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Sample records for mycobacterium tuberculosis resistant

  1. Mechanisms of fluoroquinolone resistance in Mycobacterium tuberculosis.

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    Yujiao, Zhang; Xiaojing, Li; Kaixia, Mi

    2016-10-20

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

  2. Adaptation and evolution of drug-resistant Mycobacterium tuberculosis

    NARCIS (Netherlands)

    Bergval, I.L.

    2013-01-01

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

  3. Adaptation and evolution of drug-resistant Mycobacterium tuberculosis

    NARCIS (Netherlands)

    Bergval, I.L.

    2013-01-01

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

  4. Mycobacterium tuberculosis resistance to antituberculosis drugs in Mozambique.

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

    2014-01-01

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

  5. The complex evolution of antibiotic resistance in Mycobacterium tuberculosis

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    J.D. Fonseca

    2015-03-01

    Full Text Available Multidrug-resistant and extensively drug-resistant tuberculosis (TB represent a major threat to the control of the disease worldwide. The mechanisms and pathways that result in the emergence and subsequent fixation of resistant strains of Mycobacterium tuberculosis are not fully understood and recent studies suggest that they are much more complex than initially thought. In this review, we highlight the exciting new areas of research within TB resistance that are beginning to fill these gaps in our understanding, whilst also raising new questions and providing future directions.

  6. Evaluation of DNA microarray for detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis isolates

    Institute of Scientific and Technical Information of China (English)

    王峰

    2013-01-01

    Objective To evaluate the performance of DNA microarray for rapid detection resistance to rifampin and isoniazid in Mycobacterium tuberculosis clinical isolates and identify suitable target sites for molecular genetic test. Methods Twenty-four clinical Mycobacterium

  7. Mycobacterium tuberculosis complex mycobacteria as amoeba-resistant organisms.

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    Felix Mba Medie

    Full Text Available BACKGROUND: Most environmental non-tuberculous mycobacteria have been demonstrated to invade amoebal trophozoites and cysts, but such relationships are largely unknown for members of the Mycobacterium tuberculosis complex. An environmental source has been proposed for the animal Mycobacterium bovis and the human Mycobacterium canettii. METHODOLOGY/PRINCIPAL FINDINGS: Using optic and electron microscopy and co-culture methods, we observed that 89±0.6% of M. canettii, 12.4±0.3% of M. tuberculosis, 11.7±2% of M. bovis and 11.2±0.5% of Mycobacterium avium control organisms were phagocytized by Acanthamoeba polyphaga, a ratio significantly higher for M. canettii (P = 0.03, correlating with the significantly larger size of M. canetti organisms (P = 0.035. The percentage of intraamoebal mycobacteria surviving into cytoplasmic vacuoles was 32±2% for M. canettii, 26±1% for M. tuberculosis, 28±2% for M. bovis and 36±2% for M. avium (P = 0.57. M. tuberculosis, M. bovis and M. avium mycobacteria were further entrapped within the double wall of <1% amoebal cysts, but no M. canettii organisms were observed in amoebal cysts. The number of intracystic mycobacteria was significantly (P = 10(-6 higher for M. avium than for the M. tuberculosis complex, and sub-culturing intracystic mycobacteria yielded significantly more (P = 0.02 M. avium organisms (34×10(4 CFU/mL than M. tuberculosis (42×10(1 CFU/mL and M. bovis (35×10(1 CFU/mL in the presence of a washing fluid free of mycobacteria. Mycobacteria survived in the cysts for up to 18 days and cysts protected M. tuberculosis organisms against mycobactericidal 5 mg/mL streptomycin and 2.5% glutaraldehyde. CONCLUSIONS/SIGNIFICANCE: These data indicate that M. tuberculosis complex organisms are amoeba-resistant organisms, as previously demonstrated for non-tuberculous, environmental mycobacteria. Intercystic survival of tuberculous mycobacteria, except for M. canettii, protect them

  8. Prevalence of Multidrug Resistant Mycobacterium tuberculosis by Mycobacteria growth

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    Livani S

    2012-01-01

    Full Text Available Background and objectives: Identification and monitoring ofmultidrugresistant Mycobacterium tuberculosis strains (MDR ishighlighted by the high risk of their spreading in different areas.Prevalence of these strains was evaluated in Golestan province innortheast of Iran.Material and Methods: Drug susceptibility testing to Isoniazid andrifampin was carried out for 148 clinical samples that had grown inMycobacteria growth indicator tube (MGIT system, according to themanufacturer's instructions (Becton-Dickinson, USA. The associationof drug resistance frequency with demographic characteristics andgrowth time were investigated. The appropriate statistical tests, X2 andstudent Ttest were performed for comparison of these variants. A pvalue>0.05 was considered significant in all cases.Results: The turnaround time required for growth of Mycobacteriumtuberculosis in MGIT system was between 2 to 55 days (mean16.3±10.4 days. Of all samples studied, 17.6% and 3.4% wereresistant to Isoniazid and rifampin, respectively, and 3.4% (5 sampleswere MDR (CI 95%; 1- 6%. The turnaround time required fordetermining MDR cases was 9.6 days. No statistically significantassociation was found between the resistance to the drugs and none ofthe factors including sex, age, type of clinical sample, and positivity ofthe smear.Conclusion: The prevalence of MDR in the studied region wasdetermined to be 3.4% which is similar to the country-wideevaluations. The turnaround time for Mycobacterium growth and antidrug susceptibility result can be shortened by MGIT method.Key words: Mycobacterium tuberculosis, Mycobacterium GrowthIndicator Tube, Multidrug Resistant

  9. Drug resistance of Mycobacterium tuberculosis strains in southern Brazil

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    Laynara Katize Grutzmacher

    2012-02-01

    Full Text Available INTRODUCTION: The aim of this work was to evaluate the prevalence of Mycobacterium tuberculosis (MT strains with mutations that could result in resistance to the main drugs used in treatment in a region with one of the highest numbers of tuberculosis (TB cases in southern Brazil. METHODS: Deoxyribonucleic acid (DNA from 120 sputum samples from different patients suspicious of pulmonary tuberculosis who attended the Municipal Public Laboratory for Mycobacterium sp. diagnosis was directly amplified and analyzed by PCR-SSCP. The DNA was amplified in known hotspot mutation regions of the genes rpoB, ahpC, embB, katG, inhA, and pncA. RESULTS: The percentage of samples positive by culture was 9.2% (11/120; 5% (6/120 were positive by bacilloscopy and MT-PCR, and DNA fragments of the aforementioned resistance genes could be amplified from seven (7 of the eleven (11 samples with positive results, either by culture or PCR/bacilloscopy. All presented a SSCP pattern similar to a native, nonresistant genotype, with the ATCC strain 25177 as control, except for one sample (0.01%, which presented a SSCP profile demonstrating mutation at the embB gene. CONCLUSIONS: These results are consistent with the empirical observations by physicians treating TB patients in our region of a low occurrence of cases that are refractory to conventional treatment schemes, in contrast to other parts of the country. Continued surveillance, especially molecular, is essential to detect and monitor the outbreak of MT-resistant strains.

  10. Assessment of trends of ofloxacin resistance in Mycobacterium tuberculosis

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    J S Verma

    2011-01-01

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

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

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

    2015-08-28

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

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

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    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

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

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    Fantahun Biadglegne

    2014-01-01

    Full Text Available 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.

  14. Characteristics of multidrug-resistant Mycobacterium tuberculosis in southern Brazil.

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    Perizzolo, Paulo F; Dalla Costa, Elis R; Ribeiro, Andrezza W; Spies, Fernanda S; Ribeiro, Marta O; Dias, Cláudia F; Unis, Gisela; Almeida da Silva, Pedro; Gomes, Harrison M; Suffys, Philip N; Rossetti, Maria Lucia R

    2012-01-01

    A major threat to tuberculosis (TB) control programs is the emergence of drug resistant Mycobacterium tuberculosis strains that cause TB that cannot be cured by standard anti-TB drug regimens. Because few data exist on MDR-TB in this region of the country, we performed an epidemiologic study that combined conventional and molecular analysis of MDR-TB cases from Rio Grande do Sul (RS) that were diagnosed in this period and included cases that were under treatment with second line drug schemes. Included were 121 MDR cases and sequencing of rpoB and katG showed that 106 (87.6%) strains were mutated in rpoB and 97 (80.2%) in katG. Spoligotyping demonstrated that the LAM genotype was predominant (n = 70, 57.8%) and included the largest group composed by 22 (18.1%) strains with the LAM5 ST93 genotype. Other main genotypes belonged to the families T (n = 22, 18.2%), U family (n = 16, 13.2%), Haarlem (n = 5, 4.1%) and X (n = 1, 0.8%). Genotyping by IS6110-RFLP analysis showed 51 distinct fingerprints, 38 (31.4%) of these observed only once and the other 13 patterns being shared among the rest of the isolates (n = 83, 68.6%). Among the 22 strains that were LAM5 ST93, only two had different IS6110-RFLP genotypes. In conclusion, there exists a high degree of M. Tuberculosis genotype clustering among MDR-TB cases in Rio Grande do Sul. Moreover, we observed a large MDR-TB outbreak.

  15. Multidrug resistance to Mycobacterium tuberculosis in a tertiary hospital.

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    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

  16. Implication of the RD(Rio) Mycobacterium tuberculosis sublineage in multidrug resistant tuberculosis in Portugal.

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    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.

  17. Improved Detection by Next-Generation Sequencing of Pyrazinamide Resistance in Mycobacterium tuberculosis Isolates

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    Maningi, Nontuthuko E.; Daum, Luke T.; Rodriguez, John D.; Mphahlele, Matsie; Peters, Remco P. H.; Fischer, Gerald W.; Chambers, James P.; Fourie, P. Bernard

    2015-01-01

    The technical limitations of common tests used for detecting pyrazinamide (PZA) resistance in Mycobacterium tuberculosis isolates pose challenges for comprehensive and accurate descriptions of drug resistance in patients with multidrug-resistant tuberculosis (MDR-TB). In this study, a 606-bp fragment (comprising the pncA coding region plus the promoter) was sequenced using Ion Torrent next-generation sequencing (NGS) to detect associated PZA resistance mutations in 88 recultured MDR-TB isolat...

  18. Evolution of Mycobacterium tuberculosis.

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    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.

  19. Induction in vitro and stability of Mycobacterium tuberculosis resistance to ofloxacin

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    杨华

    2014-01-01

    Objective To induct Mycobacterium tuberculosis(MTB)resistance with ofloxacin(Ofx)of stepwise increasing concentration in vitro,investigate stability to fluoroquinolone(FQs)antibiotic of MTB,and analyze the molecular mechanism and mutation specialty of drug resistance preliminarily.Methods MTB Standard strain

  20. Molecular Identification of Mycobacterium tuberculosis and Analysis of Its Resistance to Rifampin in Sputa From Tuberculosis Suspected Patients

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    M. Syaifudin

    2010-08-01

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

  1. Transmission of extensively drug-resistant and multidrug resistant Mycobacterium tuberculosis in families identified by genotyping

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    YAN Li-ping; QIN Lian-hua; ZHANG Qing; SUN Hua; HAN Min; XIAO He-ping

    2013-01-01

    Background Diagnosis and appropriate treatment of multidrug-resistant tuberculosis (MDR-TB) remain major challenges.We sought to elucidate that persons who share a household with drug resistance tuberculosis patients are at high risk for primary drug resistance tuberculosis and how to prevent these outbreaks.Methods We used 12-locus mycobacterial interspersed repetitive unit and 7-locus variable-number tandem repeat to identify household transmission of extensively drug resistant and multiple drug resistant Mycobacterium tuberculosis in three families admitted in Shanghai Pulmonary Hospital affiliated with Tongji University.Drug susceptibility tests were done by the modified proportion method in the MGIT 960 system in the same time.Clinical data were also obtained from the subjects' medical records.Results All of the six strains were defined as Beijing genotype by the deletion-targeted multiplex PCR (DTM-PCR) identification on the genomic deletion RD105.Strains from family-1 had the same minisatellite interspersed repetitive unit (MIRU) pattem (232225172531) and the same MIRU pattern (3677235).Strains from family-2 had the same MIRU pattern (2212261553323) and the same MIRU pattern (3685134).Strains from family-3 did not have the same MIRU pattern and they differed at only one locus (223326173533,223325173533),and did not have the same VNTR pattern with two locus differed (3667233,3677234).Conclusions Household transmission exists in the three families.A clear chain of tuberculosis transmission within family exists.Tuberculosis susceptibility should be considered when there is more than one tuberculosis patients in a family.Household tuberculosis transmission could be prevented with adequate treatment of source patients.

  2. High isoniazid resistance rates in rifampicin susceptible Mycobacterium tuberculosis pulmonary isolates from Pakistan.

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    Naima Fasih

    Full Text Available BACKGROUND: Rapid new diagnostic methods (including Xpert MTB/RIF assay use rifampicin resistance as a surrogate marker for multidrug resistant tuberculosis. Patients infected with rifampicin susceptible strains are prescribed first line anti-tuberculosis therapy. The roll out of such methods raises a concern that strains with resistance to other first line anti-tuberculosis drugs including isoniazid will be missed and inappropriate treatment given. To evaluate implications of using such methods review of resistance data from high burden settings such as ours is essential. OBJECTIVE: To determine resistance to first line anti-tuberculosis drugs amongst rifampicin susceptible pulmonary Mycobacterium tuberculosis (MTB isolates from Pakistan. MATERIALS AND METHODS: Data of pulmonary Mycobacterium tuberculosis strains isolated in Aga Khan University Hospital (AKUH laboratory (2009-2011 was retrospectively analyzed. Antimicrobial susceptibility profile of rifampicin susceptible isolates was evaluated for resistance to isoniazid, pyrazinamide, ethambutol, and streptomycin. RESULTS: Pulmonary specimens submitted to AKUH from 2009 to 2011 yielded 7738 strains of Mycobacterium tuberculosis. These included 54% (n 4183 rifampicin susceptible and 46% (n: 3555 rifampicin resistant strains. Analysis of rifampicin susceptible strains showed resistance to at least one of the first line drugs in 27% (n:1133 of isolates. Overall isoniazid resistance was 15.5% (n: 649, with an isoniazid mono-resistance rate of 4% (n: 174. Combined resistance to isoniazid, pyrazinamide, and ethambutol was noted in 1% (n: 40, while resistance to isoniazid, pyrazinamide, ethambutol, and streptomycin was observed in 1.7% (n: 70 of strains. CONCLUSIONS: Our data suggests that techniques (including Xpert MTB/RIF assay relying on rifampicin susceptibility as an indicator for initiating first line therapy will not detect patients infected with MTB strains resistant to other first line

  3. A Defined Tuberculosis Vaccine Candidate Boosts BCG and Protects Against Multidrug Resistant Mycobacterium tuberculosis

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    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

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

    Directory of Open Access Journals (Sweden)

    A. H. Kurniawan

    2016-01-01

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

  5. [Signal transduction and drug resistance in Mycobacterium tuberculosis--A review].

    Science.gov (United States)

    Wang, Shanshan; Feng, Yi; Zhang, Zhe

    2015-08-04

    Mycobacterium tuberculosis infection kills two million people every year, and the chemotherapy has led to significant amount of drug resistance. Signal transduction systems are used by bacteria to survive or adapt to their living environment, but the relationship to drug resistance is not well understood. In this article, we introduced the two-component signal transduction systems of M. tuberculosis and analyzed their relationship with drug resistance. We identified five two-component system pairs involved in the formation of drug resistance. Therefore, these two-component systems are good targeting sites for small biochemical drugs to target so as to reverse the drug resistance and virulence.

  6. Phenotypic and genotypic analysis of anti-tuberculosis drug resistance in Mycobacterium tuberculosis isolates in Myanmar.

    Science.gov (United States)

    Aung, Wah Wah; Ei, Phyu Win; Nyunt, Wint Wint; Swe, Thyn Lei; Lwin, Thandar; Htwe, Mi Mi; Kim, Kyung Jun; Lee, Jong Seok; Kim, Chang Ki; Cho, Sang Nae; Song, Sun Dae; Chang, Chulhun L

    2015-09-01

    Tuberculosis (TB) is one of the most serious health problems in Myanmar. Because TB drug resistance is associated with genetic mutation(s) relevant to responses to each drug, genotypic methods for detecting these mutations have been proposed to overcome the limitations of classic phenotypic drug susceptibility testing (DST). We explored the current estimates of drug-resistant TB and evaluated the usefulness of genotypic DST in Myanmar. We determined the drug susceptibility of Mycobacterium tuberculosis isolated from sputum smear-positive patients with newly diagnosed pulmonary TB at two main TB centers in Myanmar during 2013 by using conventional phenotypic DST and the GenoType MTBDRplus assay (Hain Lifescience, Germany). Discrepant results were confirmed by sequencing the genes relevant to each type of resistance (rpoB for rifampicin; katG and inhA for isoniazid). Of 191 isolates, phenotypic DST showed that 27.7% (n=53) were resistant to at least one first-line drug and 20.9% (n=40) were resistant to two or more, including 18.3% (n=35) multidrug-resistant TB (MDR-TB) strains. Monoresistant strains accounted for 6.8% (n=13) of the samples. Genotypic assay of 189 isolates showed 17.5% (n=33) MDR-TB and 5.3% (n=10) isoniazid-monoresistant strains. Genotypic susceptibility results were 99.5% (n=188) concordant and agreed almost perfectly with phenotypic DST (kappa=0.99; 95% confidence interval 0.96-1.01). The results highlight the burden of TB drug resistance and prove the usefulness of the genotypic DST in Myanmar.

  7. A defined tuberculosis vaccine candidate boosts BCG and protects against multidrug-resistant Mycobacterium tuberculosis.

    Science.gov (United States)

    Bertholet, Sylvie; Ireton, Gregory C; Ordway, Diane J; Windish, Hillarie Plessner; Pine, Samuel O; Kahn, Maria; Phan, Tony; Orme, Ian M; Vedvick, Thomas S; Baldwin, Susan L; Coler, Rhea N; Reed, Steven G

    2010-10-13

    Despite the widespread use of the childhood vaccine against tuberculosis (TB), Mycobacterium bovis bacillus Calmette-Guérin (BCG), the disease remains a serious global health problem. A successful vaccine against TB that replaces or boosts BCG would include antigens that induce or recall the appropriate T cell responses. Four Mycobacterium tuberculosis (Mtb) antigens--including members of the virulence factor families PE/PPE and EsX or antigens associated with latency--were produced as a single recombinant fusion protein (ID93). When administered together with the adjuvant GLA-SE, a stable oil-in-water nanoemulsion, the fusion protein was immunogenic in mice, guinea pigs, and cynomolgus monkeys. In mice, this fusion protein-adjuvant combination induced polyfunctional CD4 T helper 1 cell responses characterized by antigen-specific interferon-γ, tumor necrosis factor, and interleukin-2, as well as a reduction in the number of bacteria in the lungs of animals after they were subsequently infected with virulent or multidrug-resistant Mtb strains. Furthermore, boosting BCG-vaccinated guinea pigs with fusion peptide-adjuvant resulted in reduced pathology and fewer bacilli, and prevented the death of animals challenged with virulent Mtb. Finally, the fusion protein elicited polyfunctional effector CD4 and CD8 T cell responses in BCG-vaccinated or Mtb-exposed human peripheral blood mononuclear cells. This study establishes that the protein subunit vaccine consisting of the fusion protein and adjuvant protects against TB and drug-resistant TB in animals and is a candidate for boosting the protective efficacy of the childhood BCG vaccine in humans.

