Science.gov

Sample records for predicting mycobacterium tuberculosis

  1. Multiscale Model of Mycobacterium tuberculosis Infection Maps Metabolite and Gene Perturbations to Granuloma Sterilization Predictions

    PubMed Central

    Pienaar, Elsje; Matern, William M.; Linderman, Jennifer J.

    2016-01-01

    Granulomas are a hallmark of tuberculosis. Inside granulomas, the pathogen Mycobacterium tuberculosis may enter a metabolically inactive state that is less susceptible to antibiotics. Understanding M. tuberculosis metabolism within granulomas could contribute to reducing the lengthy treatment required for tuberculosis and provide additional targets for new drugs. Two key adaptations of M. tuberculosis are a nonreplicating phenotype and accumulation of lipid inclusions in response to hypoxic conditions. To explore how these adaptations influence granuloma-scale outcomes in vivo, we present a multiscale in silico model of granuloma formation in tuberculosis. The model comprises host immunity, M. tuberculosis metabolism, M. tuberculosis growth adaptation to hypoxia, and nutrient diffusion. We calibrated our model to in vivo data from nonhuman primates and rabbits and apply the model to predict M. tuberculosis population dynamics and heterogeneity within granulomas. We found that bacterial populations are highly dynamic throughout infection in response to changing oxygen levels and host immunity pressures. Our results indicate that a nonreplicating phenotype, but not lipid inclusion formation, is important for long-term M. tuberculosis survival in granulomas. We used virtual M. tuberculosis knockouts to predict the impact of both metabolic enzyme inhibitors and metabolic pathways exploited to overcome inhibition. Results indicate that knockouts whose growth rates are below ∼66% of the wild-type growth rate in a culture medium featuring lipid as the only carbon source are unable to sustain infections in granulomas. By mapping metabolite- and gene-scale perturbations to granuloma-scale outcomes and predicting mechanisms of sterilization, our method provides a powerful tool for hypothesis testing and guiding experimental searches for novel antituberculosis interventions. PMID:26975995

  2. Evolution of Mycobacterium tuberculosis.

    PubMed

    Behr, Marcel A

    2013-01-01

    Genomic studies have provided a refined understanding of the genetic diversity within the Mycobacterium genus, and more specifically within Mycobacterium tuberculosis. These results have informed a new perspective on the macro- and micro-evolution of the tubercle bacillus. In the first step, a M. kansasii-like opportunistic pathogen acquired new genes, through horizontal gene transfer, that enabled it to better exploit an intracellular niche and ultimately evolve into a professional pathogen. In the second step, different subspecies and strains of the M. tuberculosis complex emerged through mutation and deletion of unnecessary DNA. Understanding the differences between M. tuberculosis and related less pathogenic mycobacteria is expected to reveal key bacterial virulence mechanisms and provide opportunities to understand host resistance to mycobacterial infection. Understanding differences within the M. tuberculosis complex and the evolutionary forces shaping these differences is important for investigating the basis of its success as both a symbiont and a pathogen.

  3. Mycobacterial Interspersed Repetitive Unit Can Predict Drug Resistance of Mycobacterium tuberculosis in China

    PubMed Central

    Cheng, Xian-feng; Jiang, Chao; Zhang, Min; Xia, Dan; Chu, Li-li; Wen, Yu-feng; Zhu, Ming; Jiang, Yue-gen

    2016-01-01

    Background: Recently, Mycobacterial Interspersed Repetitive Unit (MIRU) was supposed to be associated with drug resistance in Mycobacterium tuberculosis (M. tuberculosis), but whether the association exists actually in local strains in China was still unknown. This research was conducted to explore that association and the predictability of MIRU to drug resistance of Tuberculosis (TB). Methods: The clinical isolates were collected and the susceptibility test were conducted with Lowenstein–Jensen (LJ) medium for five anti-TB drug. Based on PCR of MIRU-VNTR (Variable Number of Tandem Repeat) genotyping, we tested the number of the repeat unite of MIRU. Then, we used logistic regression to evaluate the association between 15 MIRU and drug resistance. In addition, we explored the most suitable MIRU locus of identified MIRU loci for drug resistance by multivariate logistic regression. Results: Of the 102 strains, one isolate was resistant to rifampicin and one isolate was resistant to streptomycin. Among these fifteen MIRU, there was a association between MIRU loci polymorphism and anti-tuberculosis drug resistance, ETRB (P = 0.03, OR = 0.19, 95% CI 0.05–0.81) and ETRC (P = 0.01, OR = 0.14, 95% CI 0.03–0.64) were negatively related to isoniazid resistance; MIRU20 (P = 0.05, OR = 2.87, 95% CI 1.01–8.12) was positively associated with ethambutol resistance; and QUB11a (P = 0.02, OR = 0.79, 95% CI 0.65–0.96) was a negative association factor of p-aminosalicylic acid resistance. Conclusion: Our research showed that MIRU loci may predict drug resistance of tuberculosis in China. However, the mechanism still needs further exploration. PMID:27047485

  4. Predicting Mycobacterium tuberculosis Complex Clades Using Knowledge-Based Bayesian Networks

    PubMed Central

    Bennett, Kristin P.

    2014-01-01

    We develop a novel approach for incorporating expert rules into Bayesian networks for classification of Mycobacterium tuberculosis complex (MTBC) clades. The proposed knowledge-based Bayesian network (KBBN) treats sets of expert rules as prior distributions on the classes. Unlike prior knowledge-based support vector machine approaches which require rules expressed as polyhedral sets, KBBN directly incorporates the rules without any modification. KBBN uses data to refine rule-based classifiers when the rule set is incomplete or ambiguous. We develop a predictive KBBN model for 69 MTBC clades found in the SITVIT international collection. We validate the approach using two testbeds that model knowledge of the MTBC obtained from two different experts and large DNA fingerprint databases to predict MTBC genetic clades and sublineages. These models represent strains of MTBC using high-throughput biomarkers called spacer oligonucleotide types (spoligotypes), since these are routinely gathered from MTBC isolates of tuberculosis (TB) patients. Results show that incorporating rules into problems can drastically increase classification accuracy if data alone are insufficient. The SITVIT KBBN is publicly available for use on the World Wide Web. PMID:24864238

  5. Predicting Mycobacterium tuberculosis complex clades using knowledge-based Bayesian networks.

    PubMed

    Aminian, Minoo; Couvin, David; Shabbeer, Amina; Hadley, Kane; Vandenberg, Scott; Rastogi, Nalin; Bennett, Kristin P

    2014-01-01

    We develop a novel approach for incorporating expert rules into Bayesian networks for classification of Mycobacterium tuberculosis complex (MTBC) clades. The proposed knowledge-based Bayesian network (KBBN) treats sets of expert rules as prior distributions on the classes. Unlike prior knowledge-based support vector machine approaches which require rules expressed as polyhedral sets, KBBN directly incorporates the rules without any modification. KBBN uses data to refine rule-based classifiers when the rule set is incomplete or ambiguous. We develop a predictive KBBN model for 69 MTBC clades found in the SITVIT international collection. We validate the approach using two testbeds that model knowledge of the MTBC obtained from two different experts and large DNA fingerprint databases to predict MTBC genetic clades and sublineages. These models represent strains of MTBC using high-throughput biomarkers called spacer oligonucleotide types (spoligotypes), since these are routinely gathered from MTBC isolates of tuberculosis (TB) patients. Results show that incorporating rules into problems can drastically increase classification accuracy if data alone are insufficient. The SITVIT KBBN is publicly available for use on the World Wide Web.

  6. Rapid antibiotic-resistance predictions from genome sequence data for Staphylococcus aureus and Mycobacterium tuberculosis

    PubMed Central

    Bradley, Phelim; Gordon, N. Claire; Walker, Timothy M.; Dunn, Laura; Heys, Simon; Huang, Bill; Earle, Sarah; Pankhurst, Louise J.; Anson, Luke; de Cesare, Mariateresa; Piazza, Paolo; Votintseva, Antonina A.; Golubchik, Tanya; Wilson, Daniel J.; Wyllie, David H.; Diel, Roland; Niemann, Stefan; Feuerriegel, Silke; Kohl, Thomas A.; Ismail, Nazir; Omar, Shaheed V.; Smith, E. Grace; Buck, David; McVean, Gil; Walker, A. Sarah; Peto, Tim E. A.; Crook, Derrick W.; Iqbal, Zamin

    2015-01-01

    The rise of antibiotic-resistant bacteria has led to an urgent need for rapid detection of drug resistance in clinical samples, and improvements in global surveillance. Here we show how de Bruijn graph representation of bacterial diversity can be used to identify species and resistance profiles of clinical isolates. We implement this method for Staphylococcus aureus and Mycobacterium tuberculosis in a software package (‘Mykrobe predictor') that takes raw sequence data as input, and generates a clinician-friendly report within 3 minutes on a laptop. For S. aureus, the error rates of our method are comparable to gold-standard phenotypic methods, with sensitivity/specificity of 99.1%/99.6% across 12 antibiotics (using an independent validation set, n=470). For M. tuberculosis, our method predicts resistance with sensitivity/specificity of 82.6%/98.5% (independent validation set, n=1,609); sensitivity is lower here, probably because of limited understanding of the underlying genetic mechanisms. We give evidence that minor alleles improve detection of extremely drug-resistant strains, and demonstrate feasibility of the use of emerging single-molecule nanopore sequencing techniques for these purposes. PMID:26686880

  7. Rapid antibiotic-resistance predictions from genome sequence data for Staphylococcus aureus and Mycobacterium tuberculosis.

    PubMed

    Bradley, Phelim; Gordon, N Claire; Walker, Timothy M; Dunn, Laura; Heys, Simon; Huang, Bill; Earle, Sarah; Pankhurst, Louise J; Anson, Luke; de Cesare, Mariateresa; Piazza, Paolo; Votintseva, Antonina A; Golubchik, Tanya; Wilson, Daniel J; Wyllie, David H; Diel, Roland; Niemann, Stefan; Feuerriegel, Silke; Kohl, Thomas A; Ismail, Nazir; Omar, Shaheed V; Smith, E Grace; Buck, David; McVean, Gil; Walker, A Sarah; Peto, Tim E A; Crook, Derrick W; Iqbal, Zamin

    2015-01-01

    The rise of antibiotic-resistant bacteria has led to an urgent need for rapid detection of drug resistance in clinical samples, and improvements in global surveillance. Here we show how de Bruijn graph representation of bacterial diversity can be used to identify species and resistance profiles of clinical isolates. We implement this method for Staphylococcus aureus and Mycobacterium tuberculosis in a software package ('Mykrobe predictor') that takes raw sequence data as input, and generates a clinician-friendly report within 3 minutes on a laptop. For S. aureus, the error rates of our method are comparable to gold-standard phenotypic methods, with sensitivity/specificity of 99.1%/99.6% across 12 antibiotics (using an independent validation set, n=470). For M. tuberculosis, our method predicts resistance with sensitivity/specificity of 82.6%/98.5% (independent validation set, n=1,609); sensitivity is lower here, probably because of limited understanding of the underlying genetic mechanisms. We give evidence that minor alleles improve detection of extremely drug-resistant strains, and demonstrate feasibility of the use of emerging single-molecule nanopore sequencing techniques for these purposes. PMID:26686880

  8. Rapid antibiotic-resistance predictions from genome sequence data for Staphylococcus aureus and Mycobacterium tuberculosis.

    PubMed

    Bradley, Phelim; Gordon, N Claire; Walker, Timothy M; Dunn, Laura; Heys, Simon; Huang, Bill; Earle, Sarah; Pankhurst, Louise J; Anson, Luke; de Cesare, Mariateresa; Piazza, Paolo; Votintseva, Antonina A; Golubchik, Tanya; Wilson, Daniel J; Wyllie, David H; Diel, Roland; Niemann, Stefan; Feuerriegel, Silke; Kohl, Thomas A; Ismail, Nazir; Omar, Shaheed V; Smith, E Grace; Buck, David; McVean, Gil; Walker, A Sarah; Peto, Tim E A; Crook, Derrick W; Iqbal, Zamin

    2015-01-01

    The rise of antibiotic-resistant bacteria has led to an urgent need for rapid detection of drug resistance in clinical samples, and improvements in global surveillance. Here we show how de Bruijn graph representation of bacterial diversity can be used to identify species and resistance profiles of clinical isolates. We implement this method for Staphylococcus aureus and Mycobacterium tuberculosis in a software package ('Mykrobe predictor') that takes raw sequence data as input, and generates a clinician-friendly report within 3 minutes on a laptop. For S. aureus, the error rates of our method are comparable to gold-standard phenotypic methods, with sensitivity/specificity of 99.1%/99.6% across 12 antibiotics (using an independent validation set, n=470). For M. tuberculosis, our method predicts resistance with sensitivity/specificity of 82.6%/98.5% (independent validation set, n=1,609); sensitivity is lower here, probably because of limited understanding of the underlying genetic mechanisms. We give evidence that minor alleles improve detection of extremely drug-resistant strains, and demonstrate feasibility of the use of emerging single-molecule nanopore sequencing techniques for these purposes.

  9. Insights from the predicted epitope similarity between Mycobacterium tuberculosis virulent factors and its human homologs

    PubMed Central

    Gutlapalli, Venkata Ravi; Sykam, Aparna; Nayarisseri, Anuraj; Suneetha, Sujai; Suneetha, Lavanya M

    2015-01-01

    Mycobacterium tuberculosis is known to be associated with several autoimmune diseases such as systemic lupus erythematous, rheumatoid arthritis and multiple sclerosis. This is attributed to sequence similarity between virulent factors and human proteins. Therefore, it is of interest to identify such regions in the virulent factors to assess potential autoimmune related information. M. tb specific virulent factors were downloaded from the VFDB database and its human homologs were identified using the sequence comparison search tool BLASTP. Both virulent proteins and their corresponding human homologs were further scanned for epitopes (B cell and HLA class I and II allele specific) using prediction programs (BCPRED and NETMHC). Data shows the presence of matching 22 B-cell, 79 HLA class II and 16 HLA class I specific predicted epitopes in these virulent factors having human homologs. A known peptide (HAFYLQYKNVKVDFA) associated with autoimmune atopic dermatitis is shown in the superoxide dismutase homolog structures of the bacterium (PDB ID: 1IDS) and human (PDB ID: 2QKC). This data provides insight into the understanding of infection-associated auto-immunity PMID:26770024

  10. Predicting extensively drug-resistant Mycobacterium tuberculosis phenotypes with genetic mutations.

    PubMed

    Rodwell, Timothy C; Valafar, Faramarz; Douglas, James; Qian, Lishi; Garfein, Richard S; Chawla, Ashu; Torres, Jessica; Zadorozhny, Victoria; Kim, Min Soo; Hoshide, Matt; Catanzaro, Donald; Jackson, Lynn; Lin, Grace; Desmond, Edward; Rodrigues, Camilla; Eisenach, Kathy; Victor, Thomas C; Ismail, Nazir; Crudu, Valeru; Gler, Maria Tarcela; Catanzaro, Antonino

    2014-03-01

    Molecular diagnostic methods based on the detection of mutations conferring drug resistance are promising technologies for rapidly detecting multidrug-/extensively drug-resistant tuberculosis (M/XDR TB), but large studies of mutations as markers of resistance are rare. The Global Consortium for Drug-Resistant TB Diagnostics analyzed 417 Mycobacterium tuberculosis isolates from multinational sites with a high prevalence of drug resistance to determine the sensitivities and specificities of mutations associated with M/XDR TB to inform the development of rapid diagnostic methods. We collected M/XDR TB isolates from regions of high TB burden in India, Moldova, the Philippines, and South Africa. The isolates underwent standardized phenotypic drug susceptibility testing (DST) to isoniazid (INH), rifampin (RIF), moxifloxacin (MOX), ofloxacin (OFX), amikacin (AMK), kanamycin (KAN), and capreomycin (CAP) using MGIT 960 and WHO-recommended critical concentrations. Eight genes (katG, inhA, rpoB, gyrA, gyrB, rrs, eis, and tlyA) were sequenced using Sanger sequencing. Three hundred seventy isolates were INHr, 356 were RIFr, 292 were MOXr/OFXr, 230 were AMKr, 219 were CAPr, and 286 were KANr. Four single nucleotide polymorphisms (SNPs) in katG/inhA had a combined sensitivity of 96% and specificities of 97 to 100% for the detection of INHr. Eleven SNPs in rpoB had a combined sensitivity of 98% for RIFr. Eight SNPs in gyrA codons 88 to 94 had sensitivities of 90% for MOXr/OFXr. The rrs 1401/1484 SNPs had 89 to 90% sensitivity for detecting AMKr/CAPr but 71% sensitivity for KANr. Adding eis promoter SNPs increased the sensitivity to 93% for detecting AMKr and to 91% for detecting KANr. Approximately 30 SNPs in six genes predicted clinically relevant XDR-TB phenotypes with 90 to 98% sensitivity and almost 100% specificity.

  11. pncA gene expression and prediction factors on pyrazinamide resistance in Mycobacterium tuberculosis.

    PubMed

    Sheen, Patricia; Lozano, Katherine; Gilman, Robert H; Valencia, Hugo J; Loli, Sebastian; Fuentes, Patricia; Grandjean, Louis; Zimic, Mirko

    2013-09-01

    Mutations in the pyrazinamidase (PZAse) coding gene, pncA, have been considered as the main cause of pyrazinamide (PZA) resistance in Mycobacterium tuberculosis. However, recent studies suggest there is no single mechanism of resistance to PZA. The pyrazinoic acid (POA) efflux rate is the basis of the PZA susceptibility Wayne test, and its quantitative measurement has been found to be a highly sensitive and specific predictor of PZA resistance. Based on biological considerations, the POA efflux rate is directly determined by the PZAse activity, the level of pncA expression, and the efficiency of the POA efflux pump system. This study analyzes the individual and the adjusted contribution of PZAse activity, pncA expression and POA efflux rate on PZA resistance. Thirty M. tuberculosis strains with known microbiological PZA susceptibility or resistance were analyzed. For each strain, PZAse was recombinantly produced and its enzymatic activity measured. The level of pncA mRNA was estimated by quantitative RT-PCR, and the POA efflux rate was determined. Mutations in the pncA promoter were detected by DNA sequencing. All factors were evaluated by multiple regression analysis to determine their adjusted effects on the level of PZA resistance. Low level of pncA expression associated to mutations in the pncA promoter region was observed in pncA wild type resistant strains. POA efflux rate was the best predictor after adjusting for the other factors, followed by PZAse activity. These results suggest that tests which rely on pncA mutations or PZAse activity are likely to be less predictive of real PZA resistance than tests which measure the rate of POA efflux. This should be further analyzed in light of the development of alternate assays to determine PZA resistance. PMID:23867321

  12. pncA gene expression and prediction factors on pyrazinamide resistance in Mycobacterium tuberculosis.

    PubMed

    Sheen, Patricia; Lozano, Katherine; Gilman, Robert H; Valencia, Hugo J; Loli, Sebastian; Fuentes, Patricia; Grandjean, Louis; Zimic, Mirko

    2013-09-01

    Mutations in the pyrazinamidase (PZAse) coding gene, pncA, have been considered as the main cause of pyrazinamide (PZA) resistance in Mycobacterium tuberculosis. However, recent studies suggest there is no single mechanism of resistance to PZA. The pyrazinoic acid (POA) efflux rate is the basis of the PZA susceptibility Wayne test, and its quantitative measurement has been found to be a highly sensitive and specific predictor of PZA resistance. Based on biological considerations, the POA efflux rate is directly determined by the PZAse activity, the level of pncA expression, and the efficiency of the POA efflux pump system. This study analyzes the individual and the adjusted contribution of PZAse activity, pncA expression and POA efflux rate on PZA resistance. Thirty M. tuberculosis strains with known microbiological PZA susceptibility or resistance were analyzed. For each strain, PZAse was recombinantly produced and its enzymatic activity measured. The level of pncA mRNA was estimated by quantitative RT-PCR, and the POA efflux rate was determined. Mutations in the pncA promoter were detected by DNA sequencing. All factors were evaluated by multiple regression analysis to determine their adjusted effects on the level of PZA resistance. Low level of pncA expression associated to mutations in the pncA promoter region was observed in pncA wild type resistant strains. POA efflux rate was the best predictor after adjusting for the other factors, followed by PZAse activity. These results suggest that tests which rely on pncA mutations or PZAse activity are likely to be less predictive of real PZA resistance than tests which measure the rate of POA efflux. This should be further analyzed in light of the development of alternate assays to determine PZA resistance.

  13. pncA gene expression and prediction factors on pyrazinamide resistance in Mycobacterium tuberculosis

    PubMed Central

    Sheen, Patricia; Lozano, Katherine; Gilman, Robert H.; Valencia, Hugo J.; Loli, Sebastian; Fuentes, Patricia; Grandjean, Louis; Zimic, Mirko

    2013-01-01

    Summary Background Mutations in the pyrazinamidase (PZAse) coding gene, pncA, have been considered as the main cause of pyrazinamide (PZA) resistance in Mycobacterium tuberculosis. However, recent studies suggest there is no single mechanism of resistance to PZA. The pyrazinoic acid (POA) efflux rate is the basis of the PZA susceptibility Wayne test, and its quantitative measurement has been found to be a highly sensitive and specific predictor of PZA resistance. Based on biological considerations, the POA efflux rate is directly determined by the PZAse activity, the level of pncA expression, and the efficiency of the POA efflux pump system. Objective This study analyzes the individual and the adjusted contribution of PZAse activity, pncA expression and POA efflux rate on PZA resistance. Methods Thirty M. tuberculosis strains with known microbiological PZA susceptibility or resistance were analyzed. For each strain, PZAse was recombinantly produced and its enzymatic activity measured. The level of pncA mRNA was estimated by quantitative RT-PCR, and the POA efflux rate was determined. Mutations in the pncA promoter were detected by DNA sequencing. All factors were evaluated by multiple regression analysis to determine their adjusted effects on the level of PZA resistance. Findings Low level of pncA expression associated to mutations in the pncA promoter region was observed in pncA wild type resistant strains. POA efflux rate was the best predictor after adjusting for the other factors, followed by PZAse activity. These results suggest that tests which rely on pncA mutations or PZAse activity are likely to be less predictive of real PZA resistance than tests which measure the rate of POA efflux. This should be further analyzed in light of the development of alternate assays to determine PZA resistance. PMID:23867321

  14. Hypoxia: a window into Mycobacterium tuberculosis latency.

    PubMed

    Rustad, Tige R; Sherrid, Ashley M; Minch, Kyle J; Sherman, David R

    2009-08-01

    Tuberculosis is a massive public health problem on a global scale and the success of Mycobacterium tuberculosis is linked to its ability to persist within humans for long periods without causing any overt disease symptoms. Hypoxia is predicted to be a key host-induced stress limiting growth of the pathogen in vivo. However, multiple studies in vitro and in vivo indicate that M. tuberculosis adapts to oxygen limitation by entering into a metabolically altered state, while awaiting the opportunity to reactivate. Molecular signatures of bacteria adapted to hypoxia in vitro are accumulating, although correlations to human disease are only now being established. Similarly, defining the mechanisms that control this adaptation is an active area of research. In this review we discuss the historical precedents linking hypoxia and latency, and the gathering knowledge of M. tuberculosis hypoxic responses. We also examine the role of these responses in tuberculosis latency, and identify promising avenues for future studies.

  15. Computational and Empirical Studies Predict Mycobacterium tuberculosis-Specific T Cells as a Biomarker for Infection Outcome.

    PubMed

    Marino, Simeone; Gideon, Hannah P; Gong, Chang; Mankad, Shawn; McCrone, John T; Lin, Philana Ling; Linderman, Jennifer J; Flynn, JoAnne L; Kirschner, Denise E

    2016-04-01

    Identifying biomarkers for tuberculosis (TB) is an ongoing challenge in developing immunological correlates of infection outcome and protection. Biomarker discovery is also necessary for aiding design and testing of new treatments and vaccines. To effectively predict biomarkers for infection progression in any disease, including TB, large amounts of experimental data are required to reach statistical power and make accurate predictions. We took a two-pronged approach using both experimental and computational modeling to address this problem. We first collected 200 blood samples over a 2- year period from 28 non-human primates (NHP) infected with a low dose of Mycobacterium tuberculosis. We identified T cells and the cytokines that they were producing (single and multiple) from each sample along with monkey status and infection progression data. Machine learning techniques were used to interrogate the experimental NHP datasets without identifying any potential TB biomarker. In parallel, we used our extensive novel NHP datasets to build and calibrate a multi-organ computational model that combines what is occurring at the site of infection (e.g., lung) at a single granuloma scale with blood level readouts that can be tracked in monkeys and humans. We then generated a large in silico repository of in silico granulomas coupled to lymph node and blood dynamics and developed an in silico tool to scale granuloma level results to a full host scale to identify what best predicts Mycobacterium tuberculosis (Mtb) infection outcomes. The analysis of in silico blood measures identifies Mtb-specific frequencies of effector T cell phenotypes at various time points post infection as promising indicators of infection outcome. We emphasize that pairing wetlab and computational approaches holds great promise to accelerate TB biomarker discovery. PMID:27065304

  16. Computational and Empirical Studies Predict Mycobacterium tuberculosis-Specific T Cells as a Biomarker for Infection Outcome

    PubMed Central

    Gong, Chang; Mankad, Shawn; McCrone, John T.; Lin, Philana Ling; Linderman, Jennifer J.; Flynn, JoAnne L.; Kirschner, Denise E.

    2016-01-01

    Identifying biomarkers for tuberculosis (TB) is an ongoing challenge in developing immunological correlates of infection outcome and protection. Biomarker discovery is also necessary for aiding design and testing of new treatments and vaccines. To effectively predict biomarkers for infection progression in any disease, including TB, large amounts of experimental data are required to reach statistical power and make accurate predictions. We took a two-pronged approach using both experimental and computational modeling to address this problem. We first collected 200 blood samples over a 2- year period from 28 non-human primates (NHP) infected with a low dose of Mycobacterium tuberculosis. We identified T cells and the cytokines that they were producing (single and multiple) from each sample along with monkey status and infection progression data. Machine learning techniques were used to interrogate the experimental NHP datasets without identifying any potential TB biomarker. In parallel, we used our extensive novel NHP datasets to build and calibrate a multi-organ computational model that combines what is occurring at the site of infection (e.g., lung) at a single granuloma scale with blood level readouts that can be tracked in monkeys and humans. We then generated a large in silico repository of in silico granulomas coupled to lymph node and blood dynamics and developed an in silico tool to scale granuloma level results to a full host scale to identify what best predicts Mycobacterium tuberculosis (Mtb) infection outcomes. The analysis of in silico blood measures identifies Mtb-specific frequencies of effector T cell phenotypes at various time points post infection as promising indicators of infection outcome. We emphasize that pairing wetlab and computational approaches holds great promise to accelerate TB biomarker discovery. PMID:27065304

  17. Computational and Empirical Studies Predict Mycobacterium tuberculosis-Specific T Cells as a Biomarker for Infection Outcome.

    PubMed

    Marino, Simeone; Gideon, Hannah P; Gong, Chang; Mankad, Shawn; McCrone, John T; Lin, Philana Ling; Linderman, Jennifer J; Flynn, JoAnne L; Kirschner, Denise E

    2016-04-01

    Identifying biomarkers for tuberculosis (TB) is an ongoing challenge in developing immunological correlates of infection outcome and protection. Biomarker discovery is also necessary for aiding design and testing of new treatments and vaccines. To effectively predict biomarkers for infection progression in any disease, including TB, large amounts of experimental data are required to reach statistical power and make accurate predictions. We took a two-pronged approach using both experimental and computational modeling to address this problem. We first collected 200 blood samples over a 2- year period from 28 non-human primates (NHP) infected with a low dose of Mycobacterium tuberculosis. We identified T cells and the cytokines that they were producing (single and multiple) from each sample along with monkey status and infection progression data. Machine learning techniques were used to interrogate the experimental NHP datasets without identifying any potential TB biomarker. In parallel, we used our extensive novel NHP datasets to build and calibrate a multi-organ computational model that combines what is occurring at the site of infection (e.g., lung) at a single granuloma scale with blood level readouts that can be tracked in monkeys and humans. We then generated a large in silico repository of in silico granulomas coupled to lymph node and blood dynamics and developed an in silico tool to scale granuloma level results to a full host scale to identify what best predicts Mycobacterium tuberculosis (Mtb) infection outcomes. The analysis of in silico blood measures identifies Mtb-specific frequencies of effector T cell phenotypes at various time points post infection as promising indicators of infection outcome. We emphasize that pairing wetlab and computational approaches holds great promise to accelerate TB biomarker discovery.

  18. Computational and empirical studies predict Mycobacterium tuberculosis-specific T cells as a biomarker for infection outcome

    DOE PAGES

    Marino, Simeone; Gideon, Hannah P.; Gong, Chang; Mankad, Shawn; McCrone, John T.; Lin, Philana Ling; Linderman, Jennifer J.; Flynn, JoAnne L.; Kirschner, Denise E.

    2016-04-11

    Identifying biomarkers for tuberculosis (TB) is an ongoing challenge in developing immunological correlates of infection outcome and protection. Biomarker discovery is also necessary for aiding design and testing of new treatments and vaccines. To effectively predict biomarkers for infection progression in any disease, including TB, large amounts of experimental data are required to reach statistical power and make accurate predictions. We took a two-pronged approach using both experimental and computational modeling to address this problem. We first collected 200 blood samples over a 2-year period from 28 non-human primates (NHP) infected with a low dose of Mycobacterium tuberculosis. We identifiedmore » T cells and the cytokines that they were producing (single and multiple) from each sample along with monkey status and infection progression data. Machine learning techniques were used to interrogate the experimental NHP datasets without identifying any potential TB biomarker. In parallel, we used our extensive novel NHP datasets to build and calibrate a multi-organ computational model that combines what is occurring at the site of infection (e.g., lung) at a single granuloma scale with blood level readouts that can be tracked in monkeys and humans. We then generated a large in silico repository of in silico granulomas coupled to lymph node and blood dynamics and developed an in silico tool to scale granuloma level results to a full host scale to identify what best predicts Mycobacterium tuberculosis (Mtb) infection outcomes. The analysis of in silico blood measures identifies Mtb-specific frequencies of effector T cell phenotypes at various time points post infection as promising indicators of infection outcome. As a result, we emphasize that pairing wetlab and computational approaches holds great promise to accelerate TB biomarker discovery.« less

  19. Predictive value of molecular drug resistance testing of Mycobacterium tuberculosis isolates in Valle del Cauca, Colombia.

    PubMed

    Ferro, Beatriz E; García, Pamela K; Nieto, Luisa Maria; van Soolingen, Dick

    2013-07-01

    Previous evaluations of the molecular GenoType tests have promoted their use to detect resistance to first- and second-line antituberculosis drugs in different geographical regions. However, there are known geographic variations in the mutations associated with drug resistance in Mycobacterium tuberculosis, and especially in South America, there is a paucity of information regarding the frequencies and types of mutations associated with resistance to first- and second-line antituberculosis drugs. We therefore evaluated the performance of the GenoType kits in this region by testing 228 M. tuberculosis isolates in Colombia, including 134 resistant and 94 pansusceptible strains. Overall, the sensitivity and specificity of the GenoType MTBDRplus test ranged from 92 to 96% and 97 to 100%, respectively; the agreement index was optimal (Cohen's kappa, >0.8). The sensitivity of the GenoType MTBDRsl test ranged from 84 to 100% and the specificity from 88 to 100%. The most common mutations were katG S315T1, rpoB S531L, embB M306V, gyrA D94G, and rrs A1401G. Our results reflect the utility of the GenoType tests in Colombia; however, as some discordance still exists between the conventional and molecular approaches in resistance testing, we adhere to the recommendation that the GenoType tests serve as early guides for therapy, followed by phenotypic drug susceptibility testing for all cases.

  20. Predictive modeling targets thymidylate synthase ThyX in Mycobacterium tuberculosis

    PubMed Central

    Djaout, Kamel; Singh, Vinayak; Boum, Yap; Katawera, Victoria; Becker, Hubert F.; Bush, Natassja G.; Hearnshaw, Stephen J.; Pritchard, Jennifer E.; Bourbon, Pauline; Madrid, Peter B.; Maxwell, Anthony; Mizrahi, Valerie; Myllykallio, Hannu; Ekins, Sean

    2016-01-01

    There is an urgent need to identify new treatments for tuberculosis (TB), a major infectious disease caused by Mycobacterium tuberculosis (Mtb), which results in 1.5 million deaths each year. We have targeted two essential enzymes in this organism that are promising for antibacterial therapy and reported to be inhibited by naphthoquinones. ThyX is an essential thymidylate synthase that is mechanistically and structurally unrelated to the human enzyme. DNA gyrase is a DNA topoisomerase present in bacteria and plants but not animals. The current study set out to understand the structure-activity relationships of these targets in Mtb using a combination of cheminformatics and in vitro screening. Here, we report the identification of new Mtb ThyX inhibitors, 2-chloro-3-(4-methanesulfonylpiperazin-1-yl)-1,4-dihydronaphthalene-1,4-dione) and idebenone, which show modest whole-cell activity and appear to act, at least in part, by targeting ThyX in Mtb. PMID:27283217

  1. Predictive Value of Molecular Drug Resistance Testing of Mycobacterium tuberculosis Isolates in Valle del Cauca, Colombia

    PubMed Central

    García, Pamela K.; Nieto, Luisa Maria; van Soolingen, Dick

    2013-01-01

    Previous evaluations of the molecular GenoType tests have promoted their use to detect resistance to first- and second-line antituberculosis drugs in different geographical regions. However, there are known geographic variations in the mutations associated with drug resistance in Mycobacterium tuberculosis, and especially in South America, there is a paucity of information regarding the frequencies and types of mutations associated with resistance to first- and second-line antituberculosis drugs. We therefore evaluated the performance of the GenoType kits in this region by testing 228 M. tuberculosis isolates in Colombia, including 134 resistant and 94 pansusceptible strains. Overall, the sensitivity and specificity of the GenoType MTBDRplus test ranged from 92 to 96% and 97 to 100%, respectively; the agreement index was optimal (Cohen's kappa, >0.8). The sensitivity of the GenoType MTBDRsl test ranged from 84 to 100% and the specificity from 88 to 100%. The most common mutations were katG S315T1, rpoB S531L, embB M306V, gyrA D94G, and rrs A1401G. Our results reflect the utility of the GenoType tests in Colombia; however, as some discordance still exists between the conventional and molecular approaches in resistance testing, we adhere to the recommendation that the GenoType tests serve as early guides for therapy, followed by phenotypic drug susceptibility testing for all cases. PMID:23658272

  2. Predictive modeling targets thymidylate synthase ThyX in Mycobacterium tuberculosis.

    PubMed

    Djaout, Kamel; Singh, Vinayak; Boum, Yap; Katawera, Victoria; Becker, Hubert F; Bush, Natassja G; Hearnshaw, Stephen J; Pritchard, Jennifer E; Bourbon, Pauline; Madrid, Peter B; Maxwell, Anthony; Mizrahi, Valerie; Myllykallio, Hannu; Ekins, Sean

    2016-01-01

    There is an urgent need to identify new treatments for tuberculosis (TB), a major infectious disease caused by Mycobacterium tuberculosis (Mtb), which results in 1.5 million deaths each year. We have targeted two essential enzymes in this organism that are promising for antibacterial therapy and reported to be inhibited by naphthoquinones. ThyX is an essential thymidylate synthase that is mechanistically and structurally unrelated to the human enzyme. DNA gyrase is a DNA topoisomerase present in bacteria and plants but not animals. The current study set out to understand the structure-activity relationships of these targets in Mtb using a combination of cheminformatics and in vitro screening. Here, we report the identification of new Mtb ThyX inhibitors, 2-chloro-3-(4-methanesulfonylpiperazin-1-yl)-1,4-dihydronaphthalene-1,4-dione) and idebenone, which show modest whole-cell activity and appear to act, at least in part, by targeting ThyX in Mtb. PMID:27283217

  3. Mycobacterium tuberculosis produces pili during human infection

    PubMed Central

    Alteri, Christopher J.; Xicohténcatl-Cortes, Juan; Hess, Sonja; Caballero-Olín, Guillermo; Girón, Jorge A.; Friedman, Richard L.

    2007-01-01

    Mycobacterium tuberculosis is responsible for nearly 3 million human deaths worldwide every year. Understanding the mechanisms and bacterial factors responsible for the ability of M. tuberculosis to cause disease in humans is critical for the development of improved treatment strategies. Many bacterial pathogens use pili as adherence factors to colonize the host. We discovered that M. tuberculosis produces fine (2- to 3-nm-wide), aggregative, flexible pili that are recognized by IgG antibodies contained in sera obtained from patients with active tuberculosis, indicating that the bacilli produce pili or pili-associated antigen during human infection. Purified M. tuberculosis pili (MTP) are composed of low-molecular-weight protein subunits encoded by the predicted M. tuberculosis H37Rv ORF, designated Rv3312A. MTP bind to the extracellular matrix protein laminin in vitro, suggesting that MTP possess adhesive properties. Isogenic mtp mutants lost the ability to produce Mtp in vitro and demonstrated decreased laminin-binding capabilities. MTP shares morphological, biochemical, and functional properties attributed to bacterial pili, especially with curli amyloid fibers. Thus, we propose that MTP are previously unidentified host-colonization factors of M. tuberculosis. PMID:17360408

  4. Whole-genome sequencing for prediction of Mycobacterium tuberculosis drug susceptibility and resistance: a retrospective cohort study

    PubMed Central

    Walker, Timothy M; Kohl, Thomas A; Omar, Shaheed V; Hedge, Jessica; Del Ojo Elias, Carlos; Bradley, Phelim; Iqbal, Zamin; Feuerriegel, Silke; Niehaus, Katherine E; Wilson, Daniel J; Clifton, David A; Kapatai, Georgia; Ip, Camilla L C; Bowden, Rory; Drobniewski, Francis A; Allix-Béguec, Caroline; Gaudin, Cyril; Parkhill, Julian; Diel, Roland; Supply, Philip; Crook, Derrick W; Smith, E Grace; Walker, A Sarah; Ismail, Nazir; Niemann, Stefan; Peto, Tim E A

    2015-01-01

    Summary Background Diagnosing drug-resistance remains an obstacle to the elimination of tuberculosis. Phenotypic drug-susceptibility testing is slow and expensive, and commercial genotypic assays screen only common resistance-determining mutations. We used whole-genome sequencing to characterise common and rare mutations predicting drug resistance, or consistency with susceptibility, for all first-line and second-line drugs for tuberculosis. Methods Between Sept 1, 2010, and Dec 1, 2013, we sequenced a training set of 2099 Mycobacterium tuberculosis genomes. For 23 candidate genes identified from the drug-resistance scientific literature, we algorithmically characterised genetic mutations as not conferring resistance (benign), resistance determinants, or uncharacterised. We then assessed the ability of these characterisations to predict phenotypic drug-susceptibility testing for an independent validation set of 1552 genomes. We sought mutations under similar selection pressure to those characterised as resistance determinants outside candidate genes to account for residual phenotypic resistance. Findings We characterised 120 training-set mutations as resistance determining, and 772 as benign. With these mutations, we could predict 89·2% of the validation-set phenotypes with a mean 92·3% sensitivity (95% CI 90·7–93·7) and 98·4% specificity (98·1–98·7). 10·8% of validation-set phenotypes could not be predicted because uncharacterised mutations were present. With an in-silico comparison, characterised resistance determinants had higher sensitivity than the mutations from three line-probe assays (85·1% vs 81·6%). No additional resistance determinants were identified among mutations under selection pressure in non-candidate genes. Interpretation A broad catalogue of genetic mutations enable data from whole-genome sequencing to be used clinically to predict drug resistance, drug susceptibility, or to identify drug phenotypes that cannot yet be genetically

  5. 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

  6. Non-human sources of Mycobacterium tuberculosis.

    PubMed

    Ghodbane, Ramzi; Drancourt, Michel

    2013-11-01

    Mycobacterium tuberculosis is a successful pathogen responsible for the vast majority of deadly tuberculosis cases in humans. It rests in a dormant form in contaminated people who constitute the reservoir with airborne interhuman transmission during pulmonary tuberculosis. M. tuberculosis is therefore regarded majoritary as a human pathogen. Here, we review the evidence for anthroponotic M. tuberculosis infection in non-human primates, other mammals and psittacines. Some infected animals may be sources for zoonotic tuberculosis caused by M. tuberculosis, with wild life trade and zoos being amplifying factors. Moreover, living animals and cadavers can scatter M. tuberculosis in the environment where it could survive for extended periods of time in soil where amoebae could play a role. Although marginal in the epidemiology of human tuberculosis, these data indicate that M. tuberculosis is not uniquely adapted to humans.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  8. Drug Resistance Mechanisms in Mycobacterium tuberculosis

    PubMed Central

    Palomino, Juan Carlos; Martin, Anandi

    2014-01-01

    Tuberculosis (TB) is a serious public health problem worldwide. Its situation is worsened by the presence of multidrug resistant (MDR) strains of Mycobacterium tuberculosis, the causative agent of the disease. In recent years, even more serious forms of drug resistance have been reported. A better knowledge of the mechanisms of drug resistance of M. tuberculosis and the relevant molecular mechanisms involved will improve the available techniques for rapid drug resistance detection and will help to explore new targets for drug activity and development. This review article discusses the mechanisms of action of anti-tuberculosis drugs and the molecular basis of drug resistance in M. tuberculosis. PMID:27025748

  9. Drug Resistance Mechanisms in Mycobacterium tuberculosis.

    PubMed

    Palomino, Juan Carlos; Martin, Anandi

    2014-01-01

    Tuberculosis (TB) is a serious public health problem worldwide. Its situation is worsened by the presence of multidrug resistant (MDR) strains of Mycobacterium tuberculosis, the causative agent of the disease. In recent years, even more serious forms of drug resistance have been reported. A better knowledge of the mechanisms of drug resistance of M. tuberculosis and the relevant molecular mechanisms involved will improve the available techniques for rapid drug resistance detection and will help to explore new targets for drug activity and development. This review article discusses the mechanisms of action of anti-tuberculosis drugs and the molecular basis of drug resistance in M. tuberculosis. PMID:27025748

  10. 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

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

    PubMed Central

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

    1987-01-01

    The in vitro susceptibility of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium avium, Mycobacterium fortuitum, and Mycobacterium chelonae (M. chelonei) to ticarcillin in combination with calvulanic acid (CA) was studied by the agar dilution method. All the M. tuberculosis, M. bovis, and M. africanum strains were inhibited at a ticarcillin concentration of 32 micrograms/ml or lower in combination with 5 micrograms of CA. M. chelonae and M. avium strains proved resistant to more than 128 micrograms of ticarcillin plus 5 micrograms of CA per ml. M. fortuitum strains needed 128 micrograms of ticarcillin plus 5 micrograms of CA to inhibit approximately 30% of the isolates. PMID:3105441

  12. Dormancy models for Mycobacterium tuberculosis: A minireview

    PubMed Central

    Alnimr, Amani M.

    2015-01-01

    Dormancy models for Mycobacterium tuberculosis play important roles in understanding various aspects of tuberculosis pathogenesis and in the testing of novel therapeutic regimens. By simulating the latent tuberculosis infection, in which the bacteria exist in a non-replicative state, the models demonstrate reduced susceptibility to antimycobacterial agents. This minireview outlines the models available for simulating latent tuberculosis both in vitro and in several animal species. Additionally, this minireview discusses the advantages and disadvantages of these models for investigating the bacterial subpopulations and susceptibilities to sterilization by various antituberculosis drugs. PMID:26413043

  13. Rapid and sensitive identification of Mycobacterium tuberculosis.

    PubMed Central

    Knisley, C V; Damato, J J; McClatchy, J K; Brennan, P J

    1985-01-01

    The fatty acid constituents of 14 species of Mycobacterium (14 isolates) and one isolate each of Corynebacterium xerosis, Nocardia asteroides, and Streptomyces albus were examined with the purpose of distinguishing Mycobacterium tuberculosis from other acid-fast bacilli. Combined thin-layer chromatography (TLC) of methyl mycolates and gas-liquid chromatography (GLC) of shorter-chain fatty acid esters provided an unequivocal identification of M. tuberculosis in a matter of 2 to 3 days. The methodology included rapid and simplified procedures for methanolysis and extraction of bacterial lipids with equally facilitated GLC and TLC analyses. These studies were performed with 0.5 to 1.0 mg of dry bacterial cells (approximately 2.5 X 10(7) CFU). When applied to 100 unknown cultures, the methodology with combined TLC-GLC correctly identified all 49 of the M. tuberculosis-Mycobacterium bovis cultures and a variety of other mycobacterium taxa. It was also interesting to note that 28 of 39 (72%) of the nontuberculous mycobacteria were correctly identified. An additional five species were tentatively identified as belonging to either of two species (Mycobacterium malmoense, Mycobacterium terrae), but in all cases, the two species belonged to the same Runyon group. All six nonmycobacterial species were differentiated from the mycobacteria studied. Images PMID:3932458

  14. Diterpene production in Mycobacterium tuberculosis

    PubMed Central

    Prach, Lisa; Kirby, James; Keasling, Jay D.; Alber, Tom

    2011-01-01

    Diterpenes are a structurally diverse class of molecules common in plants, although they are very rarely found in bacteria. We report the identification in Mycobacterium tuberculosis (Mtb) of three diterpenes proposed to promote phagolysosome maturation arrest. MS analysis reveals that these diterpenes are novel compounds not previously identified in other organisms. The diterpene with highest abundance in Mtb has a mass fragmentation pattern identical to edaxadiene, which is produced in vitro from geranylgeranyl diphosphate by the enzymes Rv3377c and Rv3378c [Mann FM et al. (2009) J Am Chem Soc 131, 17526–17527]. A second diterpene found in Mtb has a similar mass spectrum, and is always observed in the same proportion relative to edaxadiene, indicating that it is a side product of the Rv3378c reaction in vivo. We name this second diterpene olefin edaxadiene B. The least abundant of the three diterpenes in Mtb extracts is tuberculosinol, a dephosphorylated side-product of the edaxadiene pathway intermediate produced by Rv3377c [Nakano C et al. (2009) Chembiochem 10, 2060–2071; Nakano C et al. (2005) Chem Commun (Camb) 8, 1016–1018]. A frameshift in Rv3377c in Mtb completely eliminates diterpene production, whereas expression of Rv3377c and Rv3378c in the nonpathogenic M. smegmatis is sufficient to produce edaxadiene and edaxadiene B. These studies define the pathway of edaxadiene and edaxadiene B biosynthesis in vivo. Rv3377c and Rv3378c are unique to Mtb and M. bovis, making them candidates for selective therapeutics and diagnostics. PMID:20670276

  15. Radioimmunoassay of tuberculoprotein derived from Mycobacterium tuberculosis.

    PubMed Central

    Straus, E; Wu, N

    1980-01-01

    A radioimmunoassay was developed for constituent of the purified-protein derivative obtained from cultures of Mycobacterium tuberculosis. Crossreacting immunoreactive material was detected in cultures of other mycobacterial species, but no immunoreactivity was present in cultures of various fungal and bacterial species. The development of specific radioimmunoassays for tuberculoproteins offers a new research and diagnostic approach. Images PMID:6933481

  16. Functional, structural and epitopic prediction of hypothetical proteins of Mycobacterium tuberculosis H37Rv: An in silico approach for prioritizing the targets.

    PubMed

    Gazi, Md Amran; Kibria, Mohammad Golam; Mahfuz, Mustafa; Islam, Md Rezaul; Ghosh, Prakash; Afsar, Md Nure Alam; Khan, Md Arif; Ahmed, Tahmeed

    2016-10-15

    The global control of tuberculosis (TB) remains a great challenge from the standpoint of diagnosis, detection of drug resistance, and treatment. Major serodiagnostic limitations include low sensitivity and high cost in detecting TB. On the other hand, treatment measures are often hindered by low efficacies of commonly used drugs and resistance developed by the bacteria. Hence, there is a need to look into newer diagnostic and therapeutic targets. The proteome information available suggests that among the 3906 proteins in Mycobacterium tuberculosis H37Rv, about quarter remain classified as hypothetical uncharacterized set. This study involves a combination of a number of bioinformatics tools to analyze those hypothetical proteins (HPs). An entire set of 999 proteins was primarily screened for protein sequences having conserved domains with high confidence using a combination of the latest versions of protein family databases. Subsequently, 98 of such potential target proteins were extensively analyzed by means of physicochemical characteristics, protein-protein interaction, sub-cellular localization, structural similarity and functional classification. Next, we predicted antigenic proteins from the entire set and identified B and T cell epitopes of these proteins in M. tuberculosis H37Rv. We predicted the function of these HPs belong to various classes of proteins such as enzymes, transporters, receptors, structural proteins, transcription regulators and other proteins. However, the structural similarity prediction of the annotated proteins substantiated the functional classification of those proteins. Consequently, based on higher antigenicity score and sub-cellular localization, we choose two (NP_216420.1, NP_216903.1) of the antigenic proteins to exemplify B and T cell epitope prediction approach. Finally we found 15 epitopes those located partially or fully in the linear epitope region. We found 21 conformational epitopes by using Ellipro server as well. In

  17. Functional, structural and epitopic prediction of hypothetical proteins of Mycobacterium tuberculosis H37Rv: An in silico approach for prioritizing the targets.

    PubMed

    Gazi, Md Amran; Kibria, Mohammad Golam; Mahfuz, Mustafa; Islam, Md Rezaul; Ghosh, Prakash; Afsar, Md Nure Alam; Khan, Md Arif; Ahmed, Tahmeed

    2016-10-15

    The global control of tuberculosis (TB) remains a great challenge from the standpoint of diagnosis, detection of drug resistance, and treatment. Major serodiagnostic limitations include low sensitivity and high cost in detecting TB. On the other hand, treatment measures are often hindered by low efficacies of commonly used drugs and resistance developed by the bacteria. Hence, there is a need to look into newer diagnostic and therapeutic targets. The proteome information available suggests that among the 3906 proteins in Mycobacterium tuberculosis H37Rv, about quarter remain classified as hypothetical uncharacterized set. This study involves a combination of a number of bioinformatics tools to analyze those hypothetical proteins (HPs). An entire set of 999 proteins was primarily screened for protein sequences having conserved domains with high confidence using a combination of the latest versions of protein family databases. Subsequently, 98 of such potential target proteins were extensively analyzed by means of physicochemical characteristics, protein-protein interaction, sub-cellular localization, structural similarity and functional classification. Next, we predicted antigenic proteins from the entire set and identified B and T cell epitopes of these proteins in M. tuberculosis H37Rv. We predicted the function of these HPs belong to various classes of proteins such as enzymes, transporters, receptors, structural proteins, transcription regulators and other proteins. However, the structural similarity prediction of the annotated proteins substantiated the functional classification of those proteins. Consequently, based on higher antigenicity score and sub-cellular localization, we choose two (NP_216420.1, NP_216903.1) of the antigenic proteins to exemplify B and T cell epitope prediction approach. Finally we found 15 epitopes those located partially or fully in the linear epitope region. We found 21 conformational epitopes by using Ellipro server as well. In

  18. 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.

  19. 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.

  20. 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.

  1. Multidrug-resistant Mycobacterium tuberculosis: molecular perspectives.

    PubMed Central

    Rattan, A.; Kalia, A.; Ahmad, N.

    1998-01-01

    Multidrug-resistant strains of Mycobacterium tuberculosis seriously threaten tuberculosis (TB) control and prevention efforts. Molecular studies of the mechanism of action of antitubercular drugs have elucidated the genetic basis of drug resistance in M. tuberculosis. Drug resistance in M. tuberculosis is attributed primarily to the accumulation of mutations in the drug target genes; these mutations lead either to an altered target (e.g., RNA polymerase and catalase-peroxidase in rifampicin and isoniazid resistance, respectively) or to a change in titration of the drug (e.g., InhA in isoniazid resistance). Development of specific mechanism-based inhibitors and techniques to rapidly detect multidrug resistance will require further studies addressing the drug and drug-target interaction. PMID:9621190

  2. 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

  3. Systems-based approaches to probing metabolic variation within the Mycobacterium tuberculosis complex.

    PubMed

    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.

  4. 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

  5. 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

  6. 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

  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. Diagnosis and treatment of latent infection with Mycobacterium tuberculosis.

    PubMed

    Chee, Cynthia Bin-Eng; Sester, Martina; Zhang, Wenhong; Lange, Christoph

    2013-02-01

    In clinical practice, latent infection with Mycobacterium tuberculosis is defined by the presence of an M. tuberculosis-specific immune response in the absence of active tuberculosis. Targeted testing of individuals from risk groups with the tuberculin skin test or an interferon-γ release assay is currently the best method to identify those with the highest risk for progression to tuberculosis. Positive predictive values of the immunodiagnostic tests are substantially influenced by the type of test, the age of the person who is tested, the prevalence of tuberculosis in the society and the risk group to which the person belongs. As a general rule, testing should only be offered when preventive chemotherapy will be accepted in the case of a positive test result. Preventive chemotherapy can effectively protect individuals at risk from the development of tuberculosis, although at least 3 months of combination therapy or up to 9 months of monotherapy are required, and overall acceptance rate is low. Improvements of the current generation of immunodiagnostic tests could substantially lower the number of individuals that need to be treated to prevent a case of tuberculosis. If shorter treatment regimens were equally effective than those currently recommended, acceptance of preventive chemotherapy could be much improved.

  9. Targeting the histidine pathway in Mycobacterium tuberculosis.

    PubMed

    Lunardi, Juleane; Nunes, José Eduardo S; Bizarro, Cristiano V; Basso, Luiz Augusto; Santos, Diógenes Santiago; Machado, Pablo

    2013-01-01

    Worldwide, tuberculosis is the leading cause of morbidity and mortality due to a single bacterial pathogen, Mycobacterium tuberculosis (Mtb). The increasing prevalence of this disease, the emergence of multi-, extensively, and totally drug-resistant strains, complicated by co-infection with the human immunodeficiency virus, and the length of tuberculosis chemotherapy have led to an urgent and continued need for the development of new and more effective antitubercular drugs. Within this context, the L-histidine biosynthetic pathway, which converts 5-phosphoribosyl 1-pyrophosphate to L-histidine in ten enzymatic steps, has been reported as a promising target of antimicrobial agents. This pathway is found in bacteria, archaebacteria, lower eukaryotes, and plants but is absent in mammals, making these enzymes highly attractive targets for the drug design of new antimycobacterial compounds with selective toxicity. Moreover, the biosynthesis of L-histidine has been described as essential for Mtb growth in vitro. Accordingly, a comprehensive overview of Mycobacterium tuberculosis histidine pathway enzymes as attractive targets for the development of new antimycobacterial agents is provided, mainly summarizing the previously reported inhibition data for Mtb or orthologous proteins. PMID:24111909

  10. Efficacies of selected disinfectants against Mycobacterium tuberculosis.

    PubMed

    Best, M; Sattar, S A; Springthorpe, V S; Kennedy, M E

    1990-10-01

    The activities of 10 formulations as mycobactericidal agents in Mycobacterium tuberculosis-contaminated suspensions (suspension test) and stainless steel surfaces (carrier test) were investigated with sputum as the organic load. The quaternary ammonium compound, chlorhexidine gluconate, and an iodophor were ineffective in all tests. Ethanol (70%) was effective against M. tuberculosis only in suspension in the absence of sputum. Povidone-iodine was not as efficacious when the test organism was dried on a surface as it was in suspension, and its activity was further reduced in the presence of sputum. Sodium hypochlorite required a higher concentration of available chlorine to achieve an effective level of disinfection than did sodium dichloroisocyanurate. Phenol (5%) was effective under all test conditions, producing at least a 4-log10 reduction in CFU. The undiluted glutaraldehyde-phenate solution was effective against M. tuberculosis and a second test organism, Mycobacterium smegmatis, even in the presence of dried sputum, whereas the diluted solution (1:16) was only effective against M. smegmatis in the suspension test. A solution of 2% glutaraldehyde was effective against M. tuberculosis. This investigation presents tuberculocidal efficacy data generated by methods simulating actual practices of routine disinfection. PMID:2121783

  11. Implication from the predicted docked interaction of sigma H and exploration of its interaction with RNA polymerase in Mycobacterium tuberculosis.

    PubMed

    Gupta, Aayatti Mallick; Bhattacharya, Simanti; Bagchi, Angshuman; Mandal, Sukhendu

    2015-01-01

    M. tuberculosis is adapted to remain active in the extreme environmental condition due to the presence of atypical sigma factors commonly called extra cytoplasmic function (ECF) sigma factors. Among the 13 sigma factors of M. tuberculosis, 10 are regarded as the ECF sigma factor that exerts their attributes in various stress response. Therefore it is of interest to describe the structural prediction of one of the ECF sigma factors, sigma H (SigH), involved in oxidative and heat stress having interaction with the β׳ subunit of M. tuberculosis. RNA polymerase (Mtb-RNAP). The model of Mtb-SigH was build using the commercial package of Discovery Studio version 2.5 from Accelerys (San Diego, CA, USA) containing the inbuilt MODELER module and that of β׳ subunit of Mtb-RNAP using Phyre Server. Further, the protein models were docked using the fully automated web tool ClusPro (cluspro.bu.edu/login.php). Mtb-SigH is a triple helical structure having a putative DNA-binding site and the β׳ subunit of MtbRNAP consists of 18-beta sheets and 22 helices. The SigH-Mtb-RNAP β׳ interaction studies showed that Arg26, Gln19 andAsp18, residues of SigH protein are involved in binding with Arg137, Gln140, Arg152, Asn133 and Asp144 of β׳ subunit of Mtb-RNAP. The predicted model helps to explore the molecular mechanism in the control of gene regulation with a novel unique target for potential new generation inhibitor.

  12. 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

  13. 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

  14. Peptide mimotopes of Mycobacterium tuberculosis carbohydrate immunodeterminants.

    PubMed

    Gevorkian, Goar; Segura, Erika; Acero, Gonzalo; Palma, José P; Espitia, Clara; Manoutcharian, Karen; López-Marín, Luz M

    2005-04-15

    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.

  15. 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

  16. 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.

  17. 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

  18. Predicting promiscuous antigenic T cell epitopes of Mycobacterium tuberculosis mymA operon proteins binding to MHC Class I and Class II molecules.

    PubMed

    Saraav, Iti; Pandey, Kirti; Sharma, Monika; Singh, Swati; Dutta, Prasun; Bhardwaj, Anshu; Sharma, Sadhna

    2016-10-01

    Limited efficacy of Bacillus Calmette-Guérin vaccine has raised the need to explore other immunogenic candidates to develop an effective vaccine against Mycobacterium tuberculosis (Mtb). Both CD4+ and CD8+ T cells play a critical role in host immunity to Mtb. Infection of macrophages with Mtb results in upregulation of mymA operon genes thereby suggesting their importance as immune targets. In the present study, after exclusion of self-peptides mymA operon proteins of Mtb were analyzed in silico for the presence of Human Leukocyte Antigen (HLA) Class I and Class II binding peptides using Bioinformatics and molecular analysis section, NetMHC 3.4, ProPred and Immune epitope database software. Out of 56 promiscuous epitopes obtained, 41 epitopes were predicted to be antigenic for MHC Class I. In MHC Class II, out of 336 promiscuous epitopes obtained, 142 epitopes were predicted to be antigenic. The comparative bioinformatics analysis of mymA operon proteins found Rv3083 to be the best vaccine candidate. Molecular docking was performed with the most antigenic peptides of Rv3083 (LASGAASVV with alleles HLA-B51:01, HAATSGTLI with HLA-A02, IVTATGLNI and EKIHYGLKVNTA with HLA-DRB1_01:01) to study the structural basis for recognition of peptides by various HLA molecules. The software binding prediction was validated by the obtained molecular docking score of peptide-HLA complex. These peptides can be further investigated for their immunological relevance in patients of tuberculosis using major histocompatibility complex tetramer approach. PMID:27389362

  19. Disseminated Mycobacterium tuberculosis infection in a dog.

    PubMed

    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

    2013-03-01

    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

  20. Pyrimidine salvage pathway in Mycobacterium tuberculosis.

    PubMed

    Villela, A D; Sánchez-Quitian, Z A; Ducati, R G; Santos, D S; Basso, L A

    2011-01-01

    The causative agent of tuberculosis (TB), Mycobacterium tuberculosis, infects one-third of the world population. TB remains the leading cause of mortality due to a single bacterial pathogen. The worldwide increase in incidence of M. tuberculosis has been attributed to the high proliferation rates of multi and extensively drug-resistant strains, and to co-infection with the human immunodeficiency virus. There is thus a continuous requirement for studies on mycobacterial metabolism to identify promising targets for the development of new agents to combat TB. Singular characteristics of this pathogen, such as functional and structural features of enzymes involved in fundamental metabolic pathways, can be evaluated to identify possible targets for drug development. Enzymes involved in the pyrimidine salvage pathway might be attractive targets for rational drug design against TB, since this pathway is vital for all bacterial cells, and is composed of enzymes considerably different from those present in humans. Moreover, the enzymes of the pyrimidine salvage pathway might have an important role in the mycobacterial latent state, since M. tuberculosis has to recycle bases and/or nucleosides to survive in the hostile environment imposed by the host. The present review describes the enzymes of M. tuberculosis pyrimidine salvage pathway as attractive targets for the development of new antimycobacterial agents. Enzyme functional and structural data have been included to provide a broader knowledge on which to base the search for compounds with selective biological activity.

  1. 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

  2. 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.

  3. Polymorphisms of 20 regulatory proteins between Mycobacterium tuberculosis and Mycobacterium bovis.

    PubMed

    Bigi, María M; Blanco, Federico Carlos; Araújo, Flabio R; Thacker, Tyler C; Zumárraga, Martín J; Cataldi, Angel A; Soria, Marcelo A; Bigi, Fabiana

    2016-08-01

    Mycobacterium tuberculosis and Mycobacterium bovis are responsible for tuberculosis in humans and 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 other members of the MTC evolved. The genome of M. bovis is over >99.95% identical to that of M. tuberculosis but with seven deletions ranging in size from 1 to 12.7 kb. In addition, 1200 single nucleotide mutations in coding regions distinguish M. bovis from M. tuberculosis. In the present study, we assessed 75 M. tuberculosis genomes and 23 M. bovis genomes to identify non-synonymous mutations in 202 coding sequences of regulatory genes between both species. We identified species-specific variants in 20 regulatory proteins and confirmed differential expression of hypoxia-related genes between M. bovis and M. tuberculosis.

  4. 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.

  5. An Unconventional Hexacoordinated Flavohemoglobin from Mycobacterium tuberculosis*

    PubMed Central

    Gupta, Sanjay; Pawaria, Sudesh; Lu, Changyuan; Hade, Mangesh Dattu; Singh, Chaahat; Yeh, Syun-Ru; Dikshit, Kanak L.

    2012-01-01

    Being an obligate aerobe, Mycobacterium tuberculosis faces a number of energetic challenges when it encounters hypoxia and environmental stress during intracellular infection. Consequently, it has evolved innovative strategies to cope with these unfavorable conditions. Here, we report a novel flavohemoglobin (MtbFHb) from M. tuberculosis that exhibits unique features within its heme and reductase domains distinct from conventional FHbs, including the absence of the characteristic hydrogen bonding interactions within the proximal heme pocket and mutations in the FAD and NADH binding regions of the reductase domain. In contrast to conventional FHbs, it has a hexacoordinate low-spin heme with a proximal histidine ligand lacking imidazolate character and a distal heme pocket with a relatively low electrostatic potential. Additionally, MtbFHb carries a new FAD binding site in its reductase domain similar to that of d-lactate dehydrogenase (d-LDH). When overexpressed in Escherichia coli or Mycobacterium smegmatis, MtbFHb remained associated with the cell membrane and exhibited d-lactate:phenazine methosulfate reductase activity and oxidized d-lactate into pyruvate by converting the heme iron from Fe3+ to Fe2+ in a FAD-dependent manner, indicating electron transfer from d-lactate to the heme via FAD cofactor. Under oxidative stress, MtbFHb-expressing cells exhibited growth advantage with reduced levels of lipid peroxidation. Given the fact that d-lactate is a byproduct of lipid peroxidation and that M. tuberculosis lacks the gene encoding d-LDH, we propose that the novel d-lactate metabolizing activity of MtbFHb uniquely equips M. tuberculosis to balance the stress level by protecting the cell membrane from oxidative damage via cycling between the Fe3+/Fe2+ redox states. PMID:22437825

  6. Epidemiological models of Mycobacterium tuberculosis complex infections.

    PubMed

    Ozcaglar, Cagri; Shabbeer, Amina; Vandenberg, Scott L; Yener, Bülent; Bennett, Kristin P

    2012-04-01

    The resurgence of tuberculosis in the 1990s and the emergence of drug-resistant tuberculosis in the first decade of the 21st century increased the importance of epidemiological models for the disease. Due to slow progression of tuberculosis, the transmission dynamics and its long-term effects can often be better observed and predicted using simulations of epidemiological models. This study provides a review of earlier study on modeling different aspects of tuberculosis dynamics. The models simulate tuberculosis transmission dynamics, treatment, drug resistance, control strategies for increasing compliance to treatment, HIV/TB co-infection, and patient groups. The models are based on various mathematical systems, such as systems of ordinary differential equations, simulation models, and Markov Chain Monte Carlo methods. The inferences from the models are justified by case studies and statistical analysis of TB patient datasets. PMID:22387570

  7. Purine Salvage Pathway in Mycobacterium tuberculosis.

    PubMed

    Ducati, R G; Breda, A; Basso, L A; Santos, D S

    2011-01-01

    Millions of deaths worldwide are caused by the aetiological agent of tuberculosis, Mycobacterium tuberculosis. The increasing prevalence of this disease, the emergence of drug-resistant strains, and the devastating effect of human immunodeficiency virus coinfection have led to an urgent need for the development of new and more efficient antimycobacterial drugs. The modern approach to the development of new chemical compounds against complex diseases, especially the neglected endemic ones, such as tuberculosis, is based on the use of defined molecular targets. Among the advantages, this approach allows (i) the search and identification of lead compounds with defined molecular mechanisms against a specific target (e.g. enzymes from defined pathways), (ii) the analysis of a great number of compounds with a favorable cost/benefit ratio, and (iii) the development of compounds with selective toxicity. The present review describes the enzymes of the purine salvage pathway in M. tuberculosis as attractive targets for the development of new antimycobacterial agents. Enzyme kinetics and structural data have been included to provide a thorough knowledge on which to base the search for compounds with biological activity. We have focused on the mycobacterial homologues of this pathway as potential targets for the development of new antitubercular agents.

  8. 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

  9. An ELISA for the serodiagnosis of tuberculosis using a 30,000-Da native antigen of Mycobacterium tuberculosis.

    PubMed

    Sada, E; Ferguson, L E; Daniel, T M

    1990-10-01

    An ELISA was established for the measurement of IgG antibody in human serum to the 30,000-Da native antigen of Mycobacterium tuberculosis and evaluated for its utility in the diagnosis of tuberculosis at the Instituto Nacional de Enfermedades Respiratorias in Mexico City. The test had a sensitivity of 70% in patients with sputum-positive active pulmonary tuberculosis and a specificity in control subjects of 100%. The accuracy of positive prediction was 100% and of negative prediction 93% for patients with pulmonary tuberculosis at this institute. Less favorable test characteristics were obtained for patients with miliary and pleural tuberculosis, in which the test had sensitivities of 22% and 14%. These results offer the promise of an accurate serodiagnostic test for pulmonary tuberculosis using readily obtained reagents.

  10. Inhibiting Mycobacterium tuberculosis within and without.

    PubMed

    Cole, Stewart T

    2016-11-01

    Tuberculosis remains a scourge of global health with shrinking treatment options due to the spread of drug-resistant strains of Mycobacterium tuberculosis Intensive efforts have been made in the past 15 years to find leads for drug development so that better, more potent drugs inhibiting new targets could be produced and thus shorten treatment duration. Initial attempts focused on repurposing drugs that had been developed for other therapeutic areas but these agents did not meet their goals in clinical trials. Attempts to find new lead compounds employing target-based screens were unsuccessful as the leads were inactive against M. tuberculosis Greater success was achieved using phenotypic screening against live tubercle bacilli and this gave rise to the drugs bedaquiline, pretomanid and delamanid, currently in phase III trials. Subsequent phenotypic screens also uncovered new leads and targets but several of these targets proved to be promiscuous and inhibited by a variety of seemingly unrelated pharmacophores. This setback sparked an interest in alternative screening approaches that mimic the disease state more accurately. Foremost among these were cell-based screens, often involving macrophages, as these should reflect the bacterium's niche in the host more faithfully. A major advantage of this approach is its ability to uncover functions that are central to infection but not necessarily required for growth in vitro For instance, inhibition of virulence functions mediated by the ESX-1 secretion system severely attenuates intracellular M. tuberculosis, preventing intercellular spread and ultimately limiting tissue damage. Cell-based screens have highlighted the druggability of energy production via the electron transport chain and cholesterol metabolism. Here, I review the scientific progress and the pipeline, but warn against over-optimism due to the lack of industrial commitment for tuberculosis drug development and other socio-economic factors.This article is

  11. Protective efficacy of piperine against Mycobacterium tuberculosis.

    PubMed

    Sharma, Sandeep; Kalia, Nitin Pal; Suden, Pankaj; Chauhan, Prashant Singh; Kumar, Manoj; Ram, Anshu Beulah; Khajuria, Anamika; Bani, Sarang; Khan, Inshad Ali

    2014-07-01

    Piperine a trans-trans isomer of 1-piperoyl-piperidine was evaluated for its immunomodulatory activity to enhance the efficacy of rifampicin in a murine model of Mycobacterium tuberculosis infection. In-vitro immunomodulation of piperine was tested on mouse splenocytes for lymphocyte proliferation, cytokine production and macrophage activation. Protective efficacy of piperine was tested in a mice infection model of M. tuberculosis for the activation of Th-1 response and synergistic combination efficacy with rifampicin. Murine splenocytes exposed to piperine exhibited proliferation of T and B cell, increased Th-1 cytokines and enhanced macrophage activation. Piperine (1 mg/kg) in mice infected with M. tuberculosis activated the differentiation of T cells into Th-1 sub-population (CD4+ / CD8+ subsets). There was an increase in secretion of Th-1 cytokines (IFN-γ and IL-2) by these cells. The qRT-PCR studies revealed corresponding increases in the mRNA transcripts of IFN-γ and IL-2 in the infected lung tissues. Combination of piperine and rifampicin (1 mg/kg) exhibited better efficacy of and resulted in additional 1.4 to 0.8 log reduction in lung cfu as compared to rifampicin alone. The up-regulation of Th1 immunity by piperine can be synergistically combined with rifampicin to improve its therapeutic efficacy in immune-compromised TB patients.

  12. Characterization of Mycobacterium orygis as M. tuberculosis complex subspecies.

    PubMed

    van Ingen, Jakko; Rahim, Zeaur; Mulder, Arnout; Boeree, Martin J; Simeone, Roxane; Brosch, Roland; van Soolingen, Dick

    2012-04-01

    The oryx bacilli are Mycobacterium tuberculosis complex organisms for which phylogenetic position and host range are unsettled. We characterized 22 isolates by molecular methods and propose elevation to subspecies status as M. orygis. M. orygis is a causative agent of tuberculosis in animals and humans from Africa and South Asia. PMID:22469053

  13. 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.

  14. 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

  15. Post-exposure vaccination against Mycobacterium tuberculosis

    PubMed Central

    Henao-Tamayo, Marcela; Palaniswamy, Gopinath S.; Smith, Erin E.; Shanley, Crystal A.; Wang, Baolin; Orme, Ian M.; Basaraba, Randall J.; DuTeau, Nancy M.; Ordway, Diane

    2009-01-01

    Summary Enhancing immunity to tuberculosis in animal models after exposure to the infection has proved difficult. In this study we used a newly described flow cytometric technique to monitor changes in cell populations accumulating in the lungs of guinea pigs challenged by low dose aerosol infection with Mycobacterium tuberculosis and vaccinated ten days later. On day forty after infection the fusion protein F36 and a pool of Ag85A and ESAT-6 vaccines had significant effects on the bacterial load, showed increased expression of the activation marker CD45+ on CD4+ T cells, and reduced numbers of heterophils. Lung pathology and pathology scores were marginally improved in animals given these vaccines, but lymph node pathology was not influenced. Despite early effects no changes in long term survival were seen. These results suggest that a single post-exposure vaccination can initially slow the disease process. However, this effect is transient, but this could be of use in an multidrug resistant/extremely drug resistant outbreak situation because it could potentially slow the infection long enough to complete drug susceptibility testing and initiate effective chemotherapy. PMID:19264552

  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. Edaxadiene: A New Bioactive Diterpene from Mycobacterium tuberculosis

    PubMed Central

    2009-01-01

    Mycobacterium tuberculosis remains a widespread and devastating human pathogen. Presented here is the characterization of an atypical class I diterpene cyclase from M. tuberculosis that catalyzes an unusual cyclization reaction in converting the known M. tuberculosis metabolite halimadienyl diphosphate to a further cyclized novel diterpene, which we have termed edaxadiene, as it directly inhibits maturation of the phagosomal compartment in which the bacterium is taken up during infection. PMID:19583202

  18. Human immune response to Mycobacterium tuberculosis antigens.

    PubMed Central

    Havlir, D V; Wallis, R S; Boom, W H; Daniel, T M; Chervenak, K; Ellner, J J

    1991-01-01

    Little is known about the immunodominant or protective antigens of Mycobacterium tuberculosis in humans. Cell-mediated immunity is necessary for protection, and healthy tuberculin-positive individuals are relatively resistant to exogenous reinfection. We compared the targets of the cell-mediated immune response in healthy tuberculin-positive individuals to those of tuberculosis patients and tuberculin-negative persons. By using T-cell Western blotting (immunoblotting) of nitrocellulose-bound M. tuberculosis culture filtrate, peaks of T-cell blastogenic activity were identified in the healthy tuberculin reactors at 30, 37, 44, 57, 64, 71 and 88 kDa. Three of these fractions (30, 64, and 71 kDa) coincided with previously characterized proteins: antigen 6/alpha antigen, HSP60, and HSP70, respectively. The blastogenic responses to purified M. tuberculosis antigen 6/alpha antigen and BCG HSP60 were assessed. When cultured with purified antigen 6/alpha antigen, lymphocytes of healthy tuberculin reactors demonstrated greater [3H]thymidine incorporation than either healthy tuberculin-negative controls or tuberculous patients (8,113 +/- 1,939 delta cpm versus 645 +/- 425 delta cpm and 1,019 +/- 710 delta cpm, respectively; P less than 0.01). Healthy reactors also responded to HSP60, although to a lesser degree than antigen 6/alpha antigen (4,276 +/- 1,095 delta cpm; P less than 0.05). Partially purified HSP70 bound to nitrocellulose paper elicited a significant lymphocyte blastogenic response in two of six of the tuberculous patients but in none of the eight healthy tuberculin reactors. Lymphocytes of none of five tuberculin-negative controls responded to recombinant antigens at 14 or 19 kDa or to HSP70. Antibody reactivity generally was inversely correlated with blastogenic response: tuberculous sera had high titer antibody to M. tuberculosis culture filtrate in a range from 35 to 180 kDa. This is the first systematic evaluation of the human response to a panel of native

  19. 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.

  20. 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

  1. Implications of Mycobacterium Major Facilitator Superfamily for Novel Measures against Tuberculosis.

    PubMed

    Wang, Rui; Zhang, Zhen; Xie, Longxiang; Xie, Jianping

    2015-01-01

    Major facilitator superfamily (MFS) is an important secondary membrane transport protein superfamily conserved from prokaryotes to eukaryotes. The MFS proteins are widespread among bacteria and are responsible for the transfer of substrates. Pathogenic Mycobacterium MFS transporters, their distribution, function, phylogeny, and predicted crystal structures were studied to better understand the function of MFS and to discover specific inhibitors of MFS for better tuberculosis control.

  2. GSMN-TB: a web-based genome-scale network model of Mycobacterium tuberculosis metabolism

    PubMed Central

    Beste, Dany JV; Hooper, Tracy; Stewart, Graham; Bonde, Bhushan; Avignone-Rossa, Claudio; Bushell, Michael E; Wheeler, Paul; Klamt, Steffen; Kierzek, Andrzej M; McFadden, Johnjoe

    2007-01-01

    Background An impediment to the rational development of novel drugs against tuberculosis (TB) is a general paucity of knowledge concerning the metabolism of Mycobacterium tuberculosis, particularly during infection. Constraint-based modeling provides a novel approach to investigating microbial metabolism but has not yet been applied to genome-scale modeling of M. tuberculosis. Results GSMN-TB, a genome-scale metabolic model of M. tuberculosis, was constructed, consisting of 849 unique reactions and 739 metabolites, and involving 726 genes. The model was calibrated by growing Mycobacterium bovis bacille Calmette Guérin in continuous culture and steady-state growth parameters were measured. Flux balance analysis was used to calculate substrate consumption rates, which were shown to correspond closely to experimentally determined values. Predictions of gene essentiality were also made by flux balance analysis simulation and were compared with global mutagenesis data for M. tuberculosis grown in vitro. A prediction accuracy of 78% was achieved. Known drug targets were predicted to be essential by the model. The model demonstrated a potential role for the enzyme isocitrate lyase during the slow growth of mycobacteria, and this hypothesis was experimentally verified. An interactive web-based version of the model is available. Conclusion The GSMN-TB model successfully simulated many of the growth properties of M. tuberculosis. The model provides a means to examine the metabolic flexibility of bacteria and predict the phenotype of mutants, and it highlights previously unexplored features of M. tuberculosis metabolism. PMID:17521419

  3. Diversity of Mycobacterium tuberculosis across Evolutionary Scales.

    PubMed

    O'Neill, Mary B; Mortimer, Tatum D; Pepperell, Caitlin S

    2015-01-01

    Tuberculosis (TB) is a global public health emergency. Increasingly drug resistant strains of Mycobacterium tuberculosis (M.tb) continue to emerge and spread, highlighting adaptability of this pathogen. Most studies of M.tb evolution have relied on 'between-host' samples, in which each person with TB is represented by a single M.tb isolate. However, individuals with TB commonly harbor populations of M.tb numbering in the billions. Here, we use analyses of M.tb genomic data from within and between hosts to gain insight into influences shaping genetic diversity of this pathogen. We find that the amount of M.tb genetic diversity harbored by individuals with TB can vary dramatically, likely as a function of disease severity. Surprisingly, we did not find an appreciable impact of TB treatment on M.tb diversity. In examining genomic data from M.tb samples within and between hosts with TB, we find that genes involved in the regulation, synthesis, and transportation of immunomodulatory cell envelope lipids appear repeatedly in the extremes of various statistical measures of diversity. Many of these genes have been identified as possible targets of selection in other studies employing different methods and data sets. Taken together, these observations suggest that M.tb cell envelope lipids are targets of selection within hosts. Many of these lipids are specific to pathogenic mycobacteria and, in some cases, human-pathogenic mycobacteria. We speculate that rapid adaptation of cell envelope lipids is facilitated by functional redundancy, flexibility in their metabolism, and their roles mediating interactions with the host.

  4. Diversity of Mycobacterium tuberculosis across Evolutionary Scales

    PubMed Central

    O’Neill, Mary B.; Mortimer, Tatum D.; Pepperell, Caitlin S.

    2015-01-01

    Tuberculosis (TB) is a global public health emergency. Increasingly drug resistant strains of Mycobacterium tuberculosis (M.tb) continue to emerge and spread, highlighting adaptability of this pathogen. Most studies of M.tb evolution have relied on ‘between-host’ samples, in which each person with TB is represented by a single M.tb isolate. However, individuals with TB commonly harbor populations of M.tb numbering in the billions. Here, we use analyses of M.tb genomic data from within and between hosts to gain insight into influences shaping genetic diversity of this pathogen. We find that the amount of M.tb genetic diversity harbored by individuals with TB can vary dramatically, likely as a function of disease severity. Surprisingly, we did not find an appreciable impact of TB treatment on M.tb diversity. In examining genomic data from M.tb samples within and between hosts with TB, we find that genes involved in the regulation, synthesis, and transportation of immunomodulatory cell envelope lipids appear repeatedly in the extremes of various statistical measures of diversity. Many of these genes have been identified as possible targets of selection in other studies employing different methods and data sets. Taken together, these observations suggest that M.tb cell envelope lipids are targets of selection within hosts. Many of these lipids are specific to pathogenic mycobacteria and, in some cases, human-pathogenic mycobacteria. We speculate that rapid adaptation of cell envelope lipids is facilitated by functional redundancy, flexibility in their metabolism, and their roles mediating interactions with the host. PMID:26562841

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  6. Transcriptional Adaptation of Mycobacterium tuberculosis within Macrophages

    PubMed Central

    Schnappinger, Dirk; Ehrt, Sabine; Voskuil, Martin I.; Liu, Yang; Mangan, Joseph A.; Monahan, Irene M.; Dolganov, Gregory; Efron, Brad; Butcher, Philip D.; Nathan, Carl; Schoolnik, Gary K.

    2003-01-01

    Little is known about the biochemical environment in phagosomes harboring an infectious agent. To assess the state of this organelle we captured the transcriptional responses of Mycobacterium tuberculosis (MTB) in macrophages from wild-type and nitric oxide (NO) synthase 2–deficient mice before and after immunologic activation. The intraphagosomal transcriptome was compared with the transcriptome of MTB in standard broth culture and during growth in diverse conditions designed to simulate features of the phagosomal environment. Genes expressed differentially as a consequence of intraphagosomal residence included an interferon γ– and NO-induced response that intensifies an iron-scavenging program, converts the microbe from aerobic to anaerobic respiration, and induces a dormancy regulon. Induction of genes involved in the activation and β-oxidation of fatty acids indicated that fatty acids furnish carbon and energy. Induction of σE-dependent, sodium dodecyl sulfate–regulated genes and genes involved in mycolic acid modification pointed to damage and repair of the cell envelope. Sentinel genes within the intraphagosomal transcriptome were induced similarly by MTB in the lungs of mice. The microbial transcriptome thus served as a bioprobe of the MTB phagosomal environment, showing it to be nitrosative, oxidative, functionally hypoxic, carbohydrate poor, and capable of perturbing the pathogen's cell envelope. PMID:12953091

  7. A Multicopper Oxidase Is Required for Copper Resistance in Mycobacterium tuberculosis

    PubMed Central

    Rowland, Jennifer L.

    2013-01-01

    Mycobacterium tuberculosis, the causative agent of tuberculosis, is one of the most important bacterial pathogens. Recent work has revealed that the natural bactericidal properties of copper are utilized by the host immune system to combat infections with bacteria, including M. tuberculosis. However, M. tuberculosis employs multiple mechanisms to reduce the internal copper amount by efflux and sequestration, which are required for virulence of M. tuberculosis. Here, we describe an alternative mechanism of copper resistance by M. tuberculosis. Deletion of the rv0846c gene increased the susceptibility of M. tuberculosis to copper at least 10-fold, establishing Rv0846c as a major component of copper resistance in M. tuberculosis. In vitro assays showed that Rv0846c oxidized organic substrates and Fe(II). Importantly, mutation of the predicted copper-coordinating cysteine 486 resulted in inactive Rv0846c protein which did not protect M. tuberculosis against copper stress. Hence, Rv0846c is a multicopper oxidase of M. tuberculosis and was renamed mycobacterial multicopper oxidase (MmcO). MmcO is membrane associated, probably by lipidation after export across the inner membrane by the twin-arginine translocation system. However, mutation of the lipidation site did not affect the oxidase activity or the copper protective function of MmcO. Our study revealed MmcO as an important copper resistance mechanism of M. tuberculosis, which possibly acts by oxidation of toxic Cu(I) in the periplasm. PMID:23772064

  8. Pyrazinamide resistance in Mycobacterium tuberculosis fails to bite?

    PubMed

    den Hertog, Alice L; Sengstake, Sarah; Anthony, Richard M

    2015-08-01

    In contrast to most other antimycobacterial drugs where--particularly in multidrug-resistant (MDR) strains--a limited number of resistance mutations dominate, pyrazinamide (PZA) resistance associated mutations remain highly diverse with limited clustering. This apparent lack of evolutionary selection for successful PZA resistance mechanisms deserves attention. A clear understanding of the epidemiology of PZA resistance acquisition and spread would be expected to result in important insights into how PZA might be better exploited in treatment regimens to minimize the amplification of Mycobacterium tuberculosis (MTB) drug resistance. We propose that PZA resistance typically induces a fitness cost that impairs MTB transmission. This would explain the lack of extensive clustering for PZA-resistant mutants. Our hypothesis also leads to a series of testable predictions which we outline that could confirm or refute our ideas.

  9. 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/

  10. 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.

  11. DNA fingerprinting of Mycobacterium tuberculosis strains from patients with pulmonary tuberculosis in Honduras.

    PubMed Central

    Pineda-Garcia, L; Ferrera, A; Hoffner, S E

    1997-01-01

    Mycobacterium tuberculosis isolates from 84 patients with pulmonary tuberculosis in Honduras were characterized by restriction fragment length polymorphism analysis. Seventy-three different IS6110 patterns were found; 63 of these were unique and 10 were shared by two to three strains each. Thus, no ongoing spread of any specific clone of bacteria could be demonstrated. PMID:9276422

  12. 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.

  13. Atypical presentation of Mycobacterium tuberculosis in an infant.

    PubMed

    Gayathri Devi, D R; Gowri, Mangala; Padmalatha, S; Sreeja, S; Babu, Sreenivasa

    2010-12-01

    Tuberculosis of the skeletal muscle is very rare which is often missed in the early stages. This leads to delay in treatment resulting in irreversible limb deformity and loss of function. The authors describe a case of healthy child with an intramuscular cystic swelling above the elbow joint. The pus showing acid fast bacilli morphologically resembling Mycobacterium tuberculosis was also isolated in culture. Following the diagnosis and confirmation, the child was treated successfully with anti tubercular drugs. PMID:20890682

  14. Direct detection of Mycobacterium tuberculosis complex in nonrespiratory specimens by Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test.

    PubMed Central

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

    1997-01-01

    The Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test (AMTDT) was adapted for the detection of Mycobacterium tuberculosis complex in 224 nonrespiratory specimens from 188 patients. The sensitivity and specificity of the AMTDT for such specimens, after resolution of discrepant results, were 85.7 and 100%, respectively. Pretreatment of nonrespiratory specimens with sodium dodecyl (lauryl) sulfate is mandatory to obtain consistent and reproducible AMTDT results. The use of 500 microliters of decontaminated specimen improves the sensitivity of the test. Because the AMTDT detects stable rRNA from noncultivable bacilli, it is not useful for monitoring patients receiving treatment. PMID:8968935

  15. 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.

  16. 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

  17. Evaluation of Roche Amplicor PCR assay for Mycobacterium tuberculosis.

    PubMed Central

    Wobeser, W L; Krajden, M; Conly, J; Simpson, H; Yim, B; D'costa, M; Fuksa, M; Hian-Cheong, C; Patterson, M; Phillips, A; Bannatyne, R; Haddad, A; Brunton, J L; Krajden, S

    1996-01-01

    The Roche Amplicor Mycobacterium tuberculosis PCR test (RMtb-PCR) was compared with mycobacterial culture, with the BACTEC 460 system and inoculation on Lowenstein-Jensen media. Results were interpreted with an adjusted "gold standard" incorporating clinical diagnosis. A total of 1,480 clinical specimens from 1,155 patients, including tissues and fluids, as well as 141 specimens which demonstrated a positive growth index on the BACTEC 460 system were assessed. The sensitivity, specificity, and positive and negative predictive values of RMtb-PCR compared with the adjusted gold standard for clinical specimens were 79, 99, 93, and 98%, respectively. In smear-positive specimens, the sensitivity of RMtb-PCR was 98% versus 53% for smear-negative specimens. When RMtb-PCR was performed two times per week, PCR results were available an average of 21 days before the culture results. For specimens demonstrating a positive growth index on the BACTEC 460 system, RMtb-PCR had a sensitivity and specificity of 98 and 100%, respectively. This study demonstrates the value of a commercial nucleic acid amplification kit for rapid diagnosis of M. tuberculosis, particularly in smear-positive specimens or BACTEC culture-positive specimens. PMID:8748289

  18. Strength in Diversity: Hidden Genetic Depths of Mycobacterium tuberculosis.

    PubMed

    Sampson, Samantha L

    2016-02-01

    Next-generation whole genome sequencing data is currently being utilised to explore Mycobacterium tuberculosis genetic diversity. Studies have focused in particular on the evolution of drug resistance, and have revealed a surprising degree of dynamic population heterogeneity, with implications for transmission studies, treatment regimens and new drug target development.

  19. 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

  20. 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...

  1. 21 CFR 866.3370 - Mycobacterium tuberculosis immunofluorescent reagents.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 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...

  2. 21 CFR 866.3370 - Mycobacterium tuberculosis immunofluorescent reagents.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 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...

  3. 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...

  4. 21 CFR 866.3370 - Mycobacterium tuberculosis immunofluorescent reagents.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 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...

  5. 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

  6. 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. PMID:27649868

  7. Comparative analyses of the proteins from Mycobacterium tuberculosis and human genomes: Identification of potential tuberculosis drug targets.

    PubMed

    Sridhar, Settu; Dash, Pallabini; Guruprasad, Kunchur

    2016-03-15

    Tuberculosis, one of the major infectious diseases affecting human beings is caused by the bacillus Mycobacterium tuberculosis. Increased resistance to known drugs commonly used for the treatment of tuberculosis has created an urgent need to identify new targets for validation and to develop drugs. In this study, we have used various bioinformatics tools, to compare the protein sequences from twenty-three M. tuberculosis genome strains along with the known human protein sequences, in order to identify the 'conserved' M. tuberculosis proteins absent in human. Further, based on the analysis of protein interaction networks, we selected one-hundred and forty proteins that were predicted as potential M. tuberculosis drug targets and prioritized according to the ranking of 'clusters' of interacting proteins. Comparison of the predicted 140 TB targets with literature indicated that 46 of them were previously reported, thereby increasing the confidence in our predictions of the remaining 94 targets too. The analyses of the structures and functions corresponding to the predicted potential TB drug targets indicated a diverse range of proteins that included ten 'druggable' targets with some of the known drugs. PMID:26762852

  8. Comparative analyses of the proteins from Mycobacterium tuberculosis and human genomes: Identification of potential tuberculosis drug targets.

    PubMed

    Sridhar, Settu; Dash, Pallabini; Guruprasad, Kunchur

    2016-03-15

    Tuberculosis, one of the major infectious diseases affecting human beings is caused by the bacillus Mycobacterium tuberculosis. Increased resistance to known drugs commonly used for the treatment of tuberculosis has created an urgent need to identify new targets for validation and to develop drugs. In this study, we have used various bioinformatics tools, to compare the protein sequences from twenty-three M. tuberculosis genome strains along with the known human protein sequences, in order to identify the 'conserved' M. tuberculosis proteins absent in human. Further, based on the analysis of protein interaction networks, we selected one-hundred and forty proteins that were predicted as potential M. tuberculosis drug targets and prioritized according to the ranking of 'clusters' of interacting proteins. Comparison of the predicted 140 TB targets with literature indicated that 46 of them were previously reported, thereby increasing the confidence in our predictions of the remaining 94 targets too. The analyses of the structures and functions corresponding to the predicted potential TB drug targets indicated a diverse range of proteins that included ten 'druggable' targets with some of the known drugs.

  9. Insights into redox sensing metalloproteins in Mycobacterium tuberculosis.

    PubMed

    Chim, Nicholas; Johnson, Parker M; Goulding, Celia W

    2014-04-01

    Mycobacterium tuberculosis, the pathogen that causes tuberculosis, has evolved sophisticated mechanisms for evading assault by the human host. This review focuses on M. tuberculosis regulatory metalloproteins that are sensitive to exogenous stresses attributed to changes in the levels of gaseous molecules (i.e., molecular oxygen, carbon monoxide and nitric oxide) to elicit an intracellular response. In particular, we highlight recent developments on the subfamily of Whi proteins, redox sensing WhiB-like proteins that contain iron-sulfur clusters, sigma factors and their cognate anti-sigma factors of which some are zinc-regulated, and the dormancy survival regulon DosS/DosT-DosR heme sensory system. Mounting experimental evidence suggests that these systems contribute to a highly complex and interrelated regulatory network that controls M. tuberculosis biology. This review concludes with a discussion of strategies that M. tuberculosis has developed to maintain redox homeostasis, including mechanisms to regulate endogenous nitric oxide and carbon monoxide levels.

  10. 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

  11. 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.

  12. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  14. Microbial Proteome Profiling and Systems Biology: Applications to Mycobacterium tuberculosis.

    PubMed

    Schubert, Olga T; Aebersold, Ruedi

    2015-01-01

    Each year, 1.3 million people die from tuberculosis, an infectious disease caused by Mycobacterium tuberculosis. Systems biology-based strategies might significantly contribute to the knowledge-guided development of more effective vaccines and drugs to prevent and cure infectious diseases. To build models simulating the behaviour of a system in response to internal or external stimuli and to identify potential targets for therapeutic intervention, systems biology approaches require the acquisition of quantitative molecular profiles on many perturbed states. Here we review the current state of proteomic analyses in Mycobacterium tuberculosis and discuss the potential of recently emerging targeting mass spectrometry-based techniques which enable fast, sensitive and accurate protein measurements.

  15. 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

  16. 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

  17. Computer-assisted prediction of HLA-DR binding and experimental analysis for human promiscuous Th1-cell peptides in the 24 kDa secreted lipoprotein (LppX) of Mycobacterium tuberculosis.

    PubMed

    Al-Attiyah, R; Mustafa, A S

    2004-01-01

    The secreted 24 kDa lipoprotein (LppX) is an antigen that is specific for Mycobacterium tuberculosis complex and M. leprae. The present study was carried out to identify the promiscuous T helper 1 (Th1)-cell epitopes of the M. tuberculosis LppX (MT24, Rv2945c) antigen by using 15 overlapping synthetic peptides (25 mers overlapping by 10 residues) covering the sequence of the complete protein. The analysis of Rv2945c sequence for binding to 51 alleles of nine serologically defined HLA-DR molecules, by using a virtual matrix-based prediction program (propred), showed that eight of the 15 peptides of Rv2945c were predicted to bind promiscuously to >/=10 alleles from more than or equal to three serologically defined HLA-DR molecules. The Th1-cell reactivity of all the peptides was assessed in antigen-induced proliferation and interferon-gamma (IFN-gamma)-secretion assays with peripheral blood mononuclear cells (PBMCs) from 37 bacille Calmette-Guérin (BCG)-vaccinated healthy subjects. The results showed that 17 of the 37 donors, which represented an HLA-DR-heterogeneous group, responded to one or more peptides of Rv2945c in the Th1-cell assays. Although each peptide stimulated PBMCs from one or more donors in the above assays, the best positive responses (12/17 (71%) responders) were observed with the peptide p14 (aa 196-220). This suggested a highly promiscuous presentation of p14 to Th1 cells. In addition, the sequence of p14 is completely identical among the LppX of M. tuberculosis, M. bovis and M. leprae, which further supports the usefulness of Rv2945c and p14 in the subunit vaccine design against both tuberculosis and leprosy.

  18. Systems Biology-Based Identification of Mycobacterium tuberculosis Persistence Genes in Mouse Lungs

    PubMed Central

    Dutta, Noton K.; Bandyopadhyay, Nirmalya; Veeramani, Balaji; Lamichhane, Gyanu; Karakousis, Petros C.; Bader, Joel S.

    2014-01-01

    ABSTRACT Identifying Mycobacterium tuberculosis persistence genes is important for developing novel drugs to shorten the duration of tuberculosis (TB) treatment. We developed computational algorithms that predict M. tuberculosis genes required for long-term survival in mouse lungs. As the input, we used high-throughput M. tuberculosis mutant library screen data, mycobacterial global transcriptional profiles in mice and macrophages, and functional interaction networks. We selected 57 unique, genetically defined mutants (18 previously tested and 39 untested) to assess the predictive power of this approach in the murine model of TB infection. We observed a 6-fold enrichment in the predicted set of M. tuberculosis genes required for persistence in mouse lungs relative to randomly selected mutant pools. Our results also allowed us to reclassify several genes as required for M. tuberculosis persistence in vivo. Finally, the new results implicated additional high-priority candidate genes for testing. Experimental validation of computational predictions demonstrates the power of this systems biology approach for elucidating M. tuberculosis persistence genes. PMID:24549847

  19. Orchestration of pulmonary T cell immunity during Mycobacterium tuberculosis infection: immunity interruptus

    PubMed Central

    Behar, Samuel M.; Carpenter, Stephen M.; Booty, Matthew G.; Barber, Daniel L.; Jayaraman, Pushpa

    2014-01-01

    Despite the introduction almost a century ago of Mycobacterium bovis BCG (BCG), an attenuated form of M. bovis that is used as a vaccine against Mycobacterium tuberculosis, tuberculosis remains a global health threat and kills more than 1.5 million people each year. This is mostly because BCG fails to prevent pulmonary disease – the contagious form of tuberculosis. Although there have been significant advances in understanding how the immune system responds to infection, the qualities that define protective immunity against M. tuberculosis remain poorly characterized. The ability to predict who will maintain control over the infection and who will succumb to clinical disease would revolutionize our approach to surveillance, control, and treatment. Here we review the current understanding of pulmonary T cell responses following M. tuberculosis infection. While infection elicits a strong immune response that contains infection, M. tuberculosis evades eradication. Traditionally, its intracellular lifestyle and alteration of macrophage function are viewed as the dominant mechanisms of evasion. Now we appreciate that chronic inflammation leads to T cell dysfunction. While this may arise as the host balances the goals of bacterial sterilization and avoidance of tissue damage, it is becoming clear that T cell dysfunction impairs host resistance. Defining the mechanisms that lead to T cell dysfunction is crucial as memory T cell responses are likely to be subject to the same subject to the same pressures. Thus, success of T cell based vaccines is predicated on memory T cells avoiding exhaustion while at the same time not promoting overt tissue damage. PMID:25311810

  20. 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.

  1. Determination of Urinary Neopterin/Creatinine Ratio to Distinguish Active Tuberculosis from Latent Mycobacterium tuberculosis Infection

    PubMed Central

    Eisenhut, Michael; Hargreaves, Dougal S.; Scott, Anne; Housley, David; Walters, Andrew; Mulla, Rohinton

    2016-01-01

    Background. Biomarkers to distinguish latent from active Mycobacterium (M.) tuberculosis infection in clinical practice are lacking. The urinary neopterin/creatinine ratio can quantify the systemic interferon-gamma effect in patients with M. tuberculosis infection. Methods. In a prospective observational study, urinary neopterin levels were measured by enzyme linked immunosorbent assay in patients with active tuberculosis, in people with latent M. tuberculosis infection, and in healthy controls and the urinary neopterin/creatinine ratio was calculated. Results. We included a total of 44 patients with M. tuberculosis infection and nine controls. 12 patients had active tuberculosis (8 of them culture-confirmed). The median age was 15 years (range 4.5 to 49). Median urinary neopterin/creatinine ratio in patients with active tuberculosis was 374.1 micromol/mol (129.0 to 1072.3), in patients with latent M. tuberculosis infection it was 142.1 (28.0 to 384.1), and in controls it was 146.0 (40.3 to 200.0), with significantly higher levels in patients with active tuberculosis (p < 0.01). The receiver operating characteristics curve had an area under the curve of 0.84 (95% CI 0.70 to 0.97) (p < 0.01). Conclusions. Urinary neopterin/creatinine ratios are significantly higher in patients with active tuberculosis compared to patients with latent infection and may be a significant predictor of active tuberculosis in patients with M. tuberculosis infection. PMID:27433370

  2. Transmission of Mycobacterium orygis (M. tuberculosis complex species) from a tuberculosis patient to a dairy cow in New Zealand.

    PubMed

    Dawson, Kara L; Bell, Anita; Kawakami, R Pamela; Coley, Kathryn; Yates, Gary; Collins, Desmond M

    2012-09-01

    Mycobacterium orygis, previously called the oryx bacillus, is a member of the Mycobacterium tuberculosis complex and has been reported only recently as a cause of human tuberculosis in patients of South Asian origin. We present the first case documenting the transmission of this organism from a human to a cow.

  3. Transmission of Mycobacterium orygis (M. tuberculosis complex species) from a tuberculosis patient to a dairy cow in New Zealand.

    PubMed

    Dawson, Kara L; Bell, Anita; Kawakami, R Pamela; Coley, Kathryn; Yates, Gary; Collins, Desmond M

    2012-09-01

    Mycobacterium orygis, previously called the oryx bacillus, is a member of the Mycobacterium tuberculosis complex and has been reported only recently as a cause of human tuberculosis in patients of South Asian origin. We present the first case documenting the transmission of this organism from a human to a cow. PMID:22785186

  4. In Vitro and In Vivo Activities of the Nitroimidazole TBA-354 against Mycobacterium tuberculosis

    PubMed Central

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

    2014-01-01

    Nitroimidazoles are a promising new class of antitubercular agents. The nitroimidazo-oxazole delamanid (OPC-67683, Deltyba) is in phase III trials for the treatment of multidrug-resistant tuberculosis, while the nitroimidazo-oxazine PA-824 is entering phase III for drug-sensitive and drug-resistant tuberculosis. TBA-354 (SN31354[(S)-2-nitro-6-((6-(4-trifluoromethoxy)phenyl)pyridine-3-yl)methoxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine]) is a pyridine-containing biaryl compound with exceptional efficacy against chronic murine tuberculosis and favorable bioavailability in preliminary rodent studies. It was selected as a potential next-generation antituberculosis nitroimidazole following an extensive medicinal chemistry effort. Here, we further evaluate the pharmacokinetic properties and activity of TBA-354 against Mycobacterium tuberculosis. TBA-354 is narrow spectrum and bactericidal in vitro against replicating and nonreplicating Mycobacterium tuberculosis, with potency similar to that of delamanid and greater than that of PA-824. The addition of serum protein or albumin does not significantly alter this activity. TBA-354 maintains activity against Mycobacterium tuberculosis H37Rv isogenic monoresistant strains and clinical drug-sensitive and drug-resistant isolates. Spontaneous resistant mutants appear at a frequency of 3 × 10−7. In vitro studies and in vivo studies in mice confirm that TBA-354 has high bioavailability and a long elimination half-life. In vitro studies suggest a low risk of drug-drug interactions. Low-dose aerosol infection models of acute and chronic murine tuberculosis reveal time- and dose-dependent in vivo bactericidal activity that is at least as potent as that of delamanid and more potent than that of PA-824. Its superior potency and pharmacokinetic profile that predicts suitability for once-daily oral dosing suggest that TBA-354 be studied further for its potential as a next-generation nitroimidazole. PMID:25331696

  5. Integration of Published Information Into a Resistance-Associated Mutation Database for Mycobacterium tuberculosis

    PubMed Central

    Salamon, Hugh; Yamaguchi, Ken D.; Cirillo, Daniela M.; Miotto, Paolo; Schito, Marco; Posey, James; Starks, Angela M.; Niemann, Stefan; Alland, David; Hanna, Debra; Aviles, Enrique; Perkins, Mark D.; Dolinger, David L.

    2015-01-01

    Tuberculosis remains a major global public health challenge. Although incidence is decreasing, the proportion of drug-resistant cases is increasing. Technical and operational complexities prevent Mycobacterium tuberculosis drug susceptibility phenotyping in the vast majority of new and retreatment cases. The advent of molecular technologies provides an opportunity to obtain results rapidly as compared to phenotypic culture. However, correlations between genetic mutations and resistance to multiple drugs have not been systematically evaluated. Molecular testing of M. tuberculosis sampled from a typical patient continues to provide a partial picture of drug resistance. A database of phenotypic and genotypic testing results, especially where prospectively collected, could document statistically significant associations and may reveal new, predictive molecular patterns. We examine the feasibility of integrating existing molecular and phenotypic drug susceptibility data to identify associations observed across multiple studies and demonstrate potential for well-integrated M. tuberculosis mutation data to reveal actionable findings. PMID:25765106

  6. 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.

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

    PubMed

    Halim, Mohd Zakihalani A; Jaafar, Mohammad Maaruf; Teh, Lay Kek; Ismail, Mohamad Izwan; Lee, Lian Shien; Ngeow, Yun Fong; Nor, Norazmi Mohd; Zainuddin, Zainul Fadziruddin; Tang, Thean Hock; Najimudin, Mohd Nazalan Mohd; Salleh, Mohd Zaki

    2016-03-01

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

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

    PubMed Central

    Halim, Mohd Zakihalani A.; Jaafar, Mohammad Maaruf; Teh, Lay Kek; Ismail, Mohamad Izwan; Lee, Lian Shien; Ngeow, Yun Fong; Nor, Norazmi Mohd; Zainuddin, Zainul Fadziruddin; Tang, Thean Hock; Najimudin, Mohd Nazalan Mohd; Salleh, Mohd Zaki

    2016-01-01

    Here, we report the draft genome sequence and annotation of a multidrug resistant Mycobacterium tuberculosis strain PR10 (MDR-TB PR10) isolated from a patient diagnosed with tuberculosis. The size of the draft genome MDR-TB PR10 is 4.34 Mbp with 65.6% of G + C content and consists of 4637 predicted genes. The determinants were categorized by RAST into 400 subsystems with 4286 coding sequences and 50 RNAs. The whole genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number CP010968. PMID:26981419

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

    PubMed

    Halim, Mohd Zakihalani A; Jaafar, Mohammad Maaruf; Teh, Lay Kek; Ismail, Mohamad Izwan; Lee, Lian Shien; Ngeow, Yun Fong; Nor, Norazmi Mohd; Zainuddin, Zainul Fadziruddin; Tang, Thean Hock; Najimudin, Mohd Nazalan Mohd; Salleh, Mohd Zaki

    2016-03-01

    Here, we report the draft genome sequence and annotation of a multidrug resistant Mycobacterium tuberculosis strain PR10 (MDR-TB PR10) isolated from a patient diagnosed with tuberculosis. The size of the draft genome MDR-TB PR10 is 4.34 Mbp with 65.6% of G + C content and consists of 4637 predicted genes. The determinants were categorized by RAST into 400 subsystems with 4286 coding sequences and 50 RNAs. The whole genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number CP010968. PMID:26981419

  10. Detection of Rifampin Resistance in Mycobacterium tuberculosis by Double Gradient-Denaturing Gradient Gel Electrophoresis

    PubMed Central

    Scarpellini, Paolo; Braglia, Sergio; Carrera, Paola; Cedri, Maura; Cichero, Paola; Colombo, Alessia; Crucianelli, Rosella; Gori, Andrea; Ferrari, Maurizio; Lazzarin, Adriano

    1999-01-01

    We applied double gradient-denaturing gradient gel electrophoresis (DG-DGGE) for the rapid detection of rifampin (RMP) resistance from rpoB PCR products of Mycobacterium tuberculosis isolates and clinical samples. The results of this method were fully concordant with those of DNA sequencing and susceptibility testing analyses. DG-DGGE is a valid alternative to the other methods of detecting mutations for predicting RMP resistance. PMID:10508043

  11. 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

  12. Draft Genome Sequence of a New Zealand Rangipo Strain of Mycobacterium tuberculosis

    PubMed Central

    Gautam, Sanjay S.; Bower, James E.; Basu, Indira

    2016-01-01

    The Rangipo genotype of the Mycobacterium tuberculosis complex has been associated with a number of tuberculosis (TB) outbreaks in New Zealand. We report here the draft whole-genome sequence of a representative isolate of this strain. PMID:27389273

  13. 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. PMID:21464295

  14. 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

  15. 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.

  16. 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

  17. Inactivation of Mycobacterium tuberculosis for DNA Typing Analysis

    PubMed Central

    Bemer-Melchior, P.; Drugeon, H. B.

    1999-01-01

    DNA fingerprinting analysis of Mycobacterium tuberculosis is used for epidemiological studies and the control of laboratory cross-contamination. Because standardized procedures are not entirely safe for mycobacteriology laboratory staff, the paper proposes a new technique for the processing of specimens. The technique ensures the inactivation of M. tuberculosis before DNA extraction without the loss of DNA integrity. The control of inactivated cultures should be rigorous and should involve the use of two different culture media incubated for at least 4 months. PMID:10364613

  18. Mycobacterium tuberculosis virulence: insights and impact on vaccine development.

    PubMed

    Delogu, Giovanni; Provvedi, Roberta; Sali, Michela; Manganelli, Riccardo

    2015-01-01

    The existing TB vaccine, the attenuated Mycobacterium bovis strain BCG, is effective in protecting infants from severe forms of the disease, while its efficacy in protecting adults from pulmonary TB is poor. In the last two decades, a renewed interest in TB resulted in the development of several candidate vaccines that are now entering clinical trials. However, most of these vaccines are based on a common rationale and aim to induce a strong T-cell response against Mycobacterium tuberculosis. Recent advancements in the understanding of M. tuberculosis virulence determinants and associated pathogenic strategies are opening a new and broader view of the complex interaction between this remarkable pathogen and the human host, providing insights at molecular level that could lead to a new rationale for the design of novel antitubercular vaccines. A vaccination strategy that simultaneously targets different steps in TB pathogenesis may result in improved protection and reduced TB transmission.

  19. 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

  20. 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.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26259792

  2. 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. PMID:26259792

  3. Mycobacterium tuberculosis pyrazinamide resistance determinants: a multicenter study.

    PubMed

    Miotto, Paolo; 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

    2014-10-21

    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 (PZA(r)). 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 PZA(r) strains (85%), (ii) high-confidence resistance mutations found in more than 70% of PZA(r) strains, (iii) mutations with an unclear role found in less than 70% of PZA(r) 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 PZA(r); the diagnostic accuracy of such an assay would be in the range of 89.5 to 98.8%. Importance: Conventional phenotypic testing for pyrazinamide resistance in Mycobacterium tuberculosis is technically challenging and often unreliable. The development of a molecular assay for detecting pyrazinamide resistance would be a breakthrough, directly overcoming both the limitations of conventional testing and its related biosafety issues. Although the main mechanism of pyrazinamide resistance involves mutations inactivating the pncA enzyme, the highly diverse genetic variants scattered over the full length of the pncA gene and the lack of a reliable phenotypic gold standard hamper the development of molecular diagnostic

  4. Mycobacterium tuberculosis Mutations Associated with Reduced Susceptibility to Linezolid.

    PubMed

    Zhang, Shuo; Chen, Jiazhen; Cui, Peng; Shi, Wanliang; Shi, Xiaohong; Niu, Hongxia; Chan, Denise; Yew, Wing Wai; Zhang, Wenhong; Zhang, Ying

    2016-04-01

    Linezolid (LZD) has become increasingly important for the treatment of multidrug-resistant tuberculosis (MDR-TB), but its mechanisms of resistance are not well characterized. We isolated 32 mutants ofMycobacterium tuberculosiswith reduced susceptibility to LZD, which was accounted for byrrlandrplCmutations in almost equal proportions, causing lower and higher MICs, respectively. Our findings provide useful information for the rapid detection of LZD resistance for improved treatment of MDR-TB.

  5. An efficient alternative marker for specific identification of Mycobacterium tuberculosis.

    PubMed

    Zhao, Jianing; Wang, Yiwei; Li, Dairong; Liu, Jiawen; Zhang, Xuemei; He, Yujuan; Wang, Hong; Cao, Ju; Yin, Yibing; Xu, Wenchun

    2014-08-01

    Rapid and accurate identification of mycobacteria to the species level is important to provide epidemiological information and to guide the appropriate treatment, especially identification of the Mycobacterium tuberculosis (MTB) which is the leading pathogen causing tuberculosis. The genetic marker named as Mycobacterium tuberculosis specific sequence 90 (mtss90) was screened by a bioinformatics software and verified by a series of experiments. To test its specificity, 266 strains of microorganisms and human cells were used for the mtss90 conventional PCR method. Moreover, the efficiency of mtss90 was evaluated by comparing 16S rDNA (Mycobacterium genus-specific), IS6110 (specific identification of MTB complex), mtp40 (MTB-specific) and PNB/TCH method (traditional bacteriology testing) in Mycobacterium strains. All MTB isolates were mtss90 positive. No amplification was observed from any other tested strains with M. microti as an exception. Compared with the traditional PNB/TCH method, the coincidence rate was 99.1 % (233/235). All of the mtss90 positive strains were IS6110 and 16S rDNA positive, indicating a 100 % coincidence rate (216/216) between mtss90 and these two genetic markers. Additionally, mtss90 had a better specificity than mtp40 in the identification of MTB. Lastly, a real-time PCR diagnostic assay was developed for the rapid identification of MTB. In conclusion, mtss90 may be an efficient alternative marker for species-specific identification of MTB and could be used for the diagnosis of tuberculosis combined with other genetic markers.

  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. Optimization of recombinant Mycobacterium tuberculosis RNA polymerase expression and purification.

    PubMed

    Banerjee, Rajdeep; Rudra, Paulami; Prajapati, Ranjit Kumar; Sengupta, Shreya; Mukhopadhyay, Jayanta

    2014-07-01

    Mycobacterium tuberculosis, the human pathogen that causes tuberculosis, warrants enormous attention due to the emergence of multi drug resistant and extremely drug resistant strains. RNA polymerase (RNAP), the key enzyme in gene regulation, is an attractive target for anti-TB drugs. Understanding the structure-function relationship of M. tuberculosis RNAP and the mechanism of gene regulation by RNAP in conjunction with different σ factors and transcriptional regulators would provide significant information for anti-tuberculosis drug development targeting RNAP. Studies with M. tuberculosis RNAP remain tedious because of the extremely slow-growing nature of the bacteria and requirement of special laboratory facility. Here, we have developed and optimized recombinant methods to prepare M. tuberculosis RNAP core and RNAP holo enzymes assembled in vivo in Escherichia coli. These methods yield high amounts of transcriptionally active enzymes, free of E. coli RNAP contamination. The recombinant M. tuberculosis RNAP is used to develop a highly sensitive fluorescence based in vitro transcription assay that could be easily adopted in a high-throughput format to screen RNAP inhibitors. These recombinant methods would be useful to set a platform for M. tuberculosis RNAP targeted anti TB drug development, to analyse the structure/function of M. tuberculosis RNAP and to analyse the interactions among promoter DNA, RNAP, σ factors, and transcription regulators of M. tuberculosis in vitro, avoiding the hazard of handling of pathogenic bacteria.

  8. 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

  9. 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.

  10. Bisphosphonic acids as effective inhibitors of Mycobacterium tuberculosis glutamine synthetase.

    PubMed

    Kosikowska, Paulina; Bochno, Marta; Macegoniuk, Katarzyna; Forlani, Giuseppe; Kafarski, Paweł; Berlicki, Łukasz

    2016-12-01

    Inhibition of glutamine synthetase (GS) is one of the most promising strategies for the discovery of novel drugs against tuberculosis. Forty-three bisphosphonic and bis-H-phosphinic acids of various scaffolds, bearing aromatic substituents, were screened against recombinant GS from Mycobacterium tuberculosis. Most of the studied compounds exhibited activities in micromolar range, with N-(3,5-dichlorophenyl)-2-aminoethylidenebisphoshonic acid, N-(3,5-difluorophenyl)-2-aminoethylidene-bisphoshonic acid and N-(3,4-dichlorophenyl)-1-hydroxy-1,1-ethanebisphosphonic acid showing the highest potency with kinetic parameters similar to the reference compound - L-methionine-S-sulfoximine. Moreover, these inhibitors were found to be much more effective against pathogen enzyme than against the human ortholog. Thus, with the bone-targeting properties of the bisphosphonate compounds in mind, this activity/selectivity profile makes these compounds attractive agents for the treatment of bone tuberculosis.

  11. 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

  12. 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

  13. Mycobacterium tuberculosis infection in a HIV-positive patient.

    PubMed

    Montales, Maria Theresa; Beebe, Alexandria; Chaudhury, Arun; Patil, Naveen

    2015-01-01

    Mycobacterium tuberculosis (MTB) and human immunodeficiency virus (HIV) coinfection remains a global public health challenge. We report a 40 year old African American male who is a known HIV-positive patient, non-compliant with his antiretrovirals and developed pulmonary tuberculosis. His chief complaints were chronic cough, fever, night sweats and undocumented weight loss. He had a prior positive T-SPOT-TB test; however, chest radiograph and sputum smear examination revealed normal results. PCR-based GeneXPERT MTB/RIF assay was ordered and confirmed MTB infection. The sputum cultures grew MTB and sensitivities showed susceptibility to all primary anti-tuberculosis medications. A delay in diagnosis and initiation of MTB therapy, in the setting of HIV or AIDS, may result in rapid disease progression and worse clinical outcome. PMID:26744689

  14. 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

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

    PubMed

    Kumar, Anuradha; Guardia, Ana; 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; Sherman, David R

    2015-12-11

    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

  16. The efficacy of the heat killing of Mycobacterium tuberculosis.

    PubMed

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

    2002-10-01

    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 degrees 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.

  17. Prioritizing Genomic Drug Targets in Pathogens: Application to Mycobacterium tuberculosis

    PubMed Central

    Hasan, Samiul; Daugelat, Sabine; Rao, P. S. Srinivasa; Schreiber, Mark

    2006-01-01

    We have developed a software program that weights and integrates specific properties on the genes in a pathogen so that they may be ranked as drug targets. We applied this software to produce three prioritized drug target lists for Mycobacterium tuberculosis, the causative agent of tuberculosis, a disease for which a new drug is desperately needed. Each list is based on an individual criterion. The first list prioritizes metabolic drug targets by the uniqueness of their roles in the M. tuberculosis metabolome (“metabolic chokepoints”) and their similarity to known “druggable” protein classes (i.e., classes whose activity has previously been shown to be modulated by binding a small molecule). The second list prioritizes targets that would specifically impair M. tuberculosis, by weighting heavily those that are closely conserved within the Actinobacteria class but lack close homology to the host and gut flora. M. tuberculosis can survive asymptomatically in its host for many years by adapting to a dormant state referred to as “persistence.” The final list aims to prioritize potential targets involved in maintaining persistence in M. tuberculosis. The rankings of current, candidate, and proposed drug targets are highlighted with respect to these lists. Some features were found to be more accurate than others in prioritizing studied targets. It can also be shown that targets can be prioritized by using evolutionary programming to optimize the weights of each desired property. We demonstrate this approach in prioritizing persistence targets. PMID:16789813

  18. Genotype of a historic strain of Mycobacterium tuberculosis

    PubMed Central

    Bouwman, Abigail S.; Kennedy, Sandra L.; Müller, Romy; Stephens, Richard H.; Holst, Malin; Caffell, Anwen C.; Roberts, Charlotte A.; Brown, Terence A.

    2012-01-01

    The use of ancient DNA in paleopathological studies of tuberculosis has largely been restricted to confirmation of disease identifications made by skeletal analysis; few attempts at obtaining genotype data from archaeological samples have been made because of the need to perform different PCRs for each genetic locus being studied in an ancient DNA extract. We used a next generation sequencing approach involving hybridization capture directed at specific polymorphic regions of the Mycobacterium tuberculosis genome to identify a detailed genotype for a historic strain of M. tuberculosis from an individual buried in the 19th century St. George’s Crypt, Leeds, West Yorkshire, England. We obtained 664,500 sequencing by oligonucleotide ligation and detection (SOLiD) reads that mapped to the targeted regions of the M. tuberculosis genome; the coverage included 218 of 247 SNPs, 10 of 11 insertion/deletion regions, and the repeat elements IS1081 and IS6110. The accuracy of the SOLiD data was checked by conventional PCRs directed at 11 SNPs and two insertion/deletions. The data placed the historic strain of M. tuberculosis in a group that is uncommon today, but it is known to have been present in North America in the early 20th century. Our results show the use of hybridization capture followed by next generation sequencing as a means of obtaining detailed genotypes of ancient varieties of M. tuberculosis, potentially enabling meaningful comparisons between strains from different geographic locations and different periods in the past. PMID:23091009

  19. Mycobacterium tuberculosis infection within parotid gland Warthin tumor.

    PubMed

    Ozcan, Cengiz; Apa, Duygu Düşmez; Aslan, Gönül; Gülhan, Stk; Görür, Kemal

    2008-11-01

    Tuberculosis of the parotid gland is extremely unusual. Tuberculosis comprises 2.5% to 10% of parotid gland lesions. Two clinical forms of parotid gland tuberculosis infection exist. One is a diffuse parenchymatous disease (either primary or secondary to nodal disease), resembling common infection. The second is a chronic, slow-growing, painless, and firm parotid mass mimicking a neoplasm. Most of these patients were diagnosed after parotid gland surgery and histopathologic evaluation. Warthin tumor is a well-known benign neoplasm of the salivary glands. It is the second most common tumor of the parotid gland. Mycobacterium tuberculosis within Warthin tumor is also unusual. Five cases with parotid gland tuberculosis within Warthin tumor were reported in the literature. In this report, we present a new patient with parotid gland tuberculosis within the Warthin tumor. This type parotid gland pathology is an extremely rare entity, and to the best of our knowledge, this is the second documented case using polymerase chain reaction. We also discussed the possible mechanisms of development of infection within Warthin tumor.

  20. Mycobacterium tuberculosis efpA encodes an efflux protein of the QacA transporter family.

    PubMed Central

    Doran, J L; Pang, Y; Mdluli, K E; Moran, A J; Victor, T C; Stokes, R W; Mahenthiralingam, E; Kreiswirth, B N; Butt, J L; Baron, G S; Treit, J D; Kerr, V J; Van Helden, P D; Roberts, M C; Nano, F E

    1997-01-01

    The Mycobacterium tuberculosis H37Rv efpA gene encodes a putative efflux protein, EfpA, of 55,670 Da. The deduced EfpA protein was similar in secondary structure to Pur8, MmrA, TcmA, LfrA, EmrB, and other members of the QacA transporter family (QacA TF) which mediate antibiotic and chemical resistance in bacteria and yeast. The predicted EfpA sequence possessed all transporter motifs characteristic of the QacA TF, including those associated with proton-antiport function and the motif considered to be specific to exporters. The 1,590-bp efpA open reading frame was G+C rich (65%), whereas the 40-bp region immediately upstream had an A+T bias (35% G+C). Reverse transcriptase-PCR assays indicated that efpA was expressed in vitro and in situ. Putative promoter sequences were partially overlapped by the A+T-rich region and by a region capable of forming alternative secondary structures indicative of transcriptional regulation in analogous systems. PCR single-stranded conformational polymorphism analysis demonstrated that these upstream flanking sequences and the 231-bp, 5' coding region are highly conserved among both drug-sensitive and multiply-drug-resistant isolates of M. tuberculosis. The efpA gene was present in the slow-growing human pathogens M. tuberculosis, Mycobacterium leprae, and Mycobacterium bovis and in the opportunistic human pathogens Mycobacterium avium and Mycobacterium intracellular. However, efpA was not present in 17 other opportunistically pathogenic or nonpathogenic mycobacterial species. PMID:9008277

  1. Tuberculosis caused by RDRio Mycobacterium tuberculosis is not associated with differential clinical features

    PubMed Central

    Barbosa, C. de B.; Lazzarini, L. C. O.; Elias, A. R.; Leung, J. A. M.; Ribeiro, S. B.; da Silva, M. G.; Duarte, R. S.; Suffys, P.; Gomes, H. M.; Kritski, A. L.; Lapa e Silva, J. R.; Ho, J. L.; Boéchat, N.

    2013-01-01

    BACKGROUND We recently described the Mycobacterium tuberculosis RDRio genotype, a clonally derived sublineage within the Latin American–Mediterranean (LAM) family. Genetic diversity of M. tuberculosis likely affects the clinical aspects of tuberculosis (TB). Prospective studies that address this issue are scarce and remain controversial. OBJECTIVE To determine the association of differential clinical features of pulmonary TB with the RDRio M. tuberculosis etiology. METHODS Culture-proven pulmonary TB patients (n = 272) were clinically evaluated, including history, physical examination, chest X-ray and anti-human immunodeficiency virus serology. Isolates were classified as RDRio or non-RDRio M. tuberculosis by multiplex polymerase chain reaction and further spoligotyped. Clinical and M. tuberculosis genotype data were analyzed. RESULTS RDRio M. tuberculosis caused disease in 26.5% (72/270) of all TB cases. The LAM genotype, of which RDRio strains are members, was responsible for 46.0% of the TB cases. Demographic data, major signs and symptoms, radiographic presentation, microbiological features and clinical outcomes were not significantly different among patients with TB caused by RDRio and non-RDRio strains. CONCLUSIONS Disease caused by M. tuberculosis RDRio strains was not clinically distinctive or more severe than disease caused by non-RDRio strains in this series of TB patients. Larger prospective studies specifically designed to disclose differential clinical characteristics of TB caused by specific M. tuberculosis lineages are needed. PMID:22863208

  2. Identification of Functional Tat Signal Sequences in Mycobacterium tuberculosis Proteins▿ †

    PubMed Central

    McDonough, Justin A.; McCann, Jessica R.; Tekippe, Erin McElvania; Silverman, Jason S.; Rigel, Nathan W.; Braunstein, Miriam

    2008-01-01

    The twin-arginine translocation (Tat) pathway is a system used by some bacteria to export proteins out from the cytosol to the cell surface or extracellular environment. A functional Tat pathway exists in the important human pathogen Mycobacterium tuberculosis. Identification of the substrates exported by the Tat pathway can help define the role that this pathway plays in the physiology and pathogenesis of M. tuberculosis. Here we used a reporter of Tat export, a truncated β-lactamase, ′BlaC, to experimentally identify M. tuberculosis proteins with functional Tat signal sequences. Of the 13 proteins identified, one lacks the hallmark of a Tat-exported substrate, the twin-arginine dipeptide, and another is not predicted by in silico analysis of the annotated M. tuberculosis genome. Full-length versions of a subset of these proteins were tested to determine if the native proteins are Tat exported. For three proteins, expression in a Δtat mutant of Mycobacterium smegmatis revealed a defect in precursor processing compared to expression in the wild type, indicating Tat export of the full-length proteins. Conversely, two proteins showed no obvious Tat export in M. smegmatis. One of this latter group of proteins was the M. tuberculosis virulence factor phospholipase C (PlcB). Importantly, when tested in M. tuberculosis a different result was obtained and PlcB was exported in a twin-arginine-dependent manner. This suggests the existence of an M. tuberculosis-specific factor(s) for Tat export of a proven virulence protein. It also emphasizes the importance of domains beyond the Tat signal sequence and bacterium-specific factors in determining if a given protein is Tat exported. PMID:18658266

  3. 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.

  4. Mycobacterium tuberculosis Beijing genotype, northern Malawi.

    PubMed

    Glynn, Judith R; Crampin, Amelia C; Traore, Hamidou; Yates, Malcolm D; Mwaungulu, Frank D; Ngwira, Bagrey M; Chaguluka, Steven D; Mwafulirwa, Donex T; Floyd, Sian; Murphy, Caroline; Drobniewski, Francis A; Fine, Paul E M

    2005-01-01

    In a 7-year population-based study in Malawi, we showed that Beijing genotype tuberculosis (TB) increased as a proportion of TB cases. All the Beijing genotype strains were fully drug sensitive. Contact histories, TB type, and case-fatality rates were similar for Beijing and non-Beijing genotype TB.

  5. Molecular modeling of Mycobacterium tuberculosis dUTpase: docking and catalytic mechanism studies.

    PubMed

    Ramalho, Teodorico C; Caetano, Melissa S; Josa, Daniela; Luz, Gustavo P; Freitas, Elisangela A; da Cunha, Elaine F F

    2011-06-01

    Mycobacterium tuberculosis is a leading cause of infectious disease in the world today. This outlook is aggravated by a growing number of M. tuberculosis infections in individuals who are immunocompromised as a result of HIV infections. Thus, new and more potent anti-TB agents are necessary. Therefore, dUTpase was selected as a target enzyme to combat M. tuberculosis. In this work, molecular modeling methods involving docking and QM/MM calculations were carried out to investigate the binding orientation and predict binding affinities of some potential dUTpase inhibitors. Our results suggest that the best potential inhibitor investigated, among the compounds studied in this work, is the compound dUPNPP. Regarding the reaction mechanism, we concluded that the decisive stage for the reaction is the stage 1. Furthermore, it was also observed that the compounds with a -1 electrostatic charge presented lower activation energy in relation to the compounds with a -2 charge.

  6. 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

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

    PubMed

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

    2015-09-01

    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.

  8. 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

  9. 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.

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

    PubMed

    Jacobs, William R

    2014-04-01

    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.

  11. 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

  12. 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

  13. Genetic diversity of Mycobacterium tuberculosis isolates from central India

    PubMed Central

    Desikan, Prabha; Chauhan, D.S.; Sharma, Pragya; Panwalkar, Nikita; Chourey, Manju; Patidar, Mohan Lal; Yadav, Priyanka; Chandrasekaran, V.; Ohri, B.S.

    2016-01-01

    Background & objectives: There is a paucity of data available on genetic biodiversity of Mycobacterium tuberculosis isolates from central India. The present study was carried out on isolates of M. tuberculosis cultured from diagnostic clinical samples of patients from Bhopal, central India, using spoligotyping as a method of molecular typing. Methods: DNA was extracted from 340 isolates of M. tuberculosis from culture, confirmed as M. tuberculosis by molecular and biochemical methods and subjected to spoligotyping. The results were compared with the international SITVIT2 database. Results: Sixty five different spoligo international type (SIT) patterns were observed. A total of 239 (70.3%) isolates could be clustered into 25 SITs. The Central Asian (CAS) and East African Indian (EAI) families were found to be the two major circulating families in this region. SIT26/CAS1_DEL was identified as the most predominant type, followed by SIT11/EAI3_IND and SIT288/CAS2. Forty (11.8%) unique (non-clustered) and 61 (17.9%) orphan isolates were identified in the study. There was no significant association of clustering with clinical and demographic characteristics of patients. Interpretation & conclusions: Well established SITs were found to be predominant in our study. SIT26/CAS1_DEL was the most predominant type. However, the occurrence of a substantial number of orphan isolates may indicate the presence of active spatial and temporal evolutionary dynamics within the isolates of M. tuberculosis. PMID:27377505

  14. 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

  15. How B cells Shape the Immune Response against Mycobacterium tuberculosis

    PubMed Central

    Maglione, Paul J.; Chan, John

    2009-01-01

    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 nonviral 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 nonviral intracellular infections, we review recent data supporting a more significant role of B cells during M. tuberculosis infection than previously thought. PMID:19283721

  16. 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.

  17. Blood agar and Mycobacterium tuberculosis: the end of a dogma.

    PubMed

    Drancourt, M; Carrieri, P; Gévaudan, M-J; Raoult, D

    2003-04-01

    Incidental blood agar-based recovery of Mycobacterium tuberculosis led us to further investigate this routine medium for primary isolation and culture of M. tuberculosis. Fifteen respiratory tract and eight lymph node Ziehl-Neelsen-positive specimens were inoculated in parallel into tubes containing egg-based medium and 5% sheep blood agar. Colonies appeared sooner on this medium than on the egg-based medium, but this difference was not significant (P = 0.11, analysis of variance [ANOVA] test). Further experiments compared the growth of 38 respiratory and lymph node M. tuberculosis isolates when subcultured on the two media. After 6 days of incubation, 21 of 38 isolates had grown on blood agar, and the mean number of colonies was significantly greater on blood agar than on the egg-based medium (P < 0 0.001, ANOVA test). These results demonstrate that M. tuberculosis grows easily on blood agar within 1to 2 weeks, indicating that this basic medium is suitable for laboratory diagnosis of tuberculosis in addition to other media. Laboratories that routinely use prolonged incubations of blood plates, for example, for the recovery of Bartonella species, should consider the potential safety implications of encountering this highly infectious pathogen.

  18. Resistance to cellular autophagy by Mycobacterium tuberculosis Beijing strains.

    PubMed

    Haque, Md Fazlul; Boonhok, Rachasak; Prammananan, Therdsak; Chaiprasert, Angkana; Utaisincharoen, Pongsak; Sattabongkot, Jetsumon; Palittapongarnpim, Prasit; Ponpuak, Marisa

    2015-10-01

    Autophagy represents a key pathway in innate immune defense to restrict Mycobacterium tuberculosis growth inside host macrophages. Induction of autophagy has been shown to promote mycobacterial phagosome acidification and acquisition of lysosomal hydrolases, resulting in the elimination of intracellular M. tuberculosis reference strains such as H37Rv. The notorious Beijing genotype has been previously shown to be hyper-virulent and associated with increased survival in host cells and a high mortality rate in animal models, but the underlying mechanism that renders this family to have such advantages remains unclear. We hypothesize that autophagic control against M. tuberculosis Beijing strains may be altered. Here, we discovered that the Beijing strains can resist autophagic killing by host cells compared with that of the reference strain H37Rv and a strain belonging to the East African Indian genotype. Moreover, we have determined a possible underlying mechanism and found that the greater ability to evade autophagic elimination possessed by the Beijing strains stems from their higher capacity to inhibit autophagolysosome biogenesis upon autophagy induction. In summary, a previously unrecognized ability of the M. tuberculosis Beijing strains to evade host autophagy was identified, which may have important implications for tuberculosis treatment, especially in regions prevalent by the Beijing genotype. PMID:26160686

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. Triosephosphate Isomerase Is Dispensable In Vitro yet Essential for Mycobacterium tuberculosis To Establish Infection

    PubMed Central

    Trujillo, Carolina; Blumenthal, Antje; Marrero, Joeli; Rhee, Kyu Y.; Schnappinger, Dirk; Ehrt, Sabine

    2014-01-01

    ABSTRACT Triosephosphate isomerase (TPI) catalyzes the interconversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P). This reaction is required for glycolysis and gluconeogenesis, and tpi has been predicted to be essential for growth of Mycobacterium tuberculosis. However, when studying a conditionally regulated tpi knockdown mutant, we noticed that depletion of TPI reduced growth of M. tuberculosis in media containing a single carbon source but not in media that contained both a glycolytic and a gluconeogenic carbon source. We used such two-carbon-source media to isolate a tpi deletion (Δtpi) mutant. The Δtpi mutant did not survive with single carbon substrates but grew like wild-type (WT) M. tuberculosis in the presence of both a glycolytic and a gluconeogenic carbon source. 13C metabolite tracing revealed the accumulation of TPI substrates in Δtpi and the absence of alternative triosephosphate isomerases and metabolic bypass reactions, which confirmed the requirement of TPI for glycolysis and gluconeogenesis in M. tuberculosis. The Δtpi strain was furthermore severely attenuated in the mouse model of tuberculosis, suggesting that M. tuberculosis cannot simultaneously access sufficient quantities of glycolytic and gluconeogenic carbon substrates to establish infection in mice. PMID:24757211

  5. 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.

  6. MetaMerge: scaling up genome-scale metabolic reconstructions with application to Mycobacterium tuberculosis

    PubMed Central

    2012-01-01

    Reconstructed models of metabolic networks are widely used for studying metabolism in various organisms. Many different reconstructions of the same organism often exist concurrently, forcing researchers to choose one of them at the exclusion of the others. We describe MetaMerge, an algorithm for semi-automatically reconciling a pair of existing metabolic network reconstructions into a single metabolic network model. We use MetaMerge to combine two published metabolic networks for Mycobacterium tuberculosis into a single network, which allows many reactions that could not be active in the individual models to become active, and predicts essential genes with a higher positive predictive value. PMID:22292986

  7. 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

  8. 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

  9. 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.

  10. 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. PMID:27363904

  11. 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

  12. 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. PMID:25632691

  13. Antimicrobial Resistance in Mycobacterium tuberculosis: The Odd One Out.

    PubMed

    Eldholm, Vegard; Balloux, François

    2016-08-01

    Antimicrobial resistance (AMR) threats are typically represented by bacteria capable of extensive horizontal gene transfer (HGT). One clear exception is Mycobacterium tuberculosis (Mtb). It is an obligate human pathogen with limited genetic diversity and a low mutation rate which lacks any evidence for HGT. Such features should, in principle, reduce its ability to rapidly evolve AMR. We identify key features in its biology and epidemiology that allow it to overcome its low adaptive potential. We focus in particular on its innate resistance to drugs, its unusual life cycle, including an often extensive latent phase, and its ability to shelter from exposure to antimicrobial drugs within cavities it induces in the lungs. PMID:27068531

  14. Genetic regulation of vesiculogenesis and immunomodulation in Mycobacterium tuberculosis

    PubMed Central

    Rath, Poonam; Huang, Chengdong; Wang, Tao; Wang, Tianzhi; Li, Huilin; Prados-Rosales, Rafael; Elemento, Olivier; Casadevall, Arturo; Nathan, Carl F.

    2013-01-01

    Mycobacterium tuberculosis (Mtb) restrains immune responses well enough to escape eradication but elicits enough immunopathology to ensure its transmission. Here we provide evidence that this host–pathogen relationship is regulated in part by a cytosolic, membrane-associated protein with a unique structural fold, encoded by the Mtb gene rv0431. The protein acts by regulating the quantity of Mtb-derived membrane vesicles bearing Toll-like receptor 2 ligands, including the lipoproteins LpqH and SodC. We propose that rv0431 be named “vesiculogenesis and immune response regulator.” PMID:24248369

  15. 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

  16. EVOLUTION OF MYCOBACTERIUM TUBERCULOSIS AND IMPLICATIONS FOR VACCINE DEVELOPMENT.

    PubMed

    Gagneux, Sebastien

    2016-04-01

    Tuberculosis (TB) is a growing public health threat, particularly in the face of the global epidemics of multidrug resistance. Given the limited efficacy of the current TB vaccine and the recent clinical failure of the most advanced new TB vaccine candidate, novel concepts for vaccine design should be explored. Most T cell antigens in the human-adapted Mycobacterium tuberculosis complex (MTBC) are evolutionarily conserved and under strong purifying selection, indicating that host immune responses targeting these antigens might not be protective. By contrast, a few highly variable T cell epitopes have recently been discovered, which could serve as alternative vaccine antigens. Moreover, there is increasing evidence that the human-adapted MTBC has been co-evolving with the human host for a long time. Hence, studying the interaction between bacterial and human genetic diversity might help identify additional targets that could be exploited for TB vaccine development.

  17. 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.

  18. PA-824 Kills Nonreplicating Mycobacterium tuberculosis by Intracellular NO Release

    PubMed Central

    Singh, Ramandeep; Manjunatha, Ujjini; Boshoff, Helena I. M.; Ha, Young Hwan; Niyomrattanakit, Pornwaratt; Ledwidge, Richard; Dowd, Cynthia S.; Lee, Ill Young; Kim, Pilho; Zhang, Liang; Kang, Sunhee; Keller, Thomas H.; Jiricek, Jan; Barry, Clifton E.

    2009-01-01

    Bicyclic nitroimidazoles, including PA-824, are exciting candidates for the treatment of tuberculosis. These prodrugs require intracellular activation for their biological function. We found that Rv3547 is a deazaflavin-dependent nitroreductase (Ddn) that converts PA-824 into three primary metabolites; the major one is the corresponding des-nitroimidazole (des-nitro). When derivatives of PA-824 were used, the amount of des-nitro metabolite formed was highly correlated with anaerobic killing of Mycobacterium tuberculosis (Mtb). Des-nitro metabolite formation generated reactive nitrogen species, including nitric oxide (NO), which are the major effectors of the anaerobic activity of these compounds. Furthermore, NO scavengers protected the bacilli from the lethal effects of the drug. Thus, these compounds may act as intracellular NO donors and could augment a killing mechanism intrinsic to the innate immune system. PMID:19039139

  19. Iron Acquisition Mechanisms: Promising Target Against Mycobacterium tuberculosis

    PubMed Central

    Hameed, Saif; Pal, Rahul; Fatima, Zeeshan

    2015-01-01

    Continuous deployment of antitubercular drugs in treating Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) has led to the emergence of drug resistance resulting in cross-resistance to many unrelated drugs, a phenomenon termed as Multi-Drug Resistance (MDR-TB). Despite reasonable documentation of major factors which contribute to MDR mechanisms, it appears unavoidable to consider novel mechanisms combating MDR. The ability of pathogenic MTB, to sense and become accustomed to changes in the host environment is essential for its survival and confers the basis of their success as dreadful pathogen. One such significant environmental factor that MTB must surmount is iron limitation, since they encounter diverse anatomical sites during the establishment of infection within the host. Considering the importance of MTB, being the second most common cause of mortality, this review focuses on gaining insights of iron acquisition mechanisms in MTB and how it can be exploited as efficient anti-mycobacterial drug target. PMID:26464608

  20. 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

  1. 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

  2. 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.

  3. Mycobacterium tuberculosis infection of the 'non-classical immune cell'.

    PubMed

    Randall, Philippa J; Hsu, Nai-Jen; Quesniaux, Valerie; Ryffel, Bernhard; Jacobs, Muazzam

    2015-10-01

    Mycobacterium tuberculosis can infect 'non-classical immune cells', which comprise a significant constituency of cells that reside outside of those defined as 'classical immune cells' from myeloid or lymphoid origin. Here we address the influence of specific 'non-classical immune cells' in host responses and their effects in controlling mycobacterial growth or enabling an environment conducive for bacilli persistence. The interaction of M. tuberculosis with epithelial cells, endothelial cells, fibroblasts, adipocytes, glia and neurons and downstream cellular responses that often dictate immune regulation and disease outcome are discussed. Functional integration and synergy between 'classical' and 'non-classical immune cells' are highlighted as critical for determining optimal immune outcomes that favour the host. PMID:25801479

  4. Selective Mycobacterium tuberculosis Shikimate Kinase Inhibitors as Potential Antibacterials.

    PubMed

    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

  5. Progress in targeting cell envelope biogenesis in Mycobacterium tuberculosis

    PubMed Central

    Jackson, Mary; McNeil, Michael R; Brennan, Patrick J

    2013-01-01

    Most of the newly discovered compounds showing promise for the treatment of TB, notably multidrug-resistant TB, inhibit aspects of Mycobacterium tuberculosis cell envelope metabolism. This review reflects on the evolution of the knowledge that many of the front-line and emerging products inhibit aspects of cell envelope metabolism and in the process are bactericidal not only against actively replicating M. tuberculosis, but contrary to earlier impressions, are effective against latent forms of the disease. While mycolic acid and arabinogalactan synthesis are still primary targets of existing and new drugs, peptidoglycan synthesis, transport mechanisms and the synthesis of the decaprenyl-phosphate carrier lipid all show considerable promise as targets for new products, older drugs and new combinations. The advantages of whole cell- versus target-based screening in the perpetual search for new targets and products to counter multidrug-resistant TB are discussed. PMID:23841633

  6. 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

  7. A Subset of Circulating Blood Mycobacteria-Specific CD4 T Cells Can Predict the Time to Mycobacterium tuberculosis Sputum Culture Conversion

    PubMed Central

    Lugongolo, Masixole; Gwala, Thabisile; Kiravu, Agano; Deniso, Pamela; Stewart-Isherwood, Lynsey; Omar, Shaheed Vally; Grobusch, Martin P.; Coetzee, Gerrit; Conradie, Francesca; Ismail, Nazir; Kaplan, Gilla; Fallows, Dorothy

    2014-01-01

    We investigated 18 HIV-negative patients with MDR-TB for M. tuberculosis (Mtb)- and PPD-specific CD4 T cell responses and followed them over 6 months of drug therapy. Twelve of these patients were sputum culture (SC) positive and six patients were SC negative upon enrollment. Our aim was to identify a subset of mycobacteria-specific CD4 T cells that would predict time to culture conversion. The total frequency of mycobacteria-specific CD4 T cells at baseline could not distinguish patients showing positive or negative SC. However, a greater proportion of late-differentiated (LD) Mtb- and PPD-specific memory CD4 T cells was found in SC positive patients than in those who were SC negative (p = 0.004 and p = 0.0012, respectively). Similarly, a higher co-expression of HLA-DR+Ki67+ on Mtb- and PPD-specific CD4 T cells could also discriminate between sputum SC positive versus SC negative (p = 0.004 and p = 0.001, respectively). Receiver operating characteristic (ROC) analysis revealed that baseline levels of Ki67+HLA-DR+ Mtb- and PPD-specific CD4 T cells were predictive of the time to sputum culture conversion, with area-under-the-curve of 0.8 (p = 0.027). Upon treatment, there was a significant decline of these Ki67+HLA-DR+ T cell populations in the first 2 months, with a progressive increase in mycobacteria-specific polyfunctional IFNγ+IL2+TNFα+ CD4 T cells over 6 months. Thus, a subset of activated and proliferating mycobacterial-specific CD4 T cells (Ki67+HLA-DR+) may provide a valuable marker in peripheral blood that predicts time to sputum culture conversion in TB patients at the start of treatment. PMID:25048802

  8. A subset of circulating blood mycobacteria-specific CD4 T cells can predict the time to Mycobacterium tuberculosis sputum culture conversion.

    PubMed

    Riou, Catherine; Gray, Clive M; Lugongolo, Masixole; Gwala, Thabisile; Kiravu, Agano; Deniso, Pamela; Stewart-Isherwood, Lynsey; Omar, Shaheed Vally; Grobusch, Martin P; Coetzee, Gerrit; Conradie, Francesca; Ismail, Nazir; Kaplan, Gilla; Fallows, Dorothy

    2014-01-01

    We investigated 18 HIV-negative patients with MDR-TB for M. tuberculosis (Mtb)- and PPD-specific CD4 T cell responses and followed them over 6 months of drug therapy. Twelve of these patients were sputum culture (SC) positive and six patients were SC negative upon enrollment. Our aim was to identify a subset of mycobacteria-specific CD4 T cells that would predict time to culture conversion. The total frequency of mycobacteria-specific CD4 T cells at baseline could not distinguish patients showing positive or negative SC. However, a greater proportion of late-differentiated (LD) Mtb- and PPD-specific memory CD4 T cells was found in SC positive patients than in those who were SC negative (p = 0.004 and p = 0.0012, respectively). Similarly, a higher co-expression of HLA-DR+ Ki67+ on Mtb- and PPD-specific CD4 T cells could also discriminate between sputum SC positive versus SC negative (p = 0.004 and p = 0.001, respectively). Receiver operating characteristic (ROC) analysis revealed that baseline levels of Ki67+ HLA-DR+ Mtb- and PPD-specific CD4 T cells were predictive of the time to sputum culture conversion, with area-under-the-curve of 0.8 (p = 0.027). Upon treatment, there was a significant decline of these Ki67+ HLA-DR+ T cell populations in the first 2 months, with a progressive increase in mycobacteria-specific polyfunctional IFNγ+ IL2+ TNFα+ CD4 T cells over 6 months. Thus, a subset of activated and proliferating mycobacterial-specific CD4 T cells (Ki67+ HLA-DR+) may provide a valuable marker in peripheral blood that predicts time to sputum culture conversion in TB patients at the start of treatment.

  9. A subset of circulating blood mycobacteria-specific CD4 T cells can predict the time to Mycobacterium tuberculosis sputum culture conversion.

    PubMed

    Riou, Catherine; Gray, Clive M; Lugongolo, Masixole; Gwala, Thabisile; Kiravu, Agano; Deniso, Pamela; Stewart-Isherwood, Lynsey; Omar, Shaheed Vally; Grobusch, Martin P; Coetzee, Gerrit; Conradie, Francesca; Ismail, Nazir; Kaplan, Gilla; Fallows, Dorothy

    2014-01-01

    We investigated 18 HIV-negative patients with MDR-TB for M. tuberculosis (Mtb)- and PPD-specific CD4 T cell responses and followed them over 6 months of drug therapy. Twelve of these patients were sputum culture (SC) positive and six patients were SC negative upon enrollment. Our aim was to identify a subset of mycobacteria-specific CD4 T cells that would predict time to culture conversion. The total frequency of mycobacteria-specific CD4 T cells at baseline could not distinguish patients showing positive or negative SC. However, a greater proportion of late-differentiated (LD) Mtb- and PPD-specific memory CD4 T cells was found in SC positive patients than in those who were SC negative (p = 0.004 and p = 0.0012, respectively). Similarly, a higher co-expression of HLA-DR+ Ki67+ on Mtb- and PPD-specific CD4 T cells could also discriminate between sputum SC positive versus SC negative (p = 0.004 and p = 0.001, respectively). Receiver operating characteristic (ROC) analysis revealed that baseline levels of Ki67+ HLA-DR+ Mtb- and PPD-specific CD4 T cells were predictive of the time to sputum culture conversion, with area-under-the-curve of 0.8 (p = 0.027). Upon treatment, there was a significant decline of these Ki67+ HLA-DR+ T cell populations in the first 2 months, with a progressive increase in mycobacteria-specific polyfunctional IFNγ+ IL2+ TNFα+ CD4 T cells over 6 months. Thus, a subset of activated and proliferating mycobacterial-specific CD4 T cells (Ki67+ HLA-DR+) may provide a valuable marker in peripheral blood that predicts time to sputum culture conversion in TB patients at the start of treatment. PMID:25048802

  10. 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

  11. 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

    2010-07-01

    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. PMID:20512795

  12. Carbon flux rerouting during Mycobacterium tuberculosis growth arrest

    PubMed Central

    Shi, Lanbo; Sohaskey, Charles D.; Pfeiffer, Carmen; Datta, Pratik; Parks, Michael; McFadden, Johnjoe; North, Robert J.; Gennaro, Maria L.

    2010-01-01

    Summary A hallmark of the Mycobacterium tuberculosis life cycle is the pathogen’s ability to switch between replicative and non-replicative states in response to host immunity. Transcriptional profiling by qPCR of ~50 M. tuberculosis genes involved in central and lipid metabolism revealed a re-routing of carbon flow associated with bacterial growth arrest during mouse lung infection. Carbon rerouting was marked by a switch from metabolic pathways generating energy and biosynthetic precursors in growing bacilli to pathways for storage compound synthesis during growth arrest. Results of flux balance analysis using an in silico metabolic network were consistent with the transcript abundance data obtained in vivo. Similar transcriptional changes were seen in vitro when M. tuberculosis cultures were treated with bacteriostatic stressors under different nutritional conditions. Thus, altered expression of key metabolic genes reflects growth rate changes rather than changes in substrate availability. A model describing carbon flux rerouting was formulated that (i) provides a coherent interpretation of the adaptation of M. tuberculosis metabolism to immunity-induced stress and (ii) identifies features common to mycobacterial dormancy and stress responses of other organisms. PMID:21091505

  13. Molecular Diversity of Mycobacterium tuberculosis Strains in Northwestern Iran

    PubMed Central

    Pourostadi, Mahya; Rashedi, Jalil; Mahdavi Poor, Behroz; Samadi Kafil, Hossein; Shirazi, Samaneh; Asgharzadeh, Mohammad

    2016-01-01

    Background Years after the development of antituberculosis (TB) drugs, many people continue to suffer from this disease. To control the spread of TB, strains of the Mycobacterium tuberculosis complex need to be determined, and sources of infection must be identified. Such steps should help to prevent transmission of the infection. Objectives The aim of this study was to perform molecular genotyping of isolates of the M. tuberculosis complex obtained from patients in northwestern Iran. Methods One hundred ninety-four culture-positive M. tuberculosis isolates obtained from patients in northwestern Iran were analyzed using the mycobacterial interspersed repetitive unit-exact tandem repeats (MIRU-ETR) method. Results The MIRU-ETR method distinguished 162 different patterns in the 194 isolates, comprising 23 clusters and 139 unique patterns. Its discriminatory power according to the Hunter-Gaston discriminatory index (HGDI) was 0.9978. The largest cluster contained six isolates. Conclusions This research indicated that various strains of M. tuberculosis were responsible for TB and that the majority of cases were due to reactivation. PMID:27800145

  14. LAG3 Expression in Active Mycobacterium tuberculosis Infections

    PubMed Central

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

    2016-01-01

    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. PMID:25549835

  15. 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.

  16. 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. PMID:26948102

  17. 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.

  18. Population genomics of Mycobacterium tuberculosis in the Inuit.

    PubMed

    Lee, Robyn S; Radomski, Nicolas; Proulx, Jean-Francois; Levade, Ines; Shapiro, B Jesse; McIntosh, Fiona; Soualhine, Hafid; Menzies, Dick; Behr, Marcel A

    2015-11-01

    Nunavik, Québec suffers from epidemic tuberculosis (TB), with an incidence 50-fold higher than the Canadian average. Molecular studies in this region have documented limited bacterial genetic diversity among Mycobacterium tuberculosis isolates, consistent with a founder strain and/or ongoing spread. We have used whole-genome sequencing on 163 M. tuberculosis isolates from 11 geographically isolated villages to provide a high-resolution portrait of bacterial genetic diversity in this setting. All isolates were lineage 4 (Euro-American), with two sublineages present (major, n = 153; minor, n = 10). Among major sublineage isolates, there was a median of 46 pairwise single-nucleotide polymorphisms (SNPs), and the most recent common ancestor (MRCA) was in the early 20th century. Pairs of isolates within a village had significantly fewer SNPs than pairs from different villages (median: 6 vs. 47, P < 0.00005), indicating that most transmission occurs within villages. There was an excess of nonsynonymous SNPs after the diversification of M. tuberculosis within Nunavik: The ratio of nonsynonymous to synonymous substitution rates (dN/dS) was 0.534 before the MRCA but 0.777 subsequently (P = 0.010). Nonsynonymous SNPs were detected across all gene categories, arguing against positive selection and toward genetic drift with relaxation of purifying selection. Supporting the latter possibility, 28 genes were partially or completely deleted since the MRCA, including genes previously reported to be essential for M. tuberculosis growth. Our findings indicate that the epidemiologic success of M. tuberculosis in this region is more likely due to an environment conducive to TB transmission than a particularly well-adapted strain.

  19. Population genomics of Mycobacterium tuberculosis in the Inuit

    PubMed Central

    Lee, Robyn S.; Radomski, Nicolas; Proulx, Jean-Francois; Levade, Ines; Shapiro, B. Jesse; McIntosh, Fiona; Soualhine, Hafid; Menzies, Dick; Behr, Marcel A.

    2015-01-01

    Nunavik, Québec suffers from epidemic tuberculosis (TB), with an incidence 50-fold higher than the Canadian average. Molecular studies in this region have documented limited bacterial genetic diversity among Mycobacterium tuberculosis isolates, consistent with a founder strain and/or ongoing spread. We have used whole-genome sequencing on 163 M. tuberculosis isolates from 11 geographically isolated villages to provide a high-resolution portrait of bacterial genetic diversity in this setting. All isolates were lineage 4 (Euro-American), with two sublineages present (major, n = 153; minor, n = 10). Among major sublineage isolates, there was a median of 46 pairwise single-nucleotide polymorphisms (SNPs), and the most recent common ancestor (MRCA) was in the early 20th century. Pairs of isolates within a village had significantly fewer SNPs than pairs from different villages (median: 6 vs. 47, P < 0.00005), indicating that most transmission occurs within villages. There was an excess of nonsynonymous SNPs after the diversification of M. tuberculosis within Nunavik: The ratio of nonsynonymous to synonymous substitution rates (dN/dS) was 0.534 before the MRCA but 0.777 subsequently (P = 0.010). Nonsynonymous SNPs were detected across all gene categories, arguing against positive selection and toward genetic drift with relaxation of purifying selection. Supporting the latter possibility, 28 genes were partially or completely deleted since the MRCA, including genes previously reported to be essential for M. tuberculosis growth. Our findings indicate that the epidemiologic success of M. tuberculosis in this region is more likely due to an environment conducive to TB transmission than a particularly well-adapted strain. PMID:26483462

  20. 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

  1. 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

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

    PubMed Central

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

    2015-01-01

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

  3. 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

  4. 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.

  5. 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 ...

  6. Network analysis identifies Rv0324 and Rv0880 as regulators of bedaquiline tolerance in Mycobacterium tuberculosis.

    PubMed

    Peterson, Eliza J R; Ma, Shuyi; Sherman, David R; Baliga, Nitin S

    2016-01-01

    The resilience of Mycobacterium tuberculosis (MTB) emerges from its ability to effectively counteract immunological, environmental and antitubercular challenges. Here, we demonstrate that MTB can tolerate drug treatment by adopting a tolerant state that can be deciphered through systems analysis of its transcriptional responses. Specifically, we demonstrate how treatment with the antitubercular drug bedaquiline activates a regulatory network that coordinates multiple resistance mechanisms to push MTB into a tolerant state. Disruption of this network, by knocking out its predicted transcription factors, Rv0324 and Rv0880, significantly increased bedaquiline killing and enabled the discovery of a second drug, pretomanid, that potentiated killing by bedaquiline. We demonstrate that the synergistic effect of this combination emerges, in part, through disruption of the tolerance network. We discuss how this network strategy also predicts drug combinations with antagonistic interactions, potentially accelerating the discovery of new effective combination drug regimens for tuberculosis. PMID:27573104

  7. 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

  8. Essential roles for Mycobacterium tuberculosis Rel beyond the production of (p)ppGpp.

    PubMed

    Weiss, Leslie A; Stallings, Christina L

    2013-12-01

    In Mycobacterium tuberculosis, the stringent response to amino acid starvation is mediated by the M. tuberculosis Rel (RelMtb) enzyme, which transfers a pyrophosphate from ATP to GDP or GTP to synthesize ppGpp and pppGpp, respectively. (p)ppGpp then influences numerous metabolic processes. RelMtb also encodes a second, distinct catalytic domain that hydrolyzes (p)ppGpp into pyrophosphate and GDP or GTP. RelMtb is required for chronic M. tuberculosis infection in mice; however, it is unknown which catalytic activity of RelMtb mediates pathogenesis and whether (p)ppGpp itself is necessary. In order to individually investigate the roles of (p)ppGpp synthesis and hydrolysis during M. tuberculosis pathogenesis, we generated RelMtb point mutants that were either synthetase dead (RelMtb(H344Y)) or hydrolase dead (RelMtb(H80A)). M. tuberculosis strains expressing the synthetase-dead RelMtb(H344Y) mutant did not persist in mice, demonstrating that the RelMtb (p)ppGpp synthetase activity is required for maintaining bacterial titers during chronic infection. Deletion of a second predicted (p)ppGpp synthetase had no effect on pathogenesis, demonstrating that RelMtb was the major contributor to (p)ppGpp production during infection. Interestingly, expression of an allele encoding the hydrolase-dead RelMtb mutant, RelMtb(H80A), that is incapable of hydrolyzing (p)ppGpp but still able to synthesize (p)ppGpp decreased the growth rate of M. tuberculosis and changed the colony morphology of the bacteria. In addition, RelMtb(H80A) expression during acute or chronic M. tuberculosis infection in mice was lethal to the infecting bacteria. These findings highlight a distinct role for RelMtb-mediated (p)ppGpp hydrolysis that is essential for M. tuberculosis pathogenesis.

  9. Essential Roles for Mycobacterium tuberculosis Rel beyond the Production of (p)ppGpp

    PubMed Central

    Weiss, Leslie A.

    2013-01-01

    In Mycobacterium tuberculosis, the stringent response to amino acid starvation is mediated by the M. tuberculosis Rel (RelMtb) enzyme, which transfers a pyrophosphate from ATP to GDP or GTP to synthesize ppGpp and pppGpp, respectively. (p)ppGpp then influences numerous metabolic processes. RelMtb also encodes a second, distinct catalytic domain that hydrolyzes (p)ppGpp into pyrophosphate and GDP or GTP. RelMtb is required for chronic M. tuberculosis infection in mice; however, it is unknown which catalytic activity of RelMtb mediates pathogenesis and whether (p)ppGpp itself is necessary. In order to individually investigate the roles of (p)ppGpp synthesis and hydrolysis during M. tuberculosis pathogenesis, we generated RelMtb point mutants that were either synthetase dead (RelMtbH344Y) or hydrolase dead (RelMtbH80A). M. tuberculosis strains expressing the synthetase-dead RelMtbH344Y mutant did not persist in mice, demonstrating that the RelMtb (p)ppGpp synthetase activity is required for maintaining bacterial titers during chronic infection. Deletion of a second predicted (p)ppGpp synthetase had no effect on pathogenesis, demonstrating that RelMtb was the major contributor to (p)ppGpp production during infection. Interestingly, expression of an allele encoding the hydrolase-dead RelMtb mutant, RelMtbH80A, that is incapable of hydrolyzing (p)ppGpp but still able to synthesize (p)ppGpp decreased the growth rate of M. tuberculosis and changed the colony morphology of the bacteria. In addition, RelMtbH80A expression during acute or chronic M. tuberculosis infection in mice was lethal to the infecting bacteria. These findings highlight a distinct role for RelMtb-mediated (p)ppGpp hydrolysis that is essential for M. tuberculosis pathogenesis. PMID:24123821

  10. A recombinant Mycobacterium smegmatis induces potent bactericidal immunity against Mycobacterium tuberculosis

    PubMed Central

    Sweeney, Kari A; Dao, Dee N; Goldberg, Michael F; Hsu, Tsungda; Venkataswamy, Manjunatha M; Henao-Tamayo, Marcela; Ordway, Diane; Sellers, Rani S; Jain, Paras; Chen, Bing; Chen, Mei; Kim, John; Lukose, Regy; Chan, John; Orme, Ian M; Porcelli, Steven A; Jacobs, William R

    2011-01-01

    We report the involvement of an evolutionarily conserved set of mycobacterial genes, the esx-3 region, in evasion of bacterial killing by innate immunity. Whereas high-dose intravenous infections of mice with the rapidly growing mycobacterial species Mycobacterium smegmatis bearing an intact esx-3 locus were rapidly lethal, infection with an M. smegmatis Δesx-3 mutant (here designated as the IKE strain) was controlled and cleared by a MyD88-dependent bactericidal immune response. Introduction of the orthologous Mycobacterium tuberculosis esx-3 genes into the IKE strain resulted in a strain, designated IKEPLUS, that remained susceptible to innate immune killing and was highly attenuated in mice but had a marked ability to stimulate bactericidal immunity against challenge with virulent M. tuberculosis. Analysis of these adaptive immune responses indicated that the highly protective bactericidal immunity elicited by IKEPLUS was dependent on CD4+ memory T cells and involved a distinct shift in the pattern of cytokine responses by CD4+ cells. Our results establish a role for the esx-3 locus in promoting mycobacterial virulence and also identify the IKE strain as a potentially powerful candidate vaccine vector for eliciting protective immunity to M. tuberculosis. PMID:21892180

  11. [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.

  12. Crystal structure of the Mycobacterium tuberculosis transcriptional regulator Rv0302.

    PubMed

    Chou, Tsung-Han; Delmar, Jared A; Wright, Catherine C; Kumar, Nitin; Radhakrishnan, Abhijith; Doh, Julia K; Licon, Meredith H; Bolla, Jani Reddy; Lei, Hsiang-Ting; Rajashankar, Kanagalaghatta R; Su, Chih-Chia; Purdy, Georgiana E; Yu, Edward W

    2015-12-01

    Mycobacterium tuberculosis is a pathogenic bacterial species, which is neither Gram positive nor Gram negative. It has a unique cell wall, making it difficult to kill and conferring resistance to antibiotics that disrupt cell wall biosynthesis. Thus, the mycobacterial cell wall is critical to the virulence of these pathogens. Recent work shows that the mycobacterial membrane protein large (MmpL) family of transporters contributes to cell wall biosynthesis by exporting fatty acids and lipidic elements of the cell wall. The expression of the Mycobacterium tuberculosis MmpL proteins is controlled by a complicated regulatory network system. Here we report crystallographic structures of two forms of the TetR-family transcriptional regulator Rv0302, which participates in regulating the expression of MmpL proteins. The structures reveal a dimeric, two-domain molecule with architecture consistent with the TetR family of regulators. Comparison of the two Rv0302 crystal structures suggests that the conformational changes leading to derepression may be due to a rigid body rotational motion within the dimer interface of the regulator. Using fluorescence polarization and electrophoretic mobility shift assays, we demonstrate the recognition of promoter and intragenic regions of multiple mmpL genes by this protein. In addition, our isothermal titration calorimetry and electrophoretic mobility shift experiments indicate that fatty acids may be the natural ligand of this regulator. Taken together, these experiments provide new perspectives on the regulation of the MmpL family of transporters. PMID:26362239

  13. Accurate Detection of Rifampicin-Resistant Mycobacterium Tuberculosis Strains

    PubMed Central

    Song, Keum-Soo; Nimse, Satish Balasaheb; Kim, Hee Jin; Yang, Jeongseong; Kim, Taisun

    2016-01-01

    In 2013 alone, the death rate among the 9.0 million people infected with Mycobacterium tuberculosis (TB) worldwide was around 14%, which is unacceptably high. An empiric treatment of patients infected with TB or drug-resistant Mycobacterium tuberculosis (MDR-TB) strain can also result in the spread of MDR-TB. The diagnostic tools which are rapid, reliable, and have simple experimental protocols can significantly help in decreasing the prevalence rate of MDR-TB strain. We report the evaluation of the 9G technology based 9G DNAChips that allow accurate detection and discrimination of TB and MDR-TB-RIF. One hundred and thirteen known cultured samples were used to evaluate the ability of 9G DNAChip in the detection and discrimination of TB and MDR-TB-RIF strains. Hybridization of immobilized probes with the PCR products of TB and MDR-TB-RIF strains allow their detection and discrimination. The accuracy of 9G DNAChip was determined by comparing its results with sequencing analysis and drug susceptibility testing. Sequencing analysis showed 100% agreement with the results of 9G DNAChip. The 9G DNAChip showed very high sensitivity (95.4%) and specificity (100%). PMID:26999135

  14. Are the PE-PGRS proteins of Mycobacterium tuberculosis variable surface antigens?

    PubMed

    Banu, Sayera; Honoré, Nadine; Saint-Joanis, Brigitte; Philpott, Dana; Prévost, Marie-Christine; Cole, Stewart T

    2002-04-01

    Mycobacterium tuberculosis H37Rv contains 67 PE-PGRS genes, with multiple tandem repetitive sequences, encoding closely related proteins that are exceptionally rich in glycine and alanine. As no functional information was available, 10 of these genes were selected and shown to be expressed in vitro by reverse transcription-polymerase chain reaction (RT-PCR). Antibodies against five PE-PGRS proteins, raised in mice by DNA vaccination, detected single proteins when the same plasmid constructs used for immunization were expressed in epithelial cells or in reticulocyte extracts, confirming that the PE-PGRS proteins are antigenic. As expected from the conserved repetitive structure, the antibodies cross-reacted with more than one PE-PGRS protein, suggesting that different proteins share common epitopes. PE-PGRS proteins were detected by West-ern blotting in five different mycobacterial species (M. tuberculosis, M. bovis BCG, M. smegmatis, M. marinum and M. gordonae) and 11 clinical isolates of M. tuberculosis. Whole-genome comparisons of M. tuberculosis predicted allelic diversity in the PE-PGRS family, and this was confirmed by immunoblot studies as size variants were detected in clinical strains. Subcellular fractionation studies and immunoelectron microscopy localized many PE-PGRS proteins in the cell wall and cell membrane of M. tuberculosis. The data suggest that some PE-PGRS proteins are variable surface antigens.

  15. Variation of the expression of Mycobacterium tuberculosis ppe44 gene among clinical isolates.

    PubMed

    Rindi, Laura; Peroni, Irene; Lari, Nicoletta; Bonanni, Daniela; Tortoli, Enrico; Garzelli, Carlo

    2007-11-01

    PPE44 is a member of the Mycobacterium tuberculosis PPE proteins, a polymorphic family of 69 glycine-rich proteins that predictively represent a source of antigenic variation. The genetic diversity of gene ppe44 among clinical isolates has been studied. No genomic polymorphism of ppe44 was found by a PCR-restriction fragment length polymorphism assay using three restriction enzymes. Nucleotide sequencing of gene ppe44 of a number of isolates, selected to represent the major phylogenetic lineages of M. tuberculosis, showed no nucleotide substitution, with the exception of isolates of the Beijing genotype. These findings indicate that gene ppe44 is basically conserved among M. tuberculosis strains. The expression of gene ppe44 was then determined at the transcriptional level by a real-time reverse transcriptase PCR assay. Extremely high quantitative variations in ppe44 expression were found among the isolates; ppe44 expression of the Beijing strains was significantly higher than the non-Beijing strains. To test whether differential expression of gene ppe44 has the potential to provide a dynamic antigen display, antibodies to PPE44 were titered in the sera of M. tuberculosis-infected subjects. Variation of antibody response to PPE44 was found with regard to both antibody titers and the proportion of responding subjects. These results indicate that the differential expression of genes ppe could influence the host's immune responsiveness, thus having implications in the immunopathogenesis of tuberculosis. PMID:17727653

  16. Crystallographic and docking studies of purine nucleoside phosphorylase from Mycobacterium tuberculosis.

    PubMed

    Ducati, Rodrigo G; Basso, Luiz A; Santos, Diógenes S; de Azevedo, Walter F

    2010-07-01

    This work describes for the first time the structure of purine nucleoside phosphorylase from Mycobacterium tuberculosis (MtPNP) in complex with sulfate and its natural substrate, 2'-deoxyguanosine, and its application to virtual screening. We report docking studies of a set of molecules against this structure. Application of polynomial empirical scoring function was able to rank docking solutions with good predicting power which opens the possibility to apply this new criterion to analyze docking solutions and screen small-molecule databases for new chemical entities to inhibit MtPNP.

  17. Acquired Drug Resistance in Mycobacterium tuberculosis and Poor Outcomes among Patients with Multidrug-Resistant Tuberculosis

    PubMed Central

    Kipiani, Maia; Mirtskhulava, Veriko; Tukvadze, Nestani; Magee, Matthew J.; Blumberg, Henry M.

    2015-01-01

    Rates and risk factors for acquired drug resistance and association with outcomes among patients with multidrug-resistant tuberculosis (MDR TB) are not well defined. In an MDR TB cohort from the country of Georgia, drug susceptibility testing for second-line drugs (SLDs) was performed at baseline and every third month. Acquired resistance was defined as any SLD whose status changed from susceptible at baseline to resistant at follow-up. Among 141 patients, acquired resistance in Mycobacterium tuberculosis was observed in 19 (14%); prevalence was 9.1% for ofloxacin and 9.8% for capreomycin or kanamycin. Baseline cavitary disease and resistance to >6 drugs were associated with acquired resistance. Patients with M. tuberculosis that had acquired resistance were at significantly increased risk for poor treatment outcome compared with patients without these isolates (89% vs. 36%; p<0.01). Acquired resistance occurs commonly among patients with MDR TB and impedes successful treatment outcomes. PMID:25993036

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

    PubMed

    Yang, Huan; Tang, Hua; Chen, Xin-Xin; Zhang, Chang-Jian; Zhu, Pan-Pan; Ding, Hui; Chen, Wei; Lin, Hao

    2016-01-01

    Tuberculosis is killing millions of lives every year and on the blacklist of the most appalling public health problems. Recent findings suggest that secretory protein of Mycobacterium tuberculosis may serve the purpose of developing specific vaccines and drugs due to their antigenicity. Responding to global infectious disease, we focused on the identification of secretory proteins in Mycobacterium tuberculosis. A novel method called MycoSec was designed by incorporating g-gap dipeptide compositions into pseudo amino acid composition. Analysis of variance-based technique was applied in the process of feature selection and a total of 374 optimal features were obtained and used for constructing the final predicting model. In the jackknife test, MycoSec yielded a good performance with the area under the receiver operating characteristic curve of 0.93, demonstrating that the proposed system is powerful and robust. For user's convenience, the web server MycoSec was established and an obliging manual on how to use it was provided for getting around any trouble unnecessary. PMID:27597968

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

    PubMed Central

    Yang, Huan; Tang, Hua; Chen, Xin-Xin; Zhang, Chang-Jian; Zhu, Pan-Pan; Ding, Hui

    2016-01-01

    Tuberculosis is killing millions of lives every year and on the blacklist of the most appalling public health problems. Recent findings suggest that secretory protein of Mycobacterium tuberculosis may serve the purpose of developing specific vaccines and drugs due to their antigenicity. Responding to global infectious disease, we focused on the identification of secretory proteins in Mycobacterium tuberculosis. A novel method called MycoSec was designed by incorporating g-gap dipeptide compositions into pseudo amino acid composition. Analysis of variance-based technique was applied in the process of feature selection and a total of 374 optimal features were obtained and used for constructing the final predicting model. In the jackknife test, MycoSec yielded a good performance with the area under the receiver operating characteristic curve of 0.93, demonstrating that the proposed system is powerful and robust. For user's convenience, the web server MycoSec was established and an obliging manual on how to use it was provided for getting around any trouble unnecessary. PMID:27597968

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

    PubMed Central

    Yang, Huan; Tang, Hua; Chen, Xin-Xin; Zhang, Chang-Jian; Zhu, Pan-Pan; Ding, Hui

    2016-01-01

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

  1. Analysis of the Mycobacterium tuberculosis 85A antigen promoter region.

    PubMed Central

    Kremer, L; Baulard, A; Estaquier, J; Content, J; Capron, A; Locht, C

    1995-01-01

    A mycobacterial expression-secretion vector was constructed in which the Escherichia coli alkaline phosphatase (phoA) reporter gene was placed under the control of the Mycobacterium tuberculosis 85A promoter and secretion signal sequences. In recombinant Mycobacterium smegmatis and Mycobacterium bovis BCG, PhoA activity could readily be detected on the mycobacterial cell surface and in the culture supernatant, indicating that the 85A signals can drive heterologous expression and secretion in both species. In contrast to the mycobacteria, the 85A promoter did not function in E. coli. We mapped the promoter region by progressive deletions using BAL 31 exonuclease and by primer extension analysis. Insertion and deletion mutations within the promoter region indicated that, unlike most E. coli promoters but similar to Streptomyces promoters, the position of the putative -35 region was not critical for efficient promoter activity. In addition, we investigated the ability of the identified signals to drive the production and secretion in BCG of recombinant Schistosoma mansoni glutathione S-transferase (Sm28GST), a protective antigen against schistosomiasis. BALB/c mice immunized with the recombinant BCG by a single dose exhibited a weak but specific T-cell response to Sm28GST. PMID:7836298

  2. Novel multiplex real-time PCR diagnostic assay for identification and differentiation of Mycobacterium tuberculosis, Mycobacterium canettii, and Mycobacterium tuberculosis complex strains.

    PubMed

    Reddington, Kate; O'Grady, Justin; Dorai-Raj, Siobhan; Maher, Majella; van Soolingen, Dick; Barry, Thomas

    2011-02-01

    Tuberculosis (TB) in humans is caused by members of the Mycobacterium tuberculosis complex (MTC). Rapid detection of the MTC is necessary for the timely initiation of antibiotic treatment, while differentiation between members of the complex may be important to guide the appropriate antibiotic treatment and provide epidemiological information. In this study, a multiplex real-time PCR diagnostics assay using novel molecular targets was designed to identify the MTC while simultaneously differentiating between M. tuberculosis and M. canettii. The lepA gene was targeted for the detection of members of the MTC, the wbbl1 gene was used for the differentiation of M. tuberculosis and M. canettii from the remainder of the complex, and a unique region of the M. canettii genome, a possible novel region of difference (RD), was targeted for the specific identification of M. canettii. The multiplex real-time PCR assay was tested using 125 bacterial strains (64 MTC isolates, 44 nontuberculosis mycobacteria [NTM], and 17 other bacteria). The assay was determined to be 100% specific for the mycobacteria tested. Limits of detection of 2.2, 2.17, and 0.73 cell equivalents were determined for M. tuberculosis/M. canettii, the MTC, and M. canettii, respectively, using probit regression analysis. Further validation of this diagnostics assay, using clinical samples, should demonstrate its potential for the rapid, accurate, and sensitive diagnosis of TB caused by M. tuberculosis, M. canettii, and the other members of the MTC.

  3. Molecular Epidemiology of Mycobacterium tuberculosis Isolates in 100 Patients With Tuberculosis Using Pulsed Field Gel Electrophoresis

    PubMed Central

    Pooideh, Mohammad; Jabbarzadeh, Ismail; Ranjbar, Reza; Saifi, Mahnaz

    2015-01-01

    Background: Tuberculosis (TB) is a widespread infectious disease. Today, TB has created a public health crisis in the world. Genotyping of Mycobacterium tuberculosis isolates is useful for surveying the dynamics of TB infection, identifying new outbreaks, and preventing the disease. Different molecular methods for clustering of M. tuberculosis isolates have been used. Objectives: During a one year study of genotyping, 100 M. tuberculosis isolates from patients referred to Pasteur Institute of Iran were collected and their genotyping was accomplished using pulsed field gel electrophoresis (PFGE) method. Materials and Methods: Identification of all M. tuberculosis isolates was accomplished using standard biochemical and species-specific polymerase chain reaction (PCR) methods. Antibiotic susceptibility tests were performed using proportional method. After preparing PFGE plaques for each isolate of M. tuberculosis, XbaI restriction enzyme was applied for genome digestion. Finally, the digested DNA fragments were separated on 1% agarose gel and analyzed with GelCompar II software. Results: Genotyping of the studied isolates in comparison with the molecular weight marker revealed two common types; pulsotype A with 71 isolates and one multidrug resistant mycobacterium (MDR) case, and pulsotype B including 29 isolates and three MDR cases. No correlation between the antibiotypes and pulsotypes was observed. Conclusions: Molecular epidemiology studies of infectious diseases have been useful when bacterial isolates have been clustered in a period of time and in different geographical regions with variable antibiotic resistance patterns. In spite of high geographical differences and different antibiotic resistant patterns, low genetic diversity among the studied TB isolates may refer to the low rate of mutations in XbaI restriction sites in the mycobacterial genome. We also identified three MDR isolates in low-incidence pulsotype B, which could be disseminated and is highly

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

    PubMed

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

    2005-07-01

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

  5. Activity of 5-chloro-pyrazinamide in mice infected with Mycobacterium tuberculosis or Mycobacterium bovis

    PubMed Central

    Ahmad, Zahoor; Tyagi, Sandeep; Minkowski, Austin; Almeida, Deepak; Nuermberger, Eric L.; Peck, Kaitlin M.; Welch, John T.; Baughn, Anthony D.; Jacobs, Williams R.; Grosset, Jacques H.

    2012-01-01

    Background & objectives: Pyrazinamide is an essential component of first line anti-tuberculosis regimen as well as most of the second line regimens. This drug has a unique sterilizing activity against Mycobacterium tuberculosis. Its unique role in tuberculosis treatment has lead to the search and development of its structural analogues. One such analogue is 5-chloro-pyrazinamide (5-Cl-PZA) that has been tested under in vitro conditions against M. tuberculosis. The present study was designed with an aim to assess the activity of 5-Cl-PZA, alone and in combination with first-line drugs, against murine tuberculosis. Methods: The minimum inhibitory concentration (MIC) of 5-Cl-PZA in Middlebrook 7H9 broth (neutral pH) and the inhibitory titre of serum from mice that received a 300 mg/kg oral dose of 5-Cl-PZA 30 min before cardiac puncture were determined. To test the tolerability of orally administered 5-Cl-PZA, uninfected mice received doses up to 300 mg/kg for 2 wk. Four weeks after low-dose aerosol infection either with M. tuberculosis or M. bovis, mice were treated 5 days/wk with 5-Cl-PZA, at doses ranging from 37.5 to 150 mg/kg, either alone or in combination with isoniazid and rifampicin. Antimicrobial activity was assessed by colony-forming unit counts in lungs after 4 and 8 wk of treatment. Results: The MIC of 5-Cl-PZA against M. tuberculosis was between 12.5 and 25 μg/ml and the serum inhibitory titre was 1:4. Under the same experimental conditions, the MIC of pyrazinamide was >100 μg/ml and mouse serum had no inhibitory activity after a 300 mg/kg dose; 5-Cl-PZA was well tolerated in uninfected and infected mice up to 300 and 150 mg/kg, respectively. While PZA alone and in combination exhibited its usual antimicrobial activity in mice infected with M. tuberculosis and no activity in mice infected with M. bovis, 5-Cl-PZA exhibited antimicrobial activity neither in mice infected with M. tuberculosis nor in mice infected with M. bovis. Interpretation

  6. 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

  7. Molecular epidemiologic evaluation of transmissibility and virulence of Mycobacterium tuberculosis.

    PubMed

    Rhee, J T; Piatek, A S; Small, P M; Harris, L M; Chaparro, S V; Kramer, F R; Alland, D

    1999-06-01

    Discovery of genotypic markers associated with increased transmissibility in Mycobacterium tuberculosis would represent an important step in advancing mycobacterial virulence studies. M. tuberculosis strains may be classified into one of three genotypes on the basis of the presence of specific nucleotide substitutions in codon 463 of the katG gene (katG-463) and codon 95 of the gyrA gene (gyrA-95). It has previously been reported that two of these three genotypes are associated with increased IS6110-based clustering, a potential proxy of virulence. We designed a case-control analysis of U.S.-born patients with tuberculosis in San Francisco, Calif., between 1991 and 1997 to investigate associations between katG-463 and gyrA-95 genotypes and epidemiologically determined measures of strain-specific infectivity and pathogenicity and IS6110-based clustering status. We used a new class of molecular probes called molecular beacons to genotype the isolates rapidly. Infectivity was defined as the propensity of isolates to cause tuberculin skin test conversions among named contacts, and pathogenicity was defined as their propensity to cause active disease among named contacts. The molecular beacon assay was a simple and reproducible method for the detection of known single nucleotide polymorphisms in large numbers of clinical M. tuberculosis isolates. The results showed that no genotype of the katG-463- and gyrA-95-based classification system was associated with increased infectivity and pathogenicity or with increased IS6110-based clustering in San Francisco during the study period. We speculate that molecular epidemiologic studies investigating clinically relevant outcomes may contribute to the knowledge of the significance of laboratory-derived virulence factors in the propagation of tuberculosis in human communities.

  8. Use of GeneXpert Mycobacterium tuberculosis/rifampicin for rapid detection of rifampicin resistant Mycobacterium tuberculosis strains of clinically suspected multi-drug resistance tuberculosis cases

    PubMed Central

    Guenaoui, Kheira; Ouardi, Aissa; Zeggai, Soumia; Sellam, Feriel; Bekri, Farid; Cherif Touil, Sakina

    2016-01-01

    Background Multi-drug resistance (MDR) TB is defined as tuberculosis (TB) disease caused by a strain of Mycobacterium tuberculosis (MTB) that was resistant to at least isoniazid and rifampicin (RIF). Emerging Multidrug-Resistant TB is one of the major concerns of health policy and rapid detection of M. tuberculosis and detection of RIF resistance in infected patients are essential for disease management. The aim of this study was to evaluate patterns of RIF resistance in cases of sputum positive pulmonary TB by using GeneXpert MTB/RIF and comparing between phenotypic and genotypic testing of RIF resistance in MTB strains of clinically suspected MDR-TB isolated cases in western Algeria. Methods In this study 50 sputum positive cases of pulmonary TB who were potential MDR suspect were included. Their sputum samples were collected and subjected to sputum smear microscopy, culture and conventional MTB/RIF test followed by GeneXpert MTB/RIF assay. Results Of total 50 cases included in this study, MTB was detected in all patients (100%) by GeneXpert MTB/RIF. However, RIF’s resistance was detected in only 21 cases (42%) by GeneXpert MTB/RIF. All RIF resistant strains detected by GeneXpert MTB/RIF were phenotypically confirmed as MDR strains. 42.85% of cases were retreatment failure cases, retreatment cases smear positive at 4 months were 23.82%. While 19.05% of cases were retreatment cases smear positive at diagnosis, and 14.28% patient had history of contact with MDR-TB. Sensitivity, specificity, positive predictive value and negative predictive value of Xpert MTB/RIF to detect RIF resistance in comparison to conventional phenotypic drug susceptibility technique were found equal to the rates of 100%, 100%, 100% and 100%, respectively. Conclusions GeneXpert MTB/RIF assay is efficient and reliable technique for the rapid diagnostic of TB. It’s simplicity, high sensitivity and specificity for RIF resistance detection make this technique a very attractive tool for

  9. Functional assignment of Mycobacterium tuberculosis proteome revealed by genome-scale fold-recognition.

    PubMed

    Mao, Chunhong; Shukla, Maulik; Larrouy-Maumus, Gérald; Dix, Flora L; Kelley, Lawrence A; Sternberg, Michael J; Sobral, Bruno W; de Carvalho, Luiz Pedro S

    2013-01-01

    Hundreds of putative enzymes from Mycobacterium tuberculosis as well as other mycobacteria remain categorized as "conserved hypothetical proteins" or "hypothetical proteins", offering little or no information on their functional role in pathogenic and non-pathogenic processes. In this study we have predicted the fold and 3-D structure of more than 99% of all proteins encoded in the genome of M. tuberculosis H37Rv. Fold-recognition, database search, 3-D modelling was performed using Protein Homology/analogy Recognition Engine V 2.0 (Phyre2). These results are used to tentatively assign potential function for unannotated enzymes and proteins. In summary, fold-recognition and structural homology might be used as a complementary tool in genome annotation efforts and furthermore, it can deliver primary sequence-independent information regarding structure, ligands and even substrate specificity for enzymes that display low primary sequence identity with potential homologues in other species.

  10. Clinical value of the measurement of Mycobacterium tuberculosis specific antibody in pulmonary tuberculosis.

    PubMed Central

    Bothamley, G H; Rudd, R; Festenstein, F; Ivanyi, J

    1992-01-01

    BACKGROUND: A serological test that could help to diagnose tuberculosis, especially smear negative disease, would contribute to patient management. METHODS: Levels of antibody to distinct antigens of Mycobacterium tuberculosis were assessed for their value in the diagnosis and management of pulmonary tuberculosis. Serum was taken from 52 patients who were smear positive, from 27 patients who were smear negative but with evidence of active tuberculosis (sputum culture positive in 16, response to antituberculosis chemotherapy in 11), from 11 patients with old healed tuberculosis (pre-antibiotic era), and from 39 healthy subjects vaccinated with BCG. RESULTS: In smear positive tuberculosis an enzyme linked immunosorbent assay using a single 38 kDa antigen gave a diagnostic sensitivity of 80% with a 100% specificity. In smear negative pulmonary tuberculosis, however, combination of the 19 kDa antigen, lipoarabinomannan (ML 34 epitope), and hsp 65 (TB 78 epitope) was needed to achieve a sensitivity of 64% with a specificity of 95%. Recurrent and extensive radiographic disease with a poor prognosis was associated with high anti-38 kDa and low anti-14 kDa antibody levels in patients with active disease. Patients with less pulmonary cavitation had high anti-19 kDa titres. Bacteriological relapse during treatment was indicated by a rise in anti-14 kDa (TB68 epitope) antibodies. Four patients with non-tuberculous mycobacterial infection showed no anti-38 kDa antibody. CONCLUSION: Antigen or epitope specific serology may help in the diagnosis, assessment of prognosis, and monitoring of chemotherapy in patients with pulmonary tuberculosis. PMID:1585290

  11. Genome Sequence of Mycobacterium tuberculosis C2, a Cerebrospinal Fluid Clinical Isolate from Central India

    PubMed Central

    Bhullar, Shradha S.; More, Ravi P.; Puranik, Sampada; Taori, Girdhar M.; Daginawala, Hatim F.

    2014-01-01

    We report the annotated genome sequence of a Mycobacterium tuberculosis clinical isolate from the cerebrospinal fluid of a tuberculous meningitis patient admitted to the Central India Institute of Medical Sciences, Nagpur, India. PMID:25146143

  12. [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.

  13. Pulmonary Tuberculosis Caused by Mycobacterium bovis in China

    PubMed Central

    Jiang, Guanglu; Wang, Guirong; Chen, Suting; Yu, Xia; Wang, Xiaobo; Zhao, Liping; Ma, Yifeng; Dong, Lingling; Huang, Hairong

    2015-01-01

    The epidemiology of Mycobacterium bovis infection in humans in China is unknown. In this study, pulmonary tuberculosis caused by M. bovis in China was studied. A total of 4069 clinical strains isolated from sputa during the 2007–2009 nationwide surveillance of drug-resistant tuberculosis in China were analyzed. M. bovis was identified by para-nitrobenzoic acid and thiophen-2-carboxylic acid hydrazide growth tests, spoligotyping and multiplex PCR amplification. In addition, a total of 1828 clinical specimens were recruited from Beijing Chest Hospital (Beijing, China) for Löwenstein-Jensen (LJ) culture, both on standard LJ medium and LJ medium containing 4.5 mg/ml(W/V) sodium pyruvate, the latter being the preferred medium for M. bovis growth. The isolates which demonstrated more vigorous on pyruvate containing medium than on standard LJ medium were then identified by multiplex PCR amplification. Only 1 isolate from the nationwide surveillance was confirmed as M. bovis-BCG. The isolate belonged to a predominant spoligotype SB0120 (ST482). In addition, no M. bovis isolate was acquired by the continuous screening step in Beijing Chest Hospital. M. bovis has a negligible contribution to pulmonary tuberculosis in China, so neither laboratory identification nor clinical treatment of M. bovis infection need be considered in routine work. PMID:25736338

  14. Proteomic analysis of ofloxacin-mono resistant Mycobacterium tuberculosis isolates.

    PubMed

    Lata, Manju; Sharma, Divakar; Deo, Nirmala; Tiwari, Pramod Kumar; Bisht, Deepa; Venkatesan, Krishnamurthy

    2015-09-01

    Drug resistance particularly, multi drug resistance tuberculosis (MDR-TB) has emerged as a major problem in the chemotherapy of tuberculosis. Ofloxacin (OFX) has been used as second-line drug against MDR-TB. The principal target of the OFX is DNA gyrase encoded by gyrA and gyrB genes. Many explanations have been proposed for drug resistance to OFX but still some mechanisms are unknown. As proteins manifest most of the biological processes, these are attractive targets for developing drugs and diagnostics/therapeutics. We examined the OFX resistant Mycobacterium tuberculosis isolates by proteomic approach (2DE-MALDI-TOF-MS) and bioinformatic tools under OFX induced conditions. Our study showed fourteen proteins (Rv0685, Rv0363c, Rv2744c, Rv3803c, Rv2534c, Rv2140c, Rv1475c, Rv0440, Rv2245, Rv1436, Rv3551, Rv0148, Rv2882c and Rv0733) with increased intensities in OFX resistant and OFX induced as compared to susceptible isolates. Bioinformatic analysis of hypothetical proteins (Rv2744c, Rv2140c, Rv3551 and Rv0148) revealed the presence of conserved motifs and domains. Molecular docking showed proper interaction of OFX with residues of conserved motifs. These proteins might be involved in the OFX modulation/neutralization and act as novel resistance mechanisms as well as potential for diagnostics and drug targets against OFX resistance. This article is part of a Special Issue entitled: Proteomics in India.

  15. 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

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

    PubMed

    Flentie, Kelly; Garner, Ashley L; Stallings, Christina L

    2016-05-01

    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.

  17. 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

  18. Direct inhibitors of InhA active against Mycobacterium tuberculosis

    PubMed Central

    Manjunatha, Ujjini H.; Rao, Srinivasa P. S.; Kondreddi, Ravinder Reddy; Noble, Christian G.; Camacho, Luis R.; Tan, Bee H.; Ng, Seow H.; Ng, Pearly Shuyi; Ma, N. L.; Lakshminarayana, Suresh B.; Herve, Maxime; Barnes, S. Whitney; Yu, Weixuan; Kuhen, Kelli; Blasco, Francesca; Beer, David; Walker, John R.; Tonge, Peter J.; Glynne, Richard; Smith, Paul W.; Diagana, Thierry T.

    2015-01-01

    New chemotherapeutic agents are urgently required to combat the global spread of multi-drug resistant tuberculosis (MDR-TB). The mycobacterial enoyl reductase, InhA, is one of the few clinically-validated targets in tuberculosis drug discovery. Here, we report the identification of a new class of direct InhA inhibitors, the 4-hydroxy-2-pyridones, using phenotypic high-throughput whole-cell screening. This class of orally-active compounds showed potent bactericidal activity against common isoniazid-resistant TB clinical isolates. Biophysical studies revealed that 4-hydroxy-2-pyridones bound specifically to InhA in an NADH-dependent manner and blocked the enoyl-substrate binding pocket. The lead compound NITD-916 directly blocked InhA in a dose-dependent manner and showed in vivo efficacy in acute and established mouse models of infection by Mycobacterium tuberculosis. Collectively, our structural and biochemical data open up new avenues for rational structure-guided optimization of the 4-hydroxy-2-pyridone class of compounds for the treatment of MDR-TB. PMID:25568071

  19. Infection of human THP-1 cells with dormant Mycobacterium tuberculosis.

    PubMed

    Iona, Elisabetta; Pardini, Manuela; Gagliardi, Maria Cristina; Colone, Marisa; Stringaro, Anna Rita; Teloni, Raffaela; Brunori, Lara; Nisini, Roberto; Fattorini, Lanfranco; Giannoni, Federico

    2012-09-01

    Dormant, non-replicating Mycobacterium tuberculosis H37Rv strain cultured in hypoxic conditions was used to infect THP-1 cells. CFUs counting, Kinyoun staining and electron microscopy showed that dormant bacilli infected THP-1 cells at a rate similar to replicating M. tuberculosis, but failed to grow during the first 6 days of infection. The absence of growth was specific to the intracellular compartment, as demonstrated by efficient growth in liquid medium. Quantification of β-actin mRNA recovered from infected cells showed that, in contrast with log-phase bacteria, infection with dormant bacilli determined a reduced THP-1 cell death. Gene expression of intracellular non-replicating bacteria showed a pattern typical of a dormant state. Intracellular dormant bacteria induced the activation of genes associated to a proinflammatory response in THP-1 cells. Though, higher levels of TNFα, IL-1β and IL-8 mRNAs compared to aerobic H37Rv infected cells were not paralleled by increased cytokine accumulation in the supernatants. Moreover, dormant bacilli induced a higher expression of inducible cox-2 gene, accompanied by increased PGE2 secretion. Overall, our data describe a new model of in vitro infection using dormant M. tuberculosis that could provide the basis for understanding how non-replicating bacilli survive intracellularly and influence the maintenance of the hypoxic granuloma.

  20. Copper resistance is essential for virulence of Mycobacterium tuberculosis.

    PubMed

    Wolschendorf, Frank; Ackart, David; Shrestha, Tej B; Hascall-Dove, Laurel; Nolan, Scott; Lamichhane, Gyanu; Wang, Ying; Bossmann, Stefan H; Basaraba, Randall J; Niederweis, Michael

    2011-01-25

    Copper (Cu) is essential for many biological processes, but is toxic when present in excessive amounts. In this study, we provide evidence that Cu plays a crucial role in controlling tuberculosis. A Mycobacterium tuberculosis (Mtb) mutant lacking the outer membrane channel protein Rv1698 accumulated 100-fold more Cu and was more susceptible to Cu toxicity than WT Mtb. Similar phenotypes were observed for a M. smegmatis mutant lacking the homolog Ms3747, demonstrating that these mycobacterial copper transport proteins B (MctB) are essential for Cu resistance and maintenance of low intracellular Cu levels. Guinea pigs responded to infection with Mtb by increasing the Cu concentration in lung lesions. Loss of MctB resulted in a 1,000- and 100-fold reduced bacterial burden in lungs and lymph nodes, respectively, in guinea pigs infected with Mtb. In mice, the persistence defect of the Mtb mctB mutant was exacerbated by the addition of Cu to the diet. These experiments provide evidence that Cu is used by the mammalian host to control Mtb infection and that Cu resistance mechanisms are crucial for Mtb virulence. Importantly, Mtb is much more susceptible to Cu than other bacteria and is killed in vitro by Cu concentrations lower than those found in phagosomes of macrophages. Hence, this study reveals an Achilles heel of Mtb that might be a promising target for tuberculosis chemotherapy. PMID:21205886

  1. Tuberculosis from Mycobacterium bovis in Binational Communities, United States

    PubMed Central

    Moore, Marisa; Moser, Kathleen S.; Brodine, Stephanie K.; Strathdee, Steffanie A.

    2008-01-01

    The epidemiology of tuberculosis (TB) in the United States is changing as the incidence of disease becomes more concentrated in foreign-born persons. Mycobacterium bovis appears to be contributing substantially to the TB incidence in some binational communities with ties to Mexico. We conducted a retrospective analysis of TB case surveillance data from the San Diego, California, region from 1994 through 2005 to estimate incidence trends, identify correlates of M. bovis disease, and evaluate risk factors for deaths during treatment. M. bovis accounted for 45% (62/138) of all culture-positive TB cases in children (<15 years of age) and 6% (203/3,153) of adult cases. M. bovis incidence increased significantly (p = 0.002) while M. tuberculosis incidence declined (p<0.001). Almost all M. bovis cases from 2001 through 2005 were in persons of Hispanic ethnicity. Persons with M. bovis were 2.55× (p = 0.01) as likely to die during treatment than those with M. tuberculosis. PMID:18507901

  2. Soluble TNFRp75 regulates host protective immunity against Mycobacterium tuberculosis

    PubMed Central

    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

    2014-01-01

    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. PMID:24569452

  3. Rapid Whole-Genome Sequencing of Mycobacterium tuberculosis Isolates Directly from Clinical Samples

    PubMed Central

    Brown, Amanda C.; Einer-Jensen, Katja; Holdstock, Jolyon; Houniet, Darren T.; Chan, Jacqueline Z. M.; Depledge, Daniel P.; Nikolayevskyy, Vladyslav; Broda, Agnieszka; Stone, Madeline J.; Christiansen, Mette T.; Williams, Rachel; McAndrew, Michael B.; Tutill, Helena; Brown, Julianne; Melzer, Mark; Rosmarin, Caryn; McHugh, Timothy D.; Shorten, Robert J.; Drobniewski, Francis; Speight, Graham; Breuer, Judith

    2015-01-01

    The rapid identification of antimicrobial resistance is essential for effective treatment of highly resistant Mycobacterium tuberculosis. Whole-genome sequencing provides comprehensive data on resistance mutations and strain typing for monitoring transmission, but unlike for conventional molecular tests, this has previously been achievable only from cultures of M. tuberculosis. Here we describe a method utilizing biotinylated RNA baits designed specifically for M. tuberculosis DNA to capture full M. tuberculosis genomes directly from infected sputum samples, allowing whole-genome sequencing without the requirement of culture. This was carried out on 24 smear-positive sputum samples, collected from the United Kingdom and Lithuania where a matched culture sample was available, and 2 samples that had failed to grow in culture. M. tuberculosis sequencing data were obtained directly from all 24 smear-positive culture-positive sputa, of which 20 were of high quality (>20× depth and >90% of the genome covered). Results were compared with those of conventional molecular and culture-based methods, and high levels of concordance between phenotypical resistance and predicted resistance based on genotype were observed. High-quality sequence data were obtained from one smear-positive culture-negative case. This study demonstrated for the first time the successful and accurate sequencing of M. tuberculosis genomes directly from uncultured sputa. Identification of known resistance mutations within a week of sample receipt offers the prospect for personalized rather than empirical treatment of drug-resistant tuberculosis, including the use of antimicrobial-sparing regimens, leading to improved outcomes. PMID:25972414

  4. 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.

  5. Mycobacterium tuberculosis PPE protein Rv0256c induces strong B cell response in tuberculosis patients.

    PubMed

    Abraham, Philip Raj; Latha, Gaddam Suman; Valluri, Vijaya Lakshmi; Mukhopadhyay, Sangita

    2014-03-01

    Tuberculosis (TB) is one of the most important diseases of humans and major public health problem worldwide. Early and accurate diagnosis of TB is necessary for the treatment, prevention, and control of TB. Therefore, it is important to identify suitable antigens that can differentiate active tuberculosis patients from BCG-vaccinated individuals. In the present study, we have used Rv0256c (PPE2) protein of Mycobacterium tuberculosis to screen the sera of infected patients belonging to different clinical TB presentations, and BCG-vaccinated clinically healthy individuals by enzyme immunoassay. Our results demonstrated that Rv0256c displayed stronger and specific immunoreactivity against the sera obtained from clinically active tuberculosis patients compared to PPD and ESAT-6 and could differentiate the TB-patients from the BCG-vaccinated controls. Importantly, Rv0256c was also found to detect even the extrapulmonary and smear-negative pulmonary cases which often are tedious and difficult to detect using conventional diagnostic methods. This study suggests that Rv0256c can be used as a potential marker for the serodiagnosis of tuberculosis patients. PMID:23827809

  6. 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.

  7. 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

  8. Survival of Mycobacterium avium and Mycobacterium tuberculosis in Acidified Vacuoles of Murine Macrophages

    PubMed Central

    Gomes, Maria Salomé; Paul, Simon; Moreira, Andre L.; Appelberg, Rui; Rabinovitch, Michel; Kaplan, Gilla

    1999-01-01

    Despite the antimicrobial mechanisms of vertebrate phagocytes, mycobacteria can survive within the phagosomes of these cells. These organisms use various strategies to evade destruction, including inhibition of acidification of the phagosome and inhibition of phagosome-lysosome fusion. In contrast to mycobacteria, Coxiella burnetii, the etiologic agent of Q fever, inhabits a spacious acidified intracellular vacuole which is prone to fusion with other vacuoles of the host cell, including phagosomes containing mycobacteria. The Coxiella-infected cell thus provides a unique model for investigating the survival of mycobacteria in an acidified phagosome-like compartment. In the present study, murine bone marrow-derived macrophages were infected with either Mycobacterium avium or Mycobacterium tuberculosis and then coinfected with C. burnetii. We observed that the majority of phagocytosed mycobacteria colocalized to the C. burnetii-containing vacuole, which maintained its acidic properties. In coinfected macrophages, the growth of M. avium was not impaired following fusion with the acidified vacuole. In contrast, the growth rate of M. tuberculosis was reduced in acidified vacuoles. These results suggest that although both species of mycobacteria inhibit phagosome-lysosome fusion, they may be differentially susceptible to the toxic effects of the acidic environment in the mature phagolysosome. PMID:10377091

  9. Impact of β-Lactamase Inhibition on the Activity of Ceftaroline against Mycobacterium tuberculosis and Mycobacterium abscessus

    PubMed Central

    Dubée, Vincent; Soroka, Daria; Cortes, Mélanie; Lefebvre, Anne-Laure; Gutmann, Laurent; Hugonnet, Jean-Emmanuel; Arthur, Michel

    2015-01-01

    The production of β-lactamases BlaMab and BlaC contributes to β-lactam resistance in Mycobacterium abscessus and Mycobacterium tuberculosis, respectively. Ceftaroline was efficiently hydrolyzed by these enzymes. Inhibition of M. tuberculosis BlaC by clavulanate decreased the ceftaroline MIC from ≥256 to 16 to 64 μg/ml, but these values are clinically irrelevant. In contrast, the ceftaroline-avibactam combination should be evaluated against M. abscessus since it inhibited growth at lower and potentially achievable drug concentrations. PMID:25733512

  10. 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. PMID:25288092

  11. Rifampin Induces Hydroxyl Radical Formation in Mycobacterium tuberculosis

    PubMed Central

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

    2014-01-01

    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. PMID:25288092

  12. 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.

  13. [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.

  14. 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

  15. 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

  16. Functional analysis of TPM domain containing Rv2345 of Mycobacterium tuberculosis identifies its phosphatase activity.

    PubMed

    Sinha, Avni; Eniyan, Kandasamy; Sinha, Swati; Lynn, Andrew Michael; Bajpai, Urmi

    2015-07-01

    Mycobacterium tuberculosis (Mtb) is the causal agent of tuberculosis, the second largest infectious disease. With the rise of multi-drug resistant strains of M. tuberculosis, serious challenge lies ahead of us in treating the disease. The availability of complete genome sequence of Mtb has improved the scope for identifying new proteins that would not only further our understanding of biology of the organism but could also serve to discover new drug targets. In this study, Rv2345, a hypothetical membrane protein of M. tuberculosis H37Rv, which is reported to be a putative ortholog of ZipA cell division protein has been assigned function through functional annotation using bioinformatics tools followed by experimental validation. Sequence analysis showed Rv2345 to have a TPM domain at its N-terminal region and predicted it to have phosphatase activity. The TPM domain containing region of Rv2345 was cloned and expressed using pET28a vector in Escherichia coli and purified by Nickel affinity chromatography. The purified TPM domain was tested in vitro and our results confirmed it to have phosphatase activity. The enzyme activity was first checked and optimized with pNPP as substrate, followed by using ATP, which was also found to be used as substrate by the purified protein. Hence sequence analysis followed by in vitro studies characterizes TPM domain of Rv2345 to contain phosphatase activity.

  17. Evaluation of Cobas TaqMan MTB PCR for detection of Mycobacterium tuberculosis.

    PubMed

    Kim, Jeong Hyun; Kim, Young Jae; Ki, Chang-Seok; Kim, Ji-Youn; Lee, Nam Yong

    2011-01-01

    Nucleic acid-based amplification tests allow the rapid detection of Mycobacterium tuberculosis. Recently, a real-time PCR assay for M. tuberculosis complex, the Cobas TaqMan MTB test (Roche Diagnostics, Basel, Switzerland), was introduced. We performed a prospective study to evaluate the diagnostic performance of the Cobas TaqMan MTB test system. A total of 406 specimens collected from 247 patients were simultaneously tested by conventional culture, Cobas Amplicor MTB PCR, and TaqMan MTB PCR. The cross-reactivity with other Mycobacterium species and the detection limit were also evaluated. Among 406 specimens, a total of 24 specimens (5.9%) were culture positive: 14 specimens were positive by both TaqMan and Amplicor MTB PCRs, while 5 specimens were positive by only TaqMan PCR. The remaining five specimens were negative by both PCR methods. Seven specimens with negative culture results were positive by TaqMan PCR, but five of these were negative by Amplicor MTB PCR. The sensitivity, specificity, and positive (PPV) and negative (NPV) predictive values were 79.1%, 98.2%, 73.1%, and 98.7% for TaqMan and 58.3%, 99.5%, 87.5%, and 97.4% for the Amplicor MTB PCR test, respectively. There was no cross-reactivity with M. tuberculosis and nontuberculous mycobacterial species. The detection limit for the Cobas TaqMan MTB PCR test was 4.0 copies/μl. The Cobas TaqMan MTB PCR test showed higher sensitivity for detection of the M. tuberculosis complex without disturbing the specificity and NPV than the Amplicor MTB PCR test.

  18. Genomics and Machine Learning for Taxonomy Consensus: The Mycobacterium tuberculosis Complex Paradigm.

    PubMed

    Azé, Jérôme; Sola, Christophe; Zhang, Jian; Lafosse-Marin, Florian; Yasmin, Memona; Siddiqui, Rubina; Kremer, Kristin; van Soolingen, Dick; Refrégier, Guislaine

    2015-01-01

    Infra-species taxonomy is a prerequisite to compare features such as virulence in different pathogen lineages. Mycobacterium tuberculosis complex taxonomy has rapidly evolved in the last 20 years through intensive clinical isolation, advances in sequencing and in the description of fast-evolving loci (CRISPR and MIRU-VNTR). On-line tools to describe new isolates have been set up based on known diversity either on CRISPRs (also known as spoligotypes) or on MIRU-VNTR profiles. The underlying taxonomies are largely concordant but use different names and offer different depths. The objectives of this study were 1) to explicit the consensus that exists between the alternative taxonomies, and 2) to provide an on-line tool to ease classification of new isolates. Genotyping (24-VNTR, 43-spacers spoligotypes, IS6110-RFLP) was undertaken for 3,454 clinical isolates from the Netherlands (2004-2008). The resulting database was enlarged with African isolates to include most human tuberculosis diversity. Assignations were obtained using TB-Lineage, MIRU-VNTRPlus, SITVITWEB and an algorithm from Borile et al. By identifying the recurrent concordances between the alternative taxonomies, we proposed a consensus including 22 sublineages. Original and consensus assignations of the all isolates from the database were subsequently implemented into an ensemble learning approach based on Machine Learning tool Weka to derive a classification scheme. All assignations were reproduced with very good sensibilities and specificities. When applied to independent datasets, it was able to suggest new sublineages such as pseudo-Beijing. This Lineage Prediction tool, efficient on 15-MIRU, 24-VNTR and spoligotype data is available on the web interface "TBminer." Another section of this website helps summarizing key molecular epidemiological data, easing tuberculosis surveillance. Altogether, we successfully used Machine Learning on a large dataset to set up and make available the first consensual

  19. Genomics and Machine Learning for Taxonomy Consensus: The Mycobacterium tuberculosis Complex Paradigm

    PubMed Central

    Azé, Jérôme; Sola, Christophe; Zhang, Jian; Lafosse-Marin, Florian; Yasmin, Memona; Siddiqui, Rubina; Kremer, Kristin; van Soolingen, Dick; Refrégier, Guislaine

    2015-01-01

    Infra-species taxonomy is a prerequisite to compare features such as virulence in different pathogen lineages. Mycobacterium tuberculosis complex taxonomy has rapidly evolved in the last 20 years through intensive clinical isolation, advances in sequencing and in the description of fast-evolving loci (CRISPR and MIRU-VNTR). On-line tools to describe new isolates have been set up based on known diversity either on CRISPRs (also known as spoligotypes) or on MIRU-VNTR profiles. The underlying taxonomies are largely concordant but use different names and offer different depths. The objectives of this study were 1) to explicit the consensus that exists between the alternative taxonomies, and 2) to provide an on-line tool to ease classification of new isolates. Genotyping (24-VNTR, 43-spacers spoligotypes, IS6110-RFLP) was undertaken for 3,454 clinical isolates from the Netherlands (2004-2008). The resulting database was enlarged with African isolates to include most human tuberculosis diversity. Assignations were obtained using TB-Lineage, MIRU-VNTRPlus, SITVITWEB and an algorithm from Borile et al. By identifying the recurrent concordances between the alternative taxonomies, we proposed a consensus including 22 sublineages. Original and consensus assignations of the all isolates from the database were subsequently implemented into an ensemble learning approach based on Machine Learning tool Weka to derive a classification scheme. All assignations were reproduced with very good sensibilities and specificities. When applied to independent datasets, it was able to suggest new sublineages such as pseudo-Beijing. This Lineage Prediction tool, efficient on 15-MIRU, 24-VNTR and spoligotype data is available on the web interface “TBminer.” Another section of this website helps summarizing key molecular epidemiological data, easing tuberculosis surveillance. Altogether, we successfully used Machine Learning on a large dataset to set up and make available the first

  20. Mycobacterium tuberculosis infection in a canary (Serinus canana L.) and a blue-fronted Amazon parrot (Amazona amazona aestiva).

    PubMed

    Hoop, Richard K

    2002-01-01

    I report two cases of mycobacteriosis in pet birds due to Mycobacterium tuberculosis and discuss the zoonotic implications. The canary with a tuberculous knot in the lung is the first description of M. tuberculosis in a nonpsittacine bird species.

  1. Evaluation of the Speed-oligo Direct Mycobacterium tuberculosis Assay for Molecular Detection of Mycobacteria in Clinical Respiratory Specimens

    PubMed Central

    Lara-Oya, Ana; Mendoza-Lopez, Pablo; Rodriguez-Granger, Javier; Fernández-Sánchez, Ana María; Bermúdez-Ruiz, María Pilar; Toro-Peinado, Inmaculada; Palop-Borrás, Begoña; Navarro-Marí, Jose María

    2013-01-01

    We present the first evaluation of a novel molecular assay, the Speed-oligo Direct Mycobacterium tuberculosis (SO-DMT) assay, which is based on PCR combined with a dipstick for the detection of mycobacteria and the specific identification of M. tuberculosis complex (MTC) in respiratory specimens. A blind evaluation was carried out in two stages: first, under experimental conditions on convenience samples comprising 20 negative specimens, 44 smear- and culture-positive respiratory specimens, and 11 sputa inoculated with various mycobacterium-related organisms; and second, in the routine workflow of 566 fresh respiratory specimens (4.9% acid-fast bacillus [AFB] smear positives, 7.6% MTC positives, and 1.8% nontuberculous mycobacteria [NTM] culture positives) from two Mycobacterium laboratories. SO-DMT assay showed no reactivity in any of the mycobacterium-free specimens or in those with mycobacterium-related organisms. Compared to culture, the sensitivity in the selected smear-positive specimens was 0.91 (0.92 for MTC and 0.90 for NTM), and there was no molecular detection of NTM in a tuberculosis case or vice versa. With respect to culture and clinical data, the sensitivity, specificity, and positive and negative predictive values for the SO-DMT system in routine specimens were 0.76 (0.93 in smear positives [1.0 for MTC and 0.5 for NTM] and 0.56 in smear negatives [0.68 for MTC and 0.16 for NTM]), 0.99, 0.85 (1.00 in smear positives and 0.68 in smear negatives), and 0.97, respectively. Molecular misidentification of NTM cases occurred when testing 2 gastric aspirates from two children with clinically but not microbiologically confirmed lung tuberculosis. The SO-DMT assay appears to be a fast and easy alternative for detecting mycobacteria and differentiating MTC from NTM in smear-positive respiratory specimens. PMID:23100355

  2. Antigenic characterization of dimorphic surface protein in Mycobacterium tuberculosis.

    PubMed

    Matsuba, Takashi; Siddiqi, Umme Ruman; Hattori, Toshio; Nakajima, Chie; Fujii, Jun; Suzuki, Yasuhiko

    2016-05-01

    The Mycobacterium tuberculosis Rv0679c protein is a surface protein that contributes to host cell invasion. We previously showed that a single nucleotide transition of the Rv0679c gene leads to a single amino acid substitution from asparagine to lysine at codon 142 in the Beijing genotype family. In this study, we examined the immunological effect of this substitution. Several recombinant proteins were expressed in Escherichia coli and Mycobacterium smegmatis and characterized with antisera and two monoclonal antibodies named 5D4-C2 and 8G10-H2. A significant reduction of antibody binding was detected by enzyme-linked immunosorbent assay (ELISA) and western blot analysis in the Lys142-type protein. This reduction of 8G10-H2 binding was more significant, with the disappearance of a signal in the proteins expressed by recombinant mycobacteria in western blot analysis. In addition, epitope mapping analysis of the recombinant proteins showed a linear epitope by 5D4-C2 and a discontinuous epitope by 8G10-H2. The antibody recognizing the conformational epitope detected only mycobacterial Asn142-type recombinant protein. Our results suggest that a single amino acid substitution of Rv0679c has potency for antigenic change in Beijing genotype strains. PMID:27190237

  3. Increased risk of Mycobacterium tuberculosis infection in household child contacts exposed to passive tobacco smoke.

    PubMed

    Sridhar, Saranya; Karnani, Nisha; Connell, David W; Millington, Kerry A; Dosanjh, Davinder; Bakir, Mustafa; Soysal, Ahmet; Deeks, Jonathan; Lalvani, Ajit

    2014-12-01

    Risk factors associated with Mycobacterium tuberculosis infection were investigated in a prospective cohort of household child tuberculosis contacts. A significantly increased risk of acquiring infection was associated with exposure to passive cigarette smoke, higher number of index cases, younger age and reduced household monthly income.

  4. Oral therapy using nanoparticle-encapsulated antituberculosis drugs in guinea pigs infected with Mycobacterium tuberculosis.

    PubMed

    Johnson, Christine M; Pandey, Rajesh; Sharma, Sadhna; Khuller, G K; Basaraba, Randall J; Orme, Ian M; Lenaerts, Anne J

    2005-10-01

    We evaluated the efficacy of nanoparticle-encapsulated antituberculosis drugs administered every 10 days versus that of daily nonencapsulated drugs against Mycobacterium tuberculosis aerosol infection in guinea pigs. Both treatments significantly reduced the bacterial count and lung histopathology, suggesting that the nanoparticle drug delivery system has potential in intermitted treatment of tuberculosis.

  5. 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. PMID:24047678

  6. 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.

  7. STAT3 Represses Nitric Oxide Synthesis in Human Macrophages upon Mycobacterium tuberculosis Infection

    PubMed Central

    Queval, Christophe J.; Song, Ok-Ryul; Deboosère, Nathalie; Delorme, Vincent; Debrie, Anne-Sophie; Iantomasi, Raffaella; Veyron-Churlet, Romain; Jouny, Samuel; Redhage, Keely; Deloison, Gaspard; Baulard, Alain; Chamaillard, Mathias; Locht, Camille; Brodin, Priscille

    2016-01-01

    Mycobacterium tuberculosis is a successful intracellular pathogen. Numerous host innate immune responses signaling pathways are induced upon mycobacterium invasion, however their impact on M. tuberculosis replication is not fully understood. Here we reinvestigate the role of STAT3 specifically inside human macrophages shortly after M. tuberculosis uptake. We first show that STAT3 activation is mediated by IL-10 and occurs in M. tuberculosis infected cells as well as in bystander non-colonized cells. STAT3 activation results in the inhibition of IL-6, TNF-α, IFN-γ and MIP-1β. We further demonstrate that STAT3 represses iNOS expression and NO synthesis. Accordingly, the inhibition of STAT3 is detrimental for M. tuberculosis intracellular replication. Our study thus points out STAT3 as a key host factor for M. tuberculosis intracellular establishment in the early stages of macrophage infection. PMID:27384401

  8. Interaction between Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium subspecies paratuberculosis with the enteric glia and microglial cells

    PubMed Central

    2011-01-01

    Background We investigated the interaction of Mycobacterium avium subspecies paratuberculosis, M. bovis and M. tuberculosis and different glial cells (enteric glial and microglial cells) in order to evaluate the infecting ability of these microorganisms and the effects produced on these cells, such as the evaluation of cytokines expression. Results Our experiments demonstrated the adhesion of M. paratuberculosis to the enteroglial cells and the induction of IL-1A and IL-6 expression; M. tuberculosis and M. bovis showed a good adhesive capability to the enteric cell line with the expression of the following cytokines: IL-1A and IL-1B, TNF-α, G-CSF and GM-CSF; M. bovis induced the expression of IL-6 too. The experiment performed with the microglial cells confirmed the results obtained with the enteroglial cells after the infection with M. tuberculosis and M. bovis, whereas M. paratuberculosis stimulated the production of IL-1A and IL-1B. Conclusion Enteroglial and microglial cells, could be the target of pathogenic mycobacteria and, even if present in different locations (Enteric Nervous System and Central Nervous System), show to have similar mechanism of immunomodulation. PMID:22151930

  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. 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.

  11. Spoligotypes of Mycobacterium tuberculosis from Different Provinces of China▿ †

    PubMed Central

    Dong, Haiyan; Liu, Zhiguang; Lv, Bing; Zhang, Yuanyuan; Liu, Jie; Zhao, Xiuqin; Liu, Jinghua; Wan, Kanglin

    2010-01-01

    A total of 2,346 Mycobacterium tuberculosis isolates from 13 provinces in China were genotyped by spoligotyping. Two hundred seventy-eight spoligotypes were identified: 2,153 isolates were grouped into 85 clusters, and the remaining 193 isolates were orphans. Comparison with the SpolDB4.0 database revealed that 118 spoligotypes had shared international type numbers in the database and the other 160 were novel. These 160 novel spoligotypes were assigned to families and subfamilies using the SpotClust program. The most prevalent family was the Beijing family (74.08%), followed by the T family (14.11%). CAS family strains were found only in the Xinjiang and Tibet regions, while EAI family strains were found only in Fujian Province. In conclusion, the present study of the M. tuberculosis population in China demonstrated that Beijing family isolates are the most prevalent strains in China and that they exhibit geographical variation. Furthermore, many new spoligotypes were found in this study. PMID:20739484

  12. Genetic markers, genotyping methods & next generation sequencing in Mycobacterium tuberculosis

    PubMed Central

    Desikan, Srinidhi; Narayanan, Sujatha

    2015-01-01

    Molecular epidemiology (ME) is one of the main areas in tuberculosis research which is widely used to study the transmission epidemics and outbreaks of tubercle bacilli. It exploits the presence of various polymorphisms in the genome of the bacteria that can be widely used as genetic markers. Many DNA typing methods apply these genetic markers to differentiate various strains and to study the evolutionary relationships between them. The three widely used genotyping tools to differentiate Mycobacterium tuberculosis strains are IS6110 restriction fragment length polymorphism (RFLP), spacer oligotyping (Spoligotyping), and mycobacterial interspersed repeat units - variable number of tandem repeats (MIRU-VNTR). A new prospect towards ME was introduced with the development of whole genome sequencing (WGS) and the next generation sequencing (NGS) methods, where the entire genome is sequenced that not only helps in pointing out minute differences between the various sequences but also saves time and the cost. NGS is also found to be useful in identifying single nucleotide polymorphisms (SNPs), comparative genomics and also various aspects about transmission dynamics. These techniques enable the identification of mycobacterial strains and also facilitate the study of their phylogenetic and evolutionary traits. PMID:26205019

  13. SInCRe-structural interactome computational resource for Mycobacterium tuberculosis.

    PubMed

    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. PMID:26130660

  14. 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

  15. 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

  16. Tuberculosis Caused by Mycobacterium africanum, United States, 2004–2013

    PubMed Central

    Bloss, Emily; Heilig, Charles M.; Click, Eleanor S.

    2016-01-01

    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. PMID:26886258

  17. Mycobacterium tuberculosis- induced neutrophil extracellular traps activate human macrophages.

    PubMed

    Braian, Clara; Hogea, Valentin; Stendahl, Olle

    2013-01-01

    Neutrophils activated by Mycobacterium tuberculosis (Mtb) form neutrophil extracellular traps (NETs), containing DNA and several biologically active cytosolic and granular proteins. These NETs may assist in the innate immune defense against different pathogens. We investigated whether the NET-forming neutrophils mediate an activating signal to macrophages during the early multicellular inflammatory reaction and granuloma formation. Mtb-induced NETs were found to be reactive oxygen species dependent and phagocytosis dependent. A neutrophil elastase inhibitor also delayed NET formation. However, NET formation occurred independently of Mtb-induced apoptosis. We observed close interactions between macrophages and Mtb-activated neutrophils, where macrophages bound and phagocytosed NETs. Significant secretion of the cytokines interleukin (IL)-6, tumor necrosis factor-α, IL-1β and IL-10 were detected from macrophages cocultured with NETs from Mtb-activated but not phorbol myristate acetate-activated neutrophils. NETs binding heat shock protein 72 (Hsp72) or recombinant Hsp72 were able to trigger cytokine release from macrophages. Only Mtb-induced NETs contained Hsp72, suggesting that these NETs can transfer this danger signal to adjacent macrophages. We propose that Hsp72 sequestered in NETs plays an important role in the interaction between neutrophils and macrophages during the early innate immune phase of an Mtb infection. The immunomodulatory role of NETs and proteins derived from them may influence not only chronic inflammation during tuberculosis but also immune regulation and autoimmunity.

  18. Mechanism of inhibition of Mycobacterium tuberculosis antigen 85 by ebselen

    PubMed Central

    Favrot, Lorenza; Grzegorzewicz, Anna E.; Lajiness, Daniel H.; Marvin, Rachel K.; Boucau, Julie; Isailovic, Dragan; Jackson, Mary; Ronning, Donald R.

    2014-01-01

    The increasing prevalence of drug-resistant tuberculosis highlights the need for identifying new antitubercular drugs that can treat these infections. The antigen 85 (Ag85) complex has emerged as an intriguing mycobacterial drug target due to its central role in synthesizing major components of the inner and outer leaflets of the mycobacterial outer membrane. Here we identify ebselen as a potent inhibitor of the Mycobacterium tuberculosis Ag85 complex. Mass spectrometry data show that ebselen binds covalently to a cysteine residue (C209) located near the Ag85C active site. The crystal structure of Ag85C in the presence of ebselen shows that C209 modification restructures the active site, thereby disrupting the hydrogen-bonded network within the active site that is essential for enzymatic activity. C209 mutations display marked decreases in enzymatic activity. These data suggest that compounds using this mechanism of action will strongly inhibit the Ag85 complex and minimize the selection of drug resistance. PMID:24193546

  19. Amperometric immunosensor for rapid detection of Mycobacterium tuberculosis

    PubMed Central

    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

    2015-01-01

    Tuberculosis (TB) has been a major public health problem, which can be better controlled by using accurate and rapid diagnosis in low-resource settings. A simple, portable, and sensitive detection method is required for point-of-care (POC) settings. This paper studies an amperometric biosensor using a microtip immunoassay for a rapid and low cost detection of Mycobacterium Tuberculosis (MTB) in sputum. MTB in sputum is specifically captured on the functionalized microtip surface and detected by electric current. According to the numerical study, the current signal on 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 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, comparable to a more labor-intensive fluorescence detection method reported previously. PMID:26097292

  20. Biochemical characterization of uracil phosphoribosyltransferase from Mycobacterium tuberculosis.

    PubMed

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

  1. 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

  2. The Mycobacterium tuberculosis Drugome and Its Polypharmacological Implications

    PubMed Central

    Kinnings, Sarah L.; Xie, Li; Fung, Kingston H.; Jackson, Richard M.; Xie, Lei; Bourne, Philip E.

    2010-01-01

    We report a computational approach that integrates structural bioinformatics, molecular modelling and systems biology to construct a drug-target network on a structural proteome-wide scale. The approach has been applied to the genome of Mycobacterium tuberculosis (M.tb), the causative agent of one of today's most widely spread infectious diseases. The resulting drug-target interaction network for all structurally characterized approved drugs bound to putative M.tb receptors, we refer to as the ‘TB-drugome’. The TB-drugome reveals that approximately one-third of the drugs examined have the potential to be repositioned to treat tuberculosis and that many currently unexploited M.tb receptors may be chemically druggable and could serve as novel anti-tubercular targets. Furthermore, a detailed analysis of the TB-drugome has shed new light on the controversial issues surrounding drug-target networks [1]–[3]. Indeed, our results support the idea that drug-target networks are inherently modular, and further that any observed randomness is mainly caused by biased target coverage. The TB-drugome (http://funsite.sdsc.edu/drugome/TB) has the potential to be a valuable resource in the development of safe and efficient anti-tubercular drugs. More generally the methodology may be applied to other pathogens of interest with results improving as more of their structural proteomes are determined through the continued efforts of structural biology/genomics. PMID:21079673

  3. Transcriptional Adaptation of Drug-tolerant Mycobacterium tuberculosis During Treatment of Human Tuberculosis

    PubMed Central

    Walter, Nicholas D.; Dolganov, Gregory M.; Garcia, Benjamin J.; Worodria, William; Andama, Alfred; Musisi, Emmanuel; Ayakaka, Irene; Van, Tran T.; Voskuil, Martin I.; de Jong, Bouke C.; Davidson, Rebecca M.; Fingerlin, Tasha E.; Kechris, Katerina; Palmer, Claire; Nahid, Payam; Daley, Charles L.; Geraci, Mark; Huang, Laurence; Cattamanchi, Adithya; Strong, Michael; Schoolnik, Gary K.; Davis, John Lucian

    2015-01-01

    Background. Treatment initiation rapidly kills most drug-susceptible Mycobacterium tuberculosis, but a bacterial subpopulation tolerates prolonged drug exposure. We evaluated drug-tolerant bacilli in human sputum by comparing messenger RNA (mRNA) expression of drug-tolerant bacilli that survive the early bactericidal phase with treatment-naive bacilli. Methods. M. tuberculosis gene expression was quantified via reverse-transcription polymerase chain reaction in serial sputa from 17 Ugandans treated for drug-susceptible pulmonary tuberculosis. Results. Within 4 days, bacterial mRNA abundance declined >98%, indicating rapid killing. Thereafter, the rate of decline slowed >94%, indicating drug tolerance. After 14 days, 16S ribosomal RNA transcripts/genome declined 96%, indicating slow growth. Drug-tolerant bacilli displayed marked downregulation of genes associated with growth, metabolism, and lipid synthesis and upregulation in stress responses and key regulatory categories—including stress-associated sigma factors, transcription factors, and toxin-antitoxin genes. Drug efflux pumps were upregulated. The isoniazid stress signature was induced by initial drug exposure, then disappeared after 4 days. Conclusions. Transcriptional patterns suggest that drug-tolerant bacilli in sputum are in a slow-growing, metabolically and synthetically downregulated state. Absence of the isoniazid stress signature in drug-tolerant bacilli indicates that physiological state influences drug responsiveness in vivo. These results identify novel drug targets that should aid in development of novel shorter tuberculosis treatment regimens. PMID:25762787

  4. Drug resistance of Mycobacterium tuberculosis isolates from tuberculosis lymphadenitis patients in Ethiopia

    PubMed Central

    Biadglegne, Fantahun; Tessema, Belay; Sack, Ulrich; Rodloff, Arne C.

    2014-01-01

    Background & objectives: 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. Methods: 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. Results: 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). Interpretation & conclusions: 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. PMID:25222786

  5. 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

    2015-06-01

    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.

  6. Micrococcin P1 - A bactericidal thiopeptide active against Mycobacterium tuberculosis.

    PubMed

    Degiacomi, Giulia; Personne, Yoann; Mondésert, Guillaume; Ge, Xueliang; Mandava, Chandra Sekhar; Hartkoorn, Ruben C; Boldrin, Francesca; Goel, Pavitra; Peisker, Kristin; Benjak, Andrej; Barrio, Maria Belén; Ventura, Marcello; Brown, Amanda C; Leblanc, Véronique; Bauer, Armin; Sanyal, Suparna; Cole, Stewart T; Lagrange, Sophie; Parish, Tanya; Manganelli, Riccardo

    2016-09-01

    The lack of proper treatment for serious infectious diseases due to the emergence of multidrug resistance reinforces the need for the discovery of novel antibiotics. This is particularly true for tuberculosis (TB) for which 3.7% of new cases and 20% of previously treated cases are estimated to be caused by multi-drug resistant strains. In addition, in the case of TB, which claimed 1.5 million lives in 2014, the treatment of the least complicated, drug sensitive cases is lengthy and disagreeable. Therefore, new drugs with novel targets are urgently needed to control resistant Mycobacterium tuberculosis strains. In this manuscript we report the characterization of the thiopeptide micrococcin P1 as an anti-tubercular agent. Our biochemical experiments show that this antibiotic inhibits the elongation step of protein synthesis in mycobacteria. We have further identified micrococcin resistant mutations in the ribosomal protein L11 (RplK); the mutations were located in the proline loop at the N-terminus. Reintroduction of the mutations into a clean genetic background, confirmed that they conferred resistance, while introduction of the wild type RplK allele into resistant strains re-established sensitivity. We also identified a mutation in the 23S rRNA gene. These data, in good agreement with previous structural studies suggest that also in M. tuberculosis micrococcin P1 functions by binding to the cleft between the 23S rRNA and the L11 protein loop, thus interfering with the binding of elongation factors Tu and G (EF-Tu and EF-G) and inhibiting protein translocation. PMID:27553416

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

    PubMed Central

    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. PMID:25798914

  8. Assembling of the Mycobacterium tuberculosis Cell Wall Core.

    PubMed

    Grzegorzewicz, Anna E; de Sousa-d'Auria, Célia; McNeil, Michael R; Huc-Claustre, Emilie; Jones, Victoria; Petit, Cécile; Angala, Shiva Kumar; Zemanová, Júlia; Wang, Qinglan; Belardinelli, Juan Manuel; Gao, Qian; Ishizaki, Yoshimasa; Mikušová, Katarína; Brennan, Patrick J; Ronning, Donald R; Chami, Mohamed; Houssin, Christine; Jackson, Mary

    2016-09-01

    The unique cell wall of mycobacteria is essential to their viability and the target of many clinically used anti-tuberculosis drugs and inhibitors under development. Despite intensive efforts to identify the ligase(s) responsible for the covalent attachment of the two major heteropolysaccharides of the mycobacterial cell wall, arabinogalactan (AG) and peptidoglycan (PG), the enzyme or enzymes responsible have remained elusive. We here report on the identification of the two enzymes of Mycobacterium tuberculosis, CpsA1 (Rv3267) and CpsA2 (Rv3484), responsible for this function. CpsA1 and CpsA2 belong to the widespread LytR-Cps2A-Psr (LCP) family of enzymes that has been shown to catalyze a variety of glycopolymer transfer reactions in Gram-positive bacteria, including the attachment of wall teichoic acids to PG. Although individual cpsA1 and cpsA2 knock-outs of M. tuberculosis were readily obtained, the combined inactivation of both genes appears to be lethal. In the closely related microorganism Corynebacterium glutamicum, the ortholog of cpsA1 is the only gene involved in this function, and its conditional knockdown leads to dramatic changes in the cell wall composition and morphology of the bacteria due to extensive shedding of cell wall material in the culture medium as a result of defective attachment of AG to PG. This work marks an important step in our understanding of the biogenesis of the unique cell envelope of mycobacteria and opens new opportunities for drug development.

  9. 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.

  10. Rapid detection of multidrug-resistant Mycobacterium tuberculosis using the malachite green decolourisation assay.

    PubMed

    Coban, Ahmet Yilmaz; Uzun, Meltem

    2013-12-01

    Early detection of drug resistance in Mycobacterium tuberculosis isolates allows for earlier and more effective treatment of patients. The aim of this study was to investigate the performance of the malachite green decolourisation assay (MGDA) in detecting isoniazid (INH) and rifampicin (RIF) resistance in M. tuberculosis clinical isolates. Fifty M. tuberculosis isolates, including 19 multidrug-resistant, eight INH-resistant and 23 INH and RIF-susceptible samples, were tested. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and agreement of the assay for INH were 92.5%, 91.3%, 92.5%, 91.3% and 92%, respectively. Similarly, the sensitivity, specificity, PPV, NPV and agreement of the assay for RIF were 94.7%, 100%, 100%, 96.8% and 98%, respectively. There was a major discrepancy in the tests of two isolates, as they were sensitive to INH by the MGDA test, but resistant by the reference method. There was a minor discrepancy in the tests of two additional isolates, as they were sensitive to INH by the reference method, but resistant by the MGDA test. The drug susceptibility test results were obtained within eight-nine days. In conclusion, the MGDA test is a reliable and accurate method for the rapid detection of INH and RIF resistance compared with the reference method and the MGDA test additionally requires less time to obtain results.

  11. Isolation of Mycobacterium kumamotonense from a patient with pulmonary infection and latent tuberculosis.

    PubMed

    Kontos, Fanourios; Mavromanolakis, Dimitrios Nikitas; Zande, Marina Chari; Gitti, Zoe Georgios

    2016-01-01

    Mycobacterium kumamotonense is a novel, slow-growing non-chromogenic nontuberculous mycobacterium, which belongs to Mycobacterium terrae complex. We report, for the first time in Greece, the isolation of M. kumamotonense from an immunocompetent patient with pulmonary infection and latent tuberculosis. M. kumamotonense was identified by sequencing analysis of 16S rDNA and 65-kDa heat shock protein genes while by commercial molecular assays it was misidentified as Mycobacterium celatum. Antibiotic susceptibility testing was performed by the reference broth microdilution method. The strain was susceptible to amikacin, clarithromycin, rifampin, ciprofloxacin, moxifloxacin, rifabutin, ethambutol and linezolid. PMID:27080783

  12. 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

  13. Mycobacterium tuberculosis-specific cellular immune profiles suggest bacillary persistence decades after spontaneous cure in untreated tuberculosis.

    PubMed

    Millington, Kerry A; Gooding, Sarah; Hinks, Timothy S C; Reynolds, D John M; Lalvani, Ajit

    2010-12-01

    Individuals with self-healed tuberculosis from the preantibiotic era offer a unique insight into the natural history of and protective immunity to tuberculosis. In 27 such persons whose tuberculosis self-healed >50 years earlier, circulating Mycobacterium tuberculosis antigen-specific interferon γ (IFN-γ)- and interleukin 2 (IL-2)-secreting T cells were detected ex vivo in 16 and 19 individuals, respectively. The M. tuberculosis-specific T cell cytokine profile was dominated by effector memory T cells that secrete both IFN-γ and IL-2 and included T cells that secrete only IFN-γ or IL-2, suggesting persistence of antigen secreted by viable bacilli. Of 10 individuals with no M. tuberculosis antigen-specific IFN-γ-secreting T cells detectable ex vivo, 7 had evidence of central memory T cells, consistent with clearance of infection. PMID:20958211

  14. Identification of Mycobacterium tuberculosis adherence-mediating components: a review of key methods to confirm adhesin function

    PubMed Central

    Ramsugit, Saiyur; Pillay, Manormoney

    2016-01-01

    Anti-adhesion therapy represents a potentially promising avenue for the treatment and prevention of tuberculosis in a post-antibiotic era. Adhesins are surface-exposed microbial structures or molecules that enable pathogenic organisms to adhere to host surfaces, a fundamental step towards host infection. Although several Mycobacterium tuberculosis adhesins have been identified, it is predicted that numerous additional adherence-mediating components contribute to the virulence and success of this pathogen. Significant further research to discern and characterize novel M. tuberculosis adhesins is, therefore, required to gain a holistic account of M. tuberculosis adhesion to the host. This would enable the identification of potential drug and vaccine targets for attenuating M. tuberculosis adherence and infectivity. Several methods have been successfully applied to the study and identification of M. tuberculosis adhesins. In this manuscript, we review these methods, which include adherence assays that utilize wild-type and gene knockout mutant strains, epitope masking and competitive inhibition analyses, extracellular matrix protein binding assays, microsphere adhesion assays, M. tuberculosis auto-aggregation assays, and in silico analyses. PMID:27482337

  15. Cloning, expression, and immunological evaluation of two putative secreted serine protease antigens of Mycobacterium tuberculosis.

    PubMed

    Skeiky, Y A; Lodes, M J; Guderian, J A; Mohamath, R; Bement, T; Alderson, M R; Reed, S G

    1999-08-01

    Culture filtrate proteins (CFP) of Mycobacterium tuberculosis have been shown to contain immunogenic components that elicit at least partial protective immunity against Mycobacterium infection. To clone genes encoding some of the immunogenic proteins, we made a high-titer rabbit anti-CFP serum and used it to screen an M. tuberculosis genomic expression library in Escherichia coli. In this paper, we describe the molecular cloning of two new protein components of CFP and identified them as members of the serine protease gene family. Their open reading frames contain N-terminal hydrophobic secretory signals consistent with their detection in CFP. The predicted molecular masses of the mature, fully processed forms of both antigens are approximately 32 kDa, in agreement with their observed sizes on immunoblots of CFP probed with polyclonal rabbit antisera made to the recombinant proteins. Thus, these proteins have been designated MTB32A and MTB32B. Interestingly, and despite 66% amino acid sequence homology between the two proteins, polyclonal rabbit antisera made to each of the recombinant proteins were found to be specific for the respective immunizing antigens. The recombinant proteins were also evaluated in in vitro assays with donor peripheral blood mononuclear cells (PBMC) from healthy purified protein derivative (PPD)-positive individuals of diverse ethnic backgrounds. MTB32A but not MTB32B stimulated PBMC from healthy PPD-positive donors but not from PPD-negative donors to proliferate and secrete gamma interferon. MTB32A is encoded by a single-copy gene which is present in both virulent and avirulent strains of the M. tuberculosis complex and the BCG strain of Mycobacterium bovis but absent in the environmental mycobacterial species tested. In addition, nucleotide sequence comparison of mtb32a of the avirulent H37Ra strain and the virulent Erdman strain, as well as with the corresponding sequences (identified in the databases) of strain H37Rv and the clinical

  16. 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. PMID:25951404

  17. 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.

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

    PubMed Central

    Murphy, Dennis J; Brown, James R

    2007-01-01

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

  19. Hemophagocytic lymphohistiocytosis: An unusual complication in disseminated Mycobacterium tuberculosis

    PubMed Central

    Padhi, Somanath; Ravichandran, Kandasamy; Sahoo, Jayaprakash; Varghese, Renu G’Boy; Basheer, Aneesh

    2015-01-01

    Background: Hemophagocytic lymphohistiocytosis (HLH) is an uncommon, potentially fatal, hyperinflammatory syndrome that may rarely complicate the clinical course of disseminated Mycobacterium tuberculosis (MTB). The clinical course of tuberculosis-associated HLH (TB-HLH) has been reported to be unpredictable. Materials and Methods: Here we describe the clinicopathological features, laboratory parameters, management, and outcome data of a patient who satisfied the 2004 diagnostic criteria for HLH secondary to disseminated MTB; we also do a systematic review of the international literature on TB-HLH. The literature review (January 1975–March 2014) found that HLH complicated the clinical course of 63 tuberculosis patients (41 males, 22 females, mean age = 45 ± 23.5 years) with a high mortality rate of 49% (31/63 died). The mean serum ferritin level (n = 44/63) was 5963 ng/mL (range 500–38,539 ng/mL); and a higher proportion (54.2%) of patients had pancytopenia at presentation. On univariate analysis (n = 53/63), age >30 years [hazard ratio (HR): 2.79, 95% confidence interval (CI):1.03–7.56, P = 0.03], presence of comorbidities (HR 4.59, CI: 1.08–19.52, P = 0.04), marked hemophagocytosis in bone marrow (HR: 2.65, CI: 1.16–6.05, P = 0.02), and nonusage/delayed usage of antitubercular therapy (ATT) (HR: 3.44, CI: 1.51–7.87, P = 0.003) were associated with decreased survival, though none of these parameters attained statistical significance (P > 0.05) in multivariate analysis. Usage of corticosteroids and/or immunomodulator drugs (HR 1.00, CI: 0.66–3.22, P = 0.35) did not alter the outcome in these patients. Conclusion: HLH should be considered as a differential diagnosis in patients with tuberculosis who present with cytopenias, organomegaly, and coagulopathy. Strong clinical suspicion and early usage of ATT might be useful in reducing the morbidity and mortality. The utility of immunosuppressive/immunomodulator therapy lacks general concensus among

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

    DOE PAGES

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

    2015-04-07

    Racemic protein crystallography was used to determine the X-ray structure of the predicted Mycobacterium tuberculosis protein Rv1738, which had been completely recalcitrant to crystallization in its natural L-form. Native chemical ligation was used to synthesize both L-protein and D-protein enantiomers of Rv1738. Crystallization of the racemic {D-protein + L-protein} mixture was immediately successful. The resulting crystals diffracted to high resolution and also enabled facile structure determination because of the quantized phases of the data from centrosymmetric crystals. The X-ray structure of Rv1738 revealed striking similarity with bacterial hibernation factors, despite minimal sequence similarity. As a result, we predict that Rv1738,more » which is highly up-regulated in conditions that mimic the onset of persistence, helps trigger dormancy by association with the bacterial ribosome.« less

  1. A comparative modeling and molecular docking study on Mycobacterium tuberculosis targets involved in peptidoglycan biosynthesis.

    PubMed

    Fakhar, Zeynab; Naiker, Suhashni; Alves, Claudio N; Govender, Thavendran; Maguire, Glenn E M; Lameira, Jeronimo; Lamichhane, Gyanu; Kruger, Hendrik G; Honarparvar, Bahareh

    2016-11-01

    An alarming rise of multidrug-resistant Mycobacterium tuberculosis strains and the continuous high global morbidity of tuberculosis have reinvigorated the need to identify novel targets to combat the disease. The enzymes that catalyze the biosynthesis of peptidoglycan in M. tuberculosis are essential and noteworthy therapeutic targets. In this study, the biochemical function and homology modeling of MurI, MurG, MraY, DapE, DapA, Alr, and Ddl enzymes of the CDC1551 M. tuberculosis strain involved in the biosynthesis of peptidoglycan cell wall are reported. Generation of the 3D structures was achieved with Modeller 9.13. To assess the structural quality of the obtained homology modeled targets, the models were validated using PROCHECK, PDBsum, QMEAN, and ERRAT scores. Molecular dynamics simulations were performed to calculate root mean square deviation (RMSD) and radius of gyration (Rg) of MurI and MurG target proteins and their corresponding templates. For further model validation, RMSD and Rg for selected targets/templates were investigated to compare the close proximity of their dynamic behavior in terms of protein stability and average distances. To identify the potential binding mode required for molecular docking, binding site information of all modeled targets was obtained using two prediction algorithms. A docking study was performed for MurI to determine the potential mode of interaction between the inhibitor and the active site residues. This study presents the first accounts of the 3D structural information for the selected M. tuberculosis targets involved in peptidoglycan biosynthesis.

  2. PPE Antigen Rv2430c of Mycobacterium tuberculosis Induces a Strong B-Cell Response

    PubMed Central

    Choudhary, Rakesh Kumar; Mukhopadhyay, Sangita; Chakhaiyar, Prachee; Sharma, Naresh; Murthy, K. J. R.; Katoch, V. M.; Hasnain, Seyed E.

    2003-01-01

    The variation in sequence and length in the C-terminal region among members of the unique PE (Pro-Glu) and PPE (Pro-Pro-Glu) protein families of Mycobacterium tuberculosis is a likely source of antigenic variation, giving rise to the speculation that these protein families could be immunologically important. Based on in silico analysis, we selected a hypothetical open reading frame (ORF) encoding a protein belonging to the PPE family and having epitopes with predictably higher antigenic indexes. Reverse transcriptase PCR using total RNA extracted from in vitro-cultured M. tuberculosis H37Rv generated an mRNA product corresponding to this gene, indicating the expression of this ORF (Rv2430c) at the mRNA level. Recombinant protein expressed in Escherichia coli was used to screen the sera of M. tuberculosis-infected patients, as well as those of clinically healthy controls (n = 10), by enzyme-linked immunosorbent assay. The panel of patient sera comprised sera from fresh infection cases (category 1; n = 32), patients with relapsed tuberculosis (category 2; n = 30), and extrapulmonary cases (category 3; n = 30). Category 2 and 3 sera had strong antibody responses to the PPE antigen, equal to or higher than those to other well-known antigens, such as Hsp10 or purified protein derivative (PPD). However, a higher percentage of patients belonging to category 1, as opposed to clinically healthy controls, showed stronger antibody response against the PPE protein when probed with anti-immunoglobulin M (IgM) (71 versus 37.5%) or anti-IgG (62.5 versus 28.12%). Our results reveal that this PPE ORF induces a strong B-cell response compared to that generated by M. tuberculosis Hsp10 or PPD, pointing to the immunodominant nature of the protein. PMID:14573653

  3. Mycobacterium bovis infection of cattle and white-tailed deer: Translational research of relevance to human tuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  4. 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

  5. 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. PMID:26405286

  6. 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.

  7. 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

  8. Construction and application of a co-expression network in Mycobacterium tuberculosis

    PubMed Central

    Jiang, Jun; Sun, Xian; Wu, Wei; Li, Li; Wu, Hai; Zhang, Lu; Yu, Guohua; Li, Yao

    2016-01-01

    Because of its high pathogenicity and infectivity, tuberculosis is a serious threat to human health. Some information about the functions of the genes in Mycobacterium tuberculosis genome was currently available, but it was not enough to explore transcriptional regulatory mechanisms. Here, we applied the WGCNA (Weighted Gene Correlation Network Analysis) algorithm to mine pooled microarray datasets for the M. tuberculosis H37Rv strain. We constructed a co-expression network that was subdivided into 78 co-expression gene modules. The different response to two kinds of vitro models (a constant 0.2% oxygen hypoxia model and a Wayne model) were explained based on these modules. We identified potential transcription factors based on high Pearson’s correlation coefficients between the modules and genes. Three modules that may be associated with hypoxic stimulation were identified, and their potential transcription factors were predicted. In the validation experiment, we determined the expression levels of genes in the modules under hypoxic condition and under overexpression of potential transcription factors (Rv0081, furA (Rv1909c), Rv0324, Rv3334, and Rv3833). The experimental results showed that the three identified modules related to hypoxia and that the overexpression of transcription factors could significantly change the expression levels of genes in the corresponding modules. PMID:27328747

  9. Inhibition of NarL of Mycobacterium Tuberculosis: an in silico approach.

    PubMed

    Shivakumar, K V; Karunakar, Prashantha; Chatterjee, Jhinuk

    2014-12-01

    Mycobacterium tuberculosis (MTB) consumes nitrate as the alternate mechanism of respiration in the absence of oxygen, thus increasing its survival and virulence during latent stage of tuberculosis infection. NarL is a nitrate/nitrite response transcriptional regulatory protein of two-component signal transduction system which regulates nitrate reductase and formate dehydrogenase for MTB adaptation to anaerobic condition. Phosphorylation by sensor kinase (NarX) is the primary mechanism behind the activation of NarL although many response regulators get activated by small molecule phospho-donors in the absence of sensor kinase. Virtual screening was performed using Autodock suite for the compounds from ZINC database against NarL and potential inhibitors was identified to inhibit the activation of NarL by affecting its phosphorylation. Molecular dynamics simulation studies predicted the stability of 1-{1-[(3-nitrophenyl) methyl] piperidin-2-yl} ethan-1-amine in the active site of NarL over 10 ns simulation. Phosphorylation of NarL by small molecule phospho-donors is also investigated in the present study. Here we suggest that nitro benzene - amine piperidine moiety can be an effective lead candidate for developing novel anti-tuberculosis drugs. PMID:25240609

  10. In vitro time-kill curves study of three antituberculous combinations against Mycobacterium tuberculosis clinical isolates.

    PubMed

    López-Gavín, Alexandre; Tudó, Griselda; Rey-Jurado, Emma; Vergara, Andrea; Hurtado, Juan Carlos; Gonzalez-Martín, Julian

    2016-01-01

    The objective of this study was to examine the in vitro synergism of three-drug combinations against Mycobacterium tuberculosis (levofloxacin/linezolid/ethambutol, levofloxacin/amikacin/ethambutol and levofloxacin/linezolid/amikacin) using the time-kill curves method. In total, 8 multidrug-resistant and 12 drug-susceptible M. tuberculosis isolates were used. Minimum inhibitory concentrations (MICs) of the isolates for each drug were determined by the proportions method. Time-kill curves were studied for the three combinations proposed over 14 days using two different protocols. In protocol 1, 0.5× MIC for each drug was used. In protocol 2, 0.5× MIC for levofloxacin and linezolid and 0.25× MIC for amikacin and ethambutol were used. The MICs for all of the isolates studied were 0.5 mg/L for levofloxacin and linezolid and 2.5 mg/L for ethambutol and amikacin. All of the combinations displayed an additive activity compared with the most active individual drug. In conclusion, these results demonstrate that the three combinations tested were equally effective against M. tuberculosis isolates. The study of antituberculous combinations using in vitro methods is an excellent first step to predict their effect in clinical development phases as well as to test new regimens of the antituberculous drugs currently available. PMID:26691020

  11. Mixed Infections and Rifampin Heteroresistance among Mycobacterium tuberculosis Clinical Isolates.

    PubMed

    Zheng, Chao; Li, Song; Luo, Zhongyue; Pi, Rui; Sun, Honghu; He, Qingxia; Tang, Ke; Luo, Mei; Li, Yuqing; Couvin, David; Rastogi, Nalin; Sun, Qun

    2015-07-01

    Mixed infections and heteroresistance of Mycobacterium tuberculosis contribute to the difficulty of diagnosis, treatment, and control of tuberculosis. However, there is still no proper solution for these issues. This study aimed to investigate the potential relationship between mixed infections and heteroresistance and to determine the high-risk groups related to these factors. A total of 499 resistant and susceptible isolates were subjected to spoligotyping and 24-locus variable-number tandem repeat methods to analyze their genotypic lineages and the occurrence of mixed infections. Two hundred ninety-two randomly selected isolates were sequenced on their rpoB gene to examine mutations and heteroresistance. The results showed that 12 patients had mixed infections, and the corresponding isolates belonged to Manu2 (n = 8), Beijing (n = 2), T (n = 1), and unknown (n = 1) lineages. Manu2 was found to be significantly associated with mixed infections (odds ratio, 47.72; confidence interval, 9.68 to 235.23; P < 0.01). Four isolates (1.37%) were confirmed to be heteroresistant, which was caused by mixed infections in three (75%) isolates; these belonged to Manu2. Additionally, 3.8% of the rifampin-resistant isolates showing no mutation in the rpoB gene were significantly associated with mixed infections (χ(2), 56.78; P < 0.01). This study revealed for the first time that Manu2 was the predominant group in the cases of mixed infections, and this might be the main reason for heteroresistance and a possible mechanism for isolates without any mutation in the rpoB gene to become rifampin resistant. Further studies should focus on this lineage to clarify its relevance to mixed infections.

  12. Prevalence of drug resistant Mycobacterium tuberculosis among children in China.

    PubMed

    Jiao, Wei-wei; Liu, Zhi-guang; Han, Rui; Zhao, Xiu-qin; Dong, Fang; Dong, Hai-yan; Huang, Hai-rong; Li, Qin-jing; Lin, Nan; Song, Wen-qi; Wan, Kang-lin; Shen, A-dong

    2015-05-01

    The available data on the epidemic of drug resistant tuberculosis (TB) among children in China is limited. This study attempted to clarify the drug resistance profiles of clinical strains isolated from children and estimate risk factors related to acquisition of drug resistance. All Mycobacterium tuberculosis strains from children (age <15 years) and adolescent (age 15-18 years) TB patients received in the strain library of Chinese Center for Disease Control and Prevention between January 2005 and December 2012 were included in the study. A study collection included 450 clinical isolates (100 from children, 159 from adolescents, and 191 from adults) from all over China. Drug susceptibility testing was performed by a proportion method. As a result, the drug resistance and multi-drug resistance (MDR) rates in children were 55% (55/100) and 22% (22/100), respectively. In children with MDR-TB, new cases accounted for 40.9% (9/22). Compared with adults, the drug resistance rates were similar in all subgroups (new cases, previously treated cases and all cases) of children (P > 0.05), except for the lower resistance rate to isoniazid in total cases of children (P = 0.011). Patient related information was included in the MDR-TB association analysis. The treatment history was found to be strongly associated with MDR-TB in all three age groups (P < 0.05). Our results demonstrate that the prevalence of drug resistant TB in children in China is alarmingly high and similar to that seen in adults. In contrast, in adolescents, the drug resistance rate to most tested drugs was lower than in adults. Primary transmission and inadequate treatment are two equally important factors for the high MDR-TB rate in children. Thus, major efforts in the TB control in children should focus on decreasing the transmission of drug resistant TB and early testing of drug resistance.

  13. Specificity and diversity of antibodies to Mycobacterium tuberculosis arabinomannan.

    PubMed

    Navoa, Josephine Anne D; Laal, Suman; Pirofski, Liise-Anne; McLean, Gary R; Dai, Zhongdong; Robbins, John B; Schneerson, Rachel; Casadevall, Arturo; Glatman-Freedman, Aharona

    2003-01-01

    Arabinomannan (AM) is a polysaccharide antigen of the mycobacterial capsule. However, it is uncertain whether AM constitutes an immunologically distinct fraction of Mycobacterium tuberculosis. In this study, we analyzed the repertoire and specificity of antibodies to AM by using AM-binding murine monoclonal antibodies (MAbs) and human serum samples. Murine MAbs were found to be diverse in their specificity to AM and cross-reactivity with other arabinose-containing mycobacterial polysaccharides, with MAb 9d8 binding exclusively to AM. Human antibodies to AM were detected in serum samples from patients with pulmonary tuberculosis (TB), as well as in those from healthy, purified protein derivative-negative controls, with significantly higher titers among patients. The binding of human antibodies to AM was inhibited by MAb 9d8 in three patients with TB but not in controls. MAb 5c11, which recognizes other mycobacterial arabinose-containing carbohydrates in addition to AM, inhibited the binding of serum samples from 75% of patients and 76% of controls. Analysis of human antibodies with murine MAbs to human V(H) determinants demonstrated diversity among antibodies to AM with qualitative and quantitative differences compared with antibodies to lipoarabinomannan. In summary, our study suggests that antibodies to AM are diverse and heterogeneous with respect to antigen recognition and V(H) determinant expression, with human serum samples containing different subsets of antibodies to AM with the specificities of AM-binding murine MAbs. One MAb and a subset of human antibodies bind AM specifically, suggesting that this polysaccharide is antigenically distinct and is expressed in human infection.

  14. MicroRNA signatures from multidrug‑resistant Mycobacterium tuberculosis.

    PubMed

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

    2015-11-01

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

  15. Serodiagnostic Potential of Culture Filtrate Antigens of Mycobacterium tuberculosis

    PubMed Central

    Samanich, K. M.; Keen, M. A.; Vissa, V. D.; Harder, J. D.; Spencer, J. S.; Belisle, J. T.; Zolla-Pazner, S.; Laal, S.

    2000-01-01

    Our studies of the humoral responses of tuberculosis (TB) patients have defined the repertoire of culture filtrate antigens of Mycobacterium tuberculosis that are recognized by antibodies from cavitary and noncavitary TB patients and demonstrated that the profile of antigens recognized changes with disease progression (K. Samanich et al., J. Infect. Dis. 178:1534–1538, 1998). We have identified several antigens with strong serodiagnostic potential. In the present study we have evaluated the reactivity of cohorts of human immunodeficiency virus (HIV)-negative, smear-positive; HIV-negative, smear-negative; and HIV-infected TB patients, with three of the candidate antigens, an 88-kDa protein, antigen (Ag) 85C, and MPT32, and compared the reactivity of the same patient cohort with the 38-kDa antigen and Ag 85A. We have also compared the reactivity of native Ag 85C and MPT32 with their recombinant counterparts. The evaluation of the reactivity was done by a modified enzyme-linked immunosorbent assay described earlier (S. Laal et al., Clin. Diag. Lab. Immunol. 4:49–56, 1997), in which all sera are preadsorbed against Escherichia coli lysates to reduce the levels of cross-reactive antibodies. Our results demonstrate that (i) antigens identified on the basis of their reactivity with TB patients' sera provide high sensitivities for serodiagnosis, (ii) recombinant Ag 85C and MPT32, expressed in E. coli, show reduced reactivity with human TB sera, and (iii) of the panel of antigens tested, the 88-kDa protein is the most promising candidate for serodiagnosis of TB in HIV-infected individuals. Moreover, these results reaffirm that both the extent of the disease and the bacterial load may play a role in determining the antigen profile recognized by antibodies. PMID:10882669

  16. Mixed Infections and Rifampin Heteroresistance among Mycobacterium tuberculosis Clinical Isolates

    PubMed Central

    Zheng, Chao; Li, Song; Luo, Zhongyue; Pi, Rui; Sun, Honghu; He, Qingxia; Tang, Ke; Luo, Mei; Li, Yuqing; Couvin, David; Rastogi, Nalin

    2015-01-01

    Mixed infections and heteroresistance of Mycobacterium tuberculosis contribute to the difficulty of diagnosis, treatment, and control of tuberculosis. However, there is still no proper solution for these issues. This study aimed to investigate the potential relationship between mixed infections and heteroresistance and to determine the high-risk groups related to these factors. A total of 499 resistant and susceptible isolates were subjected to spoligotyping and 24-locus variable-number tandem repeat methods to analyze their genotypic lineages and the occurrence of mixed infections. Two hundred ninety-two randomly selected isolates were sequenced on their rpoB gene to examine mutations and heteroresistance. The results showed that 12 patients had mixed infections, and the corresponding isolates belonged to Manu2 (n = 8), Beijing (n = 2), T (n = 1), and unknown (n = 1) lineages. Manu2 was found to be significantly associated with mixed infections (odds ratio, 47.72; confidence interval, 9.68 to 235.23; P < 0.01). Four isolates (1.37%) were confirmed to be heteroresistant, which was caused by mixed infections in three (75%) isolates; these belonged to Manu2. Additionally, 3.8% of the rifampin-resistant isolates showing no mutation in the rpoB gene were significantly associated with mixed infections (χ2, 56.78; P < 0.01). This study revealed for the first time that Manu2 was the predominant group in the cases of mixed infections, and this might be the main reason for heteroresistance and a possible mechanism for isolates without any mutation in the rpoB gene to become rifampin resistant. Further studies should focus on this lineage to clarify its relevance to mixed infections. PMID:25903578

  17. 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.

  18. 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

  19. 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

  20. Biosynthesis and Translocation of Unsulfated Acyltrehaloses in Mycobacterium tuberculosis*

    PubMed Central

    Belardinelli, Juan Manuel; Larrouy-Maumus, Gérald; Jones, Victoria; Sorio de Carvalho, Luiz Pedro; McNeil, Michael R.; Jackson, Mary

    2014-01-01

    A number of species-specific polymethyl-branched fatty acid-containing trehalose esters populate the outer membrane of Mycobacterium tuberculosis. Among them, 2,3-diacyltrehaloses (DAT) and penta-acyltrehaloses (PAT) not only play a structural role in the cell envelope but also contribute to the ability of M. tuberculosis to multiply and persist in the infected host, promoting the intracellular survival of the bacterium and modulating host immune responses. The nature of the machinery, topology, and sequential order of the reactions leading to the biosynthesis, assembly, and export of these complex glycolipids to the cell surface are the object of the present study. Our genetic and biochemical evidence corroborates a model wherein the biosynthesis and translocation of DAT and PAT to the periplasmic space are coupled and topologically split across the plasma membrane. The formation of DAT occurs on the cytosolic face of the plasma membrane through the action of PapA3, FadD21, and Pks3/4; that of PAT occurs on the periplasmic face via transesterification reactions between DAT substrates catalyzed by the acyltransferase Chp2 (Rv1184c). The integral membrane transporter MmpL10 is essential for DAT to reach the cell surface, and its presence in the membrane is required for Chp2 to be active. Disruption of mmpL10 or chp2 leads to an important build-up of DAT inside the cells and to the formation of a novel form of unsulfated acyltrehalose esterified with polymethyl-branched fatty acids normally found in sulfolipids that is translocated to the cell surface. PMID:25124040

  1. Mycobacterium tuberculosis Zinc Metalloprotease-1 Assists Mycobacterial Dissemination in Zebrafish.

    PubMed

    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

  2. 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. PMID:27556011

  3. A chemical genetic screen in Mycobacterium tuberculosis identifies carbon-source-dependent growth inhibitors devoid of in vivo efficacy.

    PubMed

    Pethe, Kevin; Sequeira, Patricia C; Agarwalla, Sanjay; Rhee, Kyu; Kuhen, Kelli; Phong, Wai Yee; Patel, Viral; Beer, David; Walker, John R; Duraiswamy, Jeyaraj; Jiricek, Jan; Keller, Thomas H; Chatterjee, Arnab; Tan, Mai Ping; Ujjini, Manjunatha; Rao, Srinivasa P S; Camacho, Luis; Bifani, Pablo; Mak, Puiying A; Ma, Ida; Barnes, S Whitney; Chen, Zhong; Plouffe, David; Thayalan, Pamela; Ng, Seow Hwee; Au, Melvin; Lee, Boon Heng; Tan, Bee Huat; Ravindran, Sindhu; Nanjundappa, Mahesh; Lin, Xiuhua; Goh, Anne; Lakshminarayana, Suresh B; Shoen, Carolyn; Cynamon, Michael; Kreiswirth, Barry; Dartois, Veronique; Peters, Eric C; Glynne, Richard; Brenner, Sydney; Dick, Thomas

    2010-01-01

    Candidate antibacterials are usually identified on the basis of their in vitro activity. However, the apparent inhibitory activity of new leads can be misleading because most culture media do not reproduce an environment relevant to infection in vivo. In this study, while screening for novel anti-tuberculars, we uncovered how carbon metabolism can affect antimicrobial activity. Novel pyrimidine-imidazoles (PIs) were identified in a whole-cell screen against Mycobacterium tuberculosis. Lead optimization generated in vitro potent derivatives with desirable pharmacokinetic properties, yet without in vivo efficacy. Mechanism of action studies linked the PI activity to glycerol metabolism, which is not relevant for M. tuberculosis during infection. PIs induced self-poisoning of M. tuberculosis by promoting the accumulation of glycerol phosphate and rapid ATP depletion. This study underlines the importance of understanding central bacterial metabolism in vivo and of developing predictive in vitro culture conditions as a prerequisite for the rational discovery of new antibiotics. PMID:20975714

  4. 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. PMID:26416911

  5. Inadequate hospital ventilation system increases the risk of nosocomial Mycobacterium tuberculosis.

    PubMed

    Hubad, B; Lapanje, A

    2012-01-01

    The ambient air in nine locations in a pulmonary hospital and a tuberculosis diagnostic laboratory was analysed to determine the hazard posed by Mycobacterium tuberculosis. In four locations, between 6 and 20 M. tuberculosis cell equivalents/m(3) were detected. The results indicated that inadequately ventilated corridors had the highest cell equivalents. In these areas healthcare workers were less aware of infection hazard compared to areas with known sources of M. tuberculosis and the wearing of protective masks and passive ventilation were not in place. Based on these results, further infection prevention and control measures need to be implemented.

  6. 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.

  7. Structural measurements and cell line studies of the copper– PEG–Amikacin complex against Mycobacterium tuberculosis.

    PubMed

    Manning, Thomas; Patel, Hatel; Wylie, Greg; Phillips, Dennis; Jarvis, Jackie

    2015-12-15

    The bacterium responsible for causing tuberculosis is increasing its resistance to antibiotics resulting in new multidrug-resistant Mycobacterium tuberculosis (MDR-TB) and extensively drug-resistant M. tuberculosis (XDR-TB) strains. In this study, several analytical techniques including NMR, FT-ICR, MALDI-MS, and LC–MS are used to study different aspects of the Copper–polyethylene glycol (PEG)–Amikacin complex. The Cu(II) cation and the aggregate formed by PEG serve as a carrier for the antibiotic. Several Cu–PEG–Amikacin complex variations were tested against NIH-NIAID cell lines containing both resistant and nonresistant strains of M. tuberculosis.

  8. [Distribution of lysotypes of Mycobacterium tuberculosis in relation to the country of origin of the patient].

    PubMed

    Clavel-Sérès, S; Clément, F

    1984-01-01

    The diversity among the phage types of 422 strains of Mycobacterium tuberculosis isolated from patients of different geographical origins suggested that this method might prove useful in studying the propagation of tuberculosis in different types of populations. We first investigated M. tuberculosis strains isolated from France, Portugal, Romania, Algeria, Egypt, Uganda, Mali and India. We then studied in detail two groups of immigrants residing in France, from Portugal and North Africa, respectively. This investigation showed that most patients were suffering from a M. tuberculosis strain with a phage type specific of their country of origin. Factor analysis helped to display phage typing relationships with respect to the geographical origin of patients.

  9. Validation of a Multiplex Real-Time PCR Assay for Detection of Mycobacterium spp., Mycobacterium tuberculosis Complex, and Mycobacterium avium Complex Directly from Clinical Samples by Use of the BD Max Open System.

    PubMed

    Rocchetti, Talita T; Silbert, Suzane; Gostnell, Alicia; Kubasek, Carly; Widen, Raymond

    2016-06-01

    A multiplex real-time PCR was validated on the BD Max open system to detect different Mycobacterium tuberculosis complex, Mycobacterium avium complex, and Mycobacterium spp. directly from clinical samples. The PCR results were compared to those with traditional cultures. The multiplex PCR assay was found to be a specific and sensitive method for the rapid detection of mycobacteria directly from clinical specimens.

  10. A rapid detection of multidrug-resistant Mycobacterium tuberculosis by a nitrate reductase assay on blood agar.

    PubMed

    Coban, Ahmet Yilmaz; Cayci, Yeliz Tanriverdi; Deveci, Aydin; Akgunes, Alper; Uzun, Meltem; Durupinar, Belma

    2011-05-01

    The susceptibility of 49 Mycobacterium tuberculosis clinical isolates to isoniazid (INH) and rifampisin (RIF) (28 multi-drug resistant-tuberculosis samples) was determined by a nitrate reductase assay (NRA) on blood agar. Agreement between the NRA and other testing methods was found to be 93.8% for both INH and RIF. The sensitivity, specificity, positive predictive value and negative predictive value for INH were 92.8%, 94.2%, 86.6% and 97%, respectively. The sensitivity, specificity, positive predictive value and negative predictive value for RIF were 90.4%, 96.4%, 95% and 93.1%. In conclusion, we show here that blood agar can be used effectively for the NRA test.

  11. Combined use of Mycobacterium tuberculosis-specific CD4 and CD8 T-cell responses is a powerful diagnostic tool of active tuberculosis.

    PubMed

    Rozot, Virginie; Patrizia, Amelio; Vigano, Selena; Mazza-Stalder, Jesica; Idrizi, Elita; Day, Cheryl L; Perreau, Matthieu; Lazor-Blanchet, Catherine; Ohmiti, Khalid; Goletti, Delia; Bart, Pierre-Alexandre; Hanekom, Willem; Scriba, Thomas J; Nicod, Laurent; Pantaleo, Giuseppe; Harari, Alexandre

    2015-02-01

    Immune-based assays are promising tools to help to formulate diagnosis of active tuberculosis. A multiparameter flow cytometry assay assessing T-cell responses specific to Mycobacterium tuberculosis and the combination of both CD4 and CD8 T-cell responses accurately discriminated between active tuberculosis and latent infection.

  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. PMID:27570312

  13. New 1,4-di-N-oxide-quinoxaline-2-ylmethylene isonicotinic acid hydrazide derivatives as anti-Mycobacterium tuberculosis agents.

    PubMed

    Torres, Enrique; Moreno, Elsa; Ancizu, Saioa; Barea, Carlos; Galiano, Silvia; Aldana, Ignacio; Monge, Antonio; Pérez-Silanes, Silvia

    2011-06-15

    The increase in the prevalence of drug-resistant tuberculosis cases demonstrates the need of discovering new and promising compounds with antimycobacterial activity. As a continuation of our research and with the aim of identifying new antitubercular drugs candidates, a new series of quinoxaline 1,4-di-N-oxide derivatives containing isoniazid was synthesized and evaluated for in vitro anti-tuberculosis activity against Mycobacterium tuberculosis H37Rv strain. Moreover, various drug-like properties of new compounds were predicted. Taking into account the biological results and the promising drug-likeness profile of these compounds, make them valid leads for further experimental research.

  14. 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.

  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. 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.

  17. Genetic features of Mycobacterium tuberculosis modern Beijing sublineage

    PubMed Central

    Liu, Qingyun; Luo, Tao; Dong, Xinran; Sun, Gang; Liu, Zhu; Gan, Mingyun; Wu, Jie; Shen, Xin; Gao, Qian

    2016-01-01

    Mycobacterium tuberculosis (MTB) Beijing strains have caused a great concern because of their rapid emergence and increasing prevalence in worldwide regions. Great efforts have been made to investigate the pathogenic characteristics of Beijing strains such as hypervirulence, drug resistance and favoring transmission. Phylogenetically, MTB Beijing family was divided into modern and ancient sublineages. Modern Beijing strains displayed enhanced virulence and higher prevalence when compared with ancient Beijing strains, but the genetic basis for this difference remains unclear. In this study, by analyzing previously published sequencing data of 1082 MTB Beijing isolates, we determined the genetic changes that were commonly present in modern Beijing strains but absent in ancient Beijing strains. These changes include 44 single-nucleotide polymorphisms (SNPs) and two short genomic deletions. Through bioinformatics analysis, we demonstrated that these genetic changes had high probability of functional effects. For example, 4 genes were frameshifted due to premature stop mutation or genomic deletions, 19 nonsynonymous SNPs located in conservative codons, and there is a significant enrichment in regulatory network for all nonsynonymous mutations. Besides, three SNPs located in promoter regions were verified to alter downstream gene expressions. Our study precisely defined the genetic features of modern Beijing strains and provided interesting clues for future researches to elucidate the mechanisms that underlie this sublineage's successful expansion. These findings from the analysis of the modern Beijing sublineage could provide us a model to understand the dynamics of pathogenicity of MTB. PMID:26905026

  18. Role of Cathepsins in Mycobacterium tuberculosis Survival in Human Macrophages.

    PubMed

    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. Insights into battles between Mycobacterium tuberculosis and macrophages.

    PubMed

    Xu, Guanghua; Wang, Jing; Gao, George Fu; Liu, Cui Hua

    2014-10-01

    As the first line of immune defense for Mycobacterium tuberculosis (Mtb), macrophages also provide a major habitat for Mtb to reside in the host for years. The battles between Mtb and macrophages have been constant since ancient times. Triggered upon Mtb infection, multiple cellular pathways in macrophages are activated to initiate a tailored immune response toward the invading pathogen and regulate the cellular fates of the host as well. Toll-like receptors (TLRs) expressed on macrophages can recognize pathogen-associated-molecular patterns (PAMPs) on Mtb and mediate the production of immune-regulatory cytokines such as tumor necrosis factor (TNF) and type I Interferons (IFNs). In addition, Vitamin D receptor (VDR) and Vitamin D-1-hydroxylase are up-regulated in Mtb-infected macrophages, by which Vitamin D participates in innate immune responses. The signaling pathways that involve TNF, type I IFNs and Vitamin D are inter-connected, which play critical roles in the regulation of necroptosis, apoptosis, and autophagy of the infected macrophages. This review article summarizes current knowledge about the interactions between Mtb and macrophages, focusing on cellular fates of the Mtb-infected macrophages and the regulatory molecules and cellular pathways involved in those processes.

  20. 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

  1. 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

    2015-10-01

    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.

  2. 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.

  3. 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.

  4. 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

  5. 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

  6. Zinc Regulates a Switch between Primary and Alternative S18 Ribosomal Proteins in Mycobacterium tuberculosis

    PubMed Central

    Prisic, Sladjana; Hwang, Hyonson; Dow, Allexa; Barnaby, Omar; Pan, Tenny S.; Lonzanida, Jaymes A.; Chazin, Walter J.; Steen, Hanno; Husson, Robert N.

    2015-01-01

    SUMMARY The Mycobacterium tuberculosis genome encodes five putative “alternative” ribosomal proteins whose expression is repressed at high Zn2+ concentration. Each alternative protein has a primary homolog that is predicted to bind Zn2+. We hypothesized that zinc triggers a switch between these paired homologous proteins and therefore chose one of these pairs, S18-1/S18-2, to study mechanisms of the predicted competition for their incorporation into ribosomes. As predicted, our data show that Zn2+-depletion causes accumulation of both S18-2 mRNA and protein. In contrast, S18-1 mRNA levels are unchanged to slightly elevated under Zn2+-limited conditions. However the amount of S18-1 protein is markedly decreased. We further demonstrate that both S18 proteins interact with ribosomal protein S6, a committed step in ribosome biogenesis. Zn2+ is absolutely required for the S18-1/S6 interaction, while it is dispensable for S18-2/S6 dimer formation. These data suggest a model in which the S18-1 is the dominant ribosome constituent in high zinc conditions, e.g. inside of phagosomes, but that it can be replaced by S18-2 when zinc is deficient, e.g. in the extracellular milieu. Consequently, Zn2+-depletion may serve as a signal for building alternative ribosomes when M. tuberculosis is released from macrophages, to allow survival in the extracellular environment. PMID:25858183

  7. Bayesian models for screening and TB Mobile for target inference with Mycobacterium tuberculosis.

    PubMed

    Ekins, Sean; Casey, Allen C; Roberts, David; Parish, Tanya; Bunin, Barry A

    2014-03-01

    The search for compounds active against Mycobacterium tuberculosis is reliant upon high-throughput screening (HTS) in whole cells. We have used Bayesian machine learning models which can predict anti-tubercular activity to filter an internal library of over 150,000 compounds prior to in vitro testing. We used this to select and test 48 compounds in vitro; 11 were active with MIC values ranging from 0.4 μM to 10.2 μM, giving a high hit rate of 22.9%. Among the hits, we identified several compounds belonging to the same series including five quinolones (including ciprofloxacin), three molecules with long aliphatic linkers and three singletons. This approach represents a rapid method to prioritize compounds for testing that can be used alongside medicinal chemistry insight and other filters to identify active molecules. Such models can significantly increase the hit rate of HTS, above the usual 1% or lower rates seen. In addition, the potential targets for the 11 molecules were predicted using TB Mobile and clustering alongside a set of over 740 molecules with known M. tuberculosis target annotations. These predictions may serve as a mechanism for prioritizing compounds for further optimization.

  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. Updated guidelines for using Interferon Gamma Release Assays to detect Mycobacterium tuberculosis infection - United States, 2010.

    PubMed

    Mazurek, Gerald H; Jereb, John; Vernon, Andrew; LoBue, Phillip; Goldberg, Stefan; Castro, Kenneth

    2010-06-25

    n 2005, CDC published guidelines for using the QuantiFERON-TB Gold test (QFT-G) (Cellestis Limited, Carnegie, Victoria, Australia) (CDC. Guidelines for using the QuantiFERON-TB Gold test for detecting Mycobacterium tuberculosis infection, United States. MMWR;54[No. RR-15]:49-55). Subsequently, two new interferon gamma (IFN- gamma) release assays (IGRAs) were approved by the Food and Drug Administration (FDA) as aids in diagnosing M. tuberculosis infection, both latent infection and infection manifesting as active tuberculosis. These tests are the QuantiFERON-TB Gold In-Tube test (QFT-GIT) (Cellestis Limited, Carnegie, Victoria, Australia) and the T-SPOT.TB test (T-Spot) (Oxford Immunotec Limited, Abingdon, United Kingdom). The antigens, methods, and interpretation criteria for these assays differ from those for IGRAs approved previously by FDA. For assistance in developing recommendations related to IGRA use, CDC convened a group of experts to review the scientific evidence and provide opinions regarding use of IGRAs. Data submitted to FDA, published reports, and expert opinion related to IGRAs were used in preparing these guidelines. Results of studies examining sensitivity, specificity, and agreement for IGRAs and TST vary with respect to which test is better. Although data on the accuracy of IGRAs and their ability to predict subsequent active tuberculosis are limited, to date, no major deficiencies have been reported in studies involving various populations. This report provides guidance to U.S. public health officials, health-care providers, and laboratory workers for use of FDA-approved IGRAs in the diagnosis of M. tuberculosis infection in adults and children. In brief, TSTs and IGRAs (QFT-G, QFT-GIT, and T-Spot) may be used as aids in diagnosing M. tuberculosis infection. They may be used for surveillance purposes and to identify persons likely to benefit from treatment. Multiple additional recommendations are provided that address quality control, test

  10. 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.

  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. PMID:27553415

  12. Inhibition of Mycobacterium tuberculosis topoisomerase I by m-AMSA, a eukaryotic type II topoisomerase poison.

    PubMed

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

    2014-04-18

    m-AMSA, an established inhibitor of eukaryotic type II topoisomerases, exerts its cidal effect by binding to the enzyme-DNA complex thus inhibiting the DNA religation step. The molecule and its analogues have been successfully used as chemotherapeutic agents against different forms of cancer. After virtual screening using a homology model of the Mycobacterium tuberculosis topoisomerase I, we identified m-AMSA as a high scoring hit. We demonstrate that m-AMSA can inhibit the DNA relaxation activity of topoisomerase I from M. tuberculosis and Mycobacterium smegmatis. In a whole cell assay, m-AMSA inhibited the growth of both the mycobacteria.

  13. Protein targets for structure-based anti-Mycobacterium tuberculosis drug discovery.

    PubMed

    Lou, Zhiyong; Zhang, Xiaoxue

    2010-05-01

    Mycobacterium tuberculosis, which belongs to the genus Mycobacterium, is the pathogenic agent for most tuberculosis (TB). As TB remains one of the most rampant infectious diseases, causing morbidity and death with emergence of multi-drug-resistant and extensively-drug-resistant forms, it is urgent to identify new drugs with novel targets to ensure future therapeutic success. In this regards, the structural genomics of M. tuberculosis provides important information to identify potential targets, perform biochemical assays, determine crystal structures in complex with potential inhibitor(s), reveal the key sites/residues for biological activity, and thus validate drug targets and discover novel drugs. In this review, we will discuss the recent progress on novel targets for structure-based anti-M. tuberculosis drug discovery.

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

    PubMed Central

    2010-01-01

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

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

    PubMed Central

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

    2016-01-01

    The Esx and PE/PPE families of proteins are among the most immunodominant mycobacterial antigens and have thus been the focus of research to develop vaccines and immunological tests for diagnosis of bovine and human tuberculosis, mainly caused by Mycobacterium bovis and Mycobacterium tuberculosis, respectively. In non-tuberculous mycobacteria (NTM), multiple copies of genes encoding homologous proteins have mainly been identified in pathogenic Mycobacterium species phylogenically related to Mycobacterium tuberculosis and Mycobacterium bovis. Only ancestral copies of these genes have been identified in nonpathogenic NTM species like Mycobacterium smegmatis, Mycobacterium sp. KMS, Mycobacterium sp. MCS, and Mycobacterium sp. JLS. In this study we elucidated the genomes of four nonpathogenic NTM species, viz Mycobacterium komanii sp. nov., Mycobacterium malmesburii sp. nov., Mycobacterium nonchromogenicum, and Mycobacterium fortuitum ATCC 6841. These genomes were investigated for genes encoding for the Esx and PE/PPE (situated in the esx cluster) family of proteins as well as adjacent genes situated in the ESX-1 to ESX-5 regions. To identify proteins actually expressed, comparative proteomic analyses of purified protein derivatives from three of the NTM as well as Mycobacterium kansasii ATCC 12478 and the commercially available purified protein derivatives from Mycobacterium bovis and Mycobacterium avium was performed. The genomic analysis revealed the occurrence in each of the four NTM, orthologs of the genes encoding for the Esx family, the PE and PPE family proteins in M. bovis and M. tuberculosis. The identification of genes of the ESX-1, ESX-3, and ESX-4 region including esxA, esxB, ppe68, pe5, and pe35 adds to earlier reports of these genes in nonpathogenic NTM like M. smegmatis, Mycobacterium sp. JLS and Mycobacterium KMS. This report is also the first to identify esxN gene situated within the ESX-5 locus in M. nonchromogenicum. Our proteomics analysis

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

    PubMed

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

    2016-01-01

    The Esx and PE/PPE families of proteins are among the most immunodominant mycobacterial antigens and have thus been the focus of research to develop vaccines and immunological tests for diagnosis of bovine and human tuberculosis, mainly caused by Mycobacterium bovis and Mycobacterium tuberculosis, respectively. In non-tuberculous mycobacteria (NTM), multiple copies of genes encoding homologous proteins have mainly been identified in pathogenic Mycobacterium species phylogenically related to Mycobacterium tuberculosis and Mycobacterium bovis. Only ancestral copies of these genes have been identified in nonpathogenic NTM species like Mycobacterium smegmatis, Mycobacterium sp. KMS, Mycobacterium sp. MCS, and Mycobacterium sp. JLS. In this study we elucidated the genomes of four nonpathogenic NTM species, viz Mycobacterium komanii sp. nov., Mycobacterium malmesburii sp. nov., Mycobacterium nonchromogenicum, and Mycobacterium fortuitum ATCC 6841. These genomes were investigated for genes encoding for the Esx and PE/PPE (situated in the esx cluster) family of proteins as well as adjacent genes situated in the ESX-1 to ESX-5 regions. To identify proteins actually expressed, comparative proteomic analyses of purified protein derivatives from three of the NTM as well as Mycobacterium kansasii ATCC 12478 and the commercially available purified protein derivatives from Mycobacterium bovis and Mycobacterium avium was performed. The genomic analysis revealed the occurrence in each of the four NTM, orthologs of the genes encoding for the Esx family, the PE and PPE family proteins in M. bovis and M. tuberculosis. The identification of genes of the ESX-1, ESX-3, and ESX-4 region including esxA, esxB, ppe68, pe5, and pe35 adds to earlier reports of these genes in nonpathogenic NTM like M. smegmatis, Mycobacterium sp. JLS and Mycobacterium KMS. This report is also the first to identify esxN gene situated within the ESX-5 locus in M. nonchromogenicum. Our proteomics analysis

  17. ASSESSMENT OF DETECTION EFFICACY OF MYCOBACTERIUM TUBERCULOSIS IN SPUTUM SAMPLES BY REAL TIME PCR BASED METHOD.

    PubMed

    Upadhyaya, S K; Dixit, S M; Shrestha, S; Dangol, S D; Pokhrel, D; Banjara, S; Shrestha, K S; Hengoju, S; Dahal, B K

    2013-07-01

    Tuberculosis (TB) is a major public health problem in Nepal and ranks as one of the most prevalent communicable diseases throughout the country. In Nepal, 45% of total population is infected with TB and 40,000 people get TB every year. Twenty thousand new sputum positive cases are seen every year and 5000-7000 people die each year from TB. Thirty sputum samples were collected from Sukraraj Tropical and Infectious Disease Hospital, Teku, Kathmandu, Nepal and the comparative study of Acid-fast Bacilli (AFB) test and Real time PCR were conducted separately with the culture test which is regarded as gold standard by WHO. Detection of Mycobacterium tuberculosis through use of Real time PCR was found to be higher as compared to AFB and culture. Real-time PCR test showed higher sensitivity (100%) and specificity (94.11%) as compared to AFB test with sensitivity of 84.61% and specificity of 88.24%. Positive predictive value was found to be 84.61% and 92.86% for AFB and Q-PCR respectively. Negative predictive value was found to be 88.24%, and for Q-PCR, it was found to be 100%. Our statistics clearly show that TB diagnosis by Q-PCR is highly efficient and reliableover conventional methods of diagnosis and here we recommend its use in the hospitals and clinics of Nepal.

  18. Susceptibilities of Mycobacterium tuberculosis to isoniazid and rifampin on blood agar.

    PubMed

    Coban, Ahmet Yilmaz; Bilgin, Kemal; Uzun, Meltem; Tasdelen Fisgin, Nuriye; Akgunes, Alper; Cihan, Cigdem Cekic; Birinci, Asuman; Durupinar, Belma

    2005-04-01

    In this study, blood agar was used instead of 7H10 agar for the susceptibility testing of 34 clinical isolates of Mycobacterium tuberculosis to isoniazid (INH) and rifampin (RIF) in accordance with the NCCLS. The BACTEC 460 TB system (Becton Dickinson, Sparks, Md.) was used as a "gold standard." Results for both media were in agreement for RIF and INH at 100 and 94.1%, respectively. For INH, the specificity, sensitivity, positive predictive value, and negative predictive value were found to be 71.4, 100, 93.1, and 100%, respectively, while these values were 100% for RIF. In addition, the results of the susceptibility test performed with blood agar were obtained on day 14 of incubation. In conclusion, results were obtained much earlier with blood agar (2 weeks) than with 7H10 agar (3 weeks), and the results of this study suggest that blood agar may be used as an alternative medium for the susceptibility testing of M. tuberculosis to INH and RIF.

  19. Pulmonary Disease due to Mycobacterium tuberculosis in a Horse: Zoonotic Concerns and Limitations of Antemortem Testing.

    PubMed

    Lyashchenko, Konstantin P; Greenwald, Rena; Esfandiari, Javan; Lecu, Alexis; Waters, W Ray; Posthaus, Horst; Bodmer, Thomas; Janssens, Jean-Paul; Aloisio, Fabio; Graubner, Claudia; Grosclaude, Eléonore; Piersigilli, Alessandra; Schiller, Irene

    2012-01-01

    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 mycobacteriosis. In the lungs, multiple tuberculoid granulomas communicating with the bronchiolar lumen, pleural effusion, and a granulomatous lymphadenitis involving mediastinal and tracheobronchial lymph nodes were found. Serologic response to M. tuberculosis antigens was detected in the infected horse, but not in the group of 42 potentially exposed animals (18 horses, 14 alpacas, 6 donkeys, and 4 dogs) which showed no signs of disease. Diagnosis of tuberculosis in live horses remains extremely difficult. Four of 20 animal handlers at the farm were positive for tuberculous infection upon follow-up testing by interferon-gamma release assay, indicating a possibility of interspecies transmission of M. tuberculosis. PMID:22567544

  20. Local ischemia and increased expression of vascular endothelial growth factor following ocular dissemination of Mycobacterium tuberculosis.

    PubMed

    Thayil, Seema M; Albini, Thomas A; Nazari, Hossein; Moshfeghi, Andrew A; Parel, Jean-Marie A; Rao, Narsing A; Karakousis, Petros C

    2011-01-01

    The pathogenesis of intraocular tuberculosis remains poorly understood partly due to the lack of adequate animal models that accurately simulate human disease. Using a recently developed model of ocular tuberculosis following aerosol infection of guinea pigs with Mycobacterium tuberculosis, we studied the microbiological, histological, and clinical features of intraocular tuberculosis infection. Viable tubercle bacilli were cultivated from all eyes by Day 56 after aerosol delivery of ∼200 bacilli to guinea pig lungs. Choroidal tuberculous granulomas showed reduced oxygen tension, as evidenced by staining with the hypoxia-specific probe pimonidazole, and expression of vascular endothelial growth factor (VEGF) was detected in the retinal pigment epithelium (RPE) and photoreceptors. Fundoscopic examination of M. tuberculosis-infected guinea pig eyes revealed altered vascular architecture and chorioretinal hemorrhage by Day 56 after infection. This model may be useful in further elucidating the pathogenesis of ocular tuberculosis, as well as in developing tools for diagnosis and assessment of antituberculosis treatment responses in the eye.

  1. Mycobacterium tuberculosis isolates from single outpatient clinic in Panama City exhibit wide genetic diversity.

    PubMed

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

    2014-08-01

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

  2. Whole-genome sequencing to detect recent transmission of Mycobacterium tuberculosis in settings with a high burden of tuberculosis

    PubMed Central

    Luo, Tao; Yang, Chongguang; Peng, Ying; Lu, Liping; Sun, Guomei; Wu, Jie; Jin, Xiaoping; Hong, Jianjun; Li, Fabin; Mei, Jian; DeRiemer, Kathryn; Gao, Qian

    2015-01-01

    Whole genome sequencing (WGS) of Mycobacterium tuberculosis has been used to trace the transmission of Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). Previously published studies using WGS were conducted in developed countries with a low TB burden. We sought to evaluate the relative usefulness of traditional VNTR and SNP typing methods, WGS and epidemiological investigations to study the recent transmission of M. tuberculosis in a high TB burden country. We conducted epidemiological investigations of 42 TB patients whose M. tuberculosis isolates were classified into three clusters based on variable-number tandem repeat (VNTR) typing. We applied WGS to 32 (76.2%) of the 42 strains and calculated the pairwise genomic distances between strains within each cluster. Eighteen (56.3%) of the 32 strains had genomic differences ≥100 SNPs with every other strain, suggesting that direct transmission did not likely occurred. Ten strains were grouped into four WGS-based clusters with genomic distances ≤5 SNPs within each cluster, and confirmed epidemiological links were identified in two of these clusters. Our results indicate that WGS provides reliable resolution for tracing the transmission of M. tuberculosis in high TB burden settings. The high resolution of WGS is particularly useful to confirm or exclude the possibility of direct transmission events defined by traditional typing methods. PMID:24888866

  3. Viability of stressed Mycobacterium tuberculosis and association with multidrug resistance

    PubMed Central

    Martins, Maria Conceição; Giampaglia, Carmen Maria Saraiva; Chimara, Erica; Oliveira, Rosângela Siqueira; Vedovello, Danielle; Sakamoto, Sidnei Miyoshi; Ferrazoli, Lucilaine

    2013-01-01

    This study investigated biological characteristics of recovered stressed M. tuberculosis isolates that failed to grow in differential culture media for phenotypic identification and in culture media containing anti-tuberculosis drugs for drug-susceptibility testing, despite of having grown in primary culture. It represents an improvement in the diagnosis of MDR tuberculosis and tuberculosis control. PMID:24294238

  4. Antagonistic action of Streptococcus salivarius and Streptococcus faecalis to Mycobacterium tuberculosis.

    PubMed Central

    Darling, C L; Hart, G D

    1976-01-01

    Streptococcus salivarius and Streptococcus faecalis were found to inhibit the growth of Mycobacterium tuberculosis on Löwenstein-Jensen and Middlebrook 7H11 agars, but not on the latter medium when antibacterial drugs were added. S. faecalis was found to be more inhibitory than S. salivarius to 15 strains of M. tuberculosis. S. salivarius produced little or no inhibition of growth of Runyon group III organisms but was very antagonistic to Runyon group I mycobacteria. Images PMID:824304

  5. Molecular Typing of Mycobacterium Tuberculosis Strains: A Fundamental Tool for Tuberculosis Control and Elimination

    PubMed Central

    Cannas, Angela; Mazzarelli, Antonio; Di Caro, Antonino; Delogu, Giovanni; Girardi, Enrico

    2016-01-01

    Tuberculosis (TB) is still an important cause of morbidity and mortality worldwide. An improvement of the strategies for disease control is necessary in both low- and high-incidence TB countries. Clinicians, epidemiologists, laboratory specialists, and public health players should work together in order to achieve a significant reduction in TB transmission and spread of drug-resistant strains. Effective TB surveillance relies on early diagnosis of new cases, appropriate therapy, and accurate detection of outbreaks in the community, in order to implement proper TB control strategies. To achieve this goal, information from classical and molecular epidemiology, together with patient clinical data need to be combined. In this review, we summarize the methodologies currently used in molecular epidemiology, namely molecular typing. We will discuss their efficiency to phylogenetically characterize Mycobacterium tuberculosis isolates, and their ability to provide information that can be useful for disease control. We will also introduce next generation sequencing as the methodology that potentially could provide in a short time both, detection of new outbreaks and identification of resistance patterns. This could envision a potential of next generation sequencing as an important tool for accurate patient management and disease control. PMID:27403266

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

    PubMed Central

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

    2015-01-01

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

  7. Sublineages of Mycobacterium tuberculosis Beijing genotype strains and unfavorable outcomes of anti-tuberculosis treatment.

    PubMed

    Hang, Nguyen T L; Maeda, Shinji; Keicho, Naoto; Thuong, Pham H; Endo, Hiroyoshi

    2015-05-01

    The influence of Mycobacterium tuberculosis (MTB) lineages/sublineages on unfavorable tuberculosis (TB) treatment outcomes is poorly understood. We investigated the effects of Beijing genotype sublineages and other factors contributing to treatment outcome. Patients newly diagnosed with sputum smear-positive and culture-positive TB in Hanoi, Vietnam, participated in the study. After receiving anti-TB treatment, they were intensively followed up for the next 16 months. MTB isolates collected before treatment were subjected to drug susceptibility testing, and further analyzed to determine MTB (sub) lineages and their clonal similarities. Of 430 patients, 17 had treatment failure and 30 had TB recurrence. Rifampicin resistance was associated with treatment failure {adjusted odds ratio = 6.64 [95% confidence interval (CI), 1.48-29.73]}. The modern Beijing genotype was significantly associated with recurrent TB within 16 months [adjusted hazard ratio = 3.29 (95% CI, 1.17-9.27)], particularly after adjustment for the relevant antibiotic resistance. Human immunodeficiency virus coinfection and severity on chest radiographs were not significantly associated with unfavorable outcomes. Our findings provide further understanding of the influence of MTB strains on unfavorable treatment outcomes. Multiple risk factors should be considered for the optimal management of TB. PMID:25732626

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

    PubMed Central

    Lee, Yie-Vern; Wahab, Habibah A.

    2015-01-01

    Isocitrate lyase (ICL) is the first enzyme involved in glyoxylate cycle. Many plants and microorganisms are relying on glyoxylate cycle enzymes to survive upon downregulation of tricarboxylic acid cycle (TCA cycle), especially Mycobacterium tuberculosis (MTB). In fact, ICL is a potential drug target for MTB in dormancy. With the urge for new antitubercular drug to overcome tuberculosis treat such as multidrug resistant strain and HIV-coinfection, the pace of drug discovery has to be increased. There are many approaches to discovering potential inhibitor for MTB ICL and we hereby review the updated list of them. The potential inhibitors can be either a natural compound or synthetic compound. Moreover, these compounds are not necessary to be discovered only from MTB ICL, as it can also be discovered by a non-MTB ICL. Our review is categorized into four sections, namely, (a) MTB ICL with natural compounds; (b) MTB ICL with synthetic compounds; (c) non-MTB ICL with natural compounds; and (d) non-MTB ICL with synthetic compounds. Each of the approaches is capable of overcoming different challenges of inhibitor discovery. We hope that this paper will benefit the discovery of better inhibitor for ICL. PMID:25649791

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

    PubMed

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

    2014-01-01

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

  10. A Chlorhexidine- Agar Plate Culture Medium Protocol to Complement Standard Broth Culture of Mycobacterium tuberculosis

    PubMed Central

    Asmar, Shady; Chatellier, Sonia; Mirande, Caroline; van Belkum, Alex; Canard, Isabelle; Raoult, Didier; Drancourt, Michel

    2016-01-01

    The culture of Mycobacterium tuberculosis using parallel inoculation of a solid culture medium and a liquid broth provides the gold standard for the diagnosis of tuberculosis. Here, we evaluated a chlorhexidine decontamination-MOD9 solid medium protocol versus the standard NALC-NaOH-Bactec 960 MGIT protocol for the diagnosis of pulmonary tuberculosis by culture. Three-hundred clinical specimens comprising 193 sputa, 30 bronchial aspirates, 10 broncho-alveolar lavages, 47 stools, and 20 urines were prospectively submitted for the routine diagnosis of tuberculosis. The contamination rates were 5/300 (1.7%) using the MOD9 protocol and 17/300 (5.7%) with the Bactec protocol, respectively (P < 0.05, Fisher exact test). Of a total of 50 Mycobacterium isolates (48 M. tuberculosis and two Mycobacterium abscessus) were cultured. Out of these 50, 48 (96%) isolates were found using the MOD9 protocol versus 35 (70%) when using the Bactec protocol (P < 0.05, Fisher exact test). The time to positivity was 10.1 ± 3.9 days versus 14.7 ± 7.3 days, respectively, (P < 0.05, Student’s t-test). These data confirmed the usefulness of parallel inoculation of a solid culture medium with broth for the recovery of M. tuberculosis in agreement with current recommendations. More specifically, chlorhexidine decontamination and inoculation of the MOD9 solid medium could be proposed to complement the standard Bactec 960 MGIT broth protocol. PMID:26834733

  11. A Chlorhexidine- Agar Plate Culture Medium Protocol to Complement Standard Broth Culture of Mycobacterium tuberculosis.

    PubMed

    Asmar, Shady; Chatellier, Sonia; Mirande, Caroline; van Belkum, Alex; Canard, Isabelle; Raoult, Didier; Drancourt, Michel

    2016-01-01

    The culture of Mycobacterium tuberculosis using parallel inoculation of a solid culture medium and a liquid broth provides the gold standard for the diagnosis of tuberculosis. Here, we evaluated a chlorhexidine decontamination-MOD9 solid medium protocol versus the standard NALC-NaOH-Bactec 960 MGIT protocol for the diagnosis of pulmonary tuberculosis by culture. Three-hundred clinical specimens comprising 193 sputa, 30 bronchial aspirates, 10 broncho-alveolar lavages, 47 stools, and 20 urines were prospectively submitted for the routine diagnosis of tuberculosis. The contamination rates were 5/300 (1.7%) using the MOD9 protocol and 17/300 (5.7%) with the Bactec protocol, respectively (P < 0.05, Fisher exact test). Of a total of 50 Mycobacterium isolates (48 M. tuberculosis and two Mycobacterium abscessus) were cultured. Out of these 50, 48 (96%) isolates were found using the MOD9 protocol versus 35 (70%) when using the Bactec protocol (P < 0.05, Fisher exact test). The time to positivity was 10.1 ± 3.9 days versus 14.7 ± 7.3 days, respectively, (P < 0.05, Student's t-test). These data confirmed the usefulness of parallel inoculation of a solid culture medium with broth for the recovery of M. tuberculosis in agreement with current recommendations. More specifically, chlorhexidine decontamination and inoculation of the MOD9 solid medium could be proposed to complement the standard Bactec 960 MGIT broth protocol. PMID:26834733

  12. A modified visual loop-mediated isothermal amplification method for diagnosis and differentiation of main pathogens from Mycobacterium tuberculosis complex.

    PubMed

    Hong, Ming; Zha, Lei; Fu, Wenliang; Zou, Minji; Li, Wuju; Xu, Donggang

    2012-02-01

    This study was aimed to rapidly identify and differentiate two main pathogens of the Mycobacterium tuberculosis complex: Mycobacterium tuberculosis subsp. tuberculosis and Mycobacterium bovis by a modified loop-mediated isothermal amplification (LAMP) assay. The reaction results could be evaluated by naked eye with two optimized closed tube detection methods as follows: adding the modified fluorescence dye in advance into the reaction mix so as to observe the color changes or putting a tinfoil in the tube and adding the SYBR Green I dye on it, then making the dye drop into the bottom of the tube by centrifuge after reaction. The results showed that the two groups of primers used jointly in this assay could successfully identify and differentiate Mycobacterium tuberculosis subsp. tuberculosis and Mycobacterium tuberculosis bovis. Sensitivity test displayed that the modified LAMP assay with the closed tube system could determine the minimal template concentration of 1 copy/μl, which was more sensitive than that of routine PCR. The advantages of this LAMP method for detection of the Mycobacterium tuberculosis complex included high specificity, high sensitivity, simplicity, and superiority in avoidance of aerosol contamination. The modified LAMP assay would provide a potential for clinical diagnosis and therapy of tuberculosis in the developing countries and the resource-limited areas. PMID:22806847

  13. Protection by novel vaccine candidates, Mycobacterium tuberculosis ΔmosR and ΔechA7, against challenge with a Mycobacterium tuberculosis Beijing strain

    PubMed Central

    Marcus, Sarah A.; Steinberg, Howard; Talaat, Adel M.

    2015-01-01

    Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), infects over two billion people, claiming around 1.5 million lives annually. The only vaccine approved for clinical use against this disease is the Bacillus Calmette-Guérin (BCG) vaccine. Unfortunately, BCG has limited efficacy against the adult, pulmonary form of tuberculosis. This vaccine was developed from M. bovis with antigen expression and host specificity that differ from M. tuberculosis. To address these problems, we have designed two novel, live attenuated vaccine (LAV) candidates on an M. tuberculosis background: ΔmosR and ΔechA7. These targeted genes are important to M. tuberculosis pathogenicity during infection. To examine the efficacy of these strains, C57BL/6 mice were vaccinated subcutaneously with either LAV, BCG, or PBS. Both LAV strains persisted up to 16 weeks in the spleens or lungs of vaccinated mice, while eliciting minimal pathology prior to challenge. Following challenge with a selected, high virulence M. tuberculosis Beijing strain, protection was notably greater for both groups of LAV vaccinated animals as compared to BCG at both 30 and 60 days post-challenge. Additionally, vaccination with either ΔmosR or ΔechA7 elicited an immune response similar to BCG. Although these strains require further development to meet safety standards, this first evidence of protection by these two new, live attenuated vaccine candidates shows promise. PMID:26363381

  14. Whole-genome sequencing to delineate Mycobacterium tuberculosis outbreaks: a retrospective observational study

    PubMed Central

    Walker, Timothy M; Ip, Camilla LC; Harrell, Ruth H; Evans, Jason T; Kapatai, Georgia; Dedicoat, Martin J; Eyre, David W; Wilson, Daniel J; Hawkey, Peter M; Crook, Derrick W; Parkhill, Julian; Harris, David; Walker, A Sarah; Bowden, Rory; Monk, Philip; Smith, E Grace; Peto, Tim EA

    2013-01-01

    Summary Background Tuberculosis incidence in the UK has risen in the past decade. Disease control depends on epidemiological data, which can be difficult to obtain. Whole-genome sequencing can detect microevolution within Mycobacterium tuberculosis strains. We aimed to estimate the genetic diversity of related M tuberculosis strains in the UK Midlands and to investigate how this measurement might be used to investigate community outbreaks. Methods In a retrospective observational study, we used Illumina technology to sequence M tuberculosis genomes from an archive of frozen cultures. We characterised isolates into four groups: cross-sectional, longitudinal, household, and community. We measured pairwise nucleotide differences within hosts and between hosts in household outbreaks and estimated the rate of change in DNA sequences. We used the findings to interpret network diagrams constructed from 11 community clusters derived from mycobacterial interspersed repetitive-unit–variable-number tandem-repeat data. Findings We sequenced 390 separate isolates from 254 patients, including representatives from all five major lineages of M tuberculosis. The estimated rate of change in DNA sequences was 0·5 single nucleotide polymorphisms (SNPs) per genome per year (95% CI 0·3–0·7) in longitudinal isolates from 30 individuals and 25 families. Divergence is rarely higher than five SNPs in 3 years. 109 (96%) of 114 paired isolates from individuals and households differed by five or fewer SNPs. More than five SNPs separated isolates from none of 69 epidemiologically linked patients, two (15%) of 13 possibly linked patients, and 13 (17%) of 75 epidemiologically unlinked patients (three-way comparison exact p<0·0001). Genetic trees and clinical and epidemiological data suggest that super-spreaders were present in two community clusters. Interpretation Whole-genome sequencing can delineate outbreaks of tuberculosis and allows inference about direction of transmission between

  15. Catalysis and Inhibition of Mycobacterium tuberculosis Methionine Aminopeptidase

    SciTech Connect

    Lu, Jing-Ping; Chai, Sergio C.; Ye, Qi-Zhuang

    2010-09-07

    Methionine aminopeptidase (MetAP) carries out an important cotranslational N-terminal methionine excision of nascent proteins and represents a potential target to develop antibacterial and antitubercular drugs. We cloned one of the two MetAPs in Mycobacterium tuberculosis (MtMetAP1c from the mapB gene) and purified it to homogeneity as an apoenzyme. Its activity required a divalent metal ion, and Co(II), Ni(II), Mn(II), and Fe(II) were among activators of the enzyme. Co(II) and Fe(II) had the tightest binding, while Ni(II) was the most efficient cofactor for the catalysis. MtMetAP1c was also functional in E. coli cells because a plasmid-expressed MtMetAP1c complemented the essential function of MetAP in E. coli and supported the cell growth. A set of potent MtMetAP1c inhibitors were identified, and they showed high selectivity toward the Fe(II)-form, the Mn(II)-form, or the Co(II) and Ni(II) forms of the enzyme, respectively. These metalloform selective inhibitors were used to assign the metalloform of the cellular MtMetAP1c. The fact that only the Fe(II)-form selective inhibitors inhibited the cellular MtMetAP1c activity and inhibited the MtMetAP1c-complemented cell growth suggests that Fe(II) is the native metal used by MtMetAP1c in an E. coli cellular environment. Finally, X-ray structures of MtMetAP1c in complex with three metalloform-selective inhibitors were analyzed, which showed different binding modes and different interactions with metal ions and active site residues.

  16. Adenylating Enzymes in Mycobacterium tuberculosis as Drug Targets

    PubMed Central

    Duckworth, Benjamin P.; Nelson, Kathryn M.; Aldrich, Courtney C.

    2013-01-01

    Adenylation or adenylate-forming enzymes (AEs) are widely found in nature and are responsible for the activation of carboxylic acids to intermediate acyladenylates, which are mixed anhydrides of AMP. In a second reaction, AEs catalyze the transfer of the acyl group of the acyladenylate onto a nucleophilic amino, alcohol, or thiol group of an acceptor molecule leading to amide, ester, and thioester products, respectively. Mycobacterium tuberculosis encodes for more than 60 adenylating enzymes, many of which represent potential drug targets due to their confirmed essentiality or requirement for virulence. Several strategies have been used to develop potent and selective AE inhibitors including high-throughput screening, fragment-based screening, and the rationale design of bisubstrate inhibitors that mimic the acyladenylate. In this review, a comprehensive analysis of the mycobacterial adenylating enzymes will be presented with a focus on the identification of small molecule inhibitors. Specifically, this review will cover the aminoacyl tRNA-synthetases (aaRSs), MenE required for menaquinone synthesis, the FadD family of enzymes including the fatty acyl-AMP ligases (FAAL) and the fatty acyl-CoA ligases (FACLs) involved in lipid metabolism, and the nonribosomal peptide synthetase adenylation enzyme MbtA that is necessary for mycobactin synthesis. Additionally, the enzymes NadE, GuaA, PanC, and MshC involved in the respective synthesis of NAD, guanine, pantothenate, and mycothiol will be discussed as well as BirA that is responsible for biotinylation of the acyl CoA-carboxylases. PMID:22283817

  17. Structural features of the exocellular polysaccharides of Mycobacterium tuberculosis.

    PubMed Central

    Lemassu, A; Daffé, M

    1994-01-01

    The cell envelope which surrounds pathogenic mycobacteria is postulated to be a defence barrier against phagocytic cells and its outermost constituents have a tendency to accumulate in the culture medium. The present work demonstrates that the exocellular material of Mycobacterium tuberculosis contains large amounts of polysaccharides with only traces, if any at all, of lipids. Three types of polysaccharides were purified by anion-exchange and gel-filtration chromatography; all were found to be neutral compounds devoid of acyl substituents. They consisted of D-glucan, D-arabino-D-mannan and D-mannan, which were eluted from gel-filtration columns in positions corresponding to molecular masses of 123, 13 and 4 kDa respectively. Their predominant structural features were determined by the characterization of the per-O-methyl derivatives of enzymic, acetolysis and Smith-degradation products and by 1H- and 13C-n.m.r. spectroscopy of the purified polysaccharides, using mono- and two-dimensional homonuclear chemical-shift correlated spectroscopy and two-dimensional heteronuclear (1H/13C) spectroscopy. The glucan which represented up to 90% of the polysaccharides was composed of repeating units of five or six-->4-alpha-D-Glcp-1--> residues and a -->4-alpha-D-Glcp substituted at position 6 with an alpha-D-Glcp, indicating a glycogen-like highly branched structure not related to the so-called polysaccharide-II previously identified in tuberculin. The arabinomannan consisted of a mannan segment composed of a -->6-alpha-D-Man-1--> core substituted at some positions 2 with an alpha-D-Manp. The arabinan termini of the arabinomannan were found to be extensively capped with mannosyl residues. The possibility that these polysaccharides contribute to the persistence of the tubercle bacillus in the macrophage by molecular mimicry is discussed. PMID:8297342

  18. Early detection of Mycobacterium tuberculosis complex in BACTEC MGIT cultures using nucleic acid amplification.

    PubMed

    Lin, S Y; Hwang, S C; Yang, Y C; Wang, C F; Chen, Y H; Chen, T C; Lu, P L

    2016-06-01

    We evaluated the application of nucleic acid amplification (NAA) in liquid cultures for the early detection of Mycobacterium tuberculosis. The Cobas TaqMan MTB test, IS6110 real-time PCR, and hsp65 PCR-restriction fragment length polymorphism (RFLP) analysis were used to detect BACTEC MGIT 960 (MGIT) cultures on days 3, 5, 7, and 14. The procedure was initially tested with a reference strain, H37Rv (ATCC 27294). Subsequently, 200 clinical specimens, including 150 Acid Fast bacillus (AFB) smear-positive and 50 AFB smear-negative samples, were examined. The Cobas TaqMan MTB test and IS6110-based PCR analysis were able to detect M. tuberculosis after 1 day when the inoculum of H37Rv was >3 x 10(-2) CFU/ml. After a 5-day incubation in the MGIT system, all three NAA assays had a positive detection regardless of the inoculum size. After a 1-day incubation of the clinical specimens in the MGIT system, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for the Cobas TaqMan MTB assay were 70.2%, 100%, 100%, and 82.3% respectively. For IS6110-based PCR analysis, these values were 63.1%, 100%, 100%, and 78.9%, and were 88.1%, 100%, 100%, and 92.1% respectively for hsp65 PCR-RFLP analysis. After a 3-day incubation, the specificity and PPV were 100% for all three NAA tests; the Cobas TaqMan MTB assay had the best sensitivity (97.6%) and NPV (98.3%). The sensitivity, specificity, PPV, and NPV for conventional culture analysis were 98.8%, 100%, 100%, and 99.1%. Thus, NAA may be useful for the early detection of M. tuberculosis after 3 days in MGIT.

  19. Manipulation of the endocytic pathway and phagocyte functions by Mycobacterium tuberculosis lipoarabinomannan.

    PubMed

    Vergne, Isabelle; Gilleron, Martine; Nigou, Jérôme

    2014-01-01

    Lipoarabinomannan is a major immunomodulatory lipoglycan found in the cell envelope of Mycobacterium tuberculosis and related human pathogens. It reproduces several salient properties of M. tuberculosis in phagocytic cells, including inhibition of pro-inflammatory cytokine production, inhibition of phagolysosome biogenesis, and inhibition of apoptosis as well as autophagy. In this review, we present our current knowledge on lipoarabinomannan structure and ability to manipulate the endocytic pathway as well as phagocyte functions. A special focus is put on the molecular mechanisms employed and the signaling pathways hijacked. Available information is discussed in the context of M. tuberculosis pathogenesis.

  20. Structural measurements and cell line studies of the copper-PEG-Rifampicin complex against Mycobacterium tuberculosis.

    PubMed

    Manning, Thomas; Mikula, Rachel; Wylie, Greg; Phillips, Dennis; Jarvis, Jackie; Zhang, Fengli

    2015-02-01

    The bacterium responsible for tuberculosis is increasing its resistance to antibiotics resulting in new multidrug-resistant Mycobacterium tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB). In this study, several analytical techniques including NMR, FT-ICR, MALDI-MS, LC-MS and UV/Vis are used to study the copper-Rifampicin-Polyethylene glycol (PEG-3350) complex. The copper (II) cation is a carrier for the antibiotic Rifampicin as well as nutrients for the bacterium. The NIH-NIAID cell line containing several Tb strains (including antibiotic resistant strains) is tested against seven copper-PEG-RIF complex variations.

  1. [Effect of low-energy helium-neon laser on the biological properties of Mycobacterium tuberculosis].

    PubMed

    Dolzhanskiĭ, V M; Kaliuk, A N; Maliev, B M; Levchenko, T N

    1990-01-01

    The results of experimental studies of M. tuberculosis biological properties tested in guinea pigs which were subjected to different doses of helium-neon laser radiation are given. The functional evidence is compared with the results of electron microscopic study of the irradiated culture. The investigation revealed that laser radiation caused changes in biological properties of M. tuberculosis. A decrease in growth properties and virulence was found to be related to a radiation dose. It is suggested that a drop in the biological activity of M. tuberculosis under laser radiation be associated with its influence on the Mycobacterium lipid layer which contains a cord-factor and responsible for their virulence.

  2. Inverted repeats in the promoter as an autoregulatory sequence for TcrX in Mycobacterium tuberculosis

    SciTech Connect

    Bhattacharya, Monolekha; Das, Amit Kumar

    2011-11-11

    Highlights: Black-Right-Pointing-Pointer The regulatory sequences recognized by TcrX have been identified. Black-Right-Pointing-Pointer The regulatory region comprises of inverted repeats segregated by 30 bp region. Black-Right-Pointing-Pointer The mode of binding of TcrX with regulatory sequence is unique. Black-Right-Pointing-Pointer In silico TcrX-DNA docked model binds one of the inverted repeats. Black-Right-Pointing-Pointer Both phosphorylated and unphosphorylated TcrX binds regulatory sequence in vitro. -- Abstract: TcrY, a histidine kinase, and TcrX, a response regulator, constitute a two-component system in Mycobacterium tuberculosis. tcrX, which is expressed during iron scarcity, is instrumental in the survival of iron-dependent M. tuberculosis. However, the regulator of tcrX/Y has not been fully characterized. Crosslinking studies of TcrX reveal that it can form oligomers in vitro. Electrophoretic mobility shift assays (EMSAs) show that TcrX recognizes two regions in the promoter that are comprised of inverted repeats separated by {approx}30 bp. The dimeric in silico model of TcrX predicts binding to one of these inverted repeat regions. Site-directed mutagenesis and radioactive phosphorylation indicate that D54 of TcrX is phosphorylated by H256 of TcrY. However, phosphorylated and unphosphorylated TcrX bind the regulatory sequence with equal efficiency, which was shown with an EMSA using the D54A TcrX mutant.

  3. Subfamily-specific adaptations in the structures of two penicillin-binding proteins from Mycobacterium tuberculosis

    DOE PAGES

    Prigozhin, Daniil M.; Krieger, Inna V.; Huizar, John P.; Mavrici, Daniela; Waldo, Geoffrey S.; Hung, Li -Wei; Sacchettini, James C.; Terwilliger, Thomas C.; Alber, Tom; Mayer, Claudine

    2014-12-31

    Beta-lactam antibiotics target penicillin-binding proteins including several enzyme classes essential for bacterial cell-wall homeostasis. To better understand the functional and inhibitor-binding specificities of penicillin-binding proteins from the pathogen, Mycobacterium tuberculosis, we carried out structural and phylogenetic analysis of two predicted D,D-carboxypeptidases, Rv2911 and Rv3330. Optimization of Rv2911 for crystallization using directed evolution and the GFP folding reporter method yielded a soluble quadruple mutant. Structures of optimized Rv2911 bound to phenylmethylsulfonyl fluoride and Rv3330 bound to meropenem show that, in contrast to the nonspecific inhibitor, meropenem forms an extended interaction with the enzyme along a conserved surface. Phylogenetic analysis shows thatmore » Rv2911 and Rv3330 belong to different clades that emerged in Actinobacteria and are not represented in model organisms such as Escherichia coli and Bacillus subtilis. Clade-specific adaptations allow these enzymes to fulfill distinct physiological roles despite strict conservation of core catalytic residues. The characteristic differences include potential protein-protein interaction surfaces and specificity-determining residues surrounding the catalytic site. Overall, these structural insights lay the groundwork to develop improved beta-lactam therapeutics for tuberculosis.« less

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

    PubMed

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

    2016-02-18

    The transition from replication to non-replication underlies much of Mycobacterium tuberculosis (Mtb) pathogenesis, as non- or slowly replicating Mtb are responsible for persistence and poor treatment outcomes. Therapeutic targeting of non-replicating populations is a priority for tuberculosis treatment, but few drug targets in non-replicating Mtb are currently known. Here, we directly measured the activity of the highly diverse and druggable serine hydrolases (SHs) during active replication and non-replication using activity-based proteomics. We predict SH activity for 78 proteins, including 27 proteins with unknown function, and identify 37 SHs that remain active in the absence of replication, providing a set of candidate persistence targets. Non-replication was associated with major shifts in SH activity. These activity changes were largely independent of SH abundance, indicating extensive post-translational regulation of SHs. By probing a large cross-section of druggable Mtb enzyme space during replication and non-replication, we identify new SHs and suggest new persistence targets. PMID:26853625

  5. New Targets and Cofactors for the Transcription Factor LrpA from Mycobacterium tuberculosis.

    PubMed

    Song, Ningning; Cui, Yingying; Li, Zhaoli; Chen, Liping; Liu, Siguo

    2016-04-01

    Rv3291c (MtbLrpA), a transcriptional regulator, belongs to the leucine-responsive regulatory protein (Lrp) family and is thought to play an important role in Mycobacterium tuberculosis persistence. In this study, we verified 17 novel potential binding sites for MtbLrpA by in vitro binding assays on the basis of previous predictions from an in silico analysis and bacterial one-hybrid (BIH) reporter system. Amino acids, such as tyrosine, phenylalanine, tryptophan, and histidine, strongly affect the binding affinity of MtbLrpA, and vitamins, including B1, B3, B6, VC, B7, B9, B12, VA, and VK3, also decrease MtbLrpA binding affinity. This is the first report regarding that an Lrp-like protein can sense vitamins as an environmental signal. Vitamin supplementation to the environment can change the expression level of the target genes, which provides a potential mechanism for tuberculosis supplementary treatment with vitamins. PMID:26789099

  6. Subfamily-Specific Adaptations in the Structures of Two Penicillin-Binding Proteins from Mycobacterium tuberculosis

    PubMed Central

    Prigozhin, Daniil M.; Krieger, Inna V.; Huizar, John P.; Mavrici, Daniela; Waldo, Geoffrey S.; Hung, Li-Wei; Sacchettini, James C.; Terwilliger, Thomas C.; Alber, Tom

    2014-01-01

    Beta-lactam antibiotics target penicillin-binding proteins including several enzyme classes essential for bacterial cell-wall homeostasis. To better understand the functional and inhibitor-binding specificities of penicillin-binding proteins from the pathogen, Mycobacterium tuberculosis, we carried out structural and phylogenetic analysis of two predicted D,D-carboxypeptidases, Rv2911 and Rv3330. Optimization of Rv2911 for crystallization using directed evolution and the GFP folding reporter method yielded a soluble quadruple mutant. Structures of optimized Rv2911 bound to phenylmethylsulfonyl fluoride and Rv3330 bound to meropenem show that, in contrast to the nonspecific inhibitor, meropenem forms an extended interaction with the enzyme along a conserved surface. Phylogenetic analysis shows that Rv2911 and Rv3330 belong to different clades that emerged in Actinobacteria and are not represented in model organisms such as Escherichia coli and Bacillus subtilis. Clade-specific adaptations allow these enzymes to fulfill distinct physiological roles despite strict conservation of core catalytic residues. The characteristic differences include potential protein-protein interaction surfaces and specificity-determining residues surrounding the catalytic site. Overall, these structural insights lay the groundwork to develop improved beta-lactam therapeutics for tuberculosis. PMID:25551456

  7. Viral Booster Vaccines Improve Mycobacterium bovis BCG-Induced Protection Against Bovine Tuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous work in small animal laboratory models of tuberculosis have shown that vaccination strategies based on heterologous prime-boost protocols using Mycobacterium bovis bacille Calmette-Guerin (BCG) to prime and Modified Vaccinia Ankara strain (MVA85A) or recombinant attenuated adenoviruses (Ad8...

  8. 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...

  9. Measuring of Mycobacterium tuberculosis growth. A correlation of the optical measurements with colony forming units

    PubMed Central

    Peñuelas-Urquides, Katia; Villarreal-Treviño, Licet; Silva-Ramírez, Beatriz; Rivadeneyra-Espinoza, Liliana; Said-Fernández, Salvador; de León, Mario Bermúdez

    2013-01-01

    The quantification of colony forming units (cfu), turbidity, and optical density at 600 nm (OD600) measurements were used to evaluate Mycobacterium tuberculosis growth. Turbidity and OD600 measurements displayed similar growth curves, while cfu quantification showed a continuous growth curve. We determined the cfu equivalents to McFarland and OD600 units. PMID:24159318

  10. Genomes of Two Clinical Isolates of Mycobacterium tuberculosis from Odisha, India

    PubMed Central

    Majid, Mohammad; Qureshi, Asifa; Yerra, Priyadarshini; Kumar, Ashutosh; Kumar, Mandala Kiran; Tiruvayipati, Suma; Baddam, Ramani; Shaik, Sabiha; Srikantam, Aparna

    2014-01-01

    We report whole-genome sequences of two clinical isolates of Mycobacterium tuberculosis isolated from patients in Odisha, India. The sequence analysis revealed that these isolates are of an ancestral type and might represent some of the “pristine” isolates in India that have not admixed with other lineages. PMID:24652981

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

    PubMed

    Repasy, Teresa; Lee, Jinhee; Marino, Simeone; Martinez, Nuria; Kirschner, Denise E; Hendricks, Gregory; Baker, Stephen; Wilson, Andrew A; Kotton, Darrell N; Kornfeld, Hardy

    2013-02-01

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

  12. Integrated Modeling of Gene Regulatory and Metabolic Networks in Mycobacterium tuberculosis.

    PubMed

    Ma, Shuyi; Minch, Kyle J; Rustad, Tige R; Hobbs, Samuel; Zhou, Suk-Lin; Sherman, David R; Price, Nathan D

    2015-11-01

    Mycobacterium tuberculosis (MTB) is the causative bacterium of tuberculosis, a disease responsible for over a million deaths worldwide annually with a growing number of strains resistant to antibiotics. The development of better therapeutics would greatly benefit from improved understanding of the mechanisms associated with MTB responses to different genetic and environmental perturbations. Therefore, we expanded a genome-scale regulatory-metabolic model for MTB using the Probabilistic Regulation of Metabolism (PROM) framework. Our model, MTBPROM2.0, represents a substantial knowledge base update and extension of simulation capability. We incorporated a recent ChIP-seq based binding network of 2555 interactions linking to 104 transcription factors (TFs) (representing a 3.5-fold expansion of TF coverage). We integrated this expanded regulatory network with a refined genome-scale metabolic model that can correctly predict growth viability over 69 source metabolite conditions and predict metabolic gene essentiality more accurately than the original model. We used MTBPROM2.0 to simulate the metabolic consequences of knocking out and overexpressing each of the 104 TFs in the model. MTBPROM2.0 improves performance of knockout growth defect predictions compared to the original PROM MTB model, and it can successfully predict growth defects associated with TF overexpression. Moreover, condition-specific models of MTBPROM2.0 successfully predicted synergistic growth consequences of overexpressing the TF whiB4 in the presence of two standard anti-TB drugs. MTBPROM2.0 can screen in silico condition-specific transcription factor perturbations to generate putative targets of interest that can help prioritize future experiments for therapeutic development efforts.

  13. Outbreak of tuberculosis caused by Mycobacterium caprae in a zoological garden.

    PubMed

    Pate, M; Svara, T; Gombac, M; Paller, T; Zolnir-Dovc, M; Emersic, I; Prodinger, W M; Bartos, M; Zdovc, I; Krt, B; Pavlik, I; Cvetnić, Z; Pogacnik, M; Ocepek, M

    2006-10-01

    In the autumn of 2004, tuberculosis caused by Mycobacterium caprae occurred in a zoo in Slovenia. A dromedary camel (Camelus dromedarius) was killed after a history of progressive emaciation. Necropsy findings indicated disseminated tuberculosis, which was confirmed by cultivation of M. caprae. Consequently, a tuberculin skin test was performed in all epidemiologically linked animals and another dromedary camel and six bison (Bison bison) were positive and killed. Mycobacterium caprae was isolated from two bison while M. scrofulaceum and Mycobacterium spp. were found in two other bison, respectively. The second dromedary camel was found to be negative for mycobacteria under both microscopic and culture tests. The isolates were investigated with commercial identification kits, IS6110 PCR, IS6110 restriction fragment length polymorphism analysis, spoligotyping and mycobacterial interspersed repetitive units typing. Genotyping results revealed that the dromedary camel and the two bison were infected by the same M. caprae.

  14. Metabolomic signatures in guinea pigs infected with epidemic-associated W-Beijing strains of Mycobacterium tuberculosis.

    PubMed

    Somashekar, Bagganahalli S; Amin, Anita G; Tripathi, Pratima; MacKinnon, Neil; Rithner, Christopher D; Shanley, Crystal A; Basaraba, Randall; Henao-Tamayo, Marcela; Kato-Maeda, Midori; Ramamoorthy, Ayyalusamy; Orme, Ian M; Ordway, Diane J; Chatterjee, Delphi

    2012-10-01

    With the understanding that the laboratory propagated strain of Mycobacterium tuberculosis H37Rv is of modest virulence and is drug susceptible, in the present study, we performed a nuclear magnetic resonance-based metabolomic analysis of lung tissues and serum obtained from guinea pigs infected by low dose aerosol exposure to clinical isolates of Mycobacterium tuberculosis. High Resolution Magic Angle Spinning NMR coupled with multivariate statistical analysis of 159 lung tissues obtained from multiple locations of age-matched naïve and 30 and 60 days of infected guinea pig lungs revealed a wide dispersal of metabolic patterns, but within these, distinct clusters of signatures could be seen that differentiated between naive control and infected animals. Several metabolites were identified that changed in concert with the progression of each infection. Major metabolites that could be interpreted as indicating host glutaminolysis were consistent with activated host immune cells encountering increasingly hypoxic conditions in the necrotic lung lesions. Moreover, glutathione levels were constantly elevated, probably in response to oxygen radical production in these lesions. Additional distinct signatures were also seen in infected serum, with altered levels of several metabolites. Multivariate statistical analysis clearly differentiated the infected from the uninfected sera; in addition, Receiver Operator Characteristic curve generated with principal component 1 scores showed an area under the curve of 0.908. These data raise optimism that discrete metabolomic signatures can be defined that can predict the progression of the tuberculosis disease process, and form the basis of an innovative and rapid diagnostic process.

  15. Preparation, biological evaluation and molecular docking study of imidazolyl dihydropyrimidines as potential Mycobacterium tuberculosis dihydrofolate reductase inhibitors.

    PubMed

    Desai, N C; Trivedi, A R; Khedkar, Vijay M

    2016-08-15

    A series of novel dihydropyrimidine derivatives bearing an imidazole nucleus at C-4 position were synthesized in excellent yields via Biginelli multi-component reaction. The newly synthesized compounds were characterized by IR, (1)H NMR, (13)C NMR and Mass spectroscopy. In vitro antitubercular evaluation of all the newly synthesized compounds 4a-p against Mycobacterium tuberculosis (Mtb) H37Rv showed, 4j (MIC: 0.39μg/mL; SI: >25.64), 4m (MIC: 0.78μg/mL; SI: >12.82) and 4p (MIC: 0.39μg/mL; SI: 24.10) as the most promising lead analogues. Compounds 4j, 4m and 4p displayed effective reduction in residual Mtb growth within the tuberculosis-infected macrophage model. Further, molecular docking study of active molecules 4j, 4m and 4p against Mycobacterium tuberculosis dihydrofolate reductase (Mtb DHFR) proved their potency as Mtb DHFR inhibitors acting as potential leads for further development. Pharmacokinetic properties leading to drug-likeness were also predicted for most active molecules 4j, 4m and 4p. PMID:27397497

  16. Rv1698 of Mycobacterium tuberculosis Represents a New Class of Channel-forming Outer Membrane Proteins*S⃞

    PubMed Central

    Siroy, Axel; Mailaender, Claudia; Harder, Daniel; Koerber, Stephanie; Wolschendorf, Frank; Danilchanka, Olga; Wang, Ying; Heinz, Christian; Niederweis, Michael

    2008-01-01

    Mycobacteria contain an outer membrane composed of mycolic acids and a large variety of other lipids. Its protective function is an essential virulence factor of Mycobacterium tuberculosis. Only OmpA, which has numerous homologs in Gram-negative bacteria, is known to form channels in the outer membrane of M. tuberculosis so far. Rv1698 was predicted to be an outer membrane protein of unknown function. Expression of rv1698 restored the sensitivity to ampicillin and chloramphenicol of a Mycobacterium smegmatis mutant lacking the main porin MspA. Uptake experiments showed that Rv1698 partially complemented the permeability defect of the M. smegmatis porin mutant for glucose. These results indicated that Rv1698 provides an unspecific pore that can partially substitute for MspA. Lipid bilayer experiments demonstrated that purified Rv1698 is an integral membrane protein that indeed produces channels. The main single channel conductance is 4.5 ± 0.3 nanosiemens in 1 m KCl. Zero current potential measurements revealed a weak preference for cations. Whole cell digestion of recombinant M. smegmatis with proteinase K showed that Rv1698 is surface-accessible. Taken together, these experiments demonstrated that Rv1698 is a channel protein that is likely involved in transport processes across the outer membrane of M. tuberculosis. Rv1698 has single homologs of unknown functions in Corynebacterineae and thus represents the first member of a new class of channel proteins specific for mycolic acid-containing outer membranes. PMID:18434314

  17. Tuberculosis in Sudan: a study of Mycobacterium tuberculosis strain genotype and susceptibility to anti-tuberculosis drugs

    PubMed Central

    2011-01-01

    Background Sudan is a large country with a diverse population and history of civil conflict. Poverty levels are high with a gross national income per capita of less than two thousand dollars. The country has a high burden of tuberculosis (TB) with an estimated 50,000 incident cases during 2009, when the estimated prevalence was 209 cases per 100,000 of the population. Few studies have been undertaken on TB in Sudan and the prevalence of drug resistant disease is not known. Methods In this study Mycobacterium tuberculosis isolates from 235 patients attending three treatment centers in Sudan were screened for susceptibility to isoniazid, rifampicin, ethambutol and streptomycin by the proportion method on Lowenstein Jensen media. 232 isolates were also genotyped by spoligotyping. Demographic details of patients were recorded using a structured questionnaire. Statistical analyses were conducted to examine the associations between drug resistance with risk ratios computed for a set of risk factors (gender, age, case status - new or relapse, geographic origin of the patient, spoligotype, number of people per room, marital status and type of housing). Results Multi drug-resistant tuberculosis (MDR-TB), being resistance to at least rifampicin and isoniazid, was found in 5% (95% CI: 2,8) of new cases and 24% (95% CI: 14,34) of previously treated patients. Drug resistance was associated with previous treatment with risk ratios of 3.51 (95% CI: 2.69-4.60; p < 0.001) for resistance to any drug and 5.23 (95% CI: 2.30-11.90; p < 0.001) for MDR-TB. Resistance was also associated with the geographic region of origin of the patient, being most frequently observed in patients from the Northern region and least in the Eastern region with risk ratios of 7.43 (95%CI:3.42,16.18; p: < 0.001) and 14.09 (95%CI:1.80,110.53; p:0.026) for resistance to any drug and MDR-TB. The major genotype observed was of the Central Asia spoligotype family (CAS1_Delhi), representing 49% of the 232 isolates

  18. The Importance of First Impressions: Early Events in Mycobacterium tuberculosis Infection Influence Outcome

    PubMed Central

    Cadena, Anthony M.; Fortune, Sarah M.

    2016-01-01

    ABSTRACT Tuberculosis remains a major health threat in much of the world. New vaccines against Mycobacterium tuberculosis are essential for preventing infection, disease, and transmission. However, the host immune responses that need to be induced by an effective vaccine remain unclear. Increasingly, it has become clear that early events in infection are of major importance in the eventual outcome of the infection. Studying such events in humans is challenging, as they occur within the lung and thoracic lymph nodes, and any clinical signs of early infection are relatively nonspecific. Nonetheless, clinical studies and animal models of tuberculosis have provided new insights into the local events that occur in the first few weeks of tuberculosis. Development of an effective vaccine requires a clear understanding of the successful (and detrimental) early host responses against M. tuberculosis, with the goal to improve upon natural immune responses and prevent infection or disease. PMID:27048801

  19. DNA fragment length polymorphism analysis of Mycobacterium tuberculosis isolates by arbitrarily primed polymerase chain reaction.

    PubMed

    Palittapongarnpim, P; Chomyc, S; Fanning, A; Kunimoto, D

    1993-04-01

    Strain identification of Mycobacterium tuberculosis would prove whether transmission had occurred between individuals. A method to characterize strains of M. tuberculosis has been developed utilizing polymerase chain reaction (PCR). Purified chromosomal DNA of cultured clinical samples of M. tuberculosis were subjected to PCR using short (10-12 nucleotide) oligonucleotide primers. PCR products visualized after agarose gel electrophoresis and ethidium bromide staining demonstrated that different strains of M. tuberculosis give different banding patterns. This technique was used to confirm the relationship between cases of tuberculosis in several clusters, prove the lack of relationship between 2 isolates with the same antibiotic-resistance pattern, confirm a suspected mislabeling event, and suggest the source of infection in a case of tuberculous meningitis. This method is rapid and simple and does not require radioactive probes.

  20. An outer membrane channel protein of Mycobacterium tuberculosis with exotoxin activity

    PubMed Central

    Danilchanka, Olga; Sun, Jim; Pavlenok, Mikhail; Maueröder, Christian; Speer, Alexander; Siroy, Axel; Marrero, Joeli; Trujillo, Carolina; Mayhew, David L.; Doornbos, Kathryn S.; Muñoz, Luis E.; Herrmann, Martin; Ehrt, Sabine; Berens, Christian; Niederweis, Michael

    2014-01-01

    The ability to control the timing and mode of host cell death plays a pivotal role in microbial infections. Many bacteria use toxins to kill host cells and evade immune responses. Such toxins are unknown in Mycobacterium tuberculosis. Virulent M. tuberculosis strains induce necrotic cell death in macrophages by an obscure molecular mechanism. Here we show that the M. tuberculosis protein Rv3903c (channel protein with necrosis-inducing toxin, CpnT) consists of an N-terminal channel domain that is used for uptake of nutrients across the outer membrane and a secreted toxic C-terminal domain. Infection experiments revealed that CpnT is required for survival and cytotoxicity of M. tuberculosis in macrophages. Furthermore, we demonstrate that the C-terminal domain of CpnT causes necrotic cell death in eukaryotic cells. Thus, CpnT has a dual function in uptake of nutrients and induction of host cell death by M. tuberculosis. PMID:24753609

  1. The Importance of First Impressions: Early Events in Mycobacterium tuberculosis Infection Influence Outcome.

    PubMed

    Cadena, Anthony M; Flynn, JoAnne L; Fortune, Sarah M

    2016-01-01

    Tuberculosis remains a major health threat in much of the world. New vaccines against Mycobacterium tuberculosis are essential for preventing infection, disease, and transmission. However, the host immune responses that need to be induced by an effective vaccine remain unclear. Increasingly, it has become clear that early events in infection are of major importance in the eventual outcome of the infection. Studying such events in humans is challenging, as they occur within the lung and thoracic lymph nodes, and any clinical signs of early infection are relatively nonspecific. Nonetheless, clinical studies and animal models of tuberculosis have provided new insights into the local events that occur in the first few weeks of tuberculosis. Development of an effective vaccine requires a clear understanding of the successful (and detrimental) early host responses against M. tuberculosis, with the goal to improve upon natural immune responses and prevent infection or disease.

  2. Epidemiology and diagnosis of Mycobacterium tuberculosis in captive Asian elephants (Elephas maximus).

    PubMed

    Mikota, S K; Peddie, L; Peddie, J; Isaza, R; Dunker, F; West, G; Lindsay, W; Larsen, R S; Salman, M D; Chatterjee, D; Payeur, J; Whipple, D; Thoen, C; Davis, D S; Sedgwick, C; Montali, R J; Ziccardi, M; Maslow, J

    2001-03-01

    The deaths of two Asian elephants (Elephas maximus) in August 1996 led the United States Department of Agriculture to require the testing and treatment of elephants for tuberculosis. From August 1996 to September 1999. Mycobacterium tuberculosis infection was confirmed by culture in 12 of 118 elephants in six herds. Eight diagnoses were made antemortem on the basis of isolation of M. tuberculosis by culture of trunk wash samples; the remainder (including the initial two) were diagnosed postmortem. We present the case histories, epidemiologic characteristics, diagnostic test results, and therapeutic plans from these six herds. The intradermal tuberculin test, enzyme-linked immunosorbent assay serology, the blood tuberculosis test, and nucleic acid amplification and culture are compared as methods to diagnose M. tuberculosis infection in elephants.

  3. Activity against multidrug-resistant Mycobacterium tuberculosis in Mexican plants used to treat respiratory diseases.

    PubMed

    Jimenez-Arellanes, Adelina; Meckes, Mariana; Ramirez, Raquel; Torres, Javier; Luna-Herrera, Julieta

    2003-09-01

    The increase of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) demands the search for alternative antimycobacterial drugs. The aim of this study was to evaluate plants used in Mexican traditional medicine to treat respiratory diseases for activity against MDR-TB. A group of 22 plants was screened for activity against Mycobacterium tuberculosis H37Rv and Mycobacterium avium at concentrations from 50 to 200 microg/mL. The antimycobacterial effect was determined by a microcolorimetric assay with Alamar blue dye. None of the aqueous extracts had antimycobacterial activity. Hexane extracts from Artemisia ludoviciana, Chamaedora tepejilote, Lantana hispida, Juniperus communis and Malva parviflora, and methanol extracts from Artemisia ludoviciana and Juniperus communis inhibited the growth of Mycobacterium tuberculosis. Mycobacterium avium was inhibited by Juniperus communis hexane extract and by Malva parviflora methanol extract. The active extracts were tested against monoresistant variants of Mycobacterium tuberculosis H37Rv (isoniazid, rifampin, streptomycin and ethambutol resistant) and the hexane extract of Lantana hispida showed the best activity. Lantana hispida hexane extract was also active against a group of MDR-TB clinical isolates. In contrast, it did not inhibit the growth of non-tuberculous mycobacteria. The hexane extract of Lantana hispida was fractionated by column chromatography and one of its fractions (FVI) inhibited the growth of all the MDR-TB clinical isolates at concentrations up to 25 microg/mL. This study supports the fact that selecting plants by ethnobotanical criteria enhances the probability of finding species with activity against mycobacteria, and our results point to Lantana hispida as an important source of potential compounds against MDR-TB.

  4. First-Line Anti-Tubercular Drug Resistance of Mycobacterium tuberculosis in IRAN: A Systematic Review

    PubMed Central

    Pourakbari, Babak; Mamishi, Setareh; Mohammadzadeh, Mona; Mahmoudi, Shima

    2016-01-01

    Background: The spread of drug-resistant tuberculosis (TB) is one of the major public health problems through the world. Surveillance of anti-TB drug resistance is essential for monitoring of TB control strategies. The occurrence of drug resistance, particularly multi-drug resistance Mycobacterium tuberculosis (MDR), defined as resistance to at least rifampicin (RIF) and isoniazid (INH), has become a significant public health dilemma. The status of drug-resistance TB in Iran, one of the eastern Mediterranean countries locating between Azerbaijan and Armenia and high-TB burden countries (such as Afghanistan and Pakistan) has been reported inconsistently. Therefore, the aim of this study was to summarize reports of first-line anti-tubercular drug resistance in M. tuberculosis in Iran. Material and Methods: We systematically reviewed published studies on drug-resistant M. tuberculosis in Iran. The search terms were “Mycobacterium tuberculosis susceptibility” or “Mycobacterium tuberculosis resistant” and Iran. Results: Fifty-two eligible articles, published during 1998–2014, were included in this review. Most of the studies were conducted in Tehran. The most common used laboratory method for detecting M. tuberculosis drug resistant was Agar proportion. The highest resistance to first-line drugs was seen in Tehran, the capital city of Iran. The average prevalence of isoniazid (INH), rifampin (RIF), streptomycin (SM), and ethambotol (EMB) resistance via Agar proportion method in Tehran was 26, 23, 22.5, and 16%, respectively. In general, resistance to INH was more common than RIF, SM, and EMB in Tehran Conclusions: In conclusion, this systematic review summarized the prevalence and distribution of first-line anti-tubercular drug resistance of M. tuberculosis in Iran. Our results suggested that effective strategies to minimize the acquired drug resistance, to control the transmission of resistance and improve the diagnosis measures for TB control in Iran. PMID

  5. Progenitor “Mycobacterium canettii” Clone Responsible for Lymph Node Tuberculosis Epidemic, Djibouti

    PubMed Central

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

    2014-01-01

    Mycobacterium canettii,” an opportunistic human pathogen living in an unknown environmental reservoir, is the progenitor species from which Mycobacterium tuberculosis emerged. Since its discovery in 1969, most of the ≈70 known M. canettii strains were isolated in the Republic of Djibouti, frequently from expatriate children and adults. We show here, by whole-genome sequencing, that most strains collected from February 2010 through March 2013, and associated with 2 outbreaks of lymph node tuberculosis in children, belong to a unique epidemic clone within M. canettii. Evolution of this clone, which has been recovered regularly since 1983, may mimic the birth of M. tuberculosis. Thus, recognizing this organism and identifying its reservoir are clinically important. PMID:24520560

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

    PubMed

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

    2014-01-01

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

  7. Mycobacterium tuberculosis evolutionary pathogenesis and its putative impact on drug development.

    PubMed

    Le Chevalier, Fabien; Cascioferro, Alessandro; Majlessi, Laleh; Herrmann, Jean Louis; Brosch, Roland

    2014-01-01

    Mycobacterium tuberculosis, the etiological agent of human TB, is the most important mycobacterial pathogen in terms of global patient numbers and gravity of disease. The molecular mechanisms by which M. tuberculosis causes disease are complex and the result of host-pathogen coevolution that might have started already in the time of its Mycobacterium canettii-like progenitors. Despite research progress, M. tuberculosis still holds many secrets of its successful strategy for circumventing host defences, persisting in the host and developing resistance, which makes anti-TB treatment regimens extremely long and often inefficient. Here, we discuss what we have learned from recent studies on the evolution of the pathogen and its putative new drug targets that are essential for mycobacterial growth under in vitro or in vivo conditions.

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

    PubMed

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

    2014-01-01

    Mycobacterium canettii,” an opportunistic human pathogen living in an unknown environmental reservoir, is the progenitor species from which Mycobacterium tuberculosis emerged. Since its discovery in 1969, most of the ≈70 known M. canettii strains were isolated in the Republic of Djibouti, frequently from expatriate children and adults. We show here, by whole-genome sequencing, that most strains collected from February 2010 through March 2013, and associated with 2 outbreaks of lymph node tuberculosis in children, belong to a unique epidemic clone within M. canettii. Evolution of this clone, which has been recovered regularly since 1983, may mimic the birth of M. tuberculosis. Thus, recognizing this organism and identifying its reservoir are clinically important. PMID:24520560

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

    PubMed Central

    Vasconcelos, Sidra E. G.; Esteves, Leonardo S.; Gomes, Harrison M.; Almeida da Silva, Pedro; Perdigão, João; Portugal, Isabel; Viveiros, Miguel; McNerney, Ruth; Pain, Arnab; Clark, Taane G.; Rastogi, Nalin; Unis, Gisela; Rossetti, Maria Lucia R.

    2015-01-01

    We recently detected the spoligotype patterns of strains of Mycobacterium pinnipedii, a species of the Mycobacterium tuberculosis complex, in sputum samples from nine cases with pulmonary tuberculosis residing in Porto Alegre, South Brazil. Because this species is rarely encountered in humans, we further characterized these nine isolates by additional genotyping techniques, including 24-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) typing, verification of the loci TbD1, RD9, pks15/1, RDRio, and fbpC, the insertion of IS6110 at a site specific to the M. tuberculosis Latin American Mediterranean (LAM) lineage, and whole-genome sequencing. The combined analysis of these markers revealed that the isolates are in fact M. tuberculosis and more specifically belong to the LAM genotype. Most of these isolates (n = 8) were shown to be multidrug resistant (MDR), which prompted us to perform partial sequencing of the rpoA, rpoB, rpoC, katG, and inhA genes. Seven isolates (77.8%) carried the S315T mutation in katG, and one of these (11%) also presented the C(−17)T single-nucleotide polymorphism (SNP) in inhA. Interestingly, six of the MDR isolates also presented an undescribed insertion of 12 nucleotides (CCA GAA CAA CCC) in codon 516 of rpoB. No putative compensatory mutation was found in either rpoA or rpoC. This is the first report of an M. tuberculosis LAM family strain with a convergent M. pinnipedii spoligotype. These spoligotypes are observed in genotype databases at a modest frequency, highlighting that care must be taken when identifying isolates in the M. tuberculosis complex on the basis of single genetic markers. PMID:26400784

  10. Evidence of presence of Mycobacterium tuberculosis in bovine tissue samples by multiplex PCR: possible relevance to reverse zoonosis.

    PubMed

    Mittal, M; Chakravarti, S; Sharma, V; Sanjeeth, B S; Churamani, C P; Kanwar, N S

    2014-04-01

    Bovine tuberculosis, caused by Mycobacterium bovis, remains one of the most important zoonotic health concerns worldwide. The transmission of Mycobacterium tuberculosis from humans to animals also occurs especially in countries where there is close interaction of humans with the animals. In the present study, thirty bovine lung tissue autopsy samples from an organized dairy farm located in North India were screened for the presence of Mycobacterium tuberculosis complex by smear microscopy, histopathological findings and PCR. Differential diagnosis of M. tuberculosis and M. bovis was made based on the deletion of mce-3 operon in M. bovis. The present study found eight of these samples positive for M. tuberculosis by multiplex PCR. Sequencing was performed on two PCR-positive representative samples and on annotation, and BLAST analysis confirmed the presence of gene fragment specific to Mycobacterium tuberculosis. The presence of M. tuberculosis in all the positive samples raises the possibility of human-to-cattle transmission and possible adaptation of this organism in bovine tissues. This study accentuates the importance of screening and differential diagnosis of Mycobacterium tuberculosis complex in humans and livestock for adopting effective TB control and eradication programmes.

  11. Phenotypically Adapted Mycobacterium tuberculosis Populations from Sputum Are Tolerant to First-Line Drugs

    PubMed Central

    Turapov, Obolbek; O'Connor, Benjamin D.; Sarybaeva, Asel A.; Williams, Caroline; Patel, Hemu; Kadyrov, Abdullaat S.; Sarybaev, Akpay S.; Woltmann, Gerrit; Barer, Michael R.

    2016-01-01

    Tuberculous sputum contains multiple Mycobacterium tuberculosis populations with different requirements for isolation in vitro. These include cells that form colonies on solid media (plateable M. tuberculosis), cells requiring standard liquid medium for growth (nonplateable M. tuberculosis), and cells requiring supplementation of liquid medium with culture supernatant (SN) for growth (SN-dependent M. tuberculosis). Here, we describe protocols for the cryopreservation and direct assessment of antimicrobial tolerance of these M. tuberculosis populations within sputum. Our results show that first-line drugs achieved only modest bactericidal effects on all three populations over 7 days (1 to 2.5 log10 reductions), and SN-dependent M. tuberculosis was more tolerant to streptomycin and isoniazid than the plateable and nonplateable M. tuberculosis strains. Susceptibility of plateable M. tuberculosis to bactericidal drugs was significantly increased after passage in vitro; thus, tolerance observed in the sputum samples from the population groups was likely associated with mycobacterial adaptation to the host environment at some time prior to expectoration. Our findings support the use of a simple ex vivo system for testing drug efficacies against mycobacteria that have phenotypically adapted during tuberculosis infection. PMID:26883695

  12. Development of an automated MODS plate reader to detect early growth of Mycobacterium tuberculosis.

    PubMed

    Comina, G; Mendoza, D; Velazco, A; Coronel, J; Sheen, P; Gilman, R H; Moore, D A J; Zimic, M

    2011-06-01

    In this work, an automated microscopic observation drug susceptibility (MODS) plate reader has been developed. The reader automatically handles MODS plates and after autofocussing digital images are acquired of the characteristic microscopic cording structures of Mycobacterium tuberculosis, which are the identification method utilized in the MODS technique to detect tuberculosis and multidrug resistant tuberculosis. In conventional MODS, trained technicians manually move the MODS plate on the stage of an inverted microscope while trying to locate and focus upon the characteristic microscopic cording colonies. In centres with high tuberculosis diagnostic demand, sufficient time may not be available to adequately examine all cultures. An automated reader would reduce labour time and the handling of M. tuberculosis cultures by laboratory personnel. Two hundred MODS culture images (100 from tuberculosis positive and 100 from tuberculosis negative sputum samples confirmed by a standard MODS reading using a commercial microscope) were acquired randomly using the automated MODS plate reader. A specialist analysed these digital images with the help of a personal computer and designated them as M. tuberculosis present or absent. The specialist considered four images insufficiently clear to permit a definitive reading. The readings from the 196 valid images resulted in a 100% agreement with the conventional nonautomated standard reading. The automated MODS plate reader combined with open-source MODS pattern recognition software provides a novel platform for high throughput automated tuberculosis diagnosis.

  13. Revisiting the role of phospholipases C in virulence and the lifecycle of Mycobacterium tuberculosis

    PubMed Central

    Le Chevalier, Fabien; Cascioferro, Alessandro; Frigui, Wafa; Pawlik, Alexandre; Boritsch, Eva C.; Bottai, Daria; Majlessi, Laleh; Herrmann, Jean Louis; Brosch, Roland

    2015-01-01

    Mycobacterium tuberculosis, the agent of human tuberculosis has developed different virulence mechanisms and virulence-associated tools during its evolution to survive and multiply inside the host. Based on previous reports and by analogy with other bacteria, phospholipases C (PLC) of M. tuberculosis were thought to be among these tools. To get deeper insights into the function of PLCs, we investigated their putative involvement in the intracellular lifestyle of M. tuberculosis, with emphasis on phagosomal rupture and virulence, thereby re-visiting a research theme of longstanding interest. Through the construction and use of an M. tuberculosis H37Rv PLC-null mutant (ΔPLC) and control strains, we found that PLCs of M. tuberculosis were not required for induction of phagosomal rupture and only showed marginal, if any, impact on virulence of M. tuberculosis in the cellular and mouse infection models used in this study. In contrast, we found that PLC-encoding genes were strongly upregulated under phosphate starvation and that PLC-proficient M. tuberculosis strains survived better than ΔPLC mutants under conditions where phosphatidylcholine served as sole phosphate source, opening new perspectives for studies on the role of PLCs in the lifecycle of M. tuberculosis. PMID:26603639

  14. Accidental infection of veterinary personnel with Mycobacterium tuberculosis at necropsy: a case study.

    PubMed

    Posthaus, H; Bodmer, T; Alves, L; Oevermann, A; Schiller, I; Rhodes, S G; Zimmerli, S

    2011-05-01

    Mycobacterium tuberculosis is the main cause of human tuberculosis. Infection in companion animals is mainly acquired from close contact to a diseased human patient and hence rarely diagnosed in countries with low tuberculosis incidence rates. Therefore the general awareness of the disease might be low. Here we report the potential risk of infection for veterinary personnel with M. tuberculosis during the clinical and pathological examination of a dog with unexpected disseminated tuberculosis. The dog had presented with symptoms of a central nervous system disease; rapid deterioration prevented a complete clinical workup, however. Post-mortem examination revealed systemic mycobacteriosis, and M. tuberculosis was identified by PCR amplification of DNA extracts from paraffin-embedded tissue sections and spoligotyping. Contact investigations among the owners and veterinary personnel using an IFN-γ release assay indicated that the index dog did not infect humans during its lifetime. Serological and IFN-γ release assay results of one of two cats in direct contact with the index dog, however, suggested that transmission of M. tuberculosis might have occurred. Importantly, all three pathologists performing the necropsy on the dog tested positive. Accidental infection was most likely due to inhalation of M. tuberculosis containing aerosols created by using an electric saw to open the brain cavity. As a consequence routine necropsy procedures have been adapted and a disease surveillance program, including tuberculosis, has been initiated. Our results highlight the importance of disease awareness and timely diagnosis of zoonotic infectious agents in optimizing work safety for veterinary personnel. PMID:21146334

  15. Revisiting the role of phospholipases C in virulence and the lifecycle of Mycobacterium tuberculosis.

    PubMed

    Le Chevalier, Fabien; Cascioferro, Alessandro; Frigui, Wafa; Pawlik, Alexandre; Boritsch, Eva C; Bottai, Daria; Majlessi, Laleh; Herrmann, Jean Louis; Brosch, Roland

    2015-01-01

    Mycobacterium tuberculosis, the agent of human tuberculosis has developed different virulence mechanisms and virulence-associated tools during its evolution to survive and multiply inside the host. Based on previous reports and by analogy with other bacteria, phospholipases C (PLC) of M. tuberculosis were thought to be among these tools. To get deeper insights into the function of PLCs, we investigated their putative involvement in the intracellular lifestyle of M. tuberculosis, with emphasis on phagosomal rupture and virulence, thereby re-visiting a research theme of longstanding interest. Through the construction and use of an M. tuberculosis H37Rv PLC-null mutant (ΔPLC) and control strains, we found that PLCs of M. tuberculosis were not required for induction of phagosomal rupture and only showed marginal, if any, impact on virulence of M. tuberculosis in the cellular and mouse infection models used in this study. In contrast, we found that PLC-encoding genes were strongly upregulated under phosphate starvation and that PLC-proficient M. tuberculosis strains survived better than ΔPLC mutants under conditions where phosphatidylcholine served as sole phosphate source, opening new perspectives for studies on the role of PLCs in the lifecycle of M. tuberculosis.

  16. Designing of inhibitors against drug tolerant Mycobacterium tuberculosis (H37Rv)

    PubMed Central

    2013-01-01

    Background Mycobacterium tuberculosis (M.tb) is the causative agent of tuberculosis, killing ~1.7 million people annually. The remarkable capacity of this pathogen to escape the host immune system for decades and then to cause active tuberculosis disease, makes M.tb a successful pathogen. Currently available anti-mycobacterial therapy has poor compliance due to requirement of prolonged treatment resulting in accelerated emergence of drug resistant strains. Hence, there is an urgent need to identify new chemical entities with novel mechanism of action and potent activity against the drug resistant strains. Results This study describes novel computational models developed for predicting inhibitors against both replicative and non-replicative phase of drug-tolerant M.tb under carbon starvation stage. These models were trained on highly diverse dataset of 2135 compounds using four classes of binary fingerprint namely PubChem, MACCS, EState, SubStructure. We achieved the best performance Matthews correlation coefficient (MCC) of 0.45 using the model based on MACCS fingerprints for replicative phase inhibitor dataset. In case of non-replicative phase, Hybrid model based on PubChem, MACCS, EState, SubStructure fingerprints performed better with maximum MCC value of 0.28. In this study, we have shown that molecular weight, polar surface area and rotatable bond count of inhibitors (replicating and non-replicating phase) are significantly different from non-inhibitors. The fragment analysis suggests that substructures like hetero_N_nonbasic, heterocyclic, carboxylic_ester, and hetero_N_basic_no_H are predominant in replicating phase inhibitors while hetero_O, ketone, secondary_mixed_amine are preferred in the non-replicative phase inhibitors. It was observed that nitro, alkyne, and enamine are important for the molecules inhibiting bacilli residing in both the phases. In this study, we introduced a new algorithm based on Matthews correlation coefficient called MCCA for

  17. Rifoligotyping assay: an alternative method for rapid detection of rifampicin resistance in Mycobacterium tuberculosis isolates from Morocco

    PubMed Central

    Chaoui, Imane; Atalhi, Naima; Sabouni, Radia; Akrim, Mohammed; Abid, Mohammed; Amzazi, Saaid; ElMzibri, Mohammed

    2014-01-01

    One of the greatest threats to global tuberculosis (TB) control is the growing prevalence of drug resistant strains. In the past decades, considerable efforts have been made upon the development of new molecular technologies and methodologies for detection of drug resistance in Mycobacterium tuberculosis (MTB). A sensitive, specific reverse line blot assay, called rifoligotyping (RIFO), for the detection of genotypic resistance to rifampicin (RIF), was designed and evaluated. RIFO includes oligonucleotide probes specific for wild-type and mutant sequences, allowing specific and sensitive detection of both genotypes in a single assay. The RIFO was applied on 500 MTB isolates from Morocco. The results of the RIFO showed a good sensitivity (90.9%) and high specificity (100%); the positive and negative predictive values were 100% and 96.1%, respectively. This rapid, simple, economical assay provides a practical alternative for RIF genotyping, especially in low-income countries, to improve TB control and management. PMID:26740783

  18. Enriching the annotation of Mycobacterium tuberculosis H37Rv proteome using remote homology detection approaches: insights into structure and function.

    PubMed

    Ramakrishnan, Gayatri; Ochoa-Montaño, Bernardo; Raghavender, Upadhyayula S; Mudgal, Richa; Joshi, Adwait G; Chandra, Nagasuma R; Sowdhamini, Ramanathan; Blundell, Tom L; Srinivasan, Narayanaswamy

    2015-01-01

    The availability of the genome sequence of Mycobacterium tuberculosis H37Rv has encouraged determination of large numbers of protein structures and detailed definition of the biological information encoded therein; yet, the functions of many proteins in M. tuberculosis remain unknown. The emergence of multidrug resistant strains makes it a priority to exploit recent advances in homology recognition and structure prediction to re-analyse its gene products. Here we report the structural and functional characterization of gene products encoded in the M. tuberculosis genome, with the help of sensitive profile-based remote homology search and fold recognition algorithms resulting in an enhanced annotation of the proteome where 95% of the M. tuberculosis proteins were identified wholly or partly with information on structure or function. New information includes association of 244 proteins with 205 domain families and a separate set of new association of folds to 64 proteins. Extending structural information across uncharacterized protein families represented in the M. tuberculosis proteome, by determining superfamily relationships between families of known and unknown structures, has contributed to an enhancement in the knowledge of structural content. In retrospect, such superfamily relationships have facilitated recognition of probable structure and/or function for several uncharacterized protein families, eventually aiding recognition of probable functions for homologous proteins corresponding to such families. Gene products unique to mycobacteria for which no functions could be identified are 183. Of these 18 were determined to be M. tuberculosis specific. Such pathogen-specific proteins are speculated to harbour virulence factors required for pathogenesis. A re-annotated proteome of M. tuberculosis, with greater completeness of annotated proteins and domain assigned regions, provides a valuable basis for experimental endeavours designed to obtain a better

  19. Use of amplified Mycobacterium tuberculosis direct test in respiratory samples from HIV-infected patients in Brazil*

    PubMed Central

    Barreto, Leonardo Bruno Paz Ferreira; Lourenço, Maria Cristina da Silva; Rolla, Valéria Cavalcanti; Veloso, Valdiléia Gonçalves; Huf, Gisele

    2014-01-01

    OBJECTIVE: To compare the accuracy of the amplified Mycobacterium tuberculosis direct (AMTD) test with reference methods for the laboratory diagnosis of tuberculosis in HIV-infected patients. METHODS: This was a study of diagnostic accuracy comparing AMTD test results with those obtained by culture on Löwenstein-Jensen (LJ) medium and by the BACTEC Mycobacteria Growth Indicator Tube 960 (BACTEC MGIT 960) system in respiratory samples analyzed at the Bioassay and Bacteriology Laboratory of the Oswaldo Cruz Foundation Evandro Chagas Clinical Research Institute in the city of Rio de Janeiro, Brazil. RESULTS: We analyzed respiratory samples collected from 118 patients, of whom 88 (74.4%) were male. The mean age was 36.6 ± 10.6 years. Using the AMTD test, the BACTEC MGIT 960 system, and LJ culture, we identified M. tuberculosis complex in 31.0%, 29.7%, and 27.1% of the samples, respectively. In comparison with LJ culture, the AMTD test had a sensitivity, specificity, positive predictive value, and negative predictive value of 87.5%, 89.4%, 75.7%, and 95.0%, respectively, for LJ culture, whereas, in comparison with the BACTEC MGIT 960 system, it showed values of 88.6%, 92.4%, 83.8%, and 94.8%, respectively. CONCLUSIONS: The AMTD test showed good sensitivity and specificity in the population studied, enabling the laboratory detection of M. tuberculosis complex in paucibacillary respiratory specimens. PMID:24831399

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

    PubMed Central

    Andersen, Aase Bengaard; Melbye, Mads; Wohlfahrt, Jan; Andersson, Mikael; Biggar, Robert J; Ladefoged, Karin; Thomsen, Vibeke Ostergaard; Koch, Anders

    2011-01-01

    Abstract Objective To examine the risk factors for Mycobacterium tuberculosis infection (MTI) among Greenlandic children for the purpose of identifying those at highest risk of infection. Methods Between 2005 and 2007, 1797 Greenlandic schoolchildren in five different areas were tested for MTI with an interferon gamma release assay (IGRA) and a tuberculin skin test (TST). Parents or guardians were surveyed using a standardized self-administered questionnaire to obtain data on crowding in the household, parents’ educational level and the child’s health status. Demographic data for each child – i.e. parents’ place of birth, number of siblings, distance between siblings (next younger and next older), birth order and mother’s age when the child was born – were also extracted from a public registry. Logistic regression was used to check for associations between these variables and MTI, and all results were expressed as odds ratios (ORs) and 95% confidence intervals (CIs). Children were considered to have MTI if they tested positive on both the IGRA assay and the TST. Findings The overall prevalence of MTI was 8.5% (152/1797). MTI was diagnosed in 26.7% of the children with a known TB contact, as opposed to 6.4% of the children without such contact. Overall, the MTI rate was higher among Inuit children (OR: 4.22; 95% CI: 1.55–11.5) and among children born less than one year after the birth of the next older sibling (OR: 2.48; 95% CI: 1.33–4.63). Self-reported TB contact modified the profile to include household crowding and low mother’s education. Children who had an older MTI-positive sibling were much more likely to test positive for MTI themselves (OR: 14.2; 95% CI: 5.75–35.0) than children without an infected older sibling. Conclusion Ethnicity, sibling relations, number of household residents and maternal level of education are factors associated with the risk of TB infection among children in Greenland. The strong household clustering of

  1. The quiet and controversial: Ural family of Mycobacterium tuberculosis.

    PubMed

    Mokrousov, Igor

    2012-06-01

    The absence of lateral gene exchange is a characteristic feature defining the genome evolution and clonal population structure of Mycobacterium tuberculosis. Certain of its lineages have justly attracted more attention due to their global dissemination and/or remarkable pathogenic properties. In this critical review, I discuss the population structure and genetic geography of the less 'popular' but in some aspects no less noteworthy M. tuberculosis lineage, Ural family. Its specific signature was initially defined by single copy in MIRU26, and large (>6) copy number in MIRU10 loci, and by 43-spoligotyping as absence of signals 29-31 and 33-36. Here, I suggest to subdivide Ural strains with present and absent spoligosignal 2 into primary Ural-1 and secondary Ural-2 sublineages, respectively, while 1 copy in MIRU26 is specific of Ural-1. Furthermore, three copies were recently described in MIRU10 in Ural-1 strains which highlights a high diversity of this locus in Ural genotype. The data on the two Ural sublineages were extracted from SpolDB4 database and original publications in order to trace their distribution at global and within-country levels. Importantly, the rigorous reanalysis suggested the true rate of the Ural genotype in the Ural area in Russia to be only 7%. In contrast, the frequencies of the Ural sublineages peak elsewhere: in South Ukraine and Georgia/Abkhazia (Ural-1, up to 14-19%), and in southwestern Iran (Ural-2, up to 26%). However, as this name is used since 2005, it seems most parsimonious to continue its use even if misleading. The forest graph was built on the available spoligoprofiles of Ural family strains from Eurasia. It helped to suggest routes of their primary dispersal that are discussed in the context of the known human migrations also influenced by natural barriers. The north/east Pontic area may have been an area of origin and primary dispersal of the Ural (Ural-1) genotype in Eurasia, whereas political and natural borders may have

  2. The quiet and controversial: Ural family of Mycobacterium tuberculosis.

    PubMed

    Mokrousov, Igor

    2012-06-01

    The absence of lateral gene exchange is a characteristic feature defining the genome evolution and clonal population structure of Mycobacterium tuberculosis. Certain of its lineages have justly attracted more attention due to their global dissemination and/or remarkable pathogenic properties. In this critical review, I discuss the population structure and genetic geography of the less 'popular' but in some aspects no less noteworthy M. tuberculosis lineage, Ural family. Its specific signature was initially defined by single copy in MIRU26, and large (>6) copy number in MIRU10 loci, and by 43-spoligotyping as absence of signals 29-31 and 33-36. Here, I suggest to subdivide Ural strains with present and absent spoligosignal 2 into primary Ural-1 and secondary Ural-2 sublineages, respectively, while 1 copy in MIRU26 is specific of Ural-1. Furthermore, three copies were recently described in MIRU10 in Ural-1 strains which highlights a high diversity of this locus in Ural genotype. The data on the two Ural sublineages were extracted from SpolDB4 database and original publications in order to trace their distribution at global and within-country levels. Importantly, the rigorous reanalysis suggested the true rate of the Ural genotype in the Ural area in Russia to be only 7%. In contrast, the frequencies of the Ural sublineages peak elsewhere: in South Ukraine and Georgia/Abkhazia (Ural-1, up to 14-19%), and in southwestern Iran (Ural-2, up to 26%). However, as this name is used since 2005, it seems most parsimonious to continue its use even if misleading. The forest graph was built on the available spoligoprofiles of Ural family strains from Eurasia. It helped to suggest routes of their primary dispersal that are discussed in the context of the known human migrations also influenced by natural barriers. The north/east Pontic area may have been an area of origin and primary dispersal of the Ural (Ural-1) genotype in Eurasia, whereas political and natural borders may have

  3. Cloning, sequencing, and expression of the apa gene coding for the Mycobacterium tuberculosis 45/47-kilodalton secreted antigen complex.

    PubMed

    Laqueyrerie, A; Militzer, P; Romain, F; Eiglmeier, K; Cole, S; Marchal, G

    1995-10-01

    Effective protection against a virulent challenge with Mycobacterium tuberculosis is induced mainly by previous immunization with living attenuated mycobacteria, and it has been hypothesized that secreted proteins serve as major targets in the specific immune response. To identify and purify molecules present in culture medium filtrate which are dominant antigens during effective vaccination, a two-step selection procedure was used to select antigens able to interact with T lymphocytes and/or antibodies induced by immunization with living bacteria and to counterselect antigens interacting with the immune effectors induced by immunization with dead bacteria. A Mycobacterium bovis BCG 45/47-kDa antigen complex, present in BCG culture filtrate, has been previously identified and isolated (F. Romain, A. Laqueyrerie, P. Militzer, P. Pescher, P. Chavarot, M. Lagranderie, G. Auregan, M. Gheorghiu, and G. Marchal, Infect. Immun. 61:742-750, 1993). Since the cognate antibodies recognize the very same antigens present in M. tuberculosis culture medium filtrates, a project was undertaken to clone, express, and sequence the corresponding gene of M. tuberculosis. An M. tuberculosis shuttle cosmid library was transferred in Mycobacterium smegmatis and screened with a competitive enzyme-linked immunosorbent assay to detect the clones expressing the proteins. A clone containing a 40-kb DNA insert was selected, and by means of subcloning in Escherichia coli, a 2-kb fragment that coded for the molecules was identified. An open reading frame in the 2,061-nucleotide sequence codes for a secreted protein with a consensus signal peptide of 39 amino acids and a predicted molecular mass of 28,779 Da. The gene was referred to as apa because of the high percentages of proline (21.7%) and alanine (19%) in the purified protein. Southern hybridization analysis of digested total genomic DNA from M. tuberculosis (reference strains H37Rv and H37Ra) indicated that the apa gene was present as a

  4. IFNγ Response to Mycobacterium tuberculosis, Risk of Infection and Disease in Household Contacts of Tuberculosis Patients in Colombia

    PubMed Central

    Marín, Nancy D.; Marín, Diana M.; López, Lucelly; Henao, Hanna M.; Martínez, Teresita; Villa, Liliana; Barrera, Luis F.; Ortiz, Blanca L.; Ramírez, María E.; Montes, Carlos J.; Oquendo, María C.; Arango, Lisandra M.; Riaño, Felipe; Aguirre, Carlos; Bustamante, Alberto; Belisle, John T.; Dobos, Karen; Mejía, Gloria I.; Giraldo, Margarita R.; Brennan, Patrick J.; Robledo, Jaime; Arbeláez, María P.; Rojas, Carlos A.; García, Luis F.

    2009-01-01

    Objectives Household contacts (HHCs) of pulmonary tuberculosis patients are at high risk of Mycobacterium tuberculosis infection and early disease development. Identification of individuals at risk of tuberculosis disease is a desirable goal for tuberculosis control. Interferon-gamma release assays (IGRAs) using specific M. tuberculosis antigens provide an alternative to tuberculin skin testing (TST) for infection detection. Additionally, the levels of IFNγ produced in response to these antigens may have prognostic value. We estimated the prevalence of M. tuberculosis infection by IGRA and TST in HHCs and their source population (SP), and assessed whether IFNγ levels in HHCs correlate with tuberculosis development. Methods A cohort of 2060 HHCs was followed for 2–3 years after exposure to a tuberculosis case. Besides TST, IFNγ responses to mycobacterial antigens: CFP, CFP-10, HspX and Ag85A were assessed in 7-days whole blood cultures and compared to 766 individuals from the SP in Medellín, Colombia. Isoniazid prophylaxis was not offered to child contacts because Colombian tuberculosis regulations consider it only in children under 5 years, TST positive without BCG vaccination. Results Using TST 65.9% of HHCs and 42.7% subjects from the SP were positive (OR 2.60, p<0.0001). IFNγ response to CFP-10, a biomarker of M. tuberculosis infection, tested positive in 66.3% HHCs and 24.3% from the SP (OR = 6.07, p<0.0001). Tuberculosis incidence rate was 7.0/1000 person years. Children <5 years accounted for 21.6% of incident cases. No significant difference was found between positive and negative IFNγ responders to CFP-10 (HR 1.82 95% CI 0.79–4.20 p = 0.16). However, a significant trend for tuberculosis development amongst high HHC IFNγ producers was observed (trend Log rank p = 0.007). Discussion CFP-10-induced IFNγ production is useful to establish tuberculosis infection prevalence amongst HHC and identify those at highest risk of disease. The high

  5. MspA-Mycobacterium tuberculosis-transformant with reduced virulence: the "unbirthday paradigm".

    PubMed

    Lamrabet, Otmane; Ghigo, Eric; Mège, Jean-Louis; Lepidi, Hubert; Nappez, Claude; Raoult, Didier; Drancourt, Michel

    2014-11-01

    Expressing mspA porin gene from Mycobacterium smegmatis in Mycobacterium tuberculosis attenuated this pathogen. Intracellular growth of the transformants into free-living amoeba and murine and human macrophages decreased. Furthermore, transformants decreased the microbicidal program of human monocyte-derived macrophages. BALB/c mice inoculated with transformants exhibited higher weights, lower histological lesions and lower M. tuberculosis inoculum in the liver, spleen and lungs than control mice challenged with wild-type M. tuberculosis. Preliminary evaluation indicated that mice inoculated with this transformant showed higher weights and lower numbers of lung nodules and tissular mycobacteria than control mice when challenged with wild-type M. tuberculosis. Similar to the paradoxical "unbirthday" gift coined by Lewis Carroll in Alice's Adventures in Wonderland, adding mspA gene reduced the virulence of M. tuberculosis and yielded a protective effect. Lost of non-virulence genes is a mechanism for virulence in mycobacteria. Engineering non-virulence genes in M. tuberculosis may yield strains with decreased virulence and increased immunogenicity. PMID:25194334

  6. MspA-Mycobacterium tuberculosis-transformant with reduced virulence: the "unbirthday paradigm".

    PubMed

    Lamrabet, Otmane; Ghigo, Eric; Mège, Jean-Louis; Lepidi, Hubert; Nappez, Claude; Raoult, Didier; Drancourt, Michel

    2014-11-01

    Expressing mspA porin gene from Mycobacterium smegmatis in Mycobacterium tuberculosis attenuated this pathogen. Intracellular growth of the transformants into free-living amoeba and murine and human macrophages decreased. Furthermore, transformants decreased the microbicidal program of human monocyte-derived macrophages. BALB/c mice inoculated with transformants exhibited higher weights, lower histological lesions and lower M. tuberculosis inoculum in the liver, spleen and lungs than control mice challenged with wild-type M. tuberculosis. Preliminary evaluation indicated that mice inoculated with this transformant showed higher weights and lower numbers of lung nodules and tissular mycobacteria than control mice when challenged with wild-type M. tuberculosis. Similar to the paradoxical "unbirthday" gift coined by Lewis Carroll in Alice's Adventures in Wonderland, adding mspA gene reduced the virulence of M. tuberculosis and yielded a protective effect. Lost of non-virulence genes is a mechanism for virulence in mycobacteria. Engineering non-virulence genes in M. tuberculosis may yield strains with decreased virulence and increased immunogenicity.

  7. Validation of a real-time PCR assay for the molecular identification of Mycobacterium tuberculosis.

    PubMed

    Sales, Mariana L; Fonseca Júnior, Antônio Augusto; Orzil, Lívia; Alencar, Andrea Padilha; Silva, Marcio Roberto; Issa, Marina Azevedo; Soares Filho, Paulo Martins; Lage, Andrey Pereira; Heinemann, Marcos Bryan

    2014-01-01

    Mycobacterium tuberculosis is the major cause of tuberculosis in humans. This bacillus gained prominence with the occurrence of HIV, presenting itself as an important opportunistic infection associated with acquired immunodeficiency syndrome (AIDS). The current study aimed to develop a real-time PCR using Eva Green technology for molecular identification of M. tuberculosis isolates. The primers were designed to Rv1510 gene. Ninety nine samples of M. tuberculosis and sixty samples of M. bovis were tested and no sample of the bovine bacillus was detected by the qPCR. Statistical tests showed no difference between the qPCR and biochemical tests used to identify the Mycobacterium tuberculosis. The correlation between tests was perfect with Kappa index of 1.0 (p < 0.001, CI = 0.84 - 1.0). The diagnostic sensitivity and specificity were 100% (CI = 95.94% - 100%) and 100% (CI = 93.98% - 100%). This qPCR was developed with the goal of diagnosing the bacillus M. tuberculosis in samples of bacterial suspension. TB reference laboratories (health and agriculture sectors), public health programs and epidemiological studies probably may benefit from such method.

  8. The Endothelin System Has a Significant Role in the Pathogenesis and Progression of Mycobacterium tuberculosis Infection

    PubMed Central

    Correa, Andre F.; Bailão, Alexandre M.; Bastos, Izabela M. D.; Orme, Ian M.; Soares, Célia M. A.; Kipnis, Andre

    2014-01-01

    Tuberculosis (TB) remains a major global health problem, and although multiple studies have addressed the relationship between Mycobacterium tuberculosis and the host on an immunological level, few studies have addressed the impact of host physiological responses. Proteases produced by bacteria have been associated with important alterations in the host tissues, and a limited number of these enzymes have been characterized in mycobacterial species. M. tuberculosis produces a protease called Zmp1, which appears to be associated with virulence and has a putative action as an endothelin-converting enzyme. Endothelins are a family of vasoactive peptides, of which 3 distinct isoforms exist, and endothelin 1 (ET-1) is the most abundant and the best-characterized isoform. The aim of this work was to characterize the Zmp1 protease and evaluate its role in pathogenicity. Here, we have shown that M. tuberculosis produces and secretes an enzyme with ET-1 cleavage activity. These data demonstrate a possible role of Zmp1 for mycobacterium-host interactions and highlights its potential as a drug target. Moreover, the results suggest that endothelin pathways have a role in the pathogenesis of M. tuberculosis infections, and ETA or ETB receptor signaling can modulate the host response to the infection. We hypothesize that a balance between Zmp1 control of ET-1 levels and ETA/ETB signaling can allow M. tuberculosis adaptation and survival in the lung tissues. PMID:25267836

  9. Detection of Mycobacterium tuberculosis complex by nested polymerase chain reaction in pulmonary and extrapulmonary specimens* ,**

    PubMed Central

    Furini, Adriana Antônia da Cruz; Pedro, Heloisa da Silveira Paro; Rodrigues, Jean Francisco; Montenegro, Lilian Maria Lapa; Machado, Ricardo Luiz Dantas; Franco, Célia; Schindler, Haiana Charifker; Batista, Ida Maria Foschiani Dias; Rossit, Andrea Regina Baptista

    2013-01-01

    OBJECTIVE: To compare the performance of nested polymerase chain reaction (NPCR) with that of cultures in the detection of the Mycobacterium tuberculosis complex in pulmonary and extrapulmonary specimens. METHODS: We analyzed 20 and 78 pulmonary and extrapulmonary specimens, respectively, of 67 hospitalized patients suspected of having tuberculosis. An automated microbial system was used for the identification of Mycobacterium spp. cultures, and M. tuberculosis IS6110 was used as the target sequence in the NPCR. The kappa statistic was used in order to assess the level of agreement among the results. RESULTS: Among the 67 patients, 6 and 5, respectively, were diagnosed with pulmonary and extrapulmonary tuberculosis, and the NPCR was positive in all of the cases. Among the 98 clinical specimens, smear microscopy, culture, and NPCR were positive in 6.00%, 8.16%, and 13.26%, respectively. Comparing the results of NPCR with those of cultures (the gold standard), we found that NPCR had a sensitivity and specificity of 100% and 83%, respectively, in pulmonary specimens, compared with 83% and 96%, respectively, in extrapulmonary specimens, with good concordance between the tests (kappa, 0.50 and 0.6867, respectively). CONCLUSIONS: Although NPCR proved to be a very useful tool for the detection of M. tuberculosis complex, clinical, epidemiological, and other laboratory data should also be considered in the diagnosis and treatment of pulmonary and extrapulmonary tuberculosis. PMID:24473765

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

    PubMed

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

    2014-12-01

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

  11. Validation of a real-time PCR assay for the molecular identification of Mycobacterium tuberculosis

    PubMed Central

    Sales, Mariana L.; Fonseca, Antônio Augusto; Orzil, Lívia; Alencar, Andrea Padilha; Silva, Marcio Roberto; Issa, Marina Azevedo; Filho, Paulo Martins Soares; Lage, Andrey Pereira; Heinemann, Marcos Bryan

    2014-01-01

    Mycobacterium tuberculosis is the major cause of tuberculosis in humans. This bacillus gained prominence with the occurrence of HIV, presenting itself as an important opportunistic infection associated with acquired immunodeficiency syndrome (AIDS). The current study aimed to develop a real-time PCR using Eva Green technology for molecular identification of M. tuberculosis isolates. The primers were designed to Rv1510 gene. Ninety nine samples of M. tuberculosis and sixty samples of M. bovis were tested and no sample of the bovine bacillus was detected by the qPCR. Statistical tests showed no difference between the qPCR and biochemical tests used to identify the Mycobacterium tuberculosis. The correlation between tests was perfect with Kappa index of 1.0 (p < 0.001, CI = 0.84 – 1.0). The diagnostic sensitivity and specificity were 100% (CI = 95.94% – 100%) and 100% (CI = 93.98% – 100%). This qPCR was developed with the goal of diagnosing the bacillus M. tuberculosis in samples of bacterial suspension. TB reference laboratories (health and agriculture sectors), public health programs and epidemiological studies probably may benefit from such method. PMID:25763042

  12. Whole-genome sequencing to detect recent transmission of Mycobacterium tuberculosis in settings with a high burden of tuberculosis.

    PubMed

    Luo, Tao; Yang, Chongguang; Peng, Ying; Lu, Liping; Sun, Guomei; Wu, Jie; Jin, Xiaoping; Hong, Jianjun; Li, Fabin; Mei, Jian; DeRiemer, Kathryn; Gao, Qian

    2014-07-01

    Whole genome sequencing (WGS) of Mycobacterium tuberculosis has been used to trace the transmission of M. tuberculosis, the causative agent of tuberculosis (TB). Previously published studies using WGS were conducted in developed countries with a low TB burden. We sought to evaluate the relative usefulness of traditional VNTR and SNP typing methods, WGS and epidemiological investigations to study the recent transmission of M. tuberculosis in a high TB burden country. We conducted epidemiological investigations of 42 TB patients whose M. tuberculosis isolates were classified into three clusters based on variable-number tandem repeat (VNTR) typing. We applied WGS to 32 (76.2%) of the 42 strains and calculated the pairwise genomic distances between strains within each cluster. Eighteen (56.3%) of the 32 strains had genomic differences ≥100 SNPs with every other strain, suggesting that direct transmission did not likely occurred. Ten strains were grouped into four WGS-based clusters with genomic distances ≤5 SNPs within each cluster, and confirmed epidemiological links were identified in two of these clusters. Our results indicate that WGS provides reliable resolution for tracing the transmission of M. tuberculosis in high TB burden settings. The high resolution of WGS is particularly useful to confirm or exclude the possibility of direct transmission events defined by traditional typing methods.

  13. CD44 is a macrophage binding site for Mycobacterium tuberculosis that mediates macrophage recruitment and protective immunity against tuberculosis

    PubMed Central

    Leemans, Jaklien C.; Florquin, Sandrine; Heikens, Mirjam; Pals, Steven T.; Neut, Ronald van der; van der Poll, Tom

    2003-01-01

    Cell migration and phagocytosis are both important for controlling Mycobacterium tuberculosis infection and are critically dependent on the reorganization of the cytoskeleton. Since CD44 is an adhesion molecule involved in inflammatory responses and is connected to the actin cytoskeleton, we investigated the role of CD44 in both these processes. Macrophage (Mφ) recruitment into M. tuberculosis–infected lungs and delayed-type hypersensitivity sites was impaired in CD44-deficient (CD44–/–) mice. In addition, the number of T lymphocytes and the concentration of the protective key cytokine IFN-γ were reduced in the lungs of infected CD44–/– mice. The production of IFN-γ by splenocytes of CD44–/– mice was profoundly increased upon antigen-specific stimulation. Flow cytometry analysis revealed that soluble CD44 can directly bind to virulent M. tuberculosis. Mycobacteria also interacted with Mφ-associated CD44, as reflected by reduced binding and internalization of bacilli by CD44–/– Mφs. This suggests that CD44 is a receptor on Mφs for binding of M. tuberculosis. CD44–/– mice displayed a decreased survival and an enhanced mycobacterial outgrowth in lungs and liver during pulmonary tuberculosis. In summary, we have identified CD44 as a new Mφ binding site for M. tuberculosis that mediates mycobacterial phagocytosis, Mφ recruitment, and protective immunity against pulmonary tuberculosis. PMID:12618522

  14. Diminished adherence and/or ingestion of virulent Mycobacterium tuberculosis by monocyte-derived macrophages from patients with tuberculosis.

    PubMed

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

    1998-09-01

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

  15. Whole-genome sequencing to detect recent transmission of Mycobacterium tuberculosis in settings with a high burden of tuberculosis.

    PubMed

    Luo, Tao; Yang, Chongguang; Peng, Ying; Lu, Liping; Sun, Guomei; Wu, Jie; Jin, Xiaoping; Hong, Jianjun; Li, Fabin; Mei, Jian; DeRiemer, Kathryn; Gao, Qian

    2014-07-01

    Whole genome sequencing (WGS) of Mycobacterium tuberculosis has been used to trace the transmission of M. tuberculosis, the causative agent of tuberculosis (TB). Previously published studies using WGS were conducted in developed countries with a low TB burden. We sought to evaluate the relative usefulness of traditional VNTR and SNP typing methods, WGS and epidemiological investigations to study the recent transmission of M. tuberculosis in a high TB burden country. We conducted epidemiological investigations of 42 TB patients whose M. tuberculosis isolates were classified into three clusters based on variable-number tandem repeat (VNTR) typing. We applied WGS to 32 (76.2%) of the 42 strains and calculated the pairwise genomic distances between strains within each cluster. Eighteen (56.3%) of the 32 strains had genomic differences ≥100 SNPs with every other strain, suggesting that direct transmission did not likely occurred. Ten strains were grouped into four WGS-based clusters with genomic distances ≤5 SNPs within each cluster, and confirmed epidemiological links were identified in two of these clusters. Our results indicate that WGS provides reliable resolution for tracing the transmission of M. tuberculosis in high TB burden settings. The high resolution of WGS is particularly useful to confirm or exclude the possibility of direct transmission events defined by traditional typing methods. PMID:24888866

  16. Complex genetics of drug resistance in Mycobacterium tuberculosis.

    PubMed

    Warner, Digby F; Mizrahi, Valerie

    2013-10-01

    Three new studies have used whole-genome sequencing of M. tuberculosis to demonstrate unexpected complexity in the modern evolution of drug-resistant tuberculosis, and a fourth study suggests a close evolutionary relationship between the pathogen and its human host over a period of 70,000 years. Collectively, the observations in these studies suggest that future strategies to tackle drug-resistant tuberculosis must integrate host genetics with detailed strain epidemiology.

  17. Identification and survival studies of Mycobacterium tuberculosis within Laboratory-Fermented bovine milk

    PubMed Central

    2014-01-01

    Background Mycobacterium tuberculosis and Mycobacterium bovis are the classic agents causing tuberculosis (TB) in humans and animals respectively. Transmission of tuberculous bacteria to humans usually occurs by inhalation of aerosols containing droplets of tubercle bacilli or via consumption of contaminated foods and drinks, primarily milk. The practice of milk pooling, including from cows with TB of the udder, further exacerbates the situation by rendering the whole milk supply infective. The simultaneous presence of indigenous lactic acid bacteria (LAB) in Mycobacterium-contaminated milk is believed to confer protective effect when the milk is adequately fermented. This study assessed the effect of LAB on the viability of mycobacteria in inherently contaminated pool of raw milk during fermentation as a function of time. Findings Growth was obtained in the pooled raw milk culture, and identified to be M. tuberculosis. This M. tuberculosis growth was undetectable in the milk culture by day 7 as assessed by plating serial dilutions of the milk culture for up to 14 days. Conclusions Some LAB species appear to show inhibitory effect on tubercle bacilli. If proven by more rigorous, controlled experimental results regarding such effect, selected LAB (with proven safety and efficacy) may have potential applications as anti-mycobacterial agents. PMID:24666844

  18. Mycobacterium tuberculosis alters the differentiation of monocytes into macrophages in vitro.

    PubMed

    Castaño, Diana; Barrera, Luis F; Rojas, Mauricio

    2011-01-01

    This paper shows that in vitro infection of human monocytes by Mycobacterium tuberculosis affected monocyte to macrophage differentiation. Despite the low bacterial load used, M. tuberculosis-infected monocytes had fewer granules, displayed a reduced number of cytoplasmic projections and decreased HLA class II, CD68, CD86 and CD36 expression compared to cells differentiated in the absence of mycobacteria. Infected cells produced less IL-12p70, TNF-α, IL-10, IL-6 and high IL-1β in response to lipopolysaccharide and purified protein M. tuberculosis-derived. Reduced T-cell proliferative response and IFN-γ secretion in response to phytohemagglutinin and culture filtrate proteins from M. tuberculosis was also observed in infected cells when compared to non-infected ones. The ability of monocytes differentiated in the presence of M. tuberculosis to control mycobacterial growth in response to IFN-γ stimulation was attenuated, as determined by bacterial plate count; however, they had a similar ability to uptake fluorescent M. tuberculosis and latex beads compared to non-infected cells. Recombinant IL-1β partially altered monocyte differentiation into macrophages; however, treating M. tuberculosis-infected monocytes with IL-1RA did not reverse the effects of infection during differentiation. The results indicated that M. tuberculosis infection altered monocyte differentiation into macrophages and affected their ability to respond to innate stimuli and activate T-cells.

  19. Role of complement activation and antibody in the interaction between Mycobacterium tuberculosis and human macrophages.

    PubMed

    Manivannan, S; Rao, Narayan V; Ramanathan, V D

    2012-08-01

    Mycobacterium tuberculosis-specific antibodies possess immunomodulatory effects during tuberculosis infection. Prior sensitization to environmental mycobacteria is known to suppress immune responses against BCG and M. tuberculosis. Mycobacteria-induced antibodies can influence events such as complement activation and phagocytosis during infectious process. In the present study role of anti-M. tuberculosis IgG (anti-M. tb IgG) antibody during interaction between M. tuberculosis and human macrophages mediated through complement has been examined in vitro. Anti-M. tb IgG antibody significantly enhanced complement activation by M. tuberculosis. Phagocytosis of M. tuberculosis by macrophages increased significantly in the presence of complement and/or antibody. Moreover, antibody enhanced phagocytosis in the presence of complement. Addition of antibody alone or in combination with complement also augmented intracellular viability of bacilli within macrophages. Results of this study showed that anti-mycobacterial antibody enhances complement activation and anti-M. tb IgG antibody probably modulates effects of complement during early stages of tuberculosis infection.

  20. Detection of mycobacteria, Mycobacterium avium subspecies, and Mycobacterium tuberculosis complex by a novel tetraplex real-time PCR assay.

    PubMed

    Sevilla, Iker A; Molina, Elena; Elguezabal, Natalia; Pérez, Valentín; Garrido, Joseba M; Juste, Ramón A

    2015-03-01

    Mycobacterium tuberculosis complex, Mycobacterium avium, and many other nontuberculous mycobacteria are worldwide distributed microorganisms of major medical and veterinary importance. Considering the growing epidemiologic significance of wildlife-livestock-human interrelation, developing rapid detection tools of high specificity and sensitivity is vital to assess their presence and accelerate the process of diagnosing mycobacteriosis. Here we describe the development and evaluation of a novel tetraplex real-time PCR for simultaneous detection of Mycobacterium genus, M. avium subspecies, and M. tuberculosis complex in an internally monitored single assay. The method was evaluated using DNA from mycobacterial (n = 38) and nonmycobacterial (n = 28) strains, tissues spiked with different CFU amounts of three mycobacterial species (n = 57), archival clinical samples (n = 233), and strains isolated from various hosts (n = 147). The minimum detectable DNA amount per reaction was 50 fg for M. bovis BCG and M. kansasii and 5 fg for M. avium subsp. hominissuis. When spiked samples were analyzed, the method consistently detected as few as 100 to 1,000 mycobacterial CFU per gram. The sensitivity and specificity values for the panel of clinical samples were 97.5 and 100% using a verified culture-based method as the reference method. The assays performed on clinical isolates confirmed these results. This PCR was able to identify M. avium and M. tuberculosis complex in the same sample in one reaction. In conclusion, the tetraplex real-time PCR we designed represents a highly specific and sensitive tool for the detection and identification of mycobacteria in routine laboratory diagnosis with potential additional uses.

  1. Detection of Mycobacteria, Mycobacterium avium Subspecies, and Mycobacterium tuberculosis Complex by a Novel Tetraplex Real-Time PCR Assay

    PubMed Central

    Molina, Elena; Elguezabal, Natalia; Pérez, Valentín; Garrido, Joseba M.

    2015-01-01

    Mycobacterium tuberculosis complex, Mycobacterium avium, and many other nontuberculous mycobacteria are worldwide distributed microorganisms of major medical and veterinary importance. Considering the growing epidemiologic significance of wildlife-livestock-human interrelation, developing rapid detection tools of high specificity and sensitivity is vital to assess their presence and accelerate the process of diagnosing mycobacteriosis. Here we describe the development and evaluation of a novel tetraplex real-time PCR for simultaneous detection of Mycobacterium genus, M. avium subspecies, and M. tuberculosis complex in an internally monitored single assay. The method was evaluated using DNA from mycobacterial (n = 38) and nonmycobacterial (n = 28) strains, tissues spiked with different CFU amounts of three mycobacterial species (n = 57), archival clinical samples (n = 233), and strains isolated from various hosts (n = 147). The minimum detectable DNA amount per reaction was 50 fg for M. bovis BCG and M. kansasii and 5 fg for M. avium subsp. hominissuis. When spiked samples were analyzed, the method consistently detected as few as 100 to 1,000 mycobacterial CFU per gram. The sensitivity and specificity values for the panel of clinical samples were 97.5 and 100% using a verified culture-based method as the reference method. The assays performed on clinical isolates confirmed these results. This PCR was able to identify M. avium and M. tuberculosis complex in the same sample in one reaction. In conclusion, the tetraplex real-time PCR we designed represents a highly specific and sensitive tool for the detection and identification of mycobacteria in routine laboratory diagnosis with potential additional uses. PMID:25588660

  2. Rapid susceptibility testing of mycobacterium avium complex and mycobacterium tuberculosis isolated from AIDS patients

    NASA Technical Reports Server (NTRS)

    Dhople, Arvind M.

    1993-01-01

    In ominous projections issued by both U.S. Public Health Service and the World Health Organization, the epidemic of the Human Immunodeficiency Virus (HIV) infection will continue to rise more rapidly worldwide than predicted earlier. The Acquired Immunodeficiency Syndrome (AIDS) patients are susceptible to diseases called opportunistic infections of which tuberculosis and M. 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, Maryland, had adopted a biochemical indicator, adenosine triphosphate (ATP), to detect presence of life in extraterrestrial space. Therefore, we proposed to develop ATP assay technique to determine sensitivity of antibacterial compounds against MAC and M. tuberculosis. The work was initiated in June 1992. In the last report, we described our efforts in developing ATP assay method using MAC. Studies were continued further, and during the period of this report, we established the relationship between colony forming units and ATP levels of these organisms during the growth cycle. Also, we evaluated the effects of standard antimycobacterial drugs using ATP assay technique and compared the results with those obtained with conventional tube dilution proportional method.

  3. Fusing Dual-Event Datasets for Mycobacterium Tuberculosis Machine Learning Models and their Evaluation

    PubMed Central

    Ekins, Sean; Freundlich, Joel S.; Reynolds, Robert C.

    2013-01-01

    The search for new tuberculosis treatments continues as we need to find molecules that can act more quickly, be accommodated in multi-drug regimens, and overcome ever increasing levels of drug resistance. Multiple large scale phenotypic high-throughput screens against Mycobacterium tuberculosis (Mtb) have generated dose response data, enabling the generation of machine learning models. These models also incorporated cytotoxicity data and were recently validated with a large external dataset. A cheminformatics data-fusion approach followed by Bayesian machine learning, Support Vector Machine or Recursive Partitioning model development (based on publicly available Mtb screening data) was used to compare individual datasets and subsequent combined models. A set of 1924 commercially available molecules with promising antitubercular activity (and lack of relative cytotoxicity to Vero cells) were used to evaluate the predictive nature of the models. We demonstrate that combining three datasets incorporating antitubercular and cytotoxicity data in Vero cells from our previous screens results in external validation receiver operator curve (ROC) of 0.83 (Bayesian or RP Forest). Models that do not have the highest five-fold cross validation ROC scores can outperform other models in a test set dependent manner. We demonstrate with predictions for a recently published set of Mtb leads from GlaxoSmithKline that no single machine learning model may be enough to identify compounds of interest. Dataset fusion represents a further useful strategy for machine learning construction as illustrated with Mtb. Coverage of chemistry and Mtb target spaces may also be limiting factors for the whole-cell screening data generated to date. PMID:24144044

  4. Transmission of Mycobacterium tuberculosis Beijing Strains, Alberta, Canada, 1991-2007.

    PubMed

    Langlois-Klassen, Deanne; Senthilselvan, Ambikaipakan; Chui, Linda; Kunimoto, Dennis; Saunders, L Duncan; Menzies, Dick; Long, Richard

    2013-05-01

    Beijing strains are speculated to have a selective advantage over other Mycobacterium tuberculosis strains because of increased transmissibility and virulence. In Alberta, a province of Canada that receives a large number of immigrants, we conducted a population-based study to determine whether Beijing strains were associated with increased transmission leading to disease compared with non-Beijing strains. Beijing strains accounted for 258 (19%) of 1,379 pulmonary tuberculosis cases in 1991-2007; overall, 21% of Beijing cases and 37% of non-Beijing cases were associated with transmission clusters. Beijing index cases had significantly fewer secondary cases within 2 years than did non-Beijing cases, but this difference disappeared after adjustment for demographic characteristics, infectiousness, and M. tuberculosis lineage. In a province that has effective tuberculosis control, transmission of Beijing strains posed no more of a public health threat than did non-Beijing strains.

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

    PubMed

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

    2014-05-01

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

  6. IFNγ-responsive nonhematopoietic cells regulate the immune response to Mycobacterium tuberculosis

    PubMed Central

    Desvignes, Ludovic; Ernst, Joel D.

    2009-01-01

    SUMMARY Immunity to Mycobacterium tuberculosis in humans and in mice requires interferon gamma (IFNγ). While IFNγ has been studied extensively for its effects on macrophages in tuberculosis, we determined that protective immunity to tuberculosis also requires IFNγ responsive nonhematopoietic cells. Bone marrow chimeric mice with IFNγ-unresponsive lung epithelial and endothelial cells exhibited earlier mortality and higher bacterial burdens than control mice, underexpressed indoleamine-2,3-dioxygenase (Ido) in lung endothelium and epithelium and overexpressed IL-17 with massive neutrophilic inflammation in the lungs. We also found that the products of IDO catabolism of tryptophan selectively inhibit IL-17 production by Th17 cells, by inhibiting the action of IL-23. These results reveal a previously-unsuspected role for IFNγ responsiveness in nonhematopoietic cells in regulation of immunity to M. tuberculosis, and reveal a novel mechanism for IDO inhibition of Th17 responses. PMID:20064452

  7. Detection of Mycobacterium tuberculosis complex by PCR in fresh cheese from local markets in Hidalgo, Mexico.

    PubMed

    Pereira-Suárez, Ana Laura; Estrada-Chávez, Yessica; Zúñiga-Estrada, Armidá; Lopez-Rincón, Gonzálo; Hernández, David Ulises Miranda; Padilla-Ramírez, Francisco Javier; Estrada-Chávez, Ciro

    2014-05-01

    The objective of this study was to identify the presence of Mycobacterium tuberculosis complex bacterial DNA in samples extracted from fresh cheeses; 95 samples of fresh cheese were obtained from municipal markets in the state of Hidalgo, in central Mexico, and were analyzed in triplicate. The exogenous control for the amplification was the mitochondrial gene for cytochrome b (cyt-b). M. tuberculosis complex DNA was detected by nested-PCR amplification of a fragment of the mpb70 gene in six samples, four of which were obtained from regions with enzootic bovine tuberculosis. These results suggest that cheeses prepared with raw milk contaminated with M. bovis are being sold and consumed by humans, which may cause tuberculosis.

  8. Detection of Mycobacterium tuberculosis complex by PCR in fresh cheese from local markets in Hidalgo, Mexico.

    PubMed

    Pereira-Suárez, Ana Laura; Estrada-Chávez, Yessica; Zúñiga-Estrada, Armidá; Lopez-Rincón, Gonzálo; Hernández, David Ulises Miranda; Padilla-Ramírez, Francisco Javier; Estrada-Chávez, Ciro

    2014-05-01

    The objective of this study was to identify the presence of Mycobacterium tuberculosis complex bacterial DNA in samples extracted from fresh cheeses; 95 samples of fresh cheese were obtained from municipal markets in the state of Hidalgo, in central Mexico, and were analyzed in triplicate. The exogenous control for the amplification was the mitochondrial gene for cytochrome b (cyt-b). M. tuberculosis complex DNA was detected by nested-PCR amplification of a fragment of the mpb70 gene in six samples, four of which were obtained from regions with enzootic bovine tuberculosis. These results suggest that cheeses prepared with raw milk contaminated with M. bovis are being sold and consumed by humans, which may cause tuberculosis. PMID:24780344

  9. Drug resistance to Mycobacterium tuberculosis: from the traditional Chinese view to modern systems biology.

    PubMed

    Xu, Yuhui; Zhang, Zongde; Sun, Zhaogang

    2015-01-01

    The pathogen, Mycobacterium tuberculosis (M. tuberculosis) is a well-evolved, organized pathogen that has developed drug resistance, specifically multidrug resistance (MDR) and extensive drug resistance (XDR). This review primarily summarizes the mechanisms of drug resistance by M. tuberculosis according to the traditional Chinese view. The traditional Chinese view of drug resistance includes: the physical barrier of the cell wall; mutations relating to current anti-TB agents; drug efflux pumps; and drug stress, including the SOS response systems, the mismatch repair systems and the toxin-antitoxin systems. In addition, this review addresses the integrated systems biology of genomics, transcriptomics, proteomics, metabolomics and interactomics. Development of the various levels of systems biology has enabled determination of the anatomy of bacteria. Finally, the current review proposes that further investigation regarding the population of individuals with a high drug metabolic speed is vital to further understand drug resistance in M. tuberculosis.

  10. Testing chemical and genetic Modulators in Mycobacterium tuberculosis infected cells using phenotypic assays.

    PubMed

    Delorme, Vincent; Song, Ok-Ryul; Baulard, Alain; Brodin, Priscille

    2015-01-01

    Mycobacterium tuberculosis is able to colonize host cells, and it is now well admitted that the intracellular stage of the bacteria contributes to tuberculosis pathogenesis as well as to making it a persistent infection. There is still limited understanding on how the tubercle bacillus colonizes the cell and what are the factors impacting on its intracellular persistence. Recent advances in imaging technique allow rapid quantification of biological objects in complex environments. Furthermore, M. tuberculosis is a microorganism that is particularly genetically tractable and that tolerates the expression of heterologous fluorescent proteins. Thus, the intracellular distribution of M. tuberculosis expressing fluorescent proteins can be easily quantified by the use of confocal microscopy. Here we describe high-content/high-throughput imaging methods that enable tracking the bacillus inside host settings, taking into account the heterogeneity of colonization. PMID:25779330

  11. System-level strategies for studying the metabolism of Mycobacterium tuberculosis

    PubMed Central

    Beste, Dany J. V.

    2010-01-01

    Despite decades of research many aspects of the biology of Mycobacterium tuberculosis remain unclear and this is reflected in the antiquated tools available to treat and prevent tuberculosis and consequently this disease remains a serious public health problem. Important discoveries linking M. tuberculosis's metabolism and pathogenesis have renewed interest in this area of research. Previous experimental studies were limited to the analysis of individual genes or enzymes whereas recent advances in computational systems biology and high throughput experimental technologies now allow metabolism to be studied on a genome scale. Here we discuss the progress being made in applying system level approaches to studying the metabolism of this important pathogen. The information from these studies will fundamentally change our approach to tuberculosis research and lead to new targets for therapeutic drugs and vaccines. PMID:20938502

  12. Protection against Mycobacterium tuberculosis infection by adoptive immunotherapy. Requirement for T cell-deficient recipients

    SciTech Connect

    Orme, I.M.; Collins, F.M.

    1983-07-01

    The results of this study demonstrate that spleen cells taken from mice at the height of the primary immune response to intravenous infection with Mycobacterium tuberculosis possess the capacity to transfer adoptive protection to M. tuberculosis-infected recipients, but only if these recipients are first rendered T cell-deficient, either by thymectomy and gamma irradiation, or by sublethal irradiation. A similar requirement was necessary to demonstrate the adoptive protection of the lungs after exposure to an acute aerosol-delivered M. tuberculosis infection. In both infectious models successful adoptive immunotherapy was shown to be mediated by T lymphocytes, which were acquired in the donor animals in response to the immunizing infection. It is proposed that the results of this study may serve as a basic model for the subsequent analysis of t