the M. tuberculosis database (Sanger) with similarity to the enzyme isocitrate lyase of both Corynebacterium glutamicum and Rhodococcus fascians. Only marginal similarity was observed between this open reading frame (ORF) (termed icl) and a second distinct ORF (named aceA) which exhibits a low similarity to other isocitrate lyases. Both ORFs can be found as distinct genes in the various mycobacterial databases recently published. Isocitrate lyase is a key enzyme in the glyoxylate cycle and is essential as an anapleurotic enzyme for growth on acetate and certain fatty acids as carbon source. In this study we express and purify Icl, as well as AceA proteins, and show that both exhibit isocitrate lyase activity. Various known inhibitors for isocitrate lyase were effective. Furthermore, we present evidence that in both M. avium and M. tuberculosis the production and activity of the isocitrate lyase is enhanced under minimal growth conditions when supplemented with acetate or palmitate.

  11. Testing of susceptibility of Mycobacterium tuberculosis to isoniazid and rifampin by mycobacterium growth indicator tube method.

    PubMed Central

    Walters, S B; Hanna, B A

    1996-01-01

    We tested isolates of Mycobacterium tuberculosis recovered from 117 patients for their susceptibilities to isoniazid (INH) and rifampin (RIF) by the Centers for Disease Control and Prevention's disk modification of the indirect method of proportions (MOP) test and a three-tube mycobacteria growth indicator tube (MGIT; BBL) antimycobacterial susceptibility test (AST). Sixty-seven of the M. tuberculosis isolates were recovered from Lowenstein-Jensen (BBL) subcultures, and 50 of the isolates were recovered from MGIT cultures of samples from various body sites. For the MGIT AST method, 0.5 ml of test organism suspension was inoculated into an MGIT with 0.1 micrograms of INH per ml, an MGIT with 1.0 micrograms of RIF per ml, and growth control MGIT. The tubes were incubated at 37 degrees C and were examined daily. The MGIT AST results were interpreted as follows: susceptible if the tubes containing INH or RIF did not fluoresce within 2 days of the time that the positive growth control fluoresced and resistant if the tubes containing INH or RIF did fluoresce within 2 days of the time that the positive growth control fluoresced. The mean time fluorescence for the positive growth control was 5.5 days. The two methods were in agreement for 114 of the 117 isolates from patients, while for 3 isolates there were minor discordant results. PMID:8735121

  12. Differences between tuberculosis cases infected with Mycobacterium africanum, West African type 2, relative to Euro-American Mycobacterium tuberculosis: an update.

    PubMed

    de Jong, Bouke C; Adetifa, Ifedayo; Walther, Brigitte; Hill, Philip C; Antonio, Martin; Ota, Martin; Adegbola, Richard A

    2010-02-01

    Mycobacterium africanum (MAF) is a common cause of human pulmonary tuberculosis in West Africa. We previously described phenotypic differences between MAF and Mycobacterium tuberculosis (MTB) among 290 patients. In the present analysis, we compared 692 tuberculosis patients infected with the two most common lineages within the (MTB) complex found in the Gambia, namely MAF West African type 2 (39% prevalence) and Euro-American MTB (55% prevalence). We identified additional phenotypic differences between infections with these two organisms. MAF patients were more likely to be older and HIV infected. In addition, they had worse disease on chest X-ray, despite complaining of cough for an equal duration, and were more likely severely malnourished. In this cohort, the prevalence of MAF did not change significantly over a 7-year period.

  13. Rapid detection of cell-free Mycobacterium tuberculosis DNA in tuberculous pleural effusion.

    PubMed

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

    2017-03-08

    Tuberculous pleurisy is one of the most common extra-pulmonary tuberculosis, but its diagnosis remains to be difficult. In this study, we for the first time report detection of cell-free Mycobacterium tuberculosis DNA in pleural effusion and evaluation of this newly developed molecular assay. 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, Xpert MTB/RIF assay, adenosine deaminase assay, T-SPOT.TB assay, and cell-free Mycobacterium tuberculosis DNA assay were performed on all the pleural effusion samples. Cell-free Mycobacterium tuberculosis DNA assay and adenosine deaminase assay showed significantly higher sensitivities with 75.0 % and 68.3 % than those of mycobacterial culture and Xpert MTB/RIF assay with 26.7 % and 20.0 % (P < 0.01). These four tests all showed good specificities, 88.9 % for adenosine deaminase assay, and 100 % for the remaining three assays. The T-SPOT.TB assay in pleural effusion showed the highest sensitivity with 95.0 %, but the lowest specificity with 38.9 %. Cell-free Mycobacterium tuberculosis DNA assay detected as low as 1.25 copies of IS6110 in per ml of pleural effusion and showed good accordance between repeated tests (r = 0.978, P = 2.84×10(-10)). These data suggest that cell-free Mycobacterium tuberculosis DNA assay is a rapid and accurate molecular test which provides direct evidence of Mycobacterium tuberculosis etiology.

  14. Immunogenic Activity of a Ribosomal Fraction Obtained from Mycobacterium tuberculosis

    PubMed Central

    Youmans, Anne S.; Youmans, Guy P.

    1965-01-01

    Youmans, Anne S. (Northwestern University Medical School, Chicago, Ill.), and Guy P. Youmans. Immunogenic activity of a ribosomal fraction obtained from Mycobacterium tuberculosis. J. Bacteriol. 89:1291–1298. 1965.—The highly immunogenic particulate fraction obtained from mechanically ruptured cells of the H37Ra strain of Mycobacterium tuberculosis was suspended and centrifuged at 20,360 × g. The supernatant liquid from this centrifugation was centrifuged at 56,550 × g to remove the larger particles, and the supernatant liquid from this was centrifuged at 144,000 × g to obtain a ribosomal fraction. The sediments from the first two centrifugations were highly immunogenic, but the ribosomal fraction showed only slight capacity to immunize mice. However, when the ribosomal fraction was mixed with Freund's incomplete adjuvant, the immunogenic activity was equivalent to the particulate fraction from which it was prepared. To test the hypothesis that some membranous substance in the particulate fraction was acting as an adjuvant for the smaller particles in the ribosomal fraction, portions of the particulate fraction were treated separately with each of the membrane-disrupting agents, sodium deoxycholate, sodium lauryl sulfate, and 1 m sodium chloride. The treated materials were then centrifuged at 144,000 × g, and the sediments were tested for immunogenicity both with and without the addition of Freund's incomplete adjuvant. Without the adjuvant, the immunizing activities were very weak or absent; with the adjuvant, they were equivalent to that of the particulate fraction from which they were prepared. Other factors which have been found to damage or destroy membranes, such as freezing and thawing, and heat, also significantly decreased the immunogenic activity of the particulate fraction unless it was incorporated into Freund's incomplete adjuvant. The larger particles which sedimented at 56,550 × g were also treated with sodium lauryl sulfate and sodium

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

    PubMed Central

    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 Jr, 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 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. PMID:26405286

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

    PubMed

    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.

  17. Identification of Mycobacterium tuberculosis PPE68-specific HLA-A*0201-restricted epitopes for tuberculosis diagnosis.

    PubMed

    Duan, Zhi-Liang; Li, Qiang; Wang, Sina; Chen, Xin-Yu; Liu, Hui-Fang; Chen, Bo-Kun; Li, De-Zhou; Huang, Xi; Wen, Jin-Sheng

    2015-06-01

    PPE68 is a Mycobacterium tuberculosis-specific protein which is absent from the vaccine strains of BCG. A panel of 14 PPE68-derived peptides predicted to bind to HLA-A*0201 was synthesized. The HLA-A*0201 restriction of these peptides was determined in T2 cell line and HLA-A*0201 transgenic mice. The specificity of peptides was assessed in pulmonary tuberculosis (TB) patients using IFN-γ enzyme-linked immunospot (ELISPOT) assay, and immunodominant peptides were further used to evaluate their diagnostic potential in HLA-A*0201-positive pulmonary TB patients. 13 out of 14 peptides were identified as high-affinity binders. Of these peptides, 12 peptides induced significant IFN-γ-secreting T cell response in transgenic mice and 9 peptides were efficiently recognized by peripheral blood mononuclear cells of 10 HLA-A*0201-positive TB patients. Four immunodominant HLA-A*0201-restricted epitopes (PPE68126-134, PPE68133-141, PPE68140-148, and PPE68148-156) were recognized by the most of 80 HLA-A*0201-positive TB patients (81, 86, 74, and 84 %, respectively). These epitopes may be used for a potential diagnosis of M. tuberculosis infection.

  18. Validation of Mycobacterium tuberculosis Rv1681 Protein as a Diagnostic Marker of Active Pulmonary Tuberculosis

    PubMed Central

    Macovei, Lilia; Kanunfre, Kelly; Dhiman, Rakesh; Restrepo, Blanca I.; Zarate, Izelda; Pino, Paula A.; Mora-Guzman, Francisco; Fujiwara, Ricardo T.; Michel, Gerd; Kashino, Suely S.

    2013-01-01

    The development of an accurate antigen detection assay for the diagnosis of active tuberculosis (TB) would represent a major clinical advance. Here, we demonstrate that the Mycobacterium tuberculosis Rv1681 protein is a biomarker for active TB with potential diagnostic utility. We initially identified, by mass spectroscopy, peptides from the Rv1681 protein in urine specimens from 4 patients with untreated active TB. Rabbit IgG anti-recombinant Rv1681 detected Rv1681 protein in lysates and culture filtrates of M. tuberculosis and immunoprecipitated it from pooled urine specimens from two TB patients. An enzyme-linked immunosorbent assay formatted with these antibodies detected Rv1681 protein in unconcentrated urine specimens from 11/25 (44%) TB patients and 1/21 (4.8%) subjects in whom TB was initially clinically suspected but then ruled out by conventional methods. Rv1681 protein was not detected in urine specimens from 10 subjects with Escherichia coli-positive urine cultures, 26 subjects with confirmed non-TB tropical diseases (11 with schistosomiasis, 5 with Chagas' disease, and 10 with cutaneous leishmaniasis), and 14 healthy subjects. These results provide strong validation of Rv1681 protein as a promising biomarker for TB diagnosis. PMID:23390284

  19. High clustering rates of multidrug-resistant Mycobacterium tuberculosis genotypes in Panama

    PubMed Central

    2013-01-01

    Background Tuberculosis continues to be one of the leading causes of death worldwide and in the American region. Although multidrug-resistant tuberculosis (MDR-TB) remains a threat to TB control in Panama, few studies have focused in typing MDR-TB strains. The aim of our study was to characterize MDR Mycobacterium tuberculosis clinical isolates using PCR-based genetic markers. Methods From 2002 to 2004, a total of 231 Mycobacterium tuberculosis isolates from TB cases country-wide were screened for antibiotic resistance, and MDR-TB isolates were further genotyped by double repetitive element PCR (DRE-PCR), (GTG)5-PCR and spoligotyping. Results A total of 37 isolates (0.85%) were resistant to both isoniazid (INH) and rifampicin (RIF). Among these 37 isolates, only two (5.4%) were resistant to all five drugs tested. Dual genotyping using DRE-PCR and (GTG)5-PCR of MDR Mycobacterium tuberculosis isolates revealed eight clusters comprising 82.9% of the MDR-TB strain collection, and six isolates (17.1%) showed unique fingerprints. The spoligotyping of MDR-TB clinical isolates identified 68% as members of the 42 (LAM9) family genotype. Conclusion Our findings suggest that MDR Mycobacterium tuberculosis is highly clustered in Panama’s metropolitan area corresponding to Panama City and Colon City, and our study reveals the genotype distribution across the country. PMID:24053690

  20. Multidrug resistance to Mycobacterium tuberculosis in a tertiary hospital.

    PubMed Central

    Kehinde, Aderemi Oludiran; Obaseki, Felix Ariebuwa; Ishola, Oluponle Christiana; Ibrahim, Kolo Doko

    2007-01-01

    OBJECTIVE: The magnitude of drug-resistant Mycobacterium tuberculosis infection (MDR-TB) in Nigeria, the most populous country in sub-Saharan Africa, is largely unknown. This information would assist policymakers to develop intervention strategies against tuberculosis (TB) in the country. MATERIALS AND METHODS: This is a one-year laboratory-based study. Specimens from suspected new TB patients sent to the TB laboratory of the Department of Medical Microbiology, University College Hospital Ibadan, Nigeria from May 1, 2005 to April 27, 2006 were processed and analyzed. The specimens were stained with Ziehl-Neelsen (Z-N) reagents and cultured on Lowenstein-Jensen medium, incubated at 37 degrees C for 6-8 weeks. Isolates were confirmed as MDR-TB by Z-N reactions and biochemical methods. Drug susceptibility to streptomycin, ethambutol, rifampicin and isoniazid was done using Bactec 460 TB radiometric method. RESULTS: Of the 1,120 specimens processed, 80 (7.1%) were smear positive, while 56 (5.0%) were culture positive, even though the association was not statistically significant (p > 0.05). Culture contamination rate was 8.8%. Thirty (53.6%) of the culture positive isolates were resistant to both isoniazid and rifampicin, while 26 (46.4%) were susceptible. About half--53.3%--of the resistant isolates were from the antiretroviral clinic, while 10 (33.4%) were from peripheral centers. CONCLUSION: This study shows that MDR-TB is emerging in Nigeria. Further studies on MDR-TB are urgently needed in the country to ascertain the magnitude of the problem and to proffer solutions to it. PMID:17987922

  1. Self-poisoning of Mycobacterium tuberculosis by interrupting siderophore recycling.

    PubMed

    Jones, Christopher M; Wells, Ryan M; Madduri, Ashoka V R; Renfrow, Matthew B; Ratledge, Colin; Moody, D Branch; Niederweis, Michael

    2014-02-04

    Siderophores are small iron-binding molecules secreted by bacteria to scavenge iron. Mycobacterium tuberculosis (Mtb), the etiologic agent of tuberculosis, produces the siderophores mycobactin and carboxymycobactin. Complexes of the mycobacterial membrane proteins MmpS4 and MmpS5 with the transporters MmpL4 and MmpL5 are required for siderophore export and virulence in Mtb. Here we show that, surprisingly, mycobactin or carboxymycobactin did not rescue the low-iron growth defect of the export mutant but severely impaired growth. Exogenous siderophores were taken up by the export mutant, and siderophore-delivered iron was used, but the deferrated siderophores accumulated intracellularly, indicating a blockade of siderophore recycling. This hypothesis was confirmed by the observation that radiolabeled carboxymycobactin was taken up and secreted again by Mtb. Addition of iron salts to an Mtb siderophore biosynthesis mutant stimulated more growth in the presence of a limiting amount of siderophores than iron-loaded siderophores alone. Thus, recycling enables Mtb to acquire iron at lower metabolic cost because Mtb cannot use iron salts without siderophores. Exogenous siderophores were bactericidal for the export mutant in submicromolar quantities. High-resolution mass spectrometry revealed that endogenous carboxymycobactin also accumulated in the export mutant. Toxic siderophore accumulation is prevented by a drug that inhibits siderophore biosynthesis. Intracellular accumulation of siderophores was toxic despite the use of an alternative iron source such as hemin, suggesting an additional inhibitory mechanism independent of iron availability. This study indicates that targeting siderophore export/recycling would deliver a one-two punch to Mtb: restricting access to iron and causing toxic intracellular siderophore accumulation.

  2. Mycobacterium tuberculosis Zinc Metalloprotease-1 Assists Mycobacterial Dissemination in Zebrafish

    PubMed Central

    Vemula, Mani H.; Medisetti, Raghavender; Ganji, Rakesh; Jakkala, Kiran; Sankati, Swetha; Chatti, Kiranam; Banerjee, Sharmistha

    2016-01-01

    Zinc metalloprotease-1 (Zmp1) from Mycobacterium tuberculosis (M.tb), the tuberculosis (TB) causing bacillus, is a virulence factor involved in inflammasome inactivation and phagosome maturation arrest. We earlier reported that Zmp1 was secreted under granuloma-like stress conditions, induced Th2 cytokine microenvironment and was highly immunogenic in TB patients as evident from high anti-Zmp1 antibody titers in their sera. In this study, we deciphered a new physiological role of Zmp1 in mycobacterial dissemination. Exogenous treatment of THP-1 cells with 500 nM and 1 μM of recombinant Zmp1 (rZmp1) resulted in necrotic cell death. Apart from inducing secretion of necrotic cytokines, TNFα, IL-6, and IL-1β, it also induced the release of chemotactic chemokines, MCP-1, MIP-1β, and IL-8, suggesting its likely function in cell migration and mycobacterial dissemination. This was confirmed by Gap closure and Boyden chamber assays, where Zmp1 treated CHO or THP-1 cells showed ∼2 fold increased cell migration compared to the untreated cells. Additionally, Zebrafish-M. marinum based host–pathogen model was used to study mycobacterial dissemination in vivo. Td-Tomato labeled M. marinum (TdM. marinum) when injected with rZmp1 showed increased dissemination to tail region from the site of injection as compared to the untreated control fish in a dose-dependent manner. Summing up these observations along with the earlier reports, we propose that Zmp1, a multi-faceted protein, when released by mycobacteria in granuloma, may lead to necrotic cell damage and release of chemotactic chemokines by surrounding infected macrophages, attracting new immune cells, which in turn may lead to fresh cellular infections, thus assisting mycobacterial dissemination. PMID:27621726

  3. Deciphering an Outbreak of Drug-Resistant Mycobacterium tuberculosis

    PubMed Central

    Dahle, Ulf R.; Sandven, Per; Heldal, Einar; Mannsaaker, Turid; Caugant, Dominique A.

    2003-01-01

    There have been ample warnings that multidrug-resistant (MDR) tuberculosis (TB) will continue to emerge if countries do not strengthen their control of TB. In low-incidence European countries, however, these warnings have been substantiated mainly by outbreaks in association with human immunodeficiency virus (HIV)-positive patients. The aim of this study was to investigate an outbreak of infection with MDR and drug-resistant Mycobacterium tuberculosis that was diagnosed among 20 HIV-negative patients living in Norway. Of these, 19 were immigrants from East Africa and one was an ethnic Norwegian. We wanted to find out if transmission had taken place in Norway or abroad and to identify the genetic basis of drug resistance. The strains were analyzed by IS6110 restriction fragment length polymorphism, antibiotic susceptibility tests, spoligotyping, reverse hybridization to regions of the rpoB gene, and sequencing of the katG gene. Epidemiological links between the patients were mapped, and the strains were compared to those isolated in 36 other countries and regions. All strains were resistant to isoniazid and carried Ala234Gly, Ser315Thr, and Arg463Leu substitutions in the katG gene. Eleven strains were MDR and carried a Ser531Leu substitution in the rpoB gene. MDR was acquired in the index patient after arrival in Norway. Links were found among 14 patients. The strain was imported from Somalia but acquired MDR and was transmitted in Norway. This demonstrated that MDR strains are not necessarily imported from high-incidence countries and can be highly communicable. The outbreak underscores a deficiency in the TB control measures employed in many countries and challenges the adequacy of the policy of screening immigrants for TB only on arrival. PMID:12517827

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

    PubMed

    Philip, Noraini; Rodrigues, Kenneth Francis; William, Timothy; John, Daisy Vanitha

    2016-09-01

    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.

  5. Human lung hydrolases delineate Mycobacterium tuberculosis-macrophage interactions and the capacity to control infection.

    PubMed

    Arcos, Jesús; Sasindran, Smitha J; Fujiwara, Nagatoshi; Turner, Joanne; Schlesinger, Larry S; Torrelles, Jordi B

    2011-07-01

    Pulmonary surfactant contains homeostatic and antimicrobial hydrolases. When Mycobacterium tuberculosis is initially deposited in the terminal bronchioles and alveoli, as well as following release from lysed macrophages, bacilli are in intimate contact with these lung surfactant hydrolases. We identified and measured several hydrolases in human alveolar lining fluid and lung tissue that, at their physiological concentrations, dramatically modified the M. tuberculosis cell envelope. Independent of their action time (15 min to 12 h), the effects of the hydrolases on the M. tuberculosis cell envelope resulted in a significant decrease (60-80%) in M. tuberculosis association with, and intracellular growth of the bacteria within, human macrophages. The cell envelope-modifying effects of the hydrolases also led to altered M. tuberculosis intracellular trafficking and induced a protective proinflammatory response to infection. These findings add a new concept to our understanding of M. tuberculosis-macrophage interactions (i.e., the impact of lung surfactant hydrolases on M. tuberculosis infection).

  6. Mycobacterium tuberculosis is extraordinarily sensitive to killing by a vitamin C-induced Fenton reaction

    PubMed Central

    Vilchèze, Catherine; Hartman, Travis; Weinrick, Brian; Jacobs, William R.

    2013-01-01

    Drugs that kill tuberculosis more quickly could shorten chemotherapy significantly. In Escherichia coli, a common mechanism of cell death by bactericidal antibiotics involves the generation of highly reactive hydroxyl radicals via the Fenton reaction. Here we show that vitamin C, a compound known to drive the Fenton reaction, sterilizes cultures of drug-susceptible and drug-resistant Mycobacterium tuberculosis, the causative agent of tuberculosis. While M. tuberculosis is highly susceptible to killing by vitamin C, other Gram-positive and Gram-negative pathogens are not. The bactericidal activity of vitamin C against M. tuberculosis is dependent on high ferrous ion levels and reactive oxygen species production and causes a pleiotropic effect affecting several biological processes. This study enlightens the possible benefits of adding vitamin C to an anti-tuberculosis regimen and suggests that the development of drugs that generate high oxidative burst could be of great use in tuberculosis treatment. PMID:23695675

  7. Strain-dependent CNS dissemination in guinea pigs after Mycobacterium tuberculosis aerosol challenge.

    PubMed

    Be, Nicholas A; Klinkenberg, Lee G; Bishai, William R; Karakousis, Petros C; Jain, Sanjay K

    2011-09-01

    Clinical reports suggest an association of distinct Mycobacterium tuberculosis strains with CNS disease. We therefore examined CNS dissemination by different laboratory strains (two M. tuberculosis H37Rv, one CDC1551) in a guinea pig aerosol infection model. Although all strains grew exponentially in lungs, with similar bacterial burdens at the time of extrapulmonary dissemination, M. tuberculosis CDC1551 disseminated to the CNS significantly more than the H37Rv strains. No CNS lesions were observed throughout the study, with only a modest cytokine response. These data suggest that M. tuberculosis may have virulence factors that promote CNS dissemination, distinct from those required for pulmonary TB.

  8. Mycobacterium tuberculosis and Copper: A Newly Appreciated Defense against an Old Foe?

    PubMed

    Darwin, K Heran

    2015-07-31

    Several independent studies have recently converged upon the conclusion that the human bacterial pathogen Mycobacterium tuberculosis encounters copper during infections. At least three independently regulated pathways respond to excess copper and are required for the full virulence of M. tuberculosis in animals. In this review, I will discuss the functions of the best-characterized copper-responsive proteins in M. tuberculosis, the potential sources of copper during an infection, and remaining questions about the interface between copper and tuberculosis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Ins and outs of Mycobacterium tuberculosis PPE family in pathogenesis and implications for novel measures against tuberculosis.

    PubMed

    Deng, Wanyan; Xie, Jianping

    2012-04-01

    Mycobacterium tuberculosis is the most successful pathogen with multiple mechanisms to subvert host immune response, resulting in insidious disease. A unique Mycobacterium antigen family termed PPE (Pro-Pro-Glu) has long been widely speculated as "molecular mantra" to escape host immunity. Members of this family are characterized by a conserved N terminal and a variable C terminal. This family associated closely with ESAT-6(ESX) secretion system and largely located in cell wall or cell membrane. The expression of PPE protein is temporally regulated, and highly expressed during M. tuberculosis persistence. Importantly, the distribution of PPE family is so far limited to Mycobacterium genus, prevalent among pathogenic Mycobacterium species. It is tempting to explore this family due to its potential in the latency and reactivation of M. tuberculosis. The evolution, structure, and functions of most PPE proteins remain elusive. The understanding of these questions will deepen our appreciation of the pathogenesis of M. tuberculosis and accelerate novel anti-TB measures discovery. © 2011 Wiley Periodicals, Inc.

  10. Inhibition of Mycobacterium tuberculosis glutamine synthetase as a novel antibiotic strategy against tuberculosis: demonstration of efficacy in vivo.

    PubMed

    Harth, Günter; Horwitz, Marcus A

    2003-01-01

    Tuberculosis remains one of humankind's greatest killers, and new therapeutic strategies are needed to combat the causative agent, Mycobacterium tuberculosis, which is rapidly developing resistance to conventional antibiotics. Using the highly demanding guinea pig model of pulmonary tuberculosis, we have investigated the feasibility of inhibiting M. tuberculosis glutamine synthetase (GS), an enzyme that plays a key role in both nitrogen metabolism and cell wall biosynthesis, as a novel antibiotic strategy. In guinea pigs challenged by aerosol with the highly virulent Erdman strain of M. tuberculosis, the GS inhibitor L-methionine-SR-sulfoximine (MSO) protected the animals against weight loss, a hallmark of tuberculosis, and against the growth of M. tuberculosis in the lungs and spleen; MSO reduced the CFU of M. tuberculosis at 10 weeks after challenge by approximately 0.7 log unit compared with that in control animals. MSO acted synergistically with isoniazid in protecting animals against weight loss and bacterial growth, reducing the CFU in the lungs and spleen by approximately 1.5 log units below the level seen with isoniazid alone. In the presence of ascorbate, which allows treatment with a higher dose, MSO was highly efficacious, reducing the CFU in the lungs and spleen by 2.5 log units compared with that in control animals. This study demonstrates that inhibition of M. tuberculosis GS is a feasible therapeutic strategy against this pathogen and supports the concept that M. tuberculosis enzymes involved in cell wall biosynthesis, including major secretory proteins, have potential as antibiotic targets.

  11. Differential influence of nutrient-starved Mycobacterium tuberculosis on adaptive immunity results in progressive tuberculosis disease and pathology.

    PubMed

    Dietrich, Jes; Roy, Sugata; Rosenkrands, Ida; Lindenstrøm, Thomas; Filskov, Jonathan; Rasmussen, Erik Michael; Cassidy, Joseph; Andersen, Peter

    2015-12-01

    When infected with Mycobacterium tuberculosis, most individuals will remain clinically healthy but latently infected. Latent infection has been proposed to partially involve M. tuberculosis in a nonreplicating stage, which therefore represents an M. tuberculosis phenotype that the immune system most likely will encounter during latency. It is therefore relevant to examine how this particular nonreplicating form of M. tuberculosis interacts with the host immune system. To study this, we first induced a state of nonreplication through prolonged nutrient starvation of M. tuberculosis in vitro. This resulted in nonreplicating persistence even after prolonged culture in phosphate-buffered saline. Infection with either exponentially growing M. tuberculosis or nutrient-starved M. tuberculosis resulted in similar lung CFU levels in the first phase of the infection. However, between week 3 and 6 postinfection, there was a very pronounced increase in bacterial levels and associated lung pathology in nutrient-starved-M. tuberculosis-infected mice. This was associated with a shift from CD4 T cells that coexpressed gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) or IFN-γ, TNF-α, and interleukin-2 to T cells that only expressed IFN-γ. Thus, nonreplicating M. tuberculosis induced through nutrient starvation promotes a bacterial form that is genetically identical to exponentially growing M. tuberculosis yet characterized by a differential impact on the immune system that may be involved in undermining host antimycobacterial immunity and facilitate increased pathology and transmission.

  12. Mycobacterium aurum is Unable to Survive Mycobacterium tuberculosis Latency Associated Stress Conditions: Implications as Non-suitable Model Organism.

    PubMed

    Sood, Shivani; Yadav, Anant; Shrivastava, Rahul

    2016-06-01

    Mycobacterium tuberculosis manages to remain latent in the human body regardless of extensive chemotherapy. Complete eradication of tuberculosis (TB) requires treatment strategies targeted against latent form of infection, in addition to the current regimen of antimycobacterials. Many in vitro and in vivo models have been proposed to imitate latent TB infection, yet none of them is able to completely mimic latent infection state of M. tuberculosis. Highly infectious nature of the pathogen requiring BSL3 facilities and its long generation time further add to complications. M. aurum has been proposed as an important model organism for high throughput screening of drugs and exhibits high genomic similarity with that of M. tuberculosis. Thus, the present study was undertaken to explore if M. aurum could be used as a surrogate organism for studies related to M. tuberculosis latent infection. M. aurum was subjected to in vitro conditions of oxygen depletion, lack of nutrients and acidic stress encountered by latent M. tuberculosis bacteria. CFU count of M. aurum cells along with any change in cell shape and size was recorded at regular intervals during the stress conditions. M. aurum cells were unable to survive for extended periods under all three conditions used in the study. Thus, our studies suggest that M. aurum is not a suitable organism to mimic M. tuberculosis persistent infection under in vitro conditions, and further studies are required on different species for the establishment of a fast growing species as a suitable model for M. tuberculosis persistent infection.

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

    PubMed Central

    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

  14. [Case of urinary mycobacterium fortuitum in a patient with urinary tract tuberculosis posttreatment].

    PubMed

    Maehana, Takeshi; Takahashi, Satoshi; Hirobe, Megumi; Taguchi, Keisuke

    2008-11-01

    A 70-year-old male who complained of urinary frequency and a feeling of incomplete emptying was admitted to our hospital. Imaging findings showed dilation of the left renal pelvis and ureter. He was diagnosed as having urinary tuberculosis because a positive urinary Mycobacterium tuberculosis result was obtained by polymerase chain reaction (PCR). He was treated with a combination of the antituberculosis agents isoniazid, rifampicin, pyrazinamide and ethambutol for six months. The symptoms and pyuria disappeared and M. tuberculosis was negative by PCR; however, Mycobacterium fortuitum was isolated by culture. Due to asymptomatic urinary tract infection by the multidrug resistant M. fortuitum, he was followed up with observation. Currently, he remains unchanged with regard to symptoms and imaging examination. M. fortuitum is a nontubercular mycobacterium, and clinical relevance between urinary tract infection and M. fortuitum has rarely emerged. However, we should be aware that nontubercular mycobacteria such as M. fortuitum can infect the urinary tract, especially in immunocompromised patients.

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

    PubMed

    Boyle, David S; McNerney, Ruth; Teng Low, Hwee; Leader, Brandon Troy; Pérez-Osorio, Ailyn C; Meyer, Jessica C; O'Sullivan, Denise M; Brooks, David G; Piepenburg, Olaf; Forrest, Matthew S

    2014-01-01

    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 (IS1081)were 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 assays

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

    PubMed Central

    Young, Douglas B.; Comas, Iñaki; 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. 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. PMID:25988174

  17. The Regulation of Sulfur Metabolism in Mycobacterium tuberculosis

    PubMed Central

    Hatzios, Stavroula K.; Bertozzi, Carolyn R.

    2011-01-01

    Mycobacterium tuberculosis (Mtb) has evolved into a highly successful human pathogen. It deftly subverts the bactericidal mechanisms of alveolar macrophages, ultimately inducing granuloma formation and establishing long-term residence in the host. These hallmarks of Mtb infection are facilitated by the metabolic adaptation of the pathogen to its surrounding environment and the biosynthesis of molecules that mediate its interactions with host immune cells. The sulfate assimilation pathway of Mtb produces a number of sulfur-containing metabolites with important contributions to pathogenesis and survival. This pathway is regulated by diverse environmental cues and regulatory proteins that mediate sulfur transactions in the cell. Here, we discuss the transcriptional and biochemical mechanisms of sulfur metabolism regulation in Mtb and potential small molecule regulators of the sulfate assimilation pathway that are collectively poised to aid this intracellular pathogen in its expert manipulation of the host. From this global analysis, we have identified a subset of sulfur-metabolizing enzymes that are sensitive to multiple regulatory cues and may be strong candidates for therapeutic intervention. PMID:21811406

  18. Role of Cathepsins in Mycobacterium tuberculosis Survival in Human Macrophages

    PubMed Central

    Pires, David; Marques, Joana; Pombo, João Palma; Carmo, Nuno; Bettencourt, Paulo; Neyrolles, Olivier; Lugo-Villarino, Geanncarlo; Anes, Elsa

    2016-01-01

    Cathepsins are proteolytic enzymes that function in the endocytic pathway, especially in lysosomes, where they contribute directly to pathogen killing or indirectly, by their involvement in the antigen presentation pathways. Mycobacterium tuberculosis (MTB) is a facultative intracellular pathogen that survives inside the macrophage phagosomes by inhibiting their maturation to phagolysosomes and thus avoiding a low pH and protease-rich environment. We previously showed that mycobacterial inhibition of the proinflammatory transcription factor NF-κB results in impaired delivery of lysosomal enzymes to phagosomes and reduced pathogen killing. Here, we elucidate how MTB also controls cathepsins and their inhibitors, cystatins, at the level of gene expression and proteolytic activity. MTB induced a general down-regulation of cathepsin expression in infected cells, and inhibited IFNγ-mediated increase of cathepsin mRNA. We further show that a decrease in cathepsins B, S and L favours bacterial survival within human primary macrophages. A siRNA knockdown screen of a large set of cathepsins revealed that almost half of these enzymes have a role in pathogen killing, while only cathepsin F coincided with MTB resilience. Overall, we show that cathepsins are important for the control of MTB infection, and as a response, it manipulates their expression and activity to favour its intracellular survival. PMID:27572605

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

    PubMed

    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.

  20. Social networks to biological networks: systems biology of Mycobacterium tuberculosis.

    PubMed

    Vashisht, Rohit; Bhardwaj, Anshu; Osdd Consortium; Brahmachari, Samir K

    2013-07-01

    Contextualizing relevant information to construct a network that represents a given biological process presents a fundamental challenge in the network science of biology. The quality of network for the organism of interest is critically dependent on the extent of functional annotation of its genome. Mostly the automated annotation pipelines do not account for unstructured information present in volumes of literature and hence large fraction of genome remains poorly annotated. However, if used, this information could substantially enhance the functional annotation of a genome, aiding the development of a more comprehensive network. Mining unstructured information buried in volumes of literature often requires manual intervention to a great extent and thus becomes a bottleneck for most of the automated pipelines. In this review, we discuss the potential of scientific social networking as a solution for systematic manual mining of data. Focusing on Mycobacterium tuberculosis, as a case study, we discuss our open innovative approach for the functional annotation of its genome. Furthermore, we highlight the strength of such collated structured data in the context of drug target prediction based on systems level analysis of pathogen.

  1. Nucleotide triphosphate promiscuity in Mycobacterium tuberculosis dethiobiotin synthetase.

    PubMed

    Salaemae, Wanisa; Yap, Min Y; Wegener, Kate L; Booker, Grant W; Wilce, Matthew C J; Polyak, Steven W

    2015-05-01

    Dethiobiotin synthetase (DTBS) plays a crucial role in biotin biosynthesis in microorganisms, fungi, and plants. Due to its importance in bacterial pathogenesis, and the absence of a human homologue, DTBS is a promising target for the development of new antibacterials desperately needed to combat antibiotic resistance. Here we report the first X-ray structure of DTBS from Mycobacterium tuberculosis (MtDTBS) bound to a nucleotide triphosphate (CTP). The nucleoside base is stabilized in its pocket through hydrogen-bonding interactions with the protein backbone, rather than amino acid side chains. This resulted in the unexpected finding that MtDTBS could utilise ATP, CTP, GTP, ITP, TTP, or UTP with similar Km and kcat values, although the enzyme had the highest affinity for CTP in competitive binding and surface plasmon resonance assays. This is in contrast to other DTBS homologues that preferentially bind ATP primarily through hydrogen-bonds between the purine base and the carboxamide side chain of a key asparagine. Mutational analysis performed alongside in silico experiments revealed a gate-keeper role for Asn175 in Escherichia coli DTBS that excludes binding of other nucleotide triphosphates. Here we provide evidence to show that MtDTBS has a broad nucleotide specificity due to the absence of the gate-keeper residue.

  2. The trifunctional sulfate-activating complex (SAC) of Mycobacterium tuberculosis.

    PubMed

    Sun, Meihao; Andreassi, John L; Liu, Shuqing; Pinto, Rachel; Triccas, James A; Leyh, Thomas S

    2005-03-04

    The sulfate activation pathway is essential for the assimilation of sulfate and, in many bacteria, is comprised of three reactions: the synthesis of adenosine 5'-phosphosulfate (APS), the hydrolysis of GTP, and the 3'-phosphorylation of APS to produce 3'-phosphoadenosine 5'-phosphosulfate (PAPS), whose sulfuryl group is reduced or transferred to other metabolites. The entire sulfate activation pathway is organized into a single complex in Mycobacterium tuberculosis. Although present in many bacteria, these tripartite complexes have not been studied in detail. Initial rate characterization of the mycobacterial system reveals that it is poised for extremely efficient throughput: at saturating ATP, PAPS synthesis is 5800 times more efficient than APS synthesis. The APS kinase domain of the complex does not appear to form the covalent E.P intermediate observed in the closely related APS kinase from Escherichia coli. The stoichiometry of GTP hydrolysis and APS synthesis is 1:1, and the APS synthesis reaction is driven 1.1 x 10(6)-fold further during GTP hydrolysis; the system harnesses the full chemical potential of the hydrolysis reaction to the synthesis of APS. A key energy-coupling step in the mechanism is a ligand-induced isomerization that enhances the affinity of GTP and commits APS synthesis and GTP hydrolysis to the completion of the catalytic cycle. Ligand-induced increases in guanine nucleotide affinity observed in the mycobacterial system suggest that it too undergoes the energy-coupling isomerization.

  3. Different responses of human mononuclear phagocyte populations to Mycobacterium tuberculosis.

    PubMed

    Duque, Camilo; Arroyo, Leonar; Ortega, Héctor; Montúfar, Franco; Ortíz, Blanca; Rojas, Mauricio; Barrera, Luis F

    2014-03-01

    Mycobacterium tuberculosis (Mtb) infects different populations of macrophages. Alveolar macrophages (AMs) are initially infected, and their response may contribute to controlling Mtb infection and dissemination. However, Mtb infection may disseminate to other tissues, infecting a wide variety of macrophages. Given the difficulty in obtaining AMs, monocyte-derived macrophages (MDMs) are used to model macrophage-mycobacteria interactions in humans. However, the response of other tissue macrophages to Mtb infection has been poorly explored. We have compared MDMs, AMs and splenic human macrophages (SMs) for their in vitro capacity to control Mtb growth, cytokine production, and induction of cell death in response to Mtb H37Rv, and the Colombian isolate UT205, and to the virulence factor ESAT-6. Significant differences in the magnitude of cell death and cytokine production depending mainly on the Mtb strain were observed; however, no major differences in the mycobacteriostatic/mycobacteriocidal activity were detected among the macrophage populations. Infection with the clinical isolate UT205 was associated with an increased cell death with membrane damage, particularly in IFNγ-treated SMs and H37Rv induced a higher production of cytokines compared to UT205. These results are concordant with the interpretation of a differential response to Mtb infection mainly depending upon the strain of Mtb.

  4. Mycobacterium tuberculosis and cholinesterase inhibitors from Voacanga globosa.

    PubMed

    Macabeo, Allan Patrick G; Vidar, Warren S; Chen, Xinyu; Decker, Michael; Heilmann, Jörg; Wan, Baojie; Franzblau, Scott G; Galvez, Elano V; Aguinaldo, Ma Alicia M; Cordell, Geoffrey A

    2011-07-01

    Globospiramine (1), a new spirobisindole alkaloid possessing an Aspidosperma-Aspidosperma skeleton, together with deoxyvobtusine (2), deoxyvobtusine lactone (3), vobtusine lactone (4) and lupeol (5), were isolated and identified from Voacanga globosa through a bioassay-guided purification. The gross structure and absolute stereochemistry of 1 were established by circular dichroism spectroscopy, HR-MS and unambiguous NMR spectroscopic experiments. In addition, a new biogenetic pathway for the formation of the spiro-Aspidosperma-Aspidosperma skeleton is proposed. Alkaloid 1 showed potent antituberculosis activity against Mycobacterium tuberculosis H(37)Rv as evidenced in microplate Alamar blue assay (MIC = 4 μg/mL) and low-oxygen recovery assay (LORA (MIC = 5.2 μg/mL). The bisindole alkaloids also exhibited promising activity against acetylcholinesterase and, especially butyrylcholinesterase, with deoxyvobtusine (2) (IC(50) = 6.2 μM) as the most strongly inhibiting compound. This study extends the variety of alkaloid structural platforms which exhibit antimycobacterial and anticholinesterase activity. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  5. RP105 facilitates macrophage activation by Mycobacterium tuberculosis lipoproteins.

    PubMed

    Blumenthal, Antje; Kobayashi, Toshihiko; Pierini, Lynda M; Banaei, Niaz; Ernst, Joel D; Miyake, Kensuke; Ehrt, Sabine

    2009-01-22

    RP105, phylogenetically related to Toll-like receptor (TLR)-4, is reported to facilitate B cell activation by the TLR4-agonist lipopolysaccharide (LPS)--but to limit LPS-induced cytokine production by antigen-presenting cells. Here, we show that the role of RP105 extends beyond LPS recognition and that RP105 positively regulates macrophage responses to Mycobacterium tuberculosis (Mtb) lipoproteins. Mtb-infected RP105(-/-) mice exhibited impaired proinflammatory cytokine responses associated with enhanced bacterial burden and increased lung pathology. The Mtb 19 kDa lipoprotein induced release of tumor necrosis factor in a manner dependent on both TLR2 and RP105, and macrophage activation by Mtb lacking mature lipoproteins was not RP105 dependent. Thus, mycobacterial lipoproteins are RP105 agonists. RP105 physically interacted with TLR2, and both RP105 and TLR2 were required for optimal macrophage activation by Mtb. Our data identify RP105 as an accessory molecule for TLR2, forming part of the receptor complex for innate immune recognition of mycobacterial lipoproteins.

  6. Identification of widespread adenosine nucleotide binding in Mycobacterium tuberculosis

    SciTech Connect

    Ansong, Charles; Ortega, Corrie; Payne, Samuel H.; Haft, Daniel H.; Chauvigne-Hines, Lacie M.; Lewis, Michael P.; Ollodart, Anja R.; Purvine, Samuel O.; Shukla, Anil K.; Fortuin, Suereta; Smith, Richard D.; Adkins, Joshua N.; Grundner, Christoph; Wright, Aaron T.

    2013-01-24

    The annotation of protein function is almost completely performed by in silico approaches. However, computational prediction of protein function is frequently incomplete and error prone. In Mycobacterium tuberculosis (Mtb), ~25% of all genes have no predicted function and are annotated as hypothetical proteins. This lack of functional information severely limits our understanding of Mtb pathogenicity. Current tools for experimental functional annotation are limited and often do not scale to entire protein families. Here, we report a generally applicable chemical biology platform to functionally annotate bacterial proteins by combining activity-based protein profiling (ABPP) and quantitative LC-MS-based proteomics. As an example of this approach for high-throughput protein functional validation and discovery, we experimentally annotate the families of ATP-binding proteins in Mtb. Our data experimentally validate prior in silico predictions of >250 ATPases and adenosine nucleotide-binding proteins, and reveal 73 hypothetical proteins as novel ATP-binding proteins. We identify adenosine cofactor interactions with many hypothetical proteins containing a diversity of unrelated sequences, providing a new and expanded view of adenosine nucleotide binding in Mtb. Furthermore, many of these hypothetical proteins are both unique to Mycobacteria and essential for infection, suggesting specialized functions in mycobacterial physiology and pathogenicity. Thus, we provide a generally applicable approach for high throughput protein function discovery and validation, and highlight several ways in which application of activity-based proteomics data can improve the quality of functional annotations to facilitate novel biological insights.

  7. Statewide survey of laboratories performing Mycobacterium tuberculosis testing in Minnesota.

    PubMed Central

    Mills, W A; Besser-Wiek, J M; Osterholm, M T; MacDonald, K L

    1996-01-01

    Rapid and accurate laboratory detection and identification of Mycobacterium tuberculosis, particularly multidrug-resistant strains, is critical to both public health control measures and patient management. The authors surveyed microbiology laboratories to evaluate whether their methods met national guidelines. As needed, laboratories received individualized recommendations for improvement. The laboratories were resurveyed a year later to assess changes in methods. Current guidelines recommend fluorochrome acid-fast smears, broth cultures, identification by nucleic acid probe or BACTEC-NAP, and BACTEC primary susceptibility panels, which should include pyrazinamide. Of 27 laboratories performing acid-fast smears, 15 used fluorochrome methods. Six of 16 laboratories performing mycobacterial cultures used broth media. Of six laboratories performing species identification, five used nucleic acid probes or BACTEC-NAP. Of five laboratories evaluating drug sensitivity, two used BACTEC and two included pyrazinamide in their protocols. Overall, 24 (89%) laboratories needed improvements; a year later, 16 (67%) of those had altered their methods or made definite plans to do so. Survey results suggest that health departments can facilitate improvements in laboratory testing for pathogens of public health importance. PMID:8606914

  8. Machine learning and docking models for Mycobacterium tuberculosis topoisomerase I.

    PubMed

    Ekins, Sean; Godbole, Adwait Anand; Kéri, György; Orfi, Lászlo; Pato, János; Bhat, Rajeshwari Subray; Verma, Rinkee; Bradley, Erin K; Nagaraja, Valakunja

    2017-03-01

    There is a shortage of compounds that are directed towards new targets apart from those targeted by the FDA approved drugs used against Mycobacterium tuberculosis. Topoisomerase I (Mttopo I) is an essential mycobacterial enzyme and a promising target in this regard. However, it suffers from a shortage of known inhibitors. We have previously used computational approaches such as homology modeling and docking to propose 38 FDA approved drugs for testing and identified several active molecules. To follow on from this, we now describe the in vitro testing of a library of 639 compounds. These data were used to create machine learning models for Mttopo I which were further validated. The combined Mttopo I Bayesian model had a 5 fold cross validation receiver operator characteristic of 0.74 and sensitivity, specificity and concordance values above 0.76 and was used to select commercially available compounds for testing in vitro. The recently described crystal structure of Mttopo I was also compared with the previously described homology model and then used to dock the Mttopo I actives norclomipramine and imipramine. In summary, we describe our efforts to identify small molecule inhibitors of Mttopo I using a combination of machine learning modeling and docking studies in conjunction with screening of the selected molecules for enzyme inhibition. We demonstrate the experimental inhibition of Mttopo I by small molecule inhibitors and show that the enzyme can be readily targeted for lead molecule development. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Pathogen roid rage: cholesterol utilization by Mycobacterium tuberculosis.

    PubMed

    Wipperman, Matthew F; Sampson, Nicole S; Thomas, Suzanne T

    2014-01-01

    The ability of science and medicine to control the pathogen Mycobacterium tuberculosis (Mtb) requires an understanding of the complex host environment within which it resides. Pathological and biological evidence overwhelmingly demonstrate how the mammalian steroid cholesterol is present throughout the course of infection. Better understanding Mtb requires a more complete understanding of how it utilizes molecules like cholesterol in this environment to sustain the infection of the host. Cholesterol uptake, catabolism and broader utilization are important for maintenance of the pathogen in the host and it has been experimentally validated to contribute to virulence and pathogenesis. Cholesterol is catabolized by at least three distinct sub-pathways, two for the ring system and one for the side chain, yielding dozens of steroid intermediates with varying biochemical properties. Our ability to control this worldwide infectious agent requires a greater knowledge of how Mtb uses cholesterol to its advantage throughout the course of infection. Herein, the current state of knowledge of cholesterol metabolism by Mtb is reviewed from a biochemical perspective with a focus on the metabolic genes and pathways responsible for cholesterol steroid catabolism.

  10. Chronic Helminth Infection Does Not Exacerbate Mycobacterium tuberculosis Infection

    PubMed Central

    Hübner, Marc P.; Killoran, Kristin E.; Rajnik, Michael; Wilson, Samuel; Yim, Kevin C.; Torrero, Marina N.; Morris, Christopher P.; Nikonenko, Boris; Blanco, Jorge C. G.; Hemming, Val G.; Mitre, Edward

    2012-01-01

    Background Chronic helminth infections induce a Th2 immune shift and establish an immunoregulatory milieu. As both of these responses can suppress Th1 immunity, which is necessary for control of Mycobacterium tuberculosis (MTB) infection, we hypothesized that chronic helminth infections may exacerbate the course of MTB. Methodology/Principal Findings Co-infection studies were conducted in cotton rats as they are the natural host for the filarial nematode Litomosoides sigmodontis and are an excellent model for human MTB. Immunogical responses, histological studies, and quantitative mycobacterial cultures were assessed two months after MTB challenge in cotton rats with and without chronic L. sigmodontis infection. Spleen cell proliferation and interferon gamma production in response to purified protein derivative were similar between co-infected and MTB-only infected animals. In contrast to our hypothesis, MTB loads and occurrence and size of lung granulomas were not increased in co-infected animals. Conclusions/Significance These findings suggest that chronic filaria infections do not exacerbate MTB infection in the cotton rat model. While these results suggest that filaria eradication programs may not facilitate MTB control, they indicate that it may be possible to develop worm-derived therapies for autoimmune diseases that do not substantially increase the risk for infections. PMID:23285308

  11. The solution structure of acyl carrier protein from Mycobacterium tuberculosis.

    PubMed

    Wong, Hing C; Liu, Gaohua; Zhang, Yong-Mei; Rock, Charles O; Zheng, Jie

    2002-05-03

    Acyl carrier protein (ACP) performs the essential function of shuttling the intermediates between the enzymes that constitute the type II fatty acid synthase system. Mycobacterium tuberculosis is unique in producing extremely long mycolic acids, and tubercular ACP, AcpM, is also unique in possessing a longer carboxyl terminus than other ACPs. We determined the solution structure of AcpM using protein NMR spectroscopy to define the similarities and differences between AcpM and the typical structures. The amino-terminal region of the structure is well defined and consists of four helices arranged in a right-handed bundle held together by interhelical hydrophobic interactions similar to the structures of other bacterial ACPs. The unique carboxyl-terminal extension from helix IV has a "melted down" feature, and the end of the molecule is a random coil. A comparison of the apo- and holo-forms of AcpM revealed that the 4'-phosphopantetheine group oscillates between two states; in one it is bound to a hydrophobic groove on the surface of AcpM, and in another it is solvent-exposed. The similarity between AcpM and other ACPs reveals the conserved structural motif that is recognized by all type II enzymes. However, the function of the coil domain extending from helix IV to the carboxyl terminus remains enigmatic, but its structural characteristics suggest that it may interact with the very long chain intermediates in mycolic acid biosynthesis or control specific protein-protein interactions.

  12. Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

    PubMed Central

    Linger, Yvonne; Kukhtin, Alexander; Golova, Julia; Perov, Alexander; Qu, Peter; Knickerbocker, Christopher; Cooney, Christopher G.; Chandler, Darrell P.

    2014-01-01

    Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice. PMID:24796567

  13. Mycobacterium tuberculosis Pyrazinamide Resistance Determinants: a Multicenter Study

    PubMed Central

    Cabibbe, Andrea M.; Feuerriegel, Silke; Casali, Nicola; Drobniewski, Francis; Rodionova, Yulia; Bakonyte, Daiva; Stakenas, Petras; Pimkina, Edita; Augustynowicz-Kopeć, Ewa; Degano, Massimo; Ambrosi, Alessandro; Hoffner, Sven; Mansjö, Mikael; Werngren, Jim; Rüsch-Gerdes, Sabine; Niemann, Stefan; Cirillo, Daniela M.

    2014-01-01

    ABSTRACT Pyrazinamide (PZA) is a prodrug that is converted to pyrazinoic acid by the enzyme pyrazinamidase, encoded by the pncA gene in Mycobacterium tuberculosis. Molecular identification of mutations in pncA offers the potential for rapid detection of pyrazinamide resistance (PZAr). However, the genetic variants are highly variable and scattered over the full length of pncA, complicating the development of a molecular test. We performed a large multicenter study assessing pncA sequence variations in 1,950 clinical isolates, including 1,142 multidrug-resistant (MDR) strains and 483 fully susceptible strains. The results of pncA sequencing were correlated with phenotype, enzymatic activity, and structural and phylogenetic data. We identified 280 genetic variants which were divided into four classes: (i) very high confidence resistance mutations that were found only in PZAr strains (85%), (ii) high-confidence resistance mutations found in more than 70% of PZAr strains, (iii) mutations with an unclear role found in less than 70% of PZAr strains, and (iv) mutations not associated with phenotypic resistance (10%). Any future molecular diagnostic assay should be able to target and identify at least the very high and high-confidence genetic variant markers of PZAr; the diagnostic accuracy of such an assay would be in the range of 89.5 to 98.8%. PMID:25336456

  14. Global analysis of mRNA stability in Mycobacterium tuberculosis.

    PubMed

    Rustad, Tige R; Minch, Kyle J; Brabant, William; Winkler, Jessica K; Reiss, David J; Baliga, Nitin S; Sherman, David R

    2013-01-07

    Mycobacterium tuberculosis (MTB) is a highly successful pathogen that infects over a billion people. As with most organisms, MTB adapts to stress by modifying its transcriptional profile. Remodeling of the transcriptome requires both altering the transcription rate and clearing away the existing mRNA through degradation, a process that can be directly regulated in response to stress. To understand better how MTB adapts to the harsh environs of the human host, we performed a global survey of the decay rates of MTB mRNA transcripts. Decay rates were measured for 2139 of the ~4000 MTB genes, which displayed an average half-life of 9.5 min. This is nearly twice the average mRNA half-life of other prokaryotic organisms where these measurements have been made. The transcriptome was further stabilized in response to lowered temperature and hypoxic stress. The generally stable transcriptome described here, and the additional stabilization in response to physiologically relevant stresses, has far-ranging implications for how this pathogen is able to adapt in its human host.

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

    PubMed

    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.

  16. Structural Insights on the Mycobacterium tuberculosis Proteasomal ATPase Mpa

    PubMed Central

    Wang, Tao; Li, Hua; Lin, Gang; Tang, Chunyan; Li, Dongyang; Nathan, Carl; Darwin, K. Heran; Li, Huilin

    2009-01-01

    Summary Proteasome-mediated protein turnover in all domains of life is an energy-dependent process that requires ATPase activity. Mycobacterium tuberculosis (Mtb) was recently shown to possess a ubiquitin-like proteasome pathway that plays an essential role in Mtb resistance to killing by products of host macrophages. Here we report our structural and biochemical investigation of Mpa, the presumptive Mtb proteasomal ATPase. We demonstrate that Mpa binds to the Mtb proteasome in the presence of ATPγS, providing the physical evidence that Mpa is the proteasomal ATPase. X-ray crystallographic determination of the conserved inter-domain showed a five-stranded double β-barrel structure containing a Greek key motif. The structure and mutagenesis indicate a major role of the inter-domain for Mpa hexamerization. Our mutational and functional studies further suggest that the central channel in the Mpa hexamer is involved in protein substrate translocation and degradation. These studies provide insights into how a bacterial proteasomal ATPase interacts with and facilitates protein degradation by the proteasome. PMID:19836337

  17. Prokayrotic Ubiquitin-Like Protein (Pup) Proteome of Mycobacterium tuberculosis

    PubMed Central

    Mintseris, Julian; Burns, Kristin E.; Gygi, Steven P.; Darwin, K. Heran

    2010-01-01

    Prokaryotic ubiquitin-like protein (Pup) in Mycobacterium tuberculosis (Mtb) is the first known post-translational small protein modifier in prokaryotes, and targets several proteins for degradation by a bacterial proteasome in a manner akin to ubiquitin (Ub) mediated proteolysis in eukaryotes. To determine the extent of pupylation in Mtb, we used tandem affinity purification to identify its “pupylome”. Mass spectrometry identified 55 out of 604 purified proteins with confirmed pupylation sites. Forty-four proteins, including those with and without identified pupylation sites, were tested as substrates of proteolysis in Mtb. Under steady state conditions, the majority of the test proteins did not accumulate in degradation mutants, suggesting not all targets of pupylation are necessarily substrates of the proteasome under steady state conditions. Four proteins implicated in Mtb pathogenesis, Icl (isocitrate lyase), Ino1 (inositol-1-phosphate synthase), MtrA (Mtb response regulator A) and PhoP (phosphate response regulator P), showed altered levels in degradation defective Mtb. Icl, Ino1 and MtrA accumulated in Mtb degradation mutants, suggesting these proteins are targeted to the proteasome. Unexpectedly, PhoP was present in wild type Mtb but undetectable in the degradation mutants. Taken together, these data demonstrate that pupylation regulates numerous proteins in Mtb and may not always lead to degradation. PMID:20066036

  18. A Novel Copper-Responsive Regulon in Mycobacterium tuberculosis

    PubMed Central

    Festa, Richard A.; Jones, Marcus B.; Butler-Wu, Susan; Sinsimer, Daniel; Gerads, Russell; Bishai, William R.; Peterson, Scott N.; Darwin, K. Heran

    2010-01-01

    In this work we describe the identification of a copper-inducible regulon in Mycobacterium tuberculosis (Mtb). Among the regulated genes was Rv0190/MT0200, a paralogue of the copper metalloregulatory repressor CsoR. The five-locus regulon, which includes a gene that encodes the copper-protective metallothionein MymT, was highly induced in wild type Mtb treated with copper, and highly expressed in an Rv0190/MT0200 mutant. Importantly, the Rv0190/MT0200 mutant was hyper-resistant to copper. The promoters of all five loci share a palindromic motif that was recognized by the gene product of Rv0190/MT0200. For this reason we named Rv0190/MT0200 RicR for regulated in copper repressor. Intriguingly, several of the RicR-regulated genes, including MymT, are unique to pathogenic Mycobacteria. The identification of a copper-responsive regulon specific to virulent mycobacterial species suggests copper homeostasis must be maintained during an infection. Alternatively, copper may provide a cue for the expression of genes unrelated to metal homeostasis, but nonetheless necessary for survival in a host. PMID:21166899

  19. Identification of substrates of the Mycobacterium tuberculosis proteasome

    PubMed Central

    Pearce, Michael J; Arora, Pooja; Festa, Richard A; Butler-Wu, Susan M; Gokhale, Rajesh S; Darwin, K Heran

    2006-01-01

    The putative proteasome-associated proteins Mpa (Mycobaterium proteasomal ATPase) and PafA (proteasome accessory factor A) of the human pathogen Mycobacterium tuberculosis (Mtb) are essential for virulence and resistance to nitric oxide. However, a direct link between the proteasome protease and Mpa or PafA has never been demonstrated. Furthermore, protein degradation by bacterial proteasomes in vitro has not been accomplished, possibly due to the failure to find natural degradation substrates or other necessary proteasome co-factors. In this work, we identify the first bacterial proteasome substrates, malonyl Co-A acyl carrier protein transacylase and ketopantoate hydroxymethyltransferase, enzymes that are required for the biosynthesis of fatty acids and polyketides that are essential for the pathogenesis of Mtb. Maintenance of the physiological levels of these enzymes required Mpa and PafA in addition to proteasome protease activity. Mpa levels were also regulated in a proteasome-dependent manner. Finally, we found that a conserved tyrosine of Mpa was essential for function. Thus, these results suggest that Mpa, PafA, and the Mtb proteasome degrade bacterial proteins that are important for virulence in mice. PMID:17082771

  20. Zoonotic Mycobacterium bovis–induced Tuberculosis in Humans

    PubMed Central

    Dürr, Salome; Alonso, Silvia; Hattendorf, Jan; Laisse, Cláudio J.M.; Parsons, Sven D.C.; van Helden, Paul D.; Zinsstag, Jakob

    2013-01-01

    We aimed to estimate the global occurrence of zoonotic tuberculosis (TB) caused by Mycobacterium bovis or M. caprae infections in humans by performing a multilingual, systematic review and analysis of relevant scientific literature of the last 2 decades. Although information from many parts of the world was not available, data from 61 countries suggested a low global disease incidence. In regions outside Africa included in this study, overall median proportions of zoonotic TB of ≤1.4% in connection with overall TB incidence rates ≤71/100,000 population/year suggested low incidence rates. For countries of Africa included in the study, we multiplied the observed median proportion of zoonotic TB cases of 2.8% with the continental average overall TB incidence rate of 264/100,000 population/year, which resulted in a crude estimate of 7 zoonotic TB cases/100,000 population/year. These generally low incidence rates notwithstanding, available data indicated substantial consequences of this disease for some population groups and settings. PMID:23735540

  1. Demonstrating a multi-drug resistant Mycobacterium tuberculosis amplification microarray.

    PubMed

    Linger, Yvonne; Kukhtin, Alexander; Golova, Julia; Perov, Alexander; Qu, Peter; Knickerbocker, Christopher; Cooney, Christopher G; Chandler, Darrell P

    2014-04-25

    Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

  2. Proteomic definition of the cell wall of Mycobacterium tuberculosis.

    PubMed

    Wolfe, Lisa M; Mahaffey, Spencer B; Kruh, Nicole A; Dobos, Karen M

    2010-11-05

    The cell envelope of Mycobacterium tuberculosis (Mtb) is complex and diverse; composed of proteins intermingled in a matrix of peptidoglycan, mycolic acids, lipids, and carbohydrates. Proteomic studies of the Mtb cell wall have been limited; nonetheless, the characterization of resident and secreted proteins associated with the cell wall are critical to understanding bacterial survival and immune modulation in the host. In this study, the cell wall proteome was defined in order to better understand its unique biosynthetic and secretion processes. Mtb cell wall was subjected to extraction with organic solvents to remove noncovalently bound lipids and lipoglycans and remaining proteins were solubilized with either SDS, Guanidine-HCl, or TX-114. These extracts were analyzed by two-dimensional gel electrophoresis and mass-spectrometry and resulted in the identification of 234 total proteins. The lipoproteome of Mtb, enriched in the TX-114 extract, was further resolved by multidimensional chromatography and mass spectrometry to identify an additional 294 proteins. A query of the 528 total protein identifications against Neural Network or Hidden Markov model algorithms predicted secretion signals in 87 proteins. Classification of these 528 proteins also demonstrated that 35% are involved in small molecule metabolism and 25% are involved in macromolecule synthesis and degradation building upon evidence that the Mtb cell wall is actively engaged in mycobacterial survival and remodeling.

  3. Mycobacterium tuberculosis 19-kDa lipoprotein promotes neutrophil activation.

    PubMed

    Neufert, C; Pai, R K; Noss, E H; Berger, M; Boom, W H; Harding, C V

    2001-08-01

    Certain microbial substances, e.g., LPS, can activate neutrophils or prime them to enhance their response to other activating agents, e.g., fMLP. We investigated the role of the Mycobacterium tuberculosis (MTB) 19-kDa lipoprotein in activation of human neutrophils. MTB 19-kDa lipoprotein initiated phenotypic changes characteristic of neutrophil activation, including down-regulation of CD62 ligand (L-selectin) and up-regulation of CD35 (CR1) and CD11b/CD18 (CR3, Mac-1). In addition, exposure of neutrophils to MTB 19-kDa lipoprotein enhanced the subsequent oxidative burst in response to fMLP as assessed by oxidation of dihydrorhodamine 123 (determined by flow cytometry). LPS also produced these effects with similar kinetics, but an oligodeoxynucleotide containing a CpG motif failed to induce any priming or activation response. Although the effects of LPS required the presence of serum, neutrophil activation by MTB 19-kDa lipoprotein occurred independently of serum factors, suggesting the involvement of different receptors and signaling mechanisms for LPS and MTB 19-kDa lipoprotein. Thus, MTB 19-kDa lipoprotein serves as a pathogen-associated molecular pattern that promotes neutrophil priming and activation.

  4. Structural Insights on the Mycobacterium tuberculosis Proteasomal ATPase Mpa

    SciTech Connect

    Wang, T.; Li, H; Lin, G; Tang, C; Li, D; Nathan, C; Heran Darwin, K

    2009-01-01

    Proteasome-mediated protein turnover in all domains of life is an energy-dependent process that requires ATPase activity. Mycobacterium tuberculosis (Mtb) was recently shown to possess a ubiquitin-like proteasome pathway that plays an essential role in Mtb resistance to killing by products of host macrophages. Here we report our structural and biochemical investigation of Mpa, the presumptive Mtb proteasomal ATPase. We demonstrate that Mpa binds to the Mtb proteasome in the presence of ATPS, providing the physical evidence that Mpa is the proteasomal ATPase. X-ray crystallographic determination of the conserved interdomain showed a five stranded double {beta} barrel structure containing a Greek key motif. Structure and mutational analysis indicate a major role of the interdomain for Mpa hexamerization. Our mutational and functional studies further suggest that the central channel in the Mpa hexamer is involved in protein substrate translocation and degradation. These studies provide insights into how a bacterial proteasomal ATPase interacts with and facilitates protein degradation by the proteasome.

  5. Pathogen ‘Roid Rage: Cholesterol Utilization by Mycobacterium tuberculosis

    PubMed Central

    Wipperman, Matthew F.; Sampson, Nicole S.; Thomas, Suzanne, T.

    2014-01-01

    The ability of science and medicine to control the pathogen Mycobacterium tuberculosis (Mtb) requires an understanding of the complex host environment within which it resides. Pathological and biological evidence overwhelmingly demonstrate how the mammalian steroid cholesterol is present throughout the course of infection. Better understanding Mtb requires a more complete understanding of how it utilizes molecules like cholesterol in this environment to sustain the infection of the host. Cholesterol uptake, catabolism, and broader utilization are important for maintenance of the pathogen in the host and it has been experimentally validated to contribute to virulence and pathogenesis. Cholesterol is catabolized by at least three distinct sub-pathways, two for the ring system and one for the side chain, yielding dozens of steroid intermediates with varying biochemical properties. Our ability to control this worldwide infectious agent requires a greater knowledge of how Mtb uses cholesterol to its advantage throughout the course of infection. Herein, the current state of knowledge of cholesterol metabolism by Mtb is reviewed from a biochemical perspective with a focus on the metabolic genes and pathways responsible for cholesterol steroid catabolism. PMID:24611808

  6. Mycobacterium tuberculosis transmission in a country with low tuberculosis incidence: role of immigration and HIV infection.

    PubMed

    Fenner, Lukas; Gagneux, Sebastien; Helbling, Peter; Battegay, Manuel; Rieder, Hans L; Pfyffer, Gaby E; Zwahlen, Marcel; Furrer, Hansjakob; Siegrist, Hans H; Fehr, Jan; Dolina, Marisa; Calmy, Alexandra; Stucki, David; Jaton, Katia; Janssens, Jean-Paul; Stalder, Jesica Mazza; Bodmer, Thomas; Ninet, Beatrice; Böttger, Erik C; Egger, Matthias

    2012-02-01

    Immigrants from high-burden countries and HIV-coinfected individuals are risk groups for tuberculosis (TB) in countries with low TB incidence. Therefore, we studied their role in transmission of Mycobacterium tuberculosis in Switzerland. We included all TB patients from the Swiss HIV Cohort and a sample of patients from the national TB registry. We identified molecular clusters by spoligotyping and mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) analysis and used weighted logistic regression adjusted for age and sex to identify risk factors for clustering, taking sampling proportions into account. In total, we analyzed 520 TB cases diagnosed between 2000 and 2008; 401 were foreign born, and 113 were HIV coinfected. The Euro-American M. tuberculosis lineage dominated throughout the study period (378 strains; 72.7%), with no evidence for another lineage, such as the Beijing genotype, emerging. We identified 35 molecular clusters with 90 patients, indicating recent transmission; 31 clusters involved foreign-born patients, and 15 involved HIV-infected patients. Birth origin was not associated with clustering (adjusted odds ratio [aOR], 1.58; 95% confidence interval [CI], 0.73 to 3.43; P = 0.25, comparing Swiss-born with foreign-born patients), but clustering was reduced in HIV-infected patients (aOR, 0.49; 95% CI, 0.26 to 0.93; P = 0.030). Cavitary disease, male sex, and younger age were all associated with molecular clustering. In conclusion, most TB patients in Switzerland were foreign born, but transmission of M. tuberculosis was not more common among immigrants and was reduced in HIV-infected patients followed up in the national HIV cohort study. Continued access to health services and clinical follow-up will be essential to control TB in this population.

  7. Genomic Analysis of the Evolution of Fluoroquinolone Resistance in Mycobacterium tuberculosis Prior to Tuberculosis Diagnosis.

    PubMed

    Zhang, Danfeng; Gomez, James E; Chien, Jung-Yien; Haseley, Nathan; Desjardins, Christopher A; Earl, Ashlee M; Hsueh, Po-Ren; Hung, Deborah T

    2016-11-01

    Fluoroquinolones (FQs) are effective second-line drugs for treating antibiotic-resistant tuberculosis (TB) and are being considered for use as first-line agents. Because FQs are used to treat a range of infections, in a setting of undiagnosed TB, there is potential to select for drug-resistant Mycobacterium tuberculosis mutants during FQ-based treatment of other infections, including pneumonia. Here we present a detailed characterization of ofloxacin-resistant M. tuberculosis samples isolated directly from patients in Taiwan, which demonstrates that selection for FQ resistance can occur within patients who have not received FQs for the treatment of TB. Several of these samples showed no mutations in gyrA or gyrB based on PCR-based molecular assays, but genome-wide next-generation sequencing (NGS) revealed minority populations of gyrA and/or gyrB mutants. In other samples with PCR-detectable gyrA mutations, NGS revealed subpopulations containing alternative resistance-associated genotypes. Isolation of individual clones from these apparently heterogeneous samples confirmed the presence of the minority drug-resistant variants suggested by the NGS data. Further NGS of these purified clones established evolutionary links between FQ-sensitive and -resistant clones derived from the same patient, suggesting de novo emergence of FQ-resistant TB. Importantly, most of these samples were isolated from patients without a history of FQ treatment for TB. Thus, selective pressure applied by FQ monotherapy in the setting of undiagnosed TB infection appears to be able to drive the full or partial emergence of FQ-resistant M. tuberculosis, which has the potential to confound diagnostic tests for antibiotic susceptibility and limit the effectiveness of FQs in TB treatment.

  8. Mycobacterium genotypes in pulmonary tuberculosis infections and their detection by trained African giant pouched rats.

    PubMed

    Mgode, Georgies F; Cohen-Bacrie, Stéphan; Bedotto, Marielle; Weetjens, Bart J; Cox, Christophe; Jubitana, Maureen; Kuipers, Dian; Machang'u, Robert S; Kazwala, Rudovick; Mfinanga, Sayoki G; Kaufmann, Stefan H E; Drancourt, Michel

    2015-02-01

    Tuberculosis (TB) diagnosis in low-income countries is mainly done by microscopy. Hence, little is known about the diversity of Mycobacterium spp. in TB infections. Different genotypes or lineages of Mycobacterium tuberculosis vary in virulence and induce different inflammatory and immune responses. Trained Cricetomys rats show a potential for rapid diagnosis of TB. They detect over 28 % of smear-negative, culture-positive TB. However, it is unknown whether these rats can equally detect sputa from patients infected with different genotypes of M. tuberculosis. A 4-month prospective study on diversity of Mycobacterium spp. was conducted in Dar es Salaam, Tanzania. 252 sputa from 161 subjects were cultured on Lowenstein-Jensen medium and thereafter tested by rats. Mycobacterial isolates were subjected to molecular identification and multispacer sequence typing (MST) to determine species and genotypes. A total of 34 Mycobacterium spp. isolates consisting of 32 M. tuberculosis, 1 M. avium subsp. hominissuis and 1 M. intracellulare were obtained. MST analyses of 26 M. tuberculosis isolates yielded 10 distinct MST genotypes, including 3 new genotypes with two clusters of related patterns not grouped by geographic areas. Genotype MST-67, shared by one-third of M. tuberculosis isolates, was associated with the Mwananyamala clinic. This study shows that diverse M. tuberculosis genotypes (n = 10) occur in Dar es Salaam and trained rats detect 80 % of the genotypes. Sputa with two M. tuberculosis genotypes (20 %), M. avium hominissuis and M. intracellulare were not detected. Therefore, rats detect sputa with different M. tuberculosis genotypes and can be used to detect TB in resource-poor countries.

  9. Serum Therapy for Tuberculosis Revisited: Reappraisal of the Role of Antibody-Mediated Immunity against Mycobacterium tuberculosis

    PubMed Central

    Glatman-Freedman, Aharona; Casadevall, Arturo

    1998-01-01

    Fifty years after the introduction of the first effective antimicrobial agents against Mycobacterium tuberculosis, this pathogen continues to be a tremendous public health problem. The rise in the number of resistant strains and the difficulties involved in the therapy of tuberculosis in immunocompromised AIDS patients have renewed the interest in the development of effective vaccines. To evaluate whether a potential vaccine against tuberculosis could prevent infection by eliciting a protective antibody response, we reviewed the history of antibody-mediated immunity against tuberculosis. Review of the literature of the past 100 years demonstrates that there is sufficient evidence to conclude that antibody-mediated immunity can modify the course of infection in certain situations. Based on our findings and on what is known in other systems, we propose that the role of antibody-mediated immunity to M. tuberculosis be reexamined, using advanced technology. PMID:9665981

  10. Genomic Analysis of Mycobacterium tuberculosis Complex Strains Used for Production of Purified Protein Derivative

    PubMed Central

    Inwald, Jacqueline; Hinds, Jason; Palmer, Si; Dale, James; Butcher, Philip D.; Hewinson, R. Glyn; Gordon, Stephen V.

    2003-01-01

    The genomes of the tuberculin production strains Mycobacterium bovis AN5 and Mycobacterium tuberculosis DT were compared to genome-sequenced tubercle bacilli by using DNA microarrays. Neither the AN5 nor DT strain suffered extensive gene deletions during in vitro passage. This suggests that bovine tuberculin made from M. bovis AN5 is suitable to detect infection with presently prevalent M. bovis strains. PMID:12904421

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

    PubMed

    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.

  12. An outbreak of tuberculosis by Mycobacterium bovis in coatis (Nasua nasua).

    PubMed

    Murakami, Patricia Sayuri; Monego, Fernanda; Ho, John L; Gibson, Andrea; Vilani, Ricardo Guilherme D'Otaviano de Castro; Soresini, Grazielle Cristina Garcia; Brockelt, Sonia Regina; Biesdorf, Sonia Maria; Fuverki, Renata Benicio Neves; Nakatani, Sueli Massumi; Riediger, Irina Nastassja; Grazziotin, Ana Laura; do Santos, Andrea Pires; de Barros Filho, Ivan Roque; Biondo, Alexander Welker

    2012-06-01

    Mycobacterium tuberculosis complex, which includes Mycobacterium bovis, infrequently causes severe or lethal disease in captive wildlife populations. A dead coati from a wildlife triage center showing pulmonary lesions compatible with tuberculosis had raised suspicion of a potential disease caused by mycobacteria species and was further investigated. Four native coatis (Nasua nasua) with suspected mycobacterial infection were sedated, and bronchoalveolar lavages and tuberculin skin tests (TSTs) were performed. All animals tested positive upon TST. Mycobacterial culturing, Ziehl-Neelsen staining, and genetic testing were performed on postmortem samples and the etiologic agent was identified as M. bovis. Molecular genetic identification using a polymerase chain reaction panel was crucial to achieving a definitive diagnosis.

  13. High-throughput Method of One-Step DNA Isolation for PCR Diagnostics of Mycobacterium tuberculosis.

    PubMed

    Kapustin, D V; Prostyakova, A I; Alexeev, Ya I; Varlamov, D A; Zubov, V P; Zavriev, S K

    2014-04-01

    The efficiency of one-step and multi-step protocols of DNA isolation from lysed sputum samples containing the Mycobacterium tuberculosis complex has been compared. DNA was isolated using spin-cartridges containing a special silica-based sorbent modified with fluoroplast and polyaniline, or using an automated isolation system. One-step isolation using the obtained sorbent has been shown to ensure a significantly lower DNA loss and higher sensitivity in the PCR detection of Mycobacterium tuberculosis as compared to a system based on sorption and desorption of nucleic acids during the isolation.

  14. High-throughput Method of One-Step DNA Isolation for PCR Diagnostics of Mycobacterium tuberculosis

    PubMed Central

    Kapustin, D. V.; Prostyakova, A. I.; Alexeev, Ya. I.; Varlamov, D. A.; Zubov, V. P.; Zavriev, S. K.

    2014-01-01

    The efficiency of one-step and multi-step protocols of DNA isolation from lysed sputum samples containing the Mycobacterium tuberculosis complex has been compared. DNA was isolated using spin-cartridges containing a special silica-based sorbent modified with fluoroplast and polyaniline, or using an automated isolation system. One-step isolation using the obtained sorbent has been shown to ensure a significantly lower DNA loss and higher sensitivity in the PCR detection of Mycobacterium tuberculosis as compared to a system based on sorption and desorption of nucleic acids during the isolation. PMID:25093111

  15. A defined tuberculosis vaccine candidate boosts BCG and protects against multidrug-resistant Mycobacterium tuberculosis.

    PubMed

    Bertholet, Sylvie; Ireton, Gregory C; Ordway, Diane J; Windish, Hillarie Plessner; Pine, Samuel O; Kahn, Maria; Phan, Tony; Orme, Ian M; Vedvick, Thomas S; Baldwin, Susan L; Coler, Rhea N; Reed, Steven G

    2010-10-13

    Despite the widespread use of the childhood vaccine against tuberculosis (TB), Mycobacterium bovis bacillus Calmette-Guérin (BCG), the disease remains a serious global health problem. A successful vaccine against TB that replaces or boosts BCG would include antigens that induce or recall the appropriate T cell responses. Four Mycobacterium tuberculosis (Mtb) antigens--including members of the virulence factor families PE/PPE and EsX or antigens associated with latency--were produced as a single recombinant fusion protein (ID93). When administered together with the adjuvant GLA-SE, a stable oil-in-water nanoemulsion, the fusion protein was immunogenic in mice, guinea pigs, and cynomolgus monkeys. In mice, this fusion protein-adjuvant combination induced polyfunctional CD4 T helper 1 cell responses characterized by antigen-specific interferon-γ, tumor necrosis factor, and interleukin-2, as well as a reduction in the number of bacteria in the lungs of animals after they were subsequently infected with virulent or multidrug-resistant Mtb strains. Furthermore, boosting BCG-vaccinated guinea pigs with fusion peptide-adjuvant resulted in reduced pathology and fewer bacilli, and prevented the death of animals challenged with virulent Mtb. Finally, the fusion protein elicited polyfunctional effector CD4 and CD8 T cell responses in BCG-vaccinated or Mtb-exposed human peripheral blood mononuclear cells. This study establishes that the protein subunit vaccine consisting of the fusion protein and adjuvant protects against TB and drug-resistant TB in animals and is a candidate for boosting the protective efficacy of the childhood BCG vaccine in humans.