  8. Low rate of fluoroquinolone resistance in Mycobacterium tuberculosis isolates from northern Tanzania.

    Science.gov (United States)

    van den Boogaard, Jossy; Semvua, Hadija H; van Ingen, Jakko; Mwaigwisya, Solomon; van der Laan, Tridia; van Soolingen, Dick; Kibiki, Gibson S; Boeree, Martin J; Aarnoutse, Rob E

    2011-08-01

    Fluoroquinolones are used in second-line treatment of tuberculosis (TB) and have a potential role in shortening TB treatment duration. The wide use of fluoroquinolones in the treatment of other infections, including respiratory tract infections in patients with (undiagnosed) active TB, could result in fluoroquinolone-resistant Mycobacterium tuberculosis. We determined the rate of fluoroquinolone resistance in M. tuberculosis isolates obtained from Tanzanian patients and linked this to previous fluoroquinolone exposure and mycobacterial resistance to rifampicin and isoniazid. A total of 291 M. tuberculosis isolates were obtained between April 2009 and June 2010 from patients with smear-positive pulmonary TB and tested for susceptibility to ciprofloxacin, moxifloxacin, rifampicin and isoniazid. Information on previous fluoroquinolone use was obtained by interviewing patients and checking their medical files. Only 2 (0.7%) of the 291 M. tuberculosis isolates were resistant to ciprofloxacin; 1 of which was intermediately resistant to moxifloxacin as well. These two isolates were susceptible to rifampicin and isoniazid. Twenty-two (8%) of the 291 patients had a history of fluoroquinolone use (median: 7 days; interquartile range: 5-10 days). The patients from whom the fluoroquinolone-resistant M. tuberculosis isolates were obtained had no known history of previous fluoroquinolone use. Our findings indicate that the rate of fluoroquinolone-resistant M. tuberculosis in Tanzanian patients with TB is low and not related to previous, brief episodes of exposure to fluoroquinolones. The findings favour future application of fluoroquinolones in TB treatment regimens of shorter duration.

  9. [MOLECULAR CHARACTERISTICS OF THE MULTIDRUG-RESISTANT MYCOBACTERIUM TUBERCULOSIS STRAINS IN THE NORTHWEST RUSSIA].

    Science.gov (United States)

    Vyazovaya, A A; Mokrousov, I V; Zhuravlev, V Yu; Solovieva, N S; Otten, T F; Manicheva, O A; Vishnevsky, B I; Narvskaya, O V

    2016-01-01

    The goal of this work was to study the genotypic characteristics of the multidrug-resistant (MDR, i.e., resistant to at least rifampicine and isoniazid) Mycobacterium tuberculosis strains isolated in 2011-2012 from tuberculosis (TB) patients in the Northwest Russia. Spoligotyping of 195 M. tuberculosis isolates identified 14 different spoligotypes and assigned isolates to the genetic families Beijing (n = 162, 83%), LAM (n = 15), H3/URAL (n = 14), as well as T, Haarlem and X. Spoligotypes SIT1 (Beijing), SIT42 (LAM) and SIT262 (H3/URAL) were the most prevalent. Irrespective to the genotype, all the isolates were resistant to streptomycin. The multidrug resistance was accompanied by the resistance to ethionamide (56%), amikacin (31%), kanamycin (40%), and capreomycin (33%). The ethambutol resistance was found in 71% (n = 115) and 42% (n = 14) of the Beijing and non-Beijing strains, respectively (p Russia continues to be dominated by the Beijing family strains.

  10. Integration of published information into a resistance-associated mutation database for Mycobacterium tuberculosis.

    Science.gov (United States)

    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

    2015-04-01

    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.

  11. Disinfectant-susceptibility of multi-drug-resistant Mycobacterium tuberculosis isolated in Japan

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    Noriko Shinoda

    2016-02-01

    Full Text Available Abstract Background Multi-drug-resistant Mycobacterium tuberculosis has been an important problem in public health around the world. However, limited information about disinfectant-susceptibility of multi-drug-resistant strain of M. tuberculosis was available. Findings We studied susceptibility of several Japanese isolates of multi-drug-resistant M. tuberculosis against disinfectants, which are commonly used in clinical and research laboratories. We selected a laboratory reference strain (H37Rv and eight Japanese isolates, containing five drug-susceptible strains and three multi-drug-resistant strains, and determined profiles of susceptibility against eight disinfectants. The M. tuberculosis strains were distinguished into two groups by the susceptibility profile. There was no relationship between multi-drug-resistance and disinfectant-susceptibility in the M. tuberculosis strains. Cresol soap and oxydol were effective against all strains we tested, regardless of drug resistance. Conclusions Disinfectant-resistance is independent from multi-drug-resistance in M. tuberculosis. Cresol soap and oxydol were effective against all strains we tested, regardless of drug resistance.

  12. Anthelmintic Avermectins Kill Mycobacterium tuberculosis, Including Multidrug-Resistant Clinical Strains

    OpenAIRE

    Lim, Leah E.; Vilchèze, Catherine; Ng, Carol; Jacobs, William R.; Ramón-García, Santiago; Thompson, Charles J

    2013-01-01

    Avermectins are a family of macrolides known for their anthelmintic activities and traditionally believed to be inactive against all bacteria. Here we report that members of the family, ivermectin, selamectin, and moxidectin, are bactericidal against mycobacterial species, including multidrug-resistant and extensively drug-resistant clinical strains of Mycobacterium tuberculosis. Avermectins are approved for clinical and veterinary uses and have documented pharmacokinetic and safety profiles....

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

    Science.gov (United States)

    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

  14. Polymorphisms in Isoniazid and Prothionamide Resistance Genes of the Mycobacterium tuberculosis Complex

    KAUST Repository

    Projahn, M.

    2011-06-27

    Sequence analyses of 74 strains that encompassed major phylogenetic lineages of the Mycobacterium tuberculosis complex revealed 10 polymorphisms in mshA (Rv0486) and four polymorphisms in inhA (Rv1484) that were not responsible for isoniazid or prothionamide resistance. Instead, some of these mutations were phylogenetically informative. This genetic diversity must be taken into consideration for drug development and for the design of molecular tests for drug resistance.

  15. pncA Mutations in Pyrazinamide-Resistant Mycobacterium tuberculosis Isolates from Northwestern Russia

    OpenAIRE

    1999-01-01

    Thirty-six pyrazinamide-resistant and eight pyrazinamide-susceptible Mycobacterium tuberculosis isolates from Russia were analyzed for their pncA mutations. Thirty-one (86.1%) of the resistant isolates had a mutation either in pncA or upstream of the gene. Twenty of the 23 different mutations found in this study had not been described earlier. pncA genotype correlated well with pyrazinamidase activity and BACTEC 460 susceptibility test results.

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

    Science.gov (United States)

    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.

  17. Meropenem-clavulanic acid has high in vitro activity against multidrug-resistant Mycobacterium tuberculosis.

    Science.gov (United States)

    Davies Forsman, L; Giske, C G; Bruchfeld, J; Schön, T; Juréen, P; Ängeby, K

    2015-01-01

    We investigated the activity of meropenem-clavulanic acid (MEM-CLA) against 68 Mycobacterium tuberculosis isolates. We included predominantly multi- and extensively drug-resistant tuberculosis (MDR/XDR-TB) isolates, since the activity of MEM-CLA for resistant isolates has previously not been studied extensively. Using Middlebrook 7H10 medium, all but four isolates showed an MIC distribution of 0.125 to 2 mg/liter for MEM-CLA, below the non-species-related breakpoint for MEM of 2 mg/liter defined by EUCAST. MEM-CLA is a potential treatment option for MDR/XDR-TB.

  18. Cutaneous Squamous Cell Carcinoma in Lupus Vulgaris Caused by Drug Resistant Mycobacterium Tuberculosis.

    Science.gov (United States)

    Kumaran, Muthu S; Narang, Tarun; Jitendriya, Madhukara; Tirumale, Rajalakshmi; Manjunath, Suraj; Savio, Jayanthi

    2017-01-01

    Tuberculosis (TB) is still a major public health problem in the world, with many factors contributing to this burden, including poor living conditions, overcrowding, poverty, malnutrition, illiteracy, and rapid spread of human immunodeficiency virus infection. Cutaneous tuberculosis is a less common form of extrapulmonary tuberculosis, and in this paucibacillary form the diagnosis depends on histopathology, tuberculin positivity, and response to treatment. The diagnosis is even more difficult in cases with drug resistant Mycobacterium tuberculosis due to lack of awareness and lack of facilities to diagnose drug resistant tuberculosis. In this article, we describe an unusual case of multidrug resistant lupus vulgaris (LV), in a 34-year-old male who responded to anti-tubercular treatment (ATT) initially, but developed recurrent disease which failed to respond to standard four-drug ATT; subsequently, tissue culture showed growth of multidrug resistant M. tuberculosis. Subsequently, he also developed cutaneous squamous cell carcinoma. This article aims to exemplify a grave complication that can occur in long-standing case of LV, the limitations faced by clinicians in developing countries where tuberculosis is endemic, and classical methods of proving drug resistance are generally unavailable or fail.

  19. Drug-Susceptible Mycobacterium tuberculosis Beijing Genotype Does Not Develop Mutation-Conferred Resistance to Rifampin at an Elevated Rate

    OpenAIRE

    2003-01-01

    The Mycobacterium tuberculosis Beijing genotype has drawn attention because it is often strongly associated with multidrug-resistant tuberculosis (MDR-TB). A possible reason is that the Beijing strains may have an enhanced capacity to develop drug resistance. In this study, we used the Luria-Delbrück fluctuation test to investigate whether strains of Beijing and non-Beijing genotypes exhibit differences in the acquisition of drug resistance. The M. tuberculosis reference strain H37Rv and 12 f...

  20. Effect of mutation and genetic background on drug resistance in Mycobacterium tuberculosis.

    Science.gov (United States)

    Fenner, Lukas; Egger, Matthias; Bodmer, Thomas; Altpeter, Ekkehardt; Zwahlen, Marcel; Jaton, Katia; Pfyffer, Gaby E; Borrell, Sonia; Dubuis, Olivier; Bruderer, Thomas; Siegrist, Hans H; Furrer, Hansjakob; Calmy, Alexandra; Fehr, Jan; Stalder, Jesica Mazza; Ninet, Béatrice; Böttger, Erik C; Gagneux, Sebastien

    2012-06-01

    Bacterial factors may contribute to the global emergence and spread of drug-resistant tuberculosis (TB). Only a few studies have reported on the interactions between different bacterial factors. We studied drug-resistant Mycobacterium tuberculosis isolates from a nationwide study conducted from 2000 to 2008 in Switzerland. We determined quantitative drug resistance levels of first-line drugs by using Bactec MGIT-960 and drug resistance genotypes by sequencing the hot-spot regions of the relevant genes. We determined recent transmission by molecular methods and collected clinical data. Overall, we analyzed 158 isolates that were resistant to isoniazid, rifampin, or ethambutol, 48 (30.4%) of which were multidrug resistant. Among 154 isoniazid-resistant strains, katG mutations were associated with high-level and inhA promoter mutations with low-level drug resistance. Only katG(S315T) (65.6% of all isoniazid-resistant strains) and inhA promoter -15C/T (22.7%) were found in molecular clusters. M. tuberculosis lineage 2 (includes Beijing genotype) was associated with any drug resistance (adjusted odds ratio [OR], 3.0; 95% confidence interval [CI], 1.7 to 5.6; P mutations (OR, 6.4; 95% CI, 2.0 to 20.7; P = 0.002). We found that the genetic strain background influences the level of isoniazid resistance conveyed by particular mutations (interaction tests of drug resistance mutations across all lineages; P tuberculosis drug resistance mutations were associated with various levels of drug resistance and transmission, and M. tuberculosis lineages were associated with particular drug resistance-conferring mutations and phenotypic drug resistance. Our study also supports a role for epistatic interactions between different drug resistance mutations and strain genetic backgrounds in M. tuberculosis drug resistance.

  1. Association between Mycobacterium tuberculosis complex phylogenetic lineage and acquired drug resistance.

    Directory of Open Access Journals (Sweden)

    Courtney M Yuen

    Full Text Available BACKGROUND: Development of resistance to antituberculosis drugs during treatment (i.e., acquired resistance can lead to emergence of resistant strains and consequent poor clinical outcomes. However, it is unknown whether Mycobacterium tuberculosis complex species and lineage affects the likelihood of acquired resistance. METHODS: We analyzed data from the U.S. National Tuberculosis Surveillance System and National Tuberculosis Genotyping Service for tuberculosis cases during 2004-2011 with assigned species and lineage and both initial and final drug susceptibility test results. We determined univariate associations between species and lineage of Mycobacterium tuberculosis complex bacteria and acquired resistance to isoniazid, rifamycins, fluoroquinolones, and second-line injectables. We used Poisson regression with backward elimination to generate multivariable models for acquired resistance to isoniazid and rifamycins. RESULTS: M. bovis was independently associated with acquired resistance to isoniazid (adjusted prevalence ratio = 8.46, 95% CI 2.96-24.14 adjusting for HIV status, and with acquired resistance to rifamycins (adjusted prevalence ratio = 4.53, 95% CI 1.29-15.90 adjusting for homelessness, HIV status, initial resistance to isoniazid, site of disease, and administration of therapy. East Asian lineage was associated with acquired resistance to fluoroquinolones (prevalence ratio = 6.10, 95% CI 1.56-23.83. CONCLUSIONS: We found an association between mycobacterial species and lineage and acquired drug resistance using U.S. surveillance data. Prospective clinical studies are needed to determine the clinical significance of these findings, including whether rapid genotyping of isolates at the outset of treatment may benefit patient management.

  2. Pre-existing isoniazid resistance, but not the genotype of Mycobacterium tuberculosis drives rifampicin resistance codon preference in vitro.

    NARCIS (Netherlands)

    Bergval, I.; Kwok, B.; Schuitema, A.; Kremer, K.; Soolingen, D. van; Klatser, P.; Anthony, R.

    2012-01-01

    Both the probability of a mutation occurring and the ability of the mutant to persist will influence the distribution of mutants that arise in a population. We studied the interaction of these factors for the in vitro selection of rifampicin (RIF)-resistant mutants of Mycobacterium tuberculosis. We

  3. Clinical Concentrations of Thioridazine Kill Intracellular Multidrug-Resistant Mycobacterium tuberculosis

    Science.gov (United States)

    Ordway, Diane; Viveiros, Miguel; Leandro, Clara; Bettencourt, Rosário; Almeida, Josefina; Martins, Marta; Kristiansen, Jette E.; Molnar, Joseph; Amaral, Leonard

    2003-01-01

    The phenothiazines chlorpromazine (CPZ) and thioridazine (TZ) have equal in vitro activities against antibiotic-sensitive and -resistant Mycobacterium tuberculosis. These compounds have not been used as anti-M. tuberculosis agents because their in vitro activities take place at concentrations which are beyond those that are clinically achievable. In addition, chronic administration of CPZ produces frequent severe side effects. Because CPZ has been shown to enhance the killing of intracellular M. tuberculosis at concentrations in the medium that are clinically relevant, we have investigated whether TZ, a phenothiazine whose negative side effects are less frequent and serious than those associated with CPZ, kills M. tuberculosis organisms that have been phagocytosed by human macrophages, which have nominal killing activities against these bacteria. Both CPZ and TZ killed intracellular antibiotic-sensitive and -resistant M. tuberculosis organisms when they were used at concentrations in the medium well below those present in the plasma of patients treated with these agents. These concentrations in vitro were not toxic to the macrophage, nor did they affect in vitro cellular immune processes. TZ thus appears to be a serious candidate for the management of a freshly diagnosed infection of pulmonary tuberculosis or as an adjunct to conventional antituberculosis therapy if the patient originates from an area known to have a high prevalence of multidrug-resistant M. tuberculosis isolates. Nevertheless, we must await the outcomes of clinical trials to determine whether TZ itself may be safely and effectively used as an antituberculosis agent. PMID:12604522

  4. Interplay between Mutations and Efflux in Drug Resistant Clinical Isolates of Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Miguel Viveiros

    2017-04-01

    Full Text Available Numerous studies show efflux as a universal bacterial mechanism contributing to antibiotic resistance and also that the activity of the antibiotics subject to efflux can be enhanced by the combined use of efflux inhibitors. Nevertheless, the contribution of efflux to the overall drug resistance levels of clinical isolates of Mycobacterium tuberculosis is poorly understood and still is ignored by many. Here, we evaluated the contribution of drug efflux plus target-gene mutations to the drug resistance levels in clinical isolates of M. tuberculosis. A panel of 17 M. tuberculosis clinical strains were characterized for drug resistance associated mutations and antibiotic profiles in the presence and absence of efflux inhibitors. The correlation between the effect of the efflux inhibitors and the resistance levels was assessed by quantitative drug susceptibility testing. The bacterial growth/survival vs. growth inhibition was analyzed through the comparison between the time of growth in the presence and absence of an inhibitor. For the same mutation conferring antibiotic resistance, different MICs were observed and the different resistance levels found could be reduced by efflux inhibitors. Although susceptibility was not restored, the results demonstrate the existence of a broad-spectrum synergistic interaction between antibiotics and efflux inhibitors. The existence of efflux activity was confirmed by real-time fluorometry. Moreover, the efflux pump genes mmr, mmpL7, Rv1258c, p55, and efpA were shown to be overexpressed in the presence of antibiotics, demonstrating the contribution of these efflux pumps to the overall resistance phenotype of the M. tuberculosis clinical isolates studied, independently of the genotype of the strains. These results showed that the drug resistance levels of multi- and extensively-drug resistant M. tuberculosis clinical strains are a combination between drug efflux and the presence of target-gene mutations, a reality

  5. Comparative study on genotypic and phenotypic second-line drug resistance testing of Mycobacterium tuberculosis complex isolates.

    NARCIS (Netherlands)

    Ingen, J. van; Simons, S.O.; Zwaan, R. de; Laan, T. van der; Kamst-van Agterveld, M.; Boeree, M.J.; Soolingen, D. van

    2010-01-01

    The mycobacterium growth indicator tube (MGIT960) automated liquid medium testing method is becoming the international gold standard for second-line drug susceptibility testing of multidrug- and extensively drug-resistant Mycobacterium tuberculosis complex isolates. We performed a comparative study

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

    Science.gov (United States)

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

  7. Mycobacterium tuberculosis Multidrug Resistant Strain M Induces an Altered Activation of Cytotoxic CD8+ T Cells

    OpenAIRE

    Laura Geffner; Juan Ignacio Basile; Noemí Yokobori; Denise Kviatcovsky; Carmen Sabio y García; Viviana Ritacco; Beatriz López; María del Carmen Sasiain; Silvia de la Barrera

    2014-01-01

    In human tuberculosis (TB), CD8+ T cells contribute to host defense by the release of Th1 cytokines and the direct killing of Mycobacterium tuberculosis (Mtb)-infected macrophages via granule exocytosis pathway or the engagement of receptors on target cells. Previously we demonstrated that strain M, the most prevalent multidrug-resistant (MDR) Mtb strain in Argentine, is a weak inducer of IFN-γ and elicits a remarkably low CD8-dependent cytotoxic T cell activity (CTL). In contrast, the closel...

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

    Directory of Open Access Journals (Sweden)

    Mohd Zakihalani A. Halim

    2016-03-01

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

  9. Genetic diversity, transmission dynamics and drug resistance of Mycobacterium tuberculosis in Angola

    Science.gov (United States)

    Perdigão, João; Clemente, Sofia; Ramos, Jorge; Masakidi, Pedro; Machado, Diana; Silva, Carla; Couto, Isabel; Viveiros, Miguel; Taveira, Nuno; Portugal, Isabel

    2017-01-01

    Tuberculosis (TB) poses a serious public health problem in Angola. No surveillance data on drug resistance is available and nothing is known regarding the genetic diversity and population structure of circulating Mycobacterium tuberculosis strains. Here, we have genotyped and evaluated drug susceptibility of 89 Mycobacterium tuberculosis clinical isolates from Luanda. Thirty-three different spoligotype profiles corresponding to 24 different Shared International Types (SIT) and 9 orphan profiles were detected. SIT 20 (LAM1) was the most prevalent (n = 16, 18.2%) followed by SIT 42 (LAM9; n = 15, 17.1%). Overall, the M. tuberculosis population structure in this sample was dominated by LAM (64.8%) and T (33.0%) strains. Twenty-four-loci MIRU-VNTR analysis revealed that a total of 13 isolates were grouped in 5 distinct clusters. Drug susceptibility data showed that 22 (24.7%) of the 89 clinical isolates were resistant to one or more antibacillary drugs of which 4 (4.5%) were multidrug resistant. In conclusion, this study demonstrates a high predominance of LAM strains circulating in the Luanda setting and the presence of recent transmission events. The rate and the emergence dynamics of drug resistant TB found in this sample are significant and highlight the need of further studies specifically focused on MDR-TB transmission. PMID:28230095

  10. Genomic diversity of drug-resistant Mycobacterium tuberculosis isolates in Lisbon Portugal: Towards tuberculosis genomic epidemiology

    KAUST Repository

    Perdigão, João

    2015-03-01

    Multidrug- (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) present a challenge to disease control and elimination goals. Lisbon, Portugal, has a high TB incidence rate and unusual and successful XDR-TB strains that have been found in circulation for almost two decades. For the last 20. years, a continued circulation of two phylogenetic clades, Lisboa3 and Q1, which are highly associated with MDR and XDR, have been observed. In recent years, these strains have been well characterized regarding the molecular basis of drug resistance and have been inclusively subjected to whole genome sequencing (WGS). Researchers have been studying the genomic diversity of strains circulating in Lisbon and its genomic determinants through cutting-edge next generation sequencing. An enormous amount of whole genome sequence data are now available for the most prevalent and clinically relevant strains circulating in Lisbon.It is the persistence, prevalence and rapid evolution towards drug resistance that has prompted researchers to investigate the properties of these strains at the genomic level and in the future at a global transcriptomic level. Seventy Mycobacterium tuberculosis (MTB) isolates, mostly recovered in Lisbon, were genotyped by 24-. loci Mycobacterial Interspersed Repetitive Unit - Variable Number of Tandem Repeats (MIRU-VNTR) and the genomes sequenced using a next generation sequencing platform - Illumina HiSeq 2000.The genotyping data revealed three major clusters associated with MDR-TB (Lisboa3-A, Lisboa3-B and Q1), two of which are associated with XDR-TB (Lisboa3-B and Q1), whilst the genomic data contributed to elucidating the phylogenetic positioning of circulating MDR-TB strains, showing a high predominance of a single SNP cluster group 5. Furthermore, a genome-wide phylogeny analysis from these strains, together with 19 publicly available genomes of MTB clinical isolates, revealed two major clades responsible for MDR/XDR-TB in the region: Lisboa3 and Q

  11. Molecular characterisation of isoniazid- and rifampicin-resistant Mycobacterium tuberculosis in Central Tunisia.

    Science.gov (United States)

    Ben Kahla, I; Marzouk, M; Henry, M; Bedotto, M; Cohen-Bacrie, S; Ben Selma, W; Boukadida, J; Drancourt, M

    2011-12-01

    The aim of our study was to genotypically characterise isoniazid (INH) and rifampicin (RMP) resistant Mycobacterium tuberculosis isolates in Sousse, Central Tunisia, using DNA sequencing and multispacer sequence typing (MST). The results show that 27/28 (96.4%) and 1/28 (3.6%) INH-resistant isolates yielded respectively the kat G S315T and the inh A - 15C → T mutations. Two-thirds of RMP-resistant isolates yielded the rpo B D516V mutation and one sixth yielded either H526D or S531L mutations. Genotyping analysis revealed the multiclonal spread of drug-resistant isolates in Central Tunisia. Data presented here complete the previously published map of resistant M. tuberculosis isolates and highlight their regional disparity in Tunisia.

  12. Mycobacterium tuberculosis drug-resistance testing: challenges, recent developments and perspectives.

    Science.gov (United States)

    Schön, T; Miotto, P; Köser, C U; Viveiros, M; Böttger, E; Cambau, E

    2017-03-01

    Drug-resistance testing, or antimicrobial susceptibility testing (AST), is mandatory for Mycobacterium tuberculosis in cases of failure on standard therapy. We reviewed the different methods and techniques of phenotypic and genotypic approaches. Although multiresistant and extensively drug-resistant (MDR/XDR) tuberculosis is present worldwide, AST for M. tuberculosis (AST-MTB) is still mainly performed according to the resources available rather than the drug-resistance rates. Phenotypic methods, i.e. culture-based AST, are commonly used in high-income countries to confirm susceptibility of new cases of tuberculosis. They are also used to detect resistance in tuberculosis cases with risk factors, in combination with genotypic tests. In low-income countries, genotypic methods screening hot-spot mutations known to confer resistance were found to be easier to perform because they avoid the culture and biosafety constraint. Given that genotypic tests can rapidly detect the prominent mechanisms of resistance, such as the rpoB mutation for rifampicin resistance, we are facing new challenges with the observation of false-resistance (mutations not conferring resistance) and false-susceptibility (mutations different from the common mechanism) results. Phenotypic and genotypic approaches are therefore complementary for obtaining a high sensitivity and specificity for detecting drug resistances and susceptibilities to accurately predict MDR/XDR cure and to gather relevant data for resistance surveillance. Although AST-MTB was established in the 1960s, there is no consensus reference method for MIC determination against which the numerous AST-MTB techniques can be compared. This information is necessary for assessing in vitro activity and setting breakpoints for future anti-tuberculosis agents. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  13. Modification of rifamycin polyketide backbone leads to improved drug activity against rifampicin-resistant Mycobacterium tuberculosis.

    Science.gov (United States)

    Nigam, Aeshna; Almabruk, Khaled H; Saxena, Anjali; Yang, Jongtae; Mukherjee, Udita; Kaur, Hardeep; Kohli, Puneet; Kumari, Rashmi; Singh, Priya; Zakharov, Lev N; Singh, Yogendra; Mahmud, Taifo; Lal, Rup

    2014-07-25

    Rifamycin B, a product of Amycolatopsis mediterranei S699, is the precursor of clinically used antibiotics that are effective against tuberculosis, leprosy, and AIDS-related mycobacterial infections. However, prolonged usage of these antibiotics has resulted in the emergence of rifamycin-resistant strains of Mycobacterium tuberculosis. As part of our effort to generate better analogs of rifamycin, we substituted the acyltransferase domain of module 6 of rifamycin polyketide synthase with that of module 2 of rapamycin polyketide synthase. The resulting mutants (rifAT6::rapAT2) of A. mediterranei S699 produced new rifamycin analogs, 24-desmethylrifamycin B and 24-desmethylrifamycin SV, which contained modification in the polyketide backbone. 24-Desmethylrifamycin B was then converted to 24-desmethylrifamycin S, whose structure was confirmed by MS, NMR, and X-ray crystallography. Subsequently, 24-desmethylrifamycin S was converted to 24-desmethylrifampicin, which showed excellent antibacterial activity against several rifampicin-resistant M. tuberculosis strains.

  14. Molecular Genetic Analysis of Multi-drug Resistance in Indian Isolates of Mycobacterium tuberculosis

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    Noman Siddiqi

    1998-09-01

    Full Text Available A total of 116 isolates from patients attending the out-patient department at the All India Institute of Medical Sciences, New Delhi and the New Delhi Tuberculosis Centre, New Delhi, India were collected. They were analyzed for resistance to drugs prescribed in the treatment for tuberculosis. The drug resistance was initially determined by microbiological techniques. The Bactec 460TB system was employed to determine the type and level of resistance in each isolate. The isolates were further characterized at molecular level. The multi-drug loci corresponding to rpo b, gyr A, kat G were studied for mutation(s by the polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP technique. The SSCP positive samples were sequenced to characterize the mutations in rpo b, and gyr A loci. While previously reported mutations in the gyr A and rpo b loci were found to be present, several novel mutations were also scored in the rpo b locus. Interestingly, analysis of the gyr A locus showed the presence of point mutation(s that could not be detected by PCR-SSCP. Furthermore, rifampicin resistance was found to be an important marker for checking multi-drug resistance (MDR in clinical isolates of Mycobacterium tuberculosis. This is the first report on molecular genetic analysis of MDR tuberculosis one from India, highlights the increasing incidence of MDR in the Indian isolates of M. tuberculosis.

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

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

    2015-07-01

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

  16. Detection of Mycobacterium tuberculosis resistance mutations to rifampin and isoniazid by real-time PCR

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    Hristea A

    2010-01-01

    Full Text Available Objective: The objective of our study was to evaluate the use of a real-time polymerase chain reaction (PCR-based technique for the prediction of phenotypic resistance of Mycobacterium tuberculosis. Materials and Methods: We tested 67 M tuberculosis strains (26 drug resistant and 41 drug susceptible using a method recommended for the LightCycler platform. The susceptibility testing was performed by the absolute concentration method. For rifampin resistance, two regions of the rpoB gene were targeted, while for identification of isoniazid resistance, we searched for mutations in katG and inhA genes. Results: The sensitivity and specificity of this method for rapid detection of mutations for isoniazid resistance were 96% (95% CI: 88% to 100% and 95% (95% CI: 89% to 100%, respectively. For detection of rifampin resistance, the sensitivity and specificity were 92% (95% CI: 81% to 100% and 74% (95% CI: 61% to 87%, respectively. The main isoniazid resistance mechanism identified in our isolates is related to changes in the katG gene that encodes catalase. We found that for rifampin resistance the concordance between the predicted and observed phenotype was less than satisfactory. Conclusions: Using this method, the best accuracy for genotyping compared with phenotypic resistance testing was obtained for detecting isoniazid resistance mutations. Although real-time PCR assay may be a valuable diagnostic tool, it is not yet completely satisfactory for detection of drug resistance mutations in M tuberculosis.

  17. Efflux pump gene expression in multidrug-resistant Mycobacterium tuberculosis clinical isolates.

    Science.gov (United States)

    Li, Guilian; Zhang, Jingrui; Guo, Qian; Jiang, Yi; Wei, Jianhao; Zhao, Li-li; Zhao, Xiuqin; Lu, Jianxin; Wan, Kanglin

    2015-01-01

    Isoniazid (INH) and rifampicin (RIF) are the two most effective drugs in tuberculosis therapy. Understanding the molecular mechanisms of resistance to these two drugs is essential to quickly diagnose multidrug-resistant (MDR) tuberculosis and extensive drug-resistant tuberculosis. Nine clinical Mycobacterium tuberculosis isolates resistant to only INH and RIF and 10 clinical pan-sensitive isolates were included to evaluate the expression of 20 putative drug efflux pump genes and sequence mutations in rpoB (RIF), katG (INH), the inhA promoter (INH), and oxyR-ahpC (INH). Nine and three MDR isolates were induced to overexpress efflux pump genes by INH and RIF, respectively. Eight and two efflux pump genes were induced to overexpress by INH and RIF in MDR isolates, respectively. drrA, drrB, efpA, jefA (Rv2459), mmr, Rv0849, Rv1634, and Rv1250 were overexpressed under INH or RIF stress. Most efflux pump genes were overexpressed under INH stress in a MDR isolates that carried the wild-type katG, inhA, and oxyR-ahpC associated with INH resistance than in those that carried mutations. The expression levels of 11 genes (efpA, Rv0849, Rv1250, P55 (Rv1410c), Rv1634, Rv2994, stp, Rv2459, pstB, drrA, and drrB) without drug inducement were significantly higher (P < 0.05) in nine MDR isolates than in 10 pan-sensitive isolates. In conclusion, efflux pumps may play an important role in INH acquired resistance in MDR M. tuberculosis, especially in those strains having no mutations in genes associated with INH resistance; basal expression levels of some efflux pump genes are higher in MDR isolates than in pan-sensitive isolates and the basal expressional differences may be helpful to diagnose and treat resistant tuberculosis.

  18. Efflux pump gene expression in multidrug-resistant Mycobacterium tuberculosis clinical isolates.

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    Guilian Li

    Full Text Available Isoniazid (INH and rifampicin (RIF are the two most effective drugs in tuberculosis therapy. Understanding the molecular mechanisms of resistance to these two drugs is essential to quickly diagnose multidrug-resistant (MDR tuberculosis and extensive drug-resistant tuberculosis. Nine clinical Mycobacterium tuberculosis isolates resistant to only INH and RIF and 10 clinical pan-sensitive isolates were included to evaluate the expression of 20 putative drug efflux pump genes and sequence mutations in rpoB (RIF, katG (INH, the inhA promoter (INH, and oxyR-ahpC (INH. Nine and three MDR isolates were induced to overexpress efflux pump genes by INH and RIF, respectively. Eight and two efflux pump genes were induced to overexpress by INH and RIF in MDR isolates, respectively. drrA, drrB, efpA, jefA (Rv2459, mmr, Rv0849, Rv1634, and Rv1250 were overexpressed under INH or RIF stress. Most efflux pump genes were overexpressed under INH stress in a MDR isolates that carried the wild-type katG, inhA, and oxyR-ahpC associated with INH resistance than in those that carried mutations. The expression levels of 11 genes (efpA, Rv0849, Rv1250, P55 (Rv1410c, Rv1634, Rv2994, stp, Rv2459, pstB, drrA, and drrB without drug inducement were significantly higher (P < 0.05 in nine MDR isolates than in 10 pan-sensitive isolates. In conclusion, efflux pumps may play an important role in INH acquired resistance in MDR M. tuberculosis, especially in those strains having no mutations in genes associated with INH resistance; basal expression levels of some efflux pump genes are higher in MDR isolates than in pan-sensitive isolates and the basal expressional differences may be helpful to diagnose and treat resistant tuberculosis.

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

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    Srivastava S

    2008-01-01

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

  20. Differential expression of putative drug resistance genes in Mycobacterium tuberculosis clinical isolates.

    Science.gov (United States)

    González-Escalante, Laura; Peñuelas-Urquides, Katia; Said-Fernández, Salvador; Silva-Ramírez, Beatriz; Bermúdez de León, Mario

    2015-12-01

    Understanding drug resistance in Mycobacterium tuberculosis requires an integrated analysis of strain lineages, mutations and gene expression. Previously, we reported the differential expression of esxG, esxH, infA, groES, rpmI, rpsA and lipF genes in a sensitive M. tuberculosis strain and in a multidrug-resistant clinical isolate. Here, we have evaluated the expression of these genes in 24 clinical isolates that belong to different lineages and have different drug resistance profiles. In vitro, growth kinetics analysis showed no difference in the growth of the clinical isolates, and thus drug resistance occurred without a fitness cost. However, a quantitative reverse transcription PCR analysis of gene expression revealed high variability among the clinical isolates, including those with similar drug resistance profiles. Due to the complexity of gene regulation pathways and the wide diversity of M. tuberculosis lineages, the use of gene expression as a molecular signature for drug resistance is not straightforward. Therefore, we recommend that the expression of M. tuberculosis genes be performed individually, and baseline expression levels should be verified among several different clinical isolates, before any further applications of these findings.

  1. Detection of Mycobacterium isolates with different methods and their resistance ratios against anti-tuberculosis drugs

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    Mustafa Altındiş, Zafer Çetinkaya, Raike Kalaycı, Ihsan H Ciftçi, Alpaslan Arslan, Orhan C. Aktepe

    2011-06-01

    Full Text Available Objectives: The aim of the present study was to evaluate the efficacy (recovery rate, time to detection and Drug SusceptibilityTests –DST- of Mycobacteria-only B460 of new colorimetric medium, Dio-TK and to compare it with routinely used conventional media, Lowenstein Jensen (LJ and Bactec 460 TB culture system.Materials and methods: Totally 901 clinic specimens were investigated for assignment of tuberculosis by Ehrlich-Ziehl-Nielsen smear strain method, Lowenstein-Jensen, BACTEC 460TB and Dio-TK medium culture systems.Results: Nineteen of 901 clinic specimens (2.1% were positive by any of these methods. 17 (89.5% of these specimens positive found by smear strain method, 17 (89.5% by Lowenstein-Jensen, 19 (100% by BACTEC 460TB and 14 (73.7% by Dio-TK medium. NAP and Niacin identification tests were applied to Mycobacterium strains. 12 (63.1% of 19 isolates were identified as M.tuberculosis complex and 7 (36.9% were identified as Mycobacterium other than tuberculosis (MOTT bacilli. 10 (83.3% of 12 M.tuberculosis complex strains were not resistant to any major drug. But one of 2 isolate was resistant to streptomycin and the other one isolate was resistant to both streptomycin and isoniazid.Conclusion: Our data suggest that some advantages (such as an early detection and differentiation mycobacterium growth from contamination of the Dio-TK CS over other mycobacterial culture systems make it a practical and rapid system for daily use, and a suitable alternative to other currently available solid media, such as LJ, for detection time of mycobacteria and DST. J Microbiol Infect Dis 2011;1 (1 :5-9.

  2. Fluoroquinolone interactions with Mycobacterium tuberculosis gyrase: Enhancing drug activity against wild-type and resistant gyrase

    Science.gov (United States)

    Aldred, Katie J.; Kerns, Robert J.; Berger, James M.; Osheroff, Neil

    2016-01-01

    Mycobacterium tuberculosis is a significant source of global morbidity and mortality. Moxifloxacin and other fluoroquinolones are important therapeutic agents for the treatment of tuberculosis, particularly multidrug-resistant infections. To guide the development of new quinolone-based agents, it is critical to understand the basis of drug action against M. tuberculosis gyrase and how mutations in the enzyme cause resistance. Therefore, we characterized interactions of fluoroquinolones and related drugs with WT gyrase and enzymes carrying mutations at GyrAA90 and GyrAD94. M. tuberculosis gyrase lacks a conserved serine that anchors a water–metal ion bridge that is critical for quinolone interactions with other bacterial type II topoisomerases. Despite the fact that the serine is replaced by an alanine (i.e., GyrAA90) in M. tuberculosis gyrase, the bridge still forms and plays a functional role in mediating quinolone–gyrase interactions. Clinically relevant mutations at GyrAA90 and GyrAD94 cause quinolone resistance by disrupting the bridge–enzyme interaction, thereby decreasing drug affinity. Fluoroquinolone activity against WT and resistant enzymes is enhanced by the introduction of specific groups at the C7 and C8 positions. By dissecting fluoroquinolone–enzyme interactions, we determined that an 8-methyl-moxifloxacin derivative induces high levels of stable cleavage complexes with WT gyrase and two common resistant enzymes, GyrAA90V and GyrAD94G. 8-Methyl-moxifloxacin was more potent than moxifloxacin against WT M. tuberculosis gyrase and displayed higher activity against the mutant enzymes than moxifloxacin did against WT gyrase. This chemical biology approach to defining drug–enzyme interactions has the potential to identify novel drugs with improved activity against tuberculosis. PMID:26792518

  3. Fluoroquinolone interactions with Mycobacterium tuberculosis gyrase: Enhancing drug activity against wild-type and resistant gyrase.

    Science.gov (United States)

    Aldred, Katie J; Blower, Tim R; Kerns, Robert J; Berger, James M; Osheroff, Neil

    2016-02-16

    Mycobacterium tuberculosis is a significant source of global morbidity and mortality. Moxifloxacin and other fluoroquinolones are important therapeutic agents for the treatment of tuberculosis, particularly multidrug-resistant infections. To guide the development of new quinolone-based agents, it is critical to understand the basis of drug action against M. tuberculosis gyrase and how mutations in the enzyme cause resistance. Therefore, we characterized interactions of fluoroquinolones and related drugs with WT gyrase and enzymes carrying mutations at GyrA(A90) and GyrA(D94). M. tuberculosis gyrase lacks a conserved serine that anchors a water-metal ion bridge that is critical for quinolone interactions with other bacterial type II topoisomerases. Despite the fact that the serine is replaced by an alanine (i.e., GyrA(A90)) in M. tuberculosis gyrase, the bridge still forms and plays a functional role in mediating quinolone-gyrase interactions. Clinically relevant mutations at GyrA(A90) and GyrA(D94) cause quinolone resistance by disrupting the bridge-enzyme interaction, thereby decreasing drug affinity. Fluoroquinolone activity against WT and resistant enzymes is enhanced by the introduction of specific groups at the C7 and C8 positions. By dissecting fluoroquinolone-enzyme interactions, we determined that an 8-methyl-moxifloxacin derivative induces high levels of stable cleavage complexes with WT gyrase and two common resistant enzymes, GyrA(A90V) and GyrA(D94G). 8-Methyl-moxifloxacin was more potent than moxifloxacin against WT M. tuberculosis gyrase and displayed higher activity against the mutant enzymes than moxifloxacin did against WT gyrase. This chemical biology approach to defining drug-enzyme interactions has the potential to identify novel drugs with improved activity against tuberculosis.

  4. Phenotypic and Genotypic Analysis of Multidrug-Resistant Mycobacterium tuberculosis Isolates from Sudanese Patients

    Science.gov (United States)

    Salih, Mohamed Ahmed; Ali, Manasik; EL-Zaki, Salah-Eldin; Abuzeid, Nadir; Elgadi, Zeinab Abubaker Mohammed; Altayb, Hisham N.; Elegail, Asrar M. A.; Ibrahim, Nuha Y.; Elamin, Bahaeldin K.

    2017-01-01

    Background. Currently, mutations in rpoB, KatG, and rrs genes and inhA promoter were considered to be involved in conferring resistance to rifampicin, isoniazid, and streptomycin in Mycobacterium tuberculosis (MTB). Objective. The aims of this study were to detect the prevalence of first-line tuberculosis (TB) drug resistance among a group of previously treated and newly detected TB patients, to determine the association between prevalence of multidrug resistance (MDR) and demographic information (age and sex), to explain genes correlated with MDR Mycobacterium tuberculosis, and to characterize MTB via 16S ribosomal RNA (16S rRNA) analysis. Methods. A hundred MTB isolates from Sudanese pulmonary TB patients were included in the study. The proportional method of drug susceptibility test was carried out on Löwenstein-Jensen media. Multiplex PCR of rpoB and KatG genes and inhA promoter was conducted; then rrs genes were amplified by conventional PCR and were sequenced. The sequences of the PCR product were compared with known rrs gene sequences in the GenBank database by multiple sequence alignment tools. Result. The prevalence of MDR was 14.7% among old cases and 5.3% among newly diagnosed cases. Conclusion. Mutations in rrs could be considered as a diagnostic marker. PMID:28197340

  5. Phenotypic and Genotypic Analysis of Multidrug-Resistant Mycobacterium tuberculosis Isolates from Sudanese Patients

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    Solima M. A. Sabeel

    2017-01-01

    Full Text Available Background. Currently, mutations in rpoB, KatG, and rrs genes and inhA promoter were considered to be involved in conferring resistance to rifampicin, isoniazid, and streptomycin in Mycobacterium tuberculosis (MTB. Objective. The aims of this study were to detect the prevalence of first-line tuberculosis (TB drug resistance among a group of previously treated and newly detected TB patients, to determine the association between prevalence of multidrug resistance (MDR and demographic information (age and sex, to explain genes correlated with MDR Mycobacterium tuberculosis, and to characterize MTB via 16S ribosomal RNA (16S rRNA analysis. Methods. A hundred MTB isolates from Sudanese pulmonary TB patients were included in the study. The proportional method of drug susceptibility test was carried out on Löwenstein-Jensen media. Multiplex PCR of rpoB and KatG genes and inhA promoter was conducted; then rrs genes were amplified by conventional PCR and were sequenced. The sequences of the PCR product were compared with known rrs gene sequences in the GenBank database by multiple sequence alignment tools. Result. The prevalence of MDR was 14.7% among old cases and 5.3% among newly diagnosed cases. Conclusion. Mutations in rrs could be considered as a diagnostic marker.

  6. Structural insights into the quinolone resistance mechanism of Mycobacterium tuberculosis DNA gyrase.

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    Jérémie Piton

    Full Text Available Mycobacterium tuberculosis DNA gyrase, an indispensable nanomachine involved in the regulation of DNA topology, is the only type II topoisomerase present in this organism and is hence the sole target for quinolone action, a crucial drug active against multidrug-resistant tuberculosis. To understand at an atomic level the quinolone resistance mechanism, which emerges in extensively drug resistant tuberculosis, we performed combined functional, biophysical and structural studies of the two individual domains constituting the catalytic DNA gyrase reaction core, namely the Toprim and the breakage-reunion domains. This allowed us to produce a model of the catalytic reaction core in complex with DNA and a quinolone molecule, identifying original mechanistic properties of quinolone binding and clarifying the relationships between amino acid mutations and resistance phenotype of M. tuberculosis DNA gyrase. These results are compatible with our previous studies on quinolone resistance. Interestingly, the structure of the entire breakage-reunion domain revealed a new interaction, in which the Quinolone-Binding Pocket (QBP is blocked by the N-terminal helix of a symmetry-related molecule. This interaction provides useful starting points for designing peptide based inhibitors that target DNA gyrase to prevent its binding to DNA.

  7. Detection of Ethambutol - resistant Mycobacterium tuberculosis strains by MAS-PCR method and comparison with Proportion

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    M. Asgharzadeh

    2007-01-01

    Full Text Available Abstract Background and purpose: Ethambutol (EMB is one of the first - line drugs used for anti-tubercular therapy but resistance to this medicine is developed in many parts of the world. EMB resistant strains commonly have embB mutations. Purpose of this research was detection of EMB-resistant Mycobactercium tuberculosis strains isolated from patients by MAS-PCR method and comparison with Proportion procedure.Materials and Methods: One hundred and twenty M. tuberculosis strains were isolated from patients with tulerculosis in Tabriz TB research center. Susceptibility testing to EMB was performed by the Proportion method. DNA was isolated from cultivated cells by SDS-proteinase K modified method. Isolated DNA was used as the template for PCR reaction.Results: One hundred and sixteen strains were susceptible to EMB and 4 (3.33% strains were resistant to EMB. All EMB resistant strains were multidrug-resistant. The MAS-PCR method was used to evaluate of mutation in the embB306 codon. Mutation was seen at the embB306 codon in all resistant strains to ethambutol.Conclusion: The results showed that MAS-PCR method can be used as a simple and rapid procedure for detecting EMB-resistance in Mycobacterium tuberculosis strains.

  8. Contribution of Efflux to the Emergence of Isoniazid and Multidrug Resistance in Mycobacterium tuberculosis

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    Machado, Diana; Couto, Isabel; Perdigão, João; Rodrigues, Liliana; Portugal, Isabel; Baptista, Pedro; Veigas, Bruno; Amaral, Leonard; Viveiros, Miguel

    2012-01-01

    Multidrug resistant (MDR) tuberculosis is caused by Mycobacterium tuberculosis resistant to isoniazid and rifampicin, the two most effective drugs used in tuberculosis therapy. Here, we investigated the mechanism by which resistance towards isoniazid develops and how overexpression of efflux pumps favors accumulation of mutations in isoniazid targets, thus establishing a MDR phenotype. The study was based on the in vitro induction of an isoniazid resistant phenotype by prolonged serial exposure of M. tuberculosis strains to the critical concentration of isoniazid employed for determination of drug susceptibility testing in clinical isolates. Results show that susceptible and rifampicin monoresistant strains exposed to this concentration become resistant to isoniazid after three weeks; and that resistance observed for the majority of these strains could be reduced by means of efflux pumps inhibitors. RT-qPCR assessment of efflux pump genes expression showed overexpression of all tested genes. Enhanced real-time efflux of ethidium bromide, a common efflux pump substrate, was also observed, showing a clear relation between overexpression of the genes and increased efflux pump function. Further exposure to isoniazid resulted in the selection and stabilization of spontaneous mutations and deletions in the katG gene along with sustained increased efflux activity. Together, results demonstrate the relevance of efflux pumps as one of the factors of isoniazid resistance in M. tuberculosis. These results support the hypothesis that activity of efflux pumps allows the maintenance of an isoniazid resistant population in a sub-optimally treated patient from which isoniazid genetically resistant mutants emerge. Therefore, the use of inhibitors of efflux should be considered in the development of new therapeutic strategies for preventing the emergence of MDR-TB during treatment. PMID:22493700

  9. Contribution of efflux to the emergence of isoniazid and multidrug resistance in Mycobacterium tuberculosis.

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    Diana Machado

    Full Text Available Multidrug resistant (MDR tuberculosis is caused by Mycobacterium tuberculosis resistant to isoniazid and rifampicin, the two most effective drugs used in tuberculosis therapy. Here, we investigated the mechanism by which resistance towards isoniazid develops and how overexpression of efflux pumps favors accumulation of mutations in isoniazid targets, thus establishing a MDR phenotype. The study was based on the in vitro induction of an isoniazid resistant phenotype by prolonged serial exposure of M. tuberculosis strains to the critical concentration of isoniazid employed for determination of drug susceptibility testing in clinical isolates. Results show that susceptible and rifampicin monoresistant strains exposed to this concentration become resistant to isoniazid after three weeks; and that resistance observed for the majority of these strains could be reduced by means of efflux pumps inhibitors. RT-qPCR assessment of efflux pump genes expression showed overexpression of all tested genes. Enhanced real-time efflux of ethidium bromide, a common efflux pump substrate, was also observed, showing a clear relation between overexpression of the genes and increased efflux pump function. Further exposure to isoniazid resulted in the selection and stabilization of spontaneous mutations and deletions in the katG gene along with sustained increased efflux activity. Together, results demonstrate the relevance of efflux pumps as one of the factors of isoniazid resistance in M. tuberculosis. These results support the hypothesis that activity of efflux pumps allows the maintenance of an isoniazid resistant population in a sub-optimally treated patient from which isoniazid genetically resistant mutants emerge. Therefore, the use of inhibitors of efflux should be considered in the development of new therapeutic strategies for preventing the emergence of MDR-TB during treatment.

  10. Molecular characteristics of rifampin and isoniazid resistant Mycobacterium tuberculosis strains from Beijing, China

    Institute of Scientific and Technical Information of China (English)

    JIAO Wei-wei; Mokrousov Igor; SUN Gui-zhi; LI Mo; LIU Jia-wen; Narvskaya Olga; SHEN A-dong

    2007-01-01

    Background China is one of the high burden countries of Mycobacterium tuberculosis (TB) infection globally, with high incidence and mortality. We studied the molecular characteristics of rifampin (RIF) and isoniazid (INH) resistant Mycobacterium tuberculosis strains from Beijing, China, in order to find out the genetic marker for rapid detection of specific drug resistance.Methods Forty pansusceptible and 81 resistant strains of Mycobacterium tuberculosis isolated from Beijing, China during 2002-2005 were analyzed. The modified rifampin oligonucleotide (RIFO) assay based on reverse line blot hybridization was used to detect mutations in the 81 bp hot-spot region of rpoB gene, which is associated with RIF resistance. The INH resistance associated genes, regulatory region mab-inhA (-15C/T) and structural gene katG S315T were detected by reverse line blot hybridization and PCR-restriction fragment length polymorphism (RFLP) method respectively. All the strains were typed by spoligotying and the Beijing genotype was further subdivided by NTF locus analysis. The distribution of drug resistance associated mutations in the above genes was compared in these groups.Results Sixty-five (91.5%) of 71 RIF resistant and 52 (92.9%) of 56 multidrug-resistant (MDR, Ⅰ.e. Resistant to at least RIF and INH) strains were found to harbor mutations in the rpoB hot-spot region. No mutation was detected in RIF sensitive strains. The specificity and sensitivity of the modified RIFO assay were 100% and 91.5%, respectively. katG315 AGC>ACC and inhA-15C>T mutations were found in 40 (60.6%) and 10 (15.2%) of 66 Ⅰ NH resistant strains, respectively;7.6% of INH-resistant strains had mutations in both of these genes. Therefore, a combined use of both katG315 and inhA-15 identified 68.2% of INH-resistant strains. The Beijing genotype accounted for 91.7% of total strains and was further subdivided into "modern" (76.6%) and "ancestral" (23.4%) group. There is no significant difference between

  11. Clinical implications of molecular drug resistance testing for Mycobacterium tuberculosis: a TBNET/RESIST-TB consensus statement.

    Science.gov (United States)

    Domínguez, J; Boettger, E C; Cirillo, D; Cobelens, F; Eisenach, K D; Gagneux, S; Hillemann, D; Horsburgh, R; Molina-Moya, B; Niemann, S; Tortoli, E; Whitelaw, A; Lange, C

    2016-01-01

    The emergence of drug-resistant strains of Mycobacterium tuberculosis is a challenge to global tuberculosis (TB) control. Although culture-based methods have been regarded as the gold standard for drug susceptibility testing (DST), molecular methods provide rapid information on mutations in the M. tuberculosis genome associated with resistance to anti-tuberculosis drugs. We ascertained consensus on the use of the results of molecular DST for clinical treatment decisions in TB patients. This document has been developed by TBNET and RESIST-TB groups to reach a consensus about reporting standards in the clinical use of molecular DST results. Review of the available literature and the search for evidence included hand-searching journals and searching electronic databases. The panel identified single nucleotide mutations in genomic regions of M. tuberculosis coding for katG, inhA, rpoB, embB, rrs, rpsL and gyrA that are likely related to drug resistance in vivo. Identification of any of these mutations in clinical isolates of M. tuberculosis has implications for the management of TB patients, pending the results of in vitro DST. However, false-positive and false-negative results in detecting resistance-associated mutations in drugs for which there is poor or unproven correlation between phenotypic and clinical drug resistance complicate the interpretation. Reports of molecular DST results should therefore include specific information on the mutations identified and provide guidance for clinicians on interpretation and on the choice of the appropriate initial drug regimen.

  12. Au-nanoprobes for detection of SNPs associated with antibiotic resistance in Mycobacterium tuberculosis

    Science.gov (United States)

    Veigas, Bruno; Machado, Diana; Perdigão, João; Portugal, Isabel; Couto, Isabel; Viveiros, Miguel; Baptista, Pedro V.

    2010-10-01

    Tuberculosis (TB) is one of the leading causes of infection in humans, causing high morbility and mortality all over the world. The rate of new cases of multidrug resistant tuberculosis (MDRTB) continues to increase, and since these infections are very difficult to manage, they constitute a serious health problem. In most cases, drug resistance in Mycobacterium tuberculosis has been related to mutations in several loci within the pathogen's genome. The development of fast, cheap and simple screening methodologies would be of paramount relevance for the early detection of these mutations, essential for the timely and effective diagnosis and management of MDRTB patients. The use of gold nanoparticles derivatized with thiol-modified oligonucleotides (Au-nanoprobes) has led to new approaches in molecular diagnostics. Based on the differential non-cross-linking aggregation of Au-nanoprobes, we were able to develop a colorimetric method for the detection of specific sequences and to apply this approach to pathogen identification and single base mutations/single nucleotide polymorphisms (SNP) discrimination. Here we report on the development of Au-nanoprobes for the specific identification of SNPs within the beta subunit of the RNA polymerase (rpoB locus), responsible for resistance to rifampicin in over 95% of rifampicin resistant M. tuberculosis strains.

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

    Science.gov (United States)

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

    2016-03-01

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

  14. Au-nanoprobes for detection of SNPs associated with antibiotic resistance in Mycobacterium tuberculosis

    Energy Technology Data Exchange (ETDEWEB)

    Veigas, Bruno; Baptista, Pedro V [CIGMH, Departamento de Ciencias da Vida, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, Caparica (Portugal); Machado, Diana; Couto, Isabel; Viveiros, Miguel [Unidade de Micobacterias, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL) (Portugal); Perdigao, Joao; Portugal, Isabel, E-mail: pmvb@fct.unl.pt [Centro de Patogenese Molecular/URIA, Faculdade de Farmacia, Universidade de Lisboa, Lisboa (Portugal)

    2010-10-15

    Tuberculosis (TB) is one of the leading causes of infection in humans, causing high morbility and mortality all over the world. The rate of new cases of multidrug resistant tuberculosis (MDRTB) continues to increase, and since these infections are very difficult to manage, they constitute a serious health problem. In most cases, drug resistance in Mycobacterium tuberculosis has been related to mutations in several loci within the pathogen's genome. The development of fast, cheap and simple screening methodologies would be of paramount relevance for the early detection of these mutations, essential for the timely and effective diagnosis and management of MDRTB patients. The use of gold nanoparticles derivatized with thiol-modified oligonucleotides (Au-nanoprobes) has led to new approaches in molecular diagnostics. Based on the differential non-cross-linking aggregation of Au-nanoprobes, we were able to develop a colorimetric method for the detection of specific sequences and to apply this approach to pathogen identification and single base mutations/single nucleotide polymorphisms (SNP) discrimination. Here we report on the development of Au-nanoprobes for the specific identification of SNPs within the beta subunit of the RNA polymerase (rpoB locus), responsible for resistance to rifampicin in over 95% of rifampicin resistant M. tuberculosis strains.

  15. Rapid Detection of Rifampin-resistant Clinical Isolates of Mycobacterium tuberculosis by Reverse Dot Blot Hybridization

    Institute of Scientific and Technical Information of China (English)

    GUO Qian; WAN Kang Lin; YU Yan; ZHU Yan Ling; ZHAO Xiu Qin; LIU Zhi Guang; ZHANG Yuan Yuan; LI Gui Lian; WEI Jian Hao; WU Yi Mou

    2015-01-01

    Objective A PCR-reverse dot blot hybridization (RDBH) assay was developed for rapid detection of rpoB gene mutations in‘hot mutation region’ of Mycobacterium tuberculosis (M. tuberculosis). Methods 12 oligonucleotide probes based on the wild-type and mutant genotype rpoB sequences of M. tuberculosis were designed to screen the most frequent wild-type and mutant genotypes for diagnosing RIF resistance. 300 M. tuberculosis clinical isolates were detected by RDBH, conventional drug-susceptibility testing (DST) and DNA sequencing to evaluate the RDBH assay. Results The sensitivity and specificity of the RDBH assay were 91.2%(165/181) and 98.3%(117/119), respectively, as compared to DST. When compared with DNA sequencing, the accuracy, positive predictive value (PPV) and negative predictive value (NPV) of the RDBH assay were 97.7%(293/300), 98.2%(164/167), and 97.0%(129/133), respectively. Furthermore, the results indicated that the most common mutations were in codons 531 (48.6%), 526 (25.4%), 516 (8.8%), and 511 (6.6%), and the combinative mutation rate was 15 (8.3%). One and two strains of insertion and deletion were found among all strains, respectively. Conclusion Our findings demonstrate that the RDBH assay is a rapid, simple and sensitive method for diagnosing RIF-resistant tuberculosis.

  16. Mycolic Acid Cyclopropanation is Essential for Viability, Drug Resistance, and Cell Wall Integrity of Mycobacterium tuberculosis

    Energy Technology Data Exchange (ETDEWEB)

    Barkan, Daniel; Liu, Zhen; Sacchettini, James C.; Glickman, Michael S.; (MSKCC); (TAM)

    2009-12-01

    Mycobacterium tuberculosis infection remains a major global health problem complicated by escalating rates of antibiotic resistance. Despite the established role of mycolic acid cyclopropane modification in pathogenesis, the feasibility of targeting this enzyme family for antibiotic development is unknown. We show through genetics and chemical biology that mycolic acid methyltransferases are essential for M. tuberculosis viability, cell wall structure, and intrinsic resistance to antibiotics. The tool compound dioctylamine, which we show acts as a substrate mimic, directly inhibits the function of multiple mycolic acid methyltransferases, resulting in loss of cyclopropanation, cell death, loss of acid fastness, and synergistic killing with isoniazid and ciprofloxacin. These results demonstrate that mycolic acid methyltransferases are a promising antibiotic target and that a family of virulence factors can be chemically inhibited with effects not anticipated from studies of each individual enzyme.

  17. Supplementary Material for: Mycobacterium tuberculosis whole genome sequencing and protein structure modelling provides insights into anti-tuberculosis drug resistance

    KAUST Repository

    Phelan, Jody

    2016-01-01

    Abstract Background Combating the spread of drug resistant tuberculosis is a global health priority. Whole genome association studies are being applied to identify genetic determinants of resistance to anti-tuberculosis drugs. Protein structure and interaction modelling are used to understand the functional effects of putative mutations and provide insight into the molecular mechanisms leading to resistance. Methods To investigate the potential utility of these approaches, we analysed the genomes of 144 Mycobacterium tuberculosis clinical isolates from The Special Programme for Research and Training in Tropical Diseases (TDR) collection sourced from 20 countries in four continents. A genome-wide approach was applied to 127 isolates to identify polymorphisms associated with minimum inhibitory concentrations for first-line anti-tuberculosis drugs. In addition, the effect of identified candidate mutations on protein stability and interactions was assessed quantitatively with well-established computational methods. Results The analysis revealed that mutations in the genes rpoB (rifampicin), katG (isoniazid), inhA-promoter (isoniazid), rpsL (streptomycin) and embB (ethambutol) were responsible for the majority of resistance observed. A subset of the mutations identified in rpoB and katG were predicted to affect protein stability. Further, a strong direct correlation was observed between the minimum inhibitory concentration values and the distance of the mutated residues in the three-dimensional structures of rpoB and katG to their respective drugs binding sites. Conclusions Using the TDR resource, we demonstrate the usefulness of whole genome association and convergent evolution approaches to detect known and potentially novel mutations associated with drug resistance. Further, protein structural modelling could provide a means of predicting the impact of polymorphisms on drug efficacy in the absence of phenotypic data. These approaches could ultimately lead to novel

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

    KAUST Repository

    Phelan, Jody

    2016-03-23

    Background Combating the spread of drug resistant tuberculosis is a global health priority. Whole genome association studies are being applied to identify genetic determinants of resistance to anti-tuberculosis drugs. Protein structure and interaction modelling are used to understand the functional effects of putative mutations and provide insight into the molecular mechanisms leading to resistance. Methods To investigate the potential utility of these approaches, we analysed the genomes of 144 Mycobacterium tuberculosis clinical isolates from The Special Programme for Research and Training in Tropical Diseases (TDR) collection sourced from 20 countries in four continents. A genome-wide approach was applied to 127 isolates to identify polymorphisms associated with minimum inhibitory concentrations for first-line anti-tuberculosis drugs. In addition, the effect of identified candidate mutations on protein stability and interactions was assessed quantitatively with well-established computational methods. Results The analysis revealed that mutations in the genes rpoB (rifampicin), katG (isoniazid), inhA-promoter (isoniazid), rpsL (streptomycin) and embB (ethambutol) were responsible for the majority of resistance observed. A subset of the mutations identified in rpoB and katG were predicted to affect protein stability. Further, a strong direct correlation was observed between the minimum inhibitory concentration values and the distance of the mutated residues in the three-dimensional structures of rpoB and katG to their respective drugs binding sites. Conclusions Using the TDR resource, we demonstrate the usefulness of whole genome association and convergent evolution approaches to detect known and potentially novel mutations associated with drug resistance. Further, protein structural modelling could provide a means of predicting the impact of polymorphisms on drug efficacy in the absence of phenotypic data. These approaches could ultimately lead to novel resistance

  19. Interaction of CarD with RNA polymerase mediates Mycobacterium tuberculosis viability, rifampin resistance, and pathogenesis.

    Science.gov (United States)

    Weiss, Leslie A; Harrison, Phillip G; Nickels, Bryce E; Glickman, Michael S; Campbell, Elizabeth A; Darst, Seth A; Stallings, Christina L

    2012-10-01

    Mycobacterium tuberculosis infection continues to cause substantial human suffering. New chemotherapeutic strategies, which require insight into the pathways essential for M. tuberculosis pathogenesis, are imperative. We previously reported that depletion of the CarD protein in mycobacteria compromises viability, resistance to oxidative stress and fluoroquinolones, and pathogenesis. CarD associates with the RNA polymerase (RNAP), but it has been unknown which of the diverse functions of CarD are mediated through the RNAP; this question must be answered to understand the CarD mechanism of action. Herein, we describe the interaction between the M. tuberculosis CarD and the RNAP β subunit and identify point mutations that weaken this interaction. The characterization of mycobacterial strains with attenuated CarD/RNAP β interactions demonstrates that the CarD/RNAP β association is required for viability and resistance to oxidative stress but not for fluoroquinolone resistance. Weakening the CarD/RNAP β interaction also increases the sensitivity of mycobacteria to rifampin and streptomycin. Surprisingly, depletion of the CarD protein did not affect sensitivity to rifampin. These findings define the CarD/RNAP interaction as a new target for chemotherapeutic intervention that could also improve the efficacy of rifampin treatment of tuberculosis. In addition, our data demonstrate that weakening the CarD/RNAP β interaction does not completely phenocopy the depletion of CarD and support the existence of functions for CarD independent of direct RNAP binding.

  20. In vitro activity of rifampicin and verapamil combination in multidrug-resistant mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Fernanda de Oliveira Demitto

    Full Text Available The aim of the present study was to evaluate the effect of the combination of rifampicin (RIF and verapamil (VP against the Mycobacterium tuberculosis H37Rv reference strain and six multidrug-resistant (MDR M. tuberculosis clinical isolates by determining Time-Kill Curves and the ability to efflux drug by fluorometry. The RIF+VP combination showed synergism in one MDR clinical isolate. For the other five MDR clinical isolates, the drug combination showed no interaction. The MDR clinical isolate had lower ethidium bromide (EtBr accumulation when exposed to the RIF+VP combination, compared with RIF and VP exposure alone. The other MDR clinical isolates showed no significant difference in EtBr accumulation. These results suggest greater efflux action in one of the MDR clinical isolates compared with the M. tuberculosis H37Rv reference strain. The other five MDR isolates may have additional mechanisms of drug resistance to RIF. The use of the RIF+VP combination made one MDR bacillus more susceptible to RIF probably by inhibiting efflux pumps, and this combination therapy, in some cases, may contribute to a reduction of resistance to RIF in M. tuberculosis.

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

    KAUST Repository

    Dalla Costa, Elis R.

    2015-09-23

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

  2. Strong in vitro activities of two new rifabutin analogs against multidrug-resistant Mycobacterium tuberculosis.

    Science.gov (United States)

    García, Ana-Belén; Palacios, Juan J; Ruiz, María-Jesús; Barluenga, José; Aznar, Fernando; Cabal, María-Paz; García, José María; Díaz, Natalia

    2010-12-01

    Two new rifabutin analogs, RFA-1 and RFA-2, show high in vitro antimycobacterial activities against Mycobacterium tuberculosis. MIC values of RFA-1 and RFA-2 were ≤0.02 μg/ml against rifamycin-susceptible strains and 0.5 μg/ml against a wide selection of multidrug-resistant strains, compared to ≥50 μg/ml for rifampin and 10 μg/ml for rifabutin. Molecular dynamic studies indicate that the compounds may exert tighter binding to mutants of RNA polymerase that have adapted to the rifamycins.

  3. Paradoxical Hypersusceptibility of Drug-resistant Mycobacterium tuberculosis to β-lactam Antibiotics

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    Keira A. Cohen

    2016-07-01

    Full Text Available Mycobacterium tuberculosis (M. tuberculosis is considered innately resistant to β-lactam antibiotics. However, there is evidence that susceptibility to β-lactam antibiotics in combination with β–lactamase inhibitors is variable among clinical isolates, and these may present therapeutic options for drug-resistant cases. Here we report our investigation of susceptibility to β-lactam/β–lactamase inhibitor combinations among clinical isolates of M. tuberculosis, and the use of comparative genomics to understand the observed heterogeneity in susceptibility. Eighty-nine South African clinical isolates of varying first and second-line drug susceptibility patterns and two reference strains of M. tuberculosis underwent minimum inhibitory concentration (MIC determination to two β-lactams: amoxicillin and meropenem, both alone and in combination with clavulanate, a β–lactamase inhibitor. 41/91 (45% of tested isolates were found to be hypersusceptible to amoxicillin/clavulanate relative to reference strains, including 14/24 (58% of multiple drug-resistant (MDR and 22/38 (58% of extensively drug-resistant (XDR isolates. Genome-wide polymorphisms identified using whole-genome sequencing were used in a phylogenetically-aware linear mixed model to identify polymorphisms associated with amoxicillin/clavulanate susceptibility. Susceptibility to amoxicillin/clavulanate was over-represented among isolates within a specific clade (LAM4, in particular among XDR strains. Twelve sets of polymorphisms were identified as putative markers of amoxicillin/clavulanate susceptibility, five of which were confined solely to LAM4. Within the LAM4 clade, ‘paradoxical hypersusceptibility’ to amoxicillin/clavulanate has evolved in parallel to first and second-line drug resistance. Given the high prevalence of LAM4 among XDR TB in South Africa, our data support an expanded role for β-lactam/β-lactamase inhibitor combinations for treatment of drug-resistant M

  4. Genotyping and drug resistance patterns of Mycobacterium tuberculosis strains observed in a tuberculosis high-burden municipality in Northeast, Brazil

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    Roberta dos Santos Silva Luiz

    Full Text Available OBJECTIVES: This study has used a combination of clinical information, spoligotyping, and georeferencing system to elucidate the genetic diversity of the Mycobacterium tuberculosis isolates circulating in a TB-prevalent municipality of Northeast Brazil. METHODS: A total of 115 M. tuberculosis strains were isolated from pulmonary tuberculosis patients from January 2007 to March 2008 in Fortaleza. Drug susceptibility and spoligotyping assays were performed and place of residence of the patients were georeferenced. RESULTS: Of the M. tuberculosis strains studied, 51 (44.3% isolates were resistant to at least one drug (R-TB and 64 (55.7% were sensitive to all the drugs tested (S-TB. A high frequency of resistance was found in previously treated cases (84% and among new cases (16%; p < 0.001. a total of 74 (64% isolates were grouped into 22 spoligotyped lineages, while 41 (36% isolates were identified as new. among the predominant genotypes, 33% were latim american mediterranean (lam, 12% haarlem (h, and 5% u. there was no association of geographic distribution of rt-tb patients as compared to the controls and also the geographic location to the spoligotype patterns. the geospatial analysis revealed that 24 (23% patients (hot spot zones either shared the same residence or lived in a close neighborhood of a case. among these concentration zones, the patients lived in the same residence and shared a common genotype pattern and resistance pattern. DISCUSSION: it was observed that the spoligopatterns family distribution was similar to that reported for south america, prevailing the lam and h lineages. a high rate-case among the resistant TB group occurs as a result of transmitted and acquired resistance. A more effective surveillance program is needed in order to succeed in reducing tuberculosis in Northeast Brazil.

  5. [A preliminary study on the molecular characteristics of D-cycloserine resistance of Mycobacterium tuberculosis].

    Science.gov (United States)

    Li, C; Li, G L; Luo, Q; Li, S J; Wang, R B; Lou, Y L; Lyu, J X; Wan, K L

    2017-02-10

    Objective: To investigate the relationship between D-cycloserine resistance and the gene mutations of alrA, ddlA and cycA of Mycobacterium (M.) tuberculosis, as well as the association between D-cycloserine resistance and spoligotyping genotyping. Methods: A total of 145 M. tuberculosis strains were selected from the strain bank. D-cycloserine resistant phenotypes of the strains were determined by the proportion method and the minimal inhibitory concentration was determined by resazurin microtiter assay. PCR amplification and DNA direct sequencing methods were used for the analysis of gene mutations. Relationship between the resistance phenotype and genotype was analyzed by chi-square test. Results: Of the 145 clinically collected strains, 24 (16.6%) of them were D-cycloserine resistant and 121 (83.4%) were sensitive. There were only synonymous mutations noticed on alrA, ddlA and cycA in sensitive strains. Of the 24 D-cycloserine resistant strains, 3 (12.5%) isolates' cycA and 1 (4.2%) isolates' alrA happened to be non-synonymous mutations, in which the codes were 188, 318 and 508 of cycA, and 261 of alrA, respectively. Results on drug sensitivity tests confirmed the minimal inhibitory concentration of the mutant strains were all increased to some degrees. The D-cycloserine resistant rates of 88 Beijing genotype and 57 non-Beijing genotype strains were 20.5% and 10.5% , respectively, but with no statistically significant difference (χ(2) =2.47, P>0.05). Conclusions: The non-synonymous mutations of alrA and cycA might contribute to one of the mechanisms of M. tuberculosis D-cycloserine resistance. M. tuberculosis Beijing genotype or non-Beijing genotype was not considered to be associated with the D-cycloserine resistance.

  6. Genotypic detection of rifampicin and isoniazid resistant Mycobacterium tuberculosis strains by DNA sequencing: a randomized trial

    Directory of Open Access Journals (Sweden)

    El mashad Noha

    2009-01-01

    Full Text Available Abstract Background Tuberculosis is a growing international health concern. It is the biggest killer among the infectious diseases in the world today. Early detection of drug resistance allows starting of an appropriate treatment. Resistance to drugs is due to particular genomic mutations in specific genes of Mycobacterium tuberculosis(MTB. The aim of this study was to identify the presence of Isoniazid (INH and Rifampicin(RIF drug resistance in new and previously treated tuberculosis (TB cases using DNA sequencing. Methods This study was carried out on 153 tuberculous patients with positive Bactec 460 culture for acid fast bacilli. Results Of the 153 patients, 105 (68.6% were new cases and 48 (31.4% were previously treated cases. Drug susceptibility testing on Bactec revealed 50 resistant cases for one or more of the first line antituberculous. Genotypic analysis was done only for rifampicin resistant specimens (23 cases and INH resistant specimens (26 cases to detect mutations responsible for drug resistance by PCR amplification of rpoB gene for rifampicin resistant cases and KatG gene for isoniazid resistant cases. Finally, DNA sequencing was done for detection of mutation within rpoB and KatG genes. Genotypic analysis of RIF resistant cases revealed that 20/23 cases (86.9% of RIF resistance were having rpoB gene mutation versus 3 cases (13.1% having no mutation with a high statistical significant difference between them (P Conclusion We can conclude that rifampicin resistance could be used as a useful surrogate marker for estimation of multidrug resistance. In addition, Genotypic method was superior to that of the traditional phenotypic method which is time-consuming taking several weeks or longer.

  7. Genotypic and phenotypic characteristics of aminoglycoside-resistant Mycobacterium tuberculosis isolates in Latvia.

    Science.gov (United States)

    Bauskenieks, Matiss; Pole, Ilva; Skenders, Girts; Jansone, Inta; Broka, Lonija; Nodieva, Anda; Ozere, Iveta; Kalvisa, Adrija; Ranka, Renate; Baumanis, Viesturs

    2015-03-01

    Mutations causing resistance to aminoglycosides, such as kanamycin (KAN), amikacin (AMK), and streptomycin, are not completely understood. In this study, polymorphisms of aminoglycoside resistance influencing genes such as rrs, eis, rpsL, and gidB in 41 drug-resistant and 17 pan-sensitive Mycobacterium tuberculosis clinical isolates in Latvia were analyzed. Mutation A1400G in rrs gene was detected in 92% isolates with high resistance level to KAN and diverse MIC level to AMK. Mutations in promoter region of eis were detected in 80% isolates with low-level MIC of KAN. The association of K43R mutation in rpsL gene, a mutation in the rrs gene at position 513, and various polymorphisms in gidB gene with distinct genetic lineages of M. tuberculosis was observed. The results of this study suggest that association of different controversial mutations of M. tuberculosis genes to the drug resistance phenotype should be done in respect to genetic lineages.

  8. Genotypes of Mycobacterium tuberculosis in patients at risk of drug resistance in Bolivia.

    Science.gov (United States)

    Monteserin, Johana; Camacho, Mirtha; Barrera, Lucía; Palomino, Juan Carlos; Ritacco, Viviana; Martin, Anandi

    2013-07-01

    Bolivia ranks among the 10 Latin American countries with the highest rates of tuberculosis (TB) and multidrug resistant (MDR) TB. In view of this, and of the lacking information on the population structure of Mycobacterium tuberculosis in the country, we explored genotype associations with drug resistance and clustering by analyzing isolates collected in 2010 from 100 consecutive TB patients at risk of drug resistance in seven of the nine departments in which Bolivia is divided. Fourteen isolates were MDR, 29 had other drug resistance profiles, and 57 were pansusceptible. Spoligotype family distribution was: Haarlem 39.4%, LAM 26.3%, T 22.2%, S 2.0%, X 1.0%, orphan 9.1%, with very low intra-family diversity and absence of Beijing genotypes. We found 66 different MIRU-VNTR patterns; the most frequent corresponded to Multiple Locus Variable Analysis (MLVA) MtbC15 patterns 860, 372 and 873. Twelve clusters, each with identical MIRU-VNTR and spoligotypes, gathered 35 patients. We found no association of genotype with drug resistant or MDR-TB. Clustering associated with SIT 50 and the H3 subfamily to which it belongs (pBolivia. However, results should be taken cautiously because the sample is small and includes a particular subset of M. tuberculosis population. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. DRUG RESISTANCE, VIABILITY AND VIRULENCE IN VITRO ОF MYCOBACTERIUM TUBERCULOSIS STRAINS OF DIFFERENT GENOTYPES

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    O. A. Manicheva

    2011-01-01

    Full Text Available Abstract. The drug resistance, mutation spectrum caused resistance to rifampicin and isoniazid, viability, cytotoxicity were studied as well as 111 clinical isolates of Mycobacterium tuberculosis (MBT were genotyped. Spoligotyping revealed 28 spoligotypes; the greatest number of strains belonged to genetic families Beijing and LAM. The typing of 59 strains of spoligotype SIT1 (Beijing allowed to differentiate 19 variants of IS6110-RFLP-profiles: 13 of them were individual and 6 were presented by clusters. The clusters А0 and В0 included the greatest number of M. tuberculosis strains — 21 (35,6% and 17 (28,8% accordingly. The high frequency of MLU and SLU of Beijing strains was associated with mutations rpoB Ser531→Leu and katG Ser315→Thr. The strains of M. tuberculosis belonged to another genetic families (T, H, Ural, U have shown drug resistance more often. The level of resistance to isoniazid in vitro in MLU/SLU strains of MBT of different genotypes, especially, was high in case of mixed mutations such as katG Ser315→Thr и inhA_T15. The rates of viability and cytotoxicity of MBT strains of studied genotypes with different spectrum of mutations and phenotypic drug resistance were not substantially distinguished.

  10. Sulfonamide-Based Inhibitors of Aminoglycoside Acetyltransferase Eis Abolish Resistance to Kanamycin in Mycobacterium tuberculosis

    Energy Technology Data Exchange (ETDEWEB)

    Garzan, Atefeh; Willby, Melisa J.; Green, Keith D.; Gajadeera, Chathurada S.; Hou, Caixia; Tsodikov, Oleg V.; Posey, James E.; Garneau-Tsodikova, Sylvie

    2016-12-08

    A two-drug combination therapy where one drug targets an offending cell and the other targets a resistance mechanism to the first drug is a time-tested, yet underexploited approach to combat or prevent drug resistance. By high-throughput screening, we identified a sulfonamide scaffold that served as a pharmacophore to generate inhibitors of Mycobacterium tuberculosis acetyltransferase Eis, whose upregulation causes resistance to the aminoglycoside (AG) antibiotic kanamycin A (KAN) in Mycobacterium tuberculosis. Rational systematic derivatization of this scaffold to maximize Eis inhibition and abolish the Eis-mediated KAN resistance of M. tuberculosis yielded several highly potent agents. A crystal structure of Eis in complex with one of the most potent inhibitors revealed that the inhibitor bound Eis in the AG-binding pocket held by a conformationally malleable region of Eis (residues 28–37) bearing key hydrophobic residues. These Eis inhibitors are promising leads for preclinical development of innovative AG combination therapies against resistant TB.

  11. Genitourinary and pulmonary multidrug resistant Mycobacterium tuberculosis infection in an Asian elephant (Elephas maximus).

    Science.gov (United States)

    Dumonceaux, Genevieve A; St Leger, Judy; Olsen, John H; Burton, Michael S; Ashkin, David; Maslow, Joel N

    2011-12-01

    A female Asian elephant (Elephas maximus) developed vaginal and trunk discharge. Cultures were positive for pan-susceptible Mycobacterium tuberculosis. Isoniazid and pyrazinamide were given rectally and monitored by serum levels. After being trained at 10 mo to accept oral dosing, treatment was changed and rifampin was added. Oral medications were administered for another 10 mo. A year after completion of therapy, the vaginal discharge increased and cultures yielded M. tuberculosis, resistant to isoniazid and rifampin. Treatment with oral ethambutol, pyrazinamide, and enrofloxacin and intramuscular amikacin was initiated. Although followup cultures became negative, adverse reactions to medications precluded treatment completion. Due to public health concerns related to multidrug resistant M. tuberculosis (MDR-TB), the elephant was euthanized. Postmortem smears from the lung, peribronchial, and abdominal lymph nodes yielded acid-fast bacteria, although cultures were negative. This case highlights important considerations in the treatment of M. tuberculosis in animals and the need for a consistent approach to diagnosis, treatment, and follow-up.

  12. Genomic epidemiology of multidrug-resistant Mycobacterium tuberculosis during transcontinental spread.

    Science.gov (United States)

    Coscolla, Mireia; Barry, Pennan M; Oeltmann, John E; Koshinsky, Heather; Shaw, Tambi; Cilnis, Martin; Posey, Jamie; Rose, Jordan; Weber, Terry; Fofanov, Viacheslav Y; Gagneux, Sebastien; Kato-Maeda, Midori; Metcalfe, John Z

    2015-07-15

    The transcontinental spread of multidrug-resistant (MDR) tuberculosis is poorly characterized in molecular epidemiologic studies. We used genomic sequencing to understand the establishment and dispersion of MDR Mycobacterium tuberculosis within a group of immigrants to the United States. We used a genomic epidemiology approach to study a genotypically matched (by spoligotype, IS6110 restriction fragment length polymorphism, and mycobacterial interspersed repetitive units-variable number of tandem repeat signature) lineage 2/Beijing MDR strain implicated in an outbreak of tuberculosis among refugees in Thailand and consecutive cases within California. All 46 MDR M. tuberculosis genomes from both Thailand and California were highly related, with a median difference of 10 single-nucleotide polymorphisms (SNPs). The Wat Tham Krabok (WTK) strain is a new sequence type distinguished from all known Beijing strains by 55 SNPs and a genomic deletion (Rv1267c) associated with increased fitness. Sequence data revealed a highly prevalent MDR strain that included several closely related but distinct allelic variants within Thailand, rather than the occurrence of a single outbreak. In California, sequencing data supported multiple independent introductions of WTK with subsequent transmission and reactivation within the state, as well as a potential super spreader with a prolonged infectious period. Twenty-seven drug resistance-conferring mutations and 4 putative compensatory mutations were found within WTK strains. Genomic sequencing has substantial epidemiologic value in both low- and high-burden settings in understanding transmission chains of highly prevalent MDR strains.

  13. Intra- and Extracellular Activities of Trimethoprim-Sulfamethoxazole against Susceptible and Multidrug-Resistant Mycobacterium tuberculosis

    Science.gov (United States)

    Schön, T.; Simonsson, U. S. H.; Bruchfeld, J.; Larsson, M.; Juréen, P.; Sturegård, E.; Giske, C. G.; Ängeby, K.

    2014-01-01

    We investigated the activity of trimethoprim-sulfamethoxazole (SXT) against Mycobacterium tuberculosis, the pathogen that causes tuberculosis (TB). The MIC distribution of SXT was 0.125/2.4 to 2/38 mg/liter for the 100 isolates tested, including multi- and extensively drug-resistant isolates (MDR/XDR-TB), whereas the intracellular MIC90 of sulfamethoxazole (SMX) for the pansusceptible strain H37Rv was 76 mg/liter. In an exploratory analysis using a ratio of the unbound area under the concentration-time curve from 0 to 24 h over MIC (fAUC0–24/MIC) using ≥25 as a potential target, the cumulative fraction response was ≥90% at doses of ≥2,400 mg of SMX. SXT is a potential treatment option for MDR/XDR-TB. PMID:25246405

  14. Molecular characterization of rpoB gene mutations in rifampicine-resistant Mycobacterium tuberculosis isolates from tuberculosis patients in Belarus.

    Science.gov (United States)

    Titov, Leonid P; Zakerbostanabad, Saeed; Slizen, Veranika; Surkova, Larisa; Taghikhani, Mohammad; Bahrmand, Ahmadreza

    2006-12-01

    The aim of this study was to investigate the frequency, location and type of rpoB mutations in Mycobacterium tuberculosis isolated from patients in Belarus. Tuberculosis cases are increasing every year in Belarus. Moreover, resistance to anti-tuberculosis drugs, especially to rifampicine, has increased. In this study, 44 rifampicine-resistance M. tuberculosis clinical isolates (including multidrug-resistant isolates) were subjected to DNA sequencing analysis of the hypervariable region (hot-spot) of the rpoB gene originating from different geographical regions in Belarus. Sixteen different types of mutations were identified. The most common point mutations were in codons 510 (47.7%), 526 (45.5%), 523 (40.86%) and 531 (29.5%). Eleven isolates (27.7%) carried one mutation and 23 (52%), 7 (16%), 3 (7%) of isolates carried 2, 3 and 4 mutations, respectively. A characteristic, prominent finding of this study was high frequency of multiple mutations in different codons of the rpoB gene (27.7%) and also the detection of unusual types of mutations in the 510 codon, comprising CAG mutations (deletion or changing, to CTG, CAC or CAT). In our study, the change TTG in codon 531 was found in 92% of isolates and the change TGC in 8% of isolates. A TAC change in codon 526 was not found, but the GAC change was found in all isolates. Isolates of M. tuberculosis isolated in Belarus were characterized by the wide spectrum of the important mutations and might belong to the epidemic widespread clones.

  15. Beijing genotype of Mycobacterium tuberculosis is significantly associated with linezolid resistance in multidrug-resistant and extensively drug-resistant tuberculosis in China.

    Science.gov (United States)

    Zhang, Zhijian; Pang, Yu; Wang, Yufeng; Liu, Changting; Zhao, Yanlin

    2014-03-01

    Linezolid (LNZ) is a promising antimicrobial agent for the treatment of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB). To investigate the efficacy of LNZ among MDR-TB and XDR-TB in China, the LNZ susceptibility of 158 MDR-TB isolates from the national drug resistance survey was determined by the minimum inhibitory concentration method. The 158 MDR-TB isolates were also sequenced in the 23S rRNA, rplC and rplD genes conferring LNZ resistance and were typed using spoligotyping to identify the Beijing genotype of Mycobacterium tuberculosis. Overall, the prevalence of LNZ-resistant isolates was 10.8% (17/158) among MDR-TB isolates circulating in China. Beijing genotype was significantly associated with LNZ resistance in MDR-TB and XDR-TB (odds ratio=4.66, 95% confidence interval 1.03-21.16; P=0.033). In addition, a higher frequency of LNZ-resistant isolates was observed among XDR-TB strains (60%) compared with the MDR (5.6%; PMutations in 23S rRNA and rplC were responsible for only 29.4% of LNZ-resistant M. tuberculosis among MDR-TB isolates, and a novel non-synonymous substitution His155Asp in rplC was first identified to be contributing to low-level LNZ resistance (2μg/mL) in M. tuberculosis. The unsatisfactory correlation between mutant genotypes highlights the urgent need to investigate another mechanism for LNZ resistance that has not yet been described.

  16. Surveillance of pyrazinamide susceptibility among multidrug-resistant Mycobacterium tuberculosis isolates from Siriraj Hospital, Thailand

    Directory of Open Access Journals (Sweden)

    Jonmalung Jirarut

    2010-08-01

    Full Text Available Abstract Background Susceptibility testing of pyrazinamide (PZA against Mycobacterium tuberculosis is difficult to perform because the acidity of culture medium that is required for drug activity also inhibits the growth of bacteria. In Thailand, very limited information has been generated on PZA resistance, particularly among multidrug-resistant tuberculosis (MDR-TB isolated from Thailand. Only two studies on PZA susceptibility among Thai M. tuberculosis strains have been reported; one used a pyrazinamidase assay, and the other used the BACTEC 460 TB for PZA susceptibility testing. In this study, we determined the percentage of strains possessing pyrazinamide resistance among pan-susceptible M. tuberculosis and MDR-TB isolates by using the pyrazinamidase assay, BACTEC MGIT 960 PZA method and pncA sequencing, and assessed the correlation in the data generated using these methods. The type and frequency of mutations in pncA were also determined. Results Overall, 150 M. tuberculosis isolates, consisting of 50 susceptible and 100 MDR-TB isolates, were tested for PZA susceptibility by BACTEC MGIT 960 PZA, the pyrazinamidase assay and pncA sequencing. The study indicated PZA resistance in 6% and 49% of susceptible and MDR-TB isolates, respectively. In comparison to the BACTEC MGIT 960 PZA, the PZase assay showed 65.4% sensitivity and 100% specificity, whereas pncA sequencing showed 75% sensitivity and 89.8% specificity. Twenty-four mutation types were found in this study, with the most frequent mutation (16% being His71Asp. Of these mutations, eight have not been previously described. The Ile31Ser and Ile31Thr mutations were found both in PZA susceptible and resistant isolates, suggesting that mutation of this codon might not play a role on PZA resistance. Conclusions Our findings suggest that phenotypic susceptibility testing is still essential for the detection of PZA resistance, especially for MDR-TB isolates. Some mutations were not associated

  17. Genotype MTBDR plus assay for molecular detection of rifampicin and isoniazid resistance in Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Soniya Sharma

    2014-01-01

    Full Text Available Aim: This study was performed for the rapid identification of Mycobacterium tuberculosis complex and its resistance to rifampicin and isoniazid, directly from the sputum samples of pulmonary tuberculosis patients. Materials and Methods: A commercially available genotype MTBDR plus assay was used for the identification and detection of mutations in Mycobacterial isolates. A total of 100 sputum samples of pulmonary tuberculosis patients were analyzed by using the genotype MTBDR plus assay. The MTBDR plus assay is designed to detect the mutations in the hotspot region of rpoB gene, katG and regulatory region of inhA gene. Results: The genotype MTBDR plus assay detected 22% multidrug resistant (MDR, 2% rifampicin (RMP monoresistant and 1% isoniazid (INH monoresistant isolates. In 22 MDR isolates, the codons most frequently involved in RMP-associated mutations were codon 531 (54.55%, 516 (31.82% and 526 (13.63%, and 90.90% of MDR isolates showed KatG S315T mutations and 9.1% showed inhA C-15T mutations associated with INH resistance. Conclusion: The new genotype MTBDR plus assay represents a rapid, reliable tool for the detection of MDR-TB, wherein results are obtained in 5 h allowing early and appropriate treatment, which is essential to cut the transmission path and reduce the spread of MDR-TB. The genotype MTBDR plus assay can readily be included in a routine laboratory work for the early diagnosis and control of MDR-TB.

  18. Beijing genotype of Mycobacterium tuberculosis is significantly associated with high-level fluoroquinolone resistance in Vietnam.

    Science.gov (United States)

    Duong, Duy An; Nguyen, Thi Hong Duyen; Nguyen, Thi Ngoc Lan; Dai, Viet Hoa; Dang, Thi Minh Ha; Vo, Sy Kiet; Do, Dang Anh Thu; Nguyen, Van Vinh Chau; Nguyen, Huy Dung; Dinh, Ngoc Sy; Farrar, Jeremy; Caws, Maxine

    2009-11-01

    Consecutive fluoroquinolone (FQ)-resistant isolates (n = 109) identified at the Pham Ngoc Thach Hospital for Tuberculosis, Ho Chi Minh City, Vietnam, were sequenced in the quinolone resistance-determining regions of the gyrA and gyrB genes and typed by large sequence polymorphism typing and spoligotyping to identify the Beijing genotype of Mycobacterium tuberculosis. Beijing genotype prevalence was compared with 109 consecutive isolates from newly presenting patients with pulmonary tuberculosis from the hospital outpatient department. Overall, 82.6% (n = 90/109) of isolates had mutations in gyrAB. Nine novel mutations were identified in gyrB (S486F, N538T, T539P, D500A, D500H, D500N, G509A, E540V, and E540D). The influence of these novel gyrB mutations on FQ resistance is not proven. The Beijing genotype was significantly associated with FQ resistance (odds ratio [OR], 2.39 [95% confidence interval {CI}, 1.34 to 4.25]; P = 0.003). Furthermore, Beijing genotype FQ-resistant isolates were significantly more likely than FQ-resistant isolates of other genotypes to have gyrA mutations (OR, 7.75 [95% CI, 2.84 to 21.15]; P = 0.0001) and high-level (>8 microg/ml) FQ resistance (OR, 11.0 [95% CI, 2.6 to 47.0]; P = 0.001). The underlying mechanism of the association of the Beijing genotype with high-level FQ resistance in this setting remains to be determined. The association of the Beijing genotype with relatively high-level FQ resistance conferred by specific gyrA mutations reported here is of grave concern given the epidemic spread of the Beijing genotype and the current hopes for shorter first-line treatment regimens based on FQs.

  19. Cytosolic Proteome Profiling of Aminoglycosides Resistant Mycobacterium tuberculosis Clinical Isolates Using MALDI-TOF/MS

    Science.gov (United States)

    Sharma, Divakar; Lata, Manju; Singh, Rananjay; Deo, Nirmala; Venkatesan, Krishnamurthy; Bisht, Deepa

    2016-01-01

    Emergence of extensively drug resistant tuberculosis (XDR-TB) is the consequence of the failure of second line TB treatment. Aminoglycosides are the important second line anti-TB drugs used to treat the multi drug resistant tuberculosis (MDR-TB). Main known mechanism of action of aminoglycosides is to inhibit the protein synthesis by inhibiting the normal functioning of ribosome. Primary target of aminoglycosides are the ribosomal RNA and its associated proteins. Various mechanisms have been proposed for aminoglycosides resistance but still some are unsolved. As proteins are involved in most of the biological processes, these act as a potential diagnostic markers and drug targets. In the present study we analyzed the purely cytosolic proteome of amikacin (AK) and kanamycin (KM) resistant Mycobacterium tuberculosis isolates by proteomic and bioinformatic approaches. Twenty protein spots were found to have over expressed in resistant isolates and were identified. Among these Rv3208A, Rv2623, Rv1360, Rv2140c, Rv1636, and Rv2185c are six proteins with unknown functions or undefined role. Docking results showed that AK and KM binds to the conserved domain (DUF, USP-A, Luciferase, PEBP and Polyketidecyclase/dehydrase domain) of these hypothetical proteins and over expression of these proteins might neutralize/modulate the effect of drug molecules. TBPred and GPS-PUP predicted cytoplasmic nature and potential pupylation sites within these identified proteins, respectively. String analysis also suggested that over expressed proteins along with their interactive partners might be involved in aminoglycosides resistance. Cumulative effect of these over expressed proteins could be involved in AK and KM resistance by mitigating the toxicity, repression of drug target and neutralizing affect. These findings need further exploitation for the expansion of newer therapeutics or diagnostic markers against AK and KM resistance so that an extreme condition like XDR-TB can be prevented

  20. Characterization of rifampin-resistant isolates of Mycobacterium tuberculosis from Sichuan in China.

    Science.gov (United States)

    Tang, Ke; Sun, Honghu; Zhao, Yuding; Guo, Jianhua; Zhang, Congcong; Feng, Qin; He, Yongquan; Luo, Mei; Li, Yuqing; Sun, Qun

    2013-01-01

    Rifampin is a key component of standard short-course first-line therapy against Mycobacterium tuberculosis, and rifampin resistance of this pathogen has been reported to be related to rpoB gene mutations. The objective of this study was to characterize the rpoB gene mutations in rifampin-resistant M. tuberculosis isolates circulated in Sichuan. Sequencing of rpoB gene and spoligotyping were performed on 268 randomly selected isolates from January 2008 to May 2010. The results indicated that 207 (97.2%) rifampin-resistant isolates had mutations at 26 codons in the amplified region with 50 different genotypes, while 1 (1.8%) of 55 susceptible isolates had a nonsense mutation. The most common mutations were in codon 531 (55.9%), 526 (16.4%), 516 (10.3%) and 511 (8.9%). Among the 213 resistant isolates, 150 (70.4%) belonged to the Beijing family and mutation at codon 531 (TCG→TTG) was associated with Beijing genotype (χ(2), 9.8305; p, 0.0017). It is demonstrated that the frequency of 511 (CTG→CCG) mutations in Sichuan was higher than in other provinces of China, as well as other geographic regions worldwide. It is suggested that only a small portion (2.7%) of rifampin-resistant Beijing genotype isolates in Sichuan be spread by clonal expansion during the study period.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-06-30

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

  2. Drug resistance pattern ofMycobacterium tuberculosis isolates from patients of five provinces of Iran

    Institute of Scientific and Technical Information of China (English)

    Mohammad Javad Nasiri; Faranak Rezaei; Samin Zamani; Davod Darban-Sarokhalil; Abbas Ali Imani Fooladi; Hasan Shojaei; Mohammad Mehdi Feizabadi

    2014-01-01

    Objective:To determine the patterns of resistance to first line anti-tuberculosis(TB) drugs among a collection ofMycobacterium tuberculosis(MTB) isolates from5 provinces ofIran. Methods:A total of the6426 clinical specimens from patients suspected of activeTB were collected fromMarch2010 toJune2012.All specimens were subjected for microscopy and culture tests in theTB centers of studies provinces.Drug susceptibility testing to the first line anti-TB drugs for culture positiveMTB was performed onLöwenstein-Jensen(LJ) medium using proportion method.Results:Of6426 clinical specimens,261 were culture positive for mycobacteria, of which252 wereMTB and9 wereMOTT(mycobacteria other than tuberculosis). Of252MTB isolates,211(83.7%) were pan-susceptible and41(16.3%) were resistant to at least one drug.Resistance was most common to streptomycin,30 isolates(12.0%), followed by isoniazid,20 isolates(8.0%), rifampin,15 isolates(6.0%) and ethambutol,14 isolates(5.5%).Sixteen(6.3%)MTB isolates wereMDR.A clear evidence of heterogeneity amongst the5 provinces in the proportions with resistance to one or more drugs was observed [χ² =12.209(4 degrees of freedom),P values =0.0159].Conclusions:The prevalence of drug resistance in this study area underscoring the need for further enforcement ofTB control strategies in theIran.Drug susceptibility testing for allTB cases to provide optimal treatment, establishing advanced diagnostic facilities for rapid detection ofMDR-TB and continuous monitoring of drug resistance are recommended for prevention and control of drug-resistantTB.

  3. Molecular patterns of multidrug resistance of Mycobacterium tuberculosis in Georgia

    Directory of Open Access Journals (Sweden)

    N Shubladze

    2013-01-01

    Conclusions: A great majority of the Georgian MDR MTB strains have a strong preference for the drug resistance mutations carrying no or low fitness cost. Thus, it can be suggested that MDR MTB strains with such mutations will continue to arise in Georgia at a high frequency even in the absence of antibiotic pressure.

  4. Unraveling Mycobacterium tuberculosis genomic diversity and evolution in Lisbon, Portugal, a highly drug resistant setting

    KAUST Repository

    Perdigão, João

    2014-11-18

    Background Multidrug- (MDR) and extensively drug resistant (XDR) tuberculosis (TB) presents a challenge to disease control and elimination goals. In Lisbon, Portugal, specific and successful XDR-TB strains have been found in circulation for almost two decades. Results In the present study we have genotyped and sequenced the genomes of 56 Mycobacterium tuberculosis isolates recovered mostly from Lisbon. The genotyping data revealed three major clusters associated with MDR-TB, two of which are associated with XDR-TB. Whilst the genomic data contributed to elucidate the phylogenetic positioning of circulating MDR-TB strains, showing a high predominance of a single SNP cluster group 5. Furthermore, a genome-wide phylogeny analysis from these strains, together with 19 publicly available genomes of Mycobacterium tuberculosis clinical isolates, revealed two major clades responsible for M/XDR-TB in the region: Lisboa3 and Q1 (LAM). The data presented by this study yielded insights on microevolution and identification of novel compensatory mutations associated with rifampicin resistance in rpoB and rpoC. The screening for other structural variations revealed putative clade-defining variants. One deletion in PPE41, found among Lisboa3 isolates, is proposed to contribute to immune evasion and as a selective advantage. Insertion sequence (IS) mapping has also demonstrated the role of IS6110 as a major driver in mycobacterial evolution by affecting gene integrity and regulation. Conclusions Globally, this study contributes with novel genome-wide phylogenetic data and has led to the identification of new genomic variants that support the notion of a growing genomic diversity facing both setting and host adaptation.

  5. Consequences of Noncompliance for Therapy Efficacy and Emergence of Resistance in Murine Tuberculosis Caused by the Beijing Genotype of Mycobacterium tuberculosis

    OpenAIRE

    2012-01-01

    Despite great effort by health organizations worldwide in fighting tuberculosis (TB), morbidity and mortality are not declining as expected. One of the reasons is related to the evolutionary development of Mycobacterium tuberculosis, in particular the Beijing genotype strains. In a previous study, we showed the association between the Beijing genotype and an increased mutation frequency for rifampin resistance. In this study, we use a Beijing genotype strain and an East-African/Indian genotyp...

  6. rpoB Mutations in Multidrug-Resistant Strains of Mycobacterium tuberculosis Isolated in Italy

    Science.gov (United States)

    Pozzi, G.; Meloni, M.; Iona, E.; Orrù, G.; Thoresen, O. F.; Ricci, M. L.; Oggioni, M. R.; Fattorini, L.; Orefici, G.

    1999-01-01

    Mutations of rpoB associated with rifampin resistance were studied in 37 multidrug-resistant (MDR) clinical strains of Mycobacterium tuberculosis isolated in Italy. At least one mutated codon was found in each MDR strain. It was always a single-base substitution leading to an amino acid change. Nine different rpoB alleles, three of which had not been reported before, were found. The relative frequencies of specific mutations in this sample were different from those previously reported from different geographical areas, since 22 strains (59.5%) carried the mutated codon TTG in position 531 (Ser→Leu) and 11 (29.7%) had GAC in position 526 (His→Asp). PMID:10074552

  7. Mycobacterium tuberculosis Whole Genome Sequences From Southern India Suggest Novel Resistance Mechanisms and the Need for Region-Specific Diagnostics.

    Science.gov (United States)

    Manson, Abigail L; Abeel, Thomas; Galagan, James E; Sundaramurthi, Jagadish Chandrabose; Salazar, Alex; Gehrmann, Thies; Shanmugam, Siva Kumar; Palaniyandi, Kannan; Narayanan, Sujatha; Swaminathan, Soumya; Earl, Ashlee M

    2017-06-01

    India is home to 25% of all tuberculosis cases and the second highest number of multidrug resistant cases worldwide. However, little is known about the genetic diversity and resistance determinants of Indian Mycobacterium tuberculosis, particularly for the primary lineages found in India, lineages 1 and 3. We whole genome sequenced 223 randomly selected M. tuberculosis strains from 196 patients within the Tiruvallur and Madurai districts of Tamil Nadu in Southern India. Using comparative genomics, we examined genetic diversity, transmission patterns, and evolution of resistance. Genomic analyses revealed (11) prevalence of strains from lineages 1 and 3, (11) recent transmission of strains among patients from the same treatment centers, (11) emergence of drug resistance within patients over time, (11) resistance gained in an order typical of strains from different lineages and geographies, (11) underperformance of known resistance-conferring mutations to explain phenotypic resistance in Indian strains relative to studies focused on other geographies, and (11) the possibility that resistance arose through mutations not previously implicated in resistance, or through infections with multiple strains that confound genotype-based prediction of resistance. In addition to substantially expanding the genomic perspectives of lineages 1 and 3, sequencing and analysis of M. tuberculosis whole genomes from Southern India highlight challenges of infection control and rapid diagnosis of resistant tuberculosis using current technologies. Further studies are needed to fully explore the complement of diversity and resistance determinants within endemic M. tuberculosis populations.

  8. Transcriptional and proteomic analyses of two-component response regulators in multidrug-resistant Mycobacterium tuberculosis.

    Science.gov (United States)

    Zhou, Lei; Yang, Liu; Zeng, Xianfei; Danzheng, Jiacuo; Zheng, Qing; Liu, Jiayun; Liu, Feng; Xin, Yijuan; Cheng, Xiaodong; Su, Mingquan; Ma, Yueyun; Hao, Xiaoke

    2015-07-01

    Two-component systems (TCSs) have been reported to exhibit a sensing and responding role under drug stress that induces drug resistance in several bacterial species. However, the relationship between TCSs and multidrug resistance in Mycobacterium tuberculosis has not been comprehensively analysed to date. In this study, 90 M. tuberculosis clinical isolates were analysed using 15-loci mycobacterial interspersed repetitive unit (MIRU)-variable number tandem repeat (VNTR) typing and repetitive extragenic palindromic (rep)-PCR-based DNA fingerprinting. The results showed that all of the isolates were of the Beijing lineage, and strains with a drug-susceptible phenotype had not diverged into similar genotype clusters. Expression analysis of 13 response regulators of TCSs using real-time PCR and tandem mass spectrometry (MS/MS) proteomic analysis demonstrated that four response regulator genes (devR, mtrA, regX3 and Rv3143) were significantly upregulated in multidrug-resistant (MDR) strains compared with the laboratory strain H37Rv as well as drug-susceptible and isoniazid-monoresistant strains (PMycobacterium bovis BCG did not alter its sensitivity to the four antitubercular drugs. This suggests that upregulation of devR, which is common in MDR-TB strains, might be induced by drug stress and hypoxic adaptation following the acquisition of multidrug resistance.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2006-01-01

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

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

    Science.gov (United States)

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

    2016-11-01

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

  11. A study of Mycobacterium tuberculosis genotypic diversity & drug resistance mutations in Varanasi, north India

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    Anamika Gupta

    2014-01-01

    Full Text Available Background & objectives: One-fifth of the world′s new tuberculosis (TB cases and two-thirds of cases in the South East Asian region occur in India. Molecular typing of Mycobacterium tuberculosis isolates has greatly facilitated to understand the transmission of TB. This study was aimed to investigate the molecular epidemiology of M. tuberculosis genotypes in Varanasi, north India, and their association with clinical presentation among patients with pulmonary TB. Methods: M. tuberculosis isolates from 104 TB patients attending a tertiary referral hospital of north India were screened for susceptibility to isoniazid (INH, rifampicin (RIF, ethambutol (EMB and streptomycin (STR by proportion method and multiplex-allele-specific-polymerase chain reaction (MAS-PCR. These were genotyped by spoligotyping. The spoligotype patterns were compared with those in the international SITVIT2 spoligotyping database. Results: Eighty three of 104 isolates were distributed in 38 SITs, of which SIT3366 was newly created within the present study. The mass of ongoing transmission with MDR-TB isolates in Varanasi, northern India, was linked to Beijing genotype followed by the CAS1_Delhi lineage. HIV-seropositive patients had a significantly higher proportion of clustered isolates than HIV-seronegative patients and compared with the wild type(wt isolates, the isolates with katG315Thr mutation were considerably more likely to be clustered. Interpretation & conclusions: This study gives an insight into the M. tuberculosis genetic biodiversity in Varanasi, north India, the predominant spoligotypes and their impact on disease transmission. In this region of north India, TB is caused by a wide diversity of spoligotypes with predominance of four genotype lineages: Beijing, CAS, EAI and T. The Beijing genotype was the most frequent single spoligotype and strongly associated with multi drug resistant (MDR-TB isolates. These findings may have important implications for control

  12. Whole genome sequencing of Mycobacterium tuberculosis for detection of drug resistance: a systematic review.

    Science.gov (United States)

    Papaventsis, D; Casali, N; Kontsevaya, I; Drobniewski, F; Cirillo, D M; Nikolayevskyy, V

    2017-02-01

    We conducted a systematic review to determine the diagnostic accuracy of whole genome sequencing (WGS) of Mycobacterium tuberculosis for the detection of resistance to first- and second-line anti-tuberculosis (TB) drugs. The study was conducted according to the criteria of the Preferred Reporting Items for Systematic Reviews group. A total of 20 publications were included. The sensitivity, specificity, positive-predictive value and negative-predictive value of WGS using phenotypic drug susceptibility testing methods as a reference standard were determined. Anti-TB agents tested included all first-line drugs, a variety of reserve drugs, as well as new drugs. Polymorphisms in a total of 53 genes were tested for associations with drug resistance. Pooled sensitivity and specificity values for detection of resistance to selected first-line drugs were 0.98 (95% CI 0.93-0.98) and 0.98 (95% CI 0.98-1.00) for rifampicin and 0.97 (95% CI 0.94-0.99) and 0.93 (95% CI 0.91-0.96) for isoniazid, respectively. Due to high heterogeneity in study designs, lack of data, knowledge of resistance mechanisms and clarity on exclusion of phylogenetic markers, there was a significant variation in analytical performance of WGS for the remaining first-line, reserved drugs and new drugs. Whole genome sequencing could be considered a promising alternative to existing phenotypic and molecular drug susceptibility testing methods for rifampicin and isoniazid pending standardization of analytical pipelines. To ensure clinical relevance of WGS for detection of M. tuberculosis complex drug resistance, future studies should include information on clinical outcomes. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

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

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    U D Gupta

    2015-01-01

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

  14. Role of the Mmr efflux pump in drug resistance in Mycobacterium tuberculosis.

    Science.gov (United States)

    Rodrigues, Liliana; Villellas, Cristina; Bailo, Rebeca; Viveiros, Miguel; Aínsa, José A

    2013-02-01

    Efflux pumps are membrane proteins capable of actively transporting a broad range of substrates from the cytoplasm to the exterior of the cell. Increased efflux activity in response to drug treatment may be the first step in the development of bacterial drug resistance. Previous studies showed that the efflux pump Mmr was significantly overexpressed in strains exposed to isoniazid. In the work to be described, we constructed mutants lacking or overexpressing Mmr in order to clarify the role of this efflux pump in the development of resistance to isoniazid and other drugs in M. tuberculosis. The mmr knockout mutant showed an increased susceptibility to ethidium bromide, tetraphenylphosphonium, and cetyltrimethylammonium bromide (CTAB). Overexpression of mmr caused a decreased susceptibility to ethidium bromide, acriflavine, and safranin O that was obliterated in the presence of the efflux inhibitors verapamil and carbonyl cyanide m-chlorophenylhydrazone. Isoniazid susceptibility was not affected by the absence or overexpression of mmr. The fluorometric method allowed the detection of a decreased efflux of ethidium bromide in the knockout mutant, whereas the overexpressed strain showed increased efflux of this dye. This increased efflux activity was inhibited in the presence of efflux inhibitors. Under our experimental conditions, we have found that efflux pump Mmr is mainly involved in the susceptibility to quaternary compounds such as ethidium bromide and disinfectants such as CTAB. The contribution of this efflux pump to isoniazid resistance in Mycobacterium tuberculosis still needs to be further elucidated.

  15. Pyrosequencing for rapid detection of Mycobacterium tuberculosis second-line drugs and ethambutol resistance.

    Science.gov (United States)

    Lacoma, Alicia; Molina-Moya, Barbara; Prat, Cristina; Pimkina, Edita; Diaz, Jessica; Dudnyk, Andriy; García-Sierra, Nerea; Haba, Lucía; Maldonado, Jose; Samper, Sofia; Ruiz-Manzano, Juan; Ausina, Vicente; Dominguez, Jose

    2015-11-01

    The aim of this work was to study the diagnostic accuracy of pyrosequencing to detect resistance to fluoroquinolones, kanamycin, amikacin, capreomycin, and ethambutol (EMB) in Mycobacterium tuberculosis clinical strains. One hundred four clinical isolates previously characterized by BACTEC 460TB/MGIT 960 were included. Specific mutations were targeted in gyrA, rrs, eis promoter, and embB. When there was a discordant result between BACTEC and pyrosequencing, Genotype MTBDRsl (Hain Lifescience, Nehren, Germany) was performed. Sensitivity and specificity of pyrosequencing were 70.6% and 100%, respectively, for fluoroquinolones; 93.3% and 81.7%, respectively, for kanamycin; 94.1% and 95.9%, respectively, for amikacin; 90.0% and 100%, respectively, for capreomycin; and 64.8% and 87.8%, respectively, for EMB. This study shows that pyrosequencing may be a useful tool for making early decisions regarding second-line drugs and EMB resistance. However, for a correct management of patients with suspected extensively drug-resistant tuberculosis, susceptibility results obtained by molecular methods should be confirmed by a phenotypic method.

  16. Role of embCAB gene mutations in ethambutol resistance in Mycobacterium tuberculosis isolates from India.

    Science.gov (United States)

    Jadaun, G P S; Das, Ram; Upadhyay, Prashant; Chauhan, D S; Sharma, V D; Katoch, V M

    2009-05-01

    In the present study, ethambutol (EMB) resistance-associated mutations were characterised in the embCAB genes of clinical isolates of Mycobacterium tuberculosis (MTB) collected in India. Thirty MTB isolates were tested for their susceptibility to first-line antitubercular drugs using the Löwenstein-Jensen proportion method, and EMB minimum inhibitory concentrations of MTB isolates were determined by the resazurin microtitre assay. Sequencing of various regions of the embCAB genes was performed to identify EMB resistance-associated mutations. Mutations of embB306 were detected in 15 of 23 EMB-resistant MTB isolates. Three EMB-resistant isolates had mutations at codon 270 of the embC gene, two of which also harboured embB306 mutations. No mutation was identified in the embA gene. All seven EMB-sensitive MTB isolates had the wild-type embCAB sequence. In summary, embB306 mutations were associated with EMB resistance, and mutation at codon 270 of the embC gene may contribute to high-level EMB resistance in some MTB isolates.

  17. Screening for streptomycin resistance-conferring mutations in Mycobacterium tuberculosis clinical isolates from Poland.

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    Tomasz Jagielski

    Full Text Available Currently, mutations in three genes, namely rrs, rpsL, and gidB, encoding 16S rRNA, ribosomal protein S12, and 16S rRNA-specific methyltransferase, respectively, are considered to be involved in conferring resistance to streptomycin (STR in Mycobacterium tuberculosis. The aim of this study was to investigate the spectrum and frequency of these mutations in M. tuberculosis clinical isolates, both resistant and susceptible to STR. Sixty-four M. tuberculosis isolates recovered from as many TB patients from Poland in 2004 were included in the study. Within the sample were 50 multidrug-resistant (32 STR-resistant and 18 STR-susceptible and 14 pan-susceptible isolates. Preliminary testing for STR resistance was performed with the 1% proportion method. The MICs of STR were determined by the Etest method. Mutation profiling was carried out by amplifying and sequencing the entire rrs, rpsL, and gidB genes. Non-synonymous mutations in either rrs or rpsL gene were detected in 23 (71.9% of the STR-resistant and none of the STR-susceptible isolates. Mutations in the gidB gene were distributed among 12 (37.5% STR-resistant and 13 (40.6% STR-susceptible isolates. Four (12.5% STR-resistant isolates were wild-type at all three loci examined. None of the rrs, rpsL or gidB mutations could be linked to low, intermediate or high level of STR resistance. In accordance with previous findings, the gidB 47T→G (L16R mutation was associated with the Latin American-Mediterranean genotype family, whereas 276A→C (E92D and 615A→G (A205A mutations of the gidB gene were associated with the Beijing lineage. The study underlines the usefulness of rrs and rpsL mutations as molecular markers for STR resistance yet not indicative of its level. The gidB polymorphisms can serve as phylogenetic markers.

  18. Evaluation of the Genotype MTBDR Assay for Rapid Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Isolates

    OpenAIRE

    2006-01-01

    A novel PCR-based reverse hybridization method Genotype MTBDR assay (Hain Lifescience GmbH, Nehren, Germany) was evaluated for rapid detection of rifampin (RIF) and isoniazid (INH) resistance in Turkish Mycobacterium tuberculosis isolates. The Genotype MTBDR assay is designed to detect mutations within the 81-bp hotspot region of rpoB and mutations at katG codon 315. A total of 41 RIF-resistant M. tuberculosis isolates with rpoB mutations that were previously tested by the INNO-LiPA Rif.TB ki...

  19. Improved Detection by Next-Generation Sequencing of Pyrazinamide Resistance in Mycobacterium tuberculosis Isolates.

    Science.gov (United States)

    Maningi, Nontuthuko E; Daum, Luke T; Rodriguez, John D; Mphahlele, Matsie; Peters, Remco P H; Fischer, Gerald W; Chambers, James P; Fourie, P Bernard

    2015-12-01

    The technical limitations of common tests used for detecting pyrazinamide (PZA) resistance in Mycobacterium tuberculosis isolates pose challenges for comprehensive and accurate descriptions of drug resistance in patients with multidrug-resistant tuberculosis (MDR-TB). In this study, a 606-bp fragment (comprising the pncA coding region plus the promoter) was sequenced using Ion Torrent next-generation sequencing (NGS) to detect associated PZA resistance mutations in 88 recultured MDR-TB isolates from an archived series collected in 2001. These 88 isolates were previously Sanger sequenced, with 55 (61%) designated as carrying the wild-type pncA gene and 33 (37%) showing mutations. PZA susceptibility of the isolates was also determined using the Bactec 460 TB system and the Wayne test. In this study, isolates were recultured and susceptibility testing was performed in Bactec 960 MGIT. Concordance between NGS and MGIT results was 93% (n = 88), and concordance values between the Bactec 460, the Wayne test, or pncA gene Sanger sequencing and NGS results were 82% (n = 88), 83% (n = 88), and 89% (n = 88), respectively. NGS confirmed the majority of pncA mutations detected by Sanger sequencing but revealed several new and mixed-strain mutations that resolved discordancy in other phenotypic results. Importantly, in 53% (18/34) of these isolates, pncA mutations were located in the 151 to 360 region and warrant further exploration. In these isolates, with their known resistance to rifampin, NGS of pncA improved PZA resistance detection sensitivity to 97% and specificity to 94% using NGS as the gold standard and helped to resolve discordant results from conventional methodologies.

  20. Influence of antituberculosis drug resistance and Mycobacterium tuberculosis lineage on outcome in HIV-associated tuberculous meningitis.

    Science.gov (United States)

    Tho, Dau Quang; Török, M Estée; Yen, Nguyen Thi Bich; Bang, Nguyen Duc; Lan, Nguyen Thi Ngoc; Kiet, Vo Sy; van Vinh Chau, Nguyen; Dung, Nguyen Huy; Day, Jeremy; Farrar, Jeremy; Wolbers, Marcel; Caws, Maxine

    2012-06-01

    HIV-associated tuberculous meningitis (TBM) has high mortality. Aside from the devastating impact of multidrug resistance (MDR) on survival, little is understood about the influence of other bacterial factors on outcome. This study examined the influence of Mycobacterium tuberculosis drug resistance, bacterial lineage, and host vaccination status on outcome in patients with HIV-associated TBM. Mycobacterium tuberculosis isolates from the cerebrospinal fluid of 186 patients enrolled in two studies of HIV-associated TBM in Ho Chi Minh City, Vietnam, were tested for resistance to first-line antituberculosis drugs. Lineage genotyping was available for 122 patients. The influence of antituberculosis drug resistance and M. tuberculosis lineage on 9-month mortality was analyzed using Kaplan-Meier survival analysis and Cox multiple regression models. Isoniazid (INH) resistance without rifampin resistance was associated with increased mortality (adjusted hazard ratio [HR], 1.78, 95% confidence interval [CI], 1.18 to 2.66; P = 0.005), and multidrug resistance was uniformly fatal (n = 8/8; adjusted HR, 5.21, 95% CI, 2.38 to 11.42; P tuberculosis lineage are important determinants of mortality in patients with HIV-associated TBM. Interventions which target these factors may help reduce the unacceptably high mortality in patients with TBM.

  1. Drug resistant Mycobacterium tuberculosis of the Beijing genotype does not spread in Sweden.

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    Solomon Ghebremichael

    Full Text Available BACKGROUND: Drug resistant (DR and multi-drug resistant (MDR tuberculosis (TB is increasing worldwide. In some parts of the world 10% or more of new TB cases are MDR. The Beijing genotype is a distinct genetic lineage of Mycobacterium tuberculosis, which is distributed worldwide, and has caused large outbreaks of MDR-TB. It has been proposed that certain lineages of M. tuberculosis, such as the Beijing lineage, may have specific adaptive advantages. We have investigated the presence and transmission of DR Beijing strains in the Swedish population. METHODOLOGY/PRINCIPAL FINDINGS: All DR M. tuberculosis complex isolates between 1994 and 2008 were studied. Isolates that were of Beijing genotype were investigated for specific resistance mutations and phylogenetic markers. Seventy (13% of 536 DR strains were of Beijing genotype. The majority of the patients with Beijing strains were foreign born, and their country of origin reflects the countries where the Beijing genotype is most prevalent. Multidrug-resistance was significantly more common in Beijing strains than in non-Beijing strains. There was a correlation between the Beijing genotype and specific resistance mutations in the katG gene, the mabA-inhA-promotor and the rpoB gene. By a combined use of RD deletions, spoligotyping, IS1547, mutT gene polymorphism and Rv3135 gene analysis the Beijing strains could be divided into 11 genomic sublineages. Of the patients with Beijing strains 28 (41% were found in altogether 10 clusters (2-5 per cluster, as defined by RFLP IS6110, while 52% of the patients with non-Beijing strains were in clusters. By 24 loci MIRU-VNTR 31 (45% of the patients with Beijing strains were found in altogether 7 clusters (2-11 per cluster. Contact tracing established possible epidemiological linkage between only two patients with Beijing strains. CONCLUSIONS/SIGNIFICANCE: Although extensive outbreaks with non-Beijing TB strains have occurred in Sweden, Beijing strains have not

  2. Characterization of the genetic diversity of extensively-drug resistant Mycobacterium tuberculosis clinical isolates from pulmonary tuberculosis patients in Peru.

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    Omar Cáceres

    Full Text Available BACKGROUND: Peru holds the fourth highest burden of tuberculosis in the Americas. Despite an apparently well-functioning DOTS control program, the prevalence of multidrug resistant tuberculosis (MDR-TB continues to increase. To worsen this situation, cases of extensively drug resistance tuberculosis (XDR-TB have been detected. Little information exists about the genetic diversity of drug-susceptible vs. MDR-TB and XDR-TB. METHODS: Cryopreserved samples of XDR strains from 2007 to 2009 (second semester, were identified and collected. Starting from 227 frozen samples, a total of 142 XDR-TB strains of Mycobacterium tuberculosis complex (MTBC; 1 isolate per patient were retained for this study. Each strain DNA was analyzed by spoligotyping and the 15-loci Mycobacterial Interspersed Repetitive Unit (MIRU-15. RESULTS: Among the 142 isolates analyzed, only 2 samples (1.41% could not be matched to any lineage. The most prevalent sublineage was Haarlem (43.66%, followed by T (27.46%, LAM (16.2%, Beijing (9.15%, and X clade (1.41%. Spoligotype analysis identified clustering for 128/142 (90.1% isolates vs. 49/142 (34.5% with MIRUs. Of the samples, 90.85% belonged to retreated patients. The drug resistant profile demonstrated that 62.67% showed resistance to injectable drugs capreomycin (CAP and kanamycin (KAN vs. 15.5% to CAP alone and 21.8% to KAN alone. The SIT219/T1 and SIT50/H3 were the most prevalent patterns in our study. The spoligoforest analysis showed that SIT53/T1 was at the origin of many of the T lineage strains as well as a big proportion of Haarlem lineage strains (SIT50/H3, followed by SIT47/H1, SIT49/H3, and SIT2375/H1, as opposed to the SIT1/Beijing strains that did not appear to evolve into minor Beijing sublineages among the XDR-TB strains. CONCLUSION: In contrast with other Latin-American countries where LAM sublineage is the most predominant, we found the Haarlem to be the most common followed by T sublineage among the XDR-TB strains.

  3. Performance assessment of the GenoType MTBDRplus test and DNA sequencing in detection of multidrug-resistant Mycobacterium tuberculosis.

    Science.gov (United States)

    Huang, Wei-Lun; Chen, Huang-Yau; Kuo, Yuh-Min; Jou, Ruwen

    2009-08-01

    To facilitate the management of multidrug-resistant (MDR) tuberculosis, two nucleic acid sequence-based methods, the GenoType MTBDRplus test and DNA sequencing, were assessed for the rapid detection of drug-resistant Mycobacterium tuberculosis for the first time in the Asia-Pacific region. The performances of these two assays in detecting the presence of rifampin (rifampicin) (RIF) and isoniazid (INH) resistance-associated mutations in the rpoB, katG, inhA regulatory region, inhA, and oxyR-ahpC genes were compared to that of a conventional agar proportion drug susceptibility test. A total of 242 MDR and 30 pansusceptible M. tuberculosis isolates were evaluated in this study. The sensitivities obtained for RIF-resistant detection by the GenoType MTBDRplus test and by resistance gene sequencing were 95.5% and 97.9%, respectively. The sensitivities for INH resistance detection by the GenoType MTBDRplus test and by resistance gene sequencing were 81.8% and 93.4%, respectively. Together, the sensitivity for MDR tuberculosis detection was 78.5% with the GenoType MTBDRplus test and 91.3% by resistance gene sequencing. The specificity for RIF resistance, INH resistance, and MDR detection was 100% by both methods. The GenoType MTBDRplus test has the advantage of a short turnaround time for drug-resistant M. tuberculosis detection. Overall, the two assays performed equally well in detecting RIF resistance (P = 0.13). However, DNA sequencing demonstrated superior performance in detecting INH resistance (P tuberculosis (P GenoType MTBDRplus test, especially for different geographic areas with genetically diverse M. tuberculosis strains.

  4. QSAR studies, synthesis and antibacterial assessment of new inhibitors against multidrug-resistant Mycobacterium tuberculosis.

    Science.gov (United States)

    Kovalishyn, Vasyl; Brovarets, Volodymyr; Blagodatnyi, Volodymyr; Kopernyk, Iryna; Hodyna, Diana; Chumachenko, Svitlana; Shablykin, Oleg; Kozachenko, Oleksandr; Vovk, Myhailo; Barus, Marianna; Bratenko, Myhailo; Metelytsia, Larysa

    2016-11-08

    This paper describes Quantitative Structure-Activity Relationships (QSAR) studies using Artificial Neural Networks (ANN), synthesis and in vitro antitubercular activity of several potent compounds against H37Rv and resistant Mycobacterium tuberculosis (Mtb) strains. Eight QSAR models were built using various types of descriptors with four publicly available structurally diverse datasets, including recent data from PubChem and ChEMBL. The predictive power of the obtained QSAR models was evaluated with a cross-validation procedure, giving a q2=0.74-0.78 for regression models and overall accuracy 78.9-94.4% for classification models. The external test sets were predicted with accuracies in the range of 84.1-95.0% (for the active/inactive classifications) and q2=0.80-0.83 for regressions. The 15 synthesized compounds showed inhibitory activity against H37Rv strain whereas the compounds 1-7 were also active against resistant Mtb strain (resistant to isoniazid and rifampicin). The results indicated that compounds 1-7 could serve as promising leads for further optimization as novel antibacterial inhibitors, in particular, for the treatment of drug resistance of Mtb forms.

  5. Detecting katG Drug Resistant Genetic Mutation among pneumoconiosis patients complicated with tuberculosis in Mycobacterium tuberculosis L-forms by PCR-SSCP

    Institute of Scientific and Technical Information of China (English)

    Jun Lu; Zhao Zheng; Song Ye; Chaopin Li

    2006-01-01

    Objective: To study the relationship between mutation in the katG gene and drug resistance of INH in Mycobacterium tuberculosis L-forms among patients of pneumoconiosis complicated with pulmonary tuberculosis, and to explore the clinical application of PCR-SSCP. Methods:A total of 52 clinical isolated strains of Mycobacterium tuberculosis L-forms were collected. Mutation in the katG genes was detected by PCR-SSCP and traditional antimicrobial susceptibility test (AST). Results:The results by AST showed that there were 40 persisters in 52 clinical isolated strains. The drug resistant rate was 76.92%(40/52),and the gene mutation rate of katG was 57.70% (30/52)by.PCR-SSCP, the difference was no quite significance (χ2 = 2.8507, P >0.05). The coincidence rate of two methods was 75.00% (30/40). Conclusion: The detectionrate of katG resistant strains in Mycobacterium tuberculosis L-forms was high by PCR-SSCP. The combined application of PCR-SSCP and traditional antimicrobial susceptibility test can improve the detecting rate.

  6. Identification of new drug targets and resistance mechanisms in Mycobacterium tuberculosis.

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    Thomas R Ioerger

    Full Text Available Identification of new drug targets is vital for the advancement of drug discovery against Mycobacterium tuberculosis, especially given the increase of resistance worldwide to first- and second-line drugs. Because traditional target-based screening has largely proven unsuccessful for antibiotic discovery, we have developed a scalable platform for target identification in M. tuberculosis that is based on whole-cell screening, coupled with whole-genome sequencing of resistant mutants and recombineering to confirm. The method yields targets paired with whole-cell active compounds, which can serve as novel scaffolds for drug development, molecular tools for validation, and/or as ligands for co-crystallization. It may also reveal other information about mechanisms of action, such as activation or efflux. Using this method, we identified resistance-linked genes for eight compounds with anti-tubercular activity. Four of the genes have previously been shown to be essential: AspS, aspartyl-tRNA synthetase, Pks13, a polyketide synthase involved in mycolic acid biosynthesis, MmpL3, a membrane transporter, and EccB3, a component of the ESX-3 type VII secretion system. AspS and Pks13 represent novel targets in protein translation and cell-wall biosynthesis. Both MmpL3 and EccB3 are involved in membrane transport. Pks13, AspS, and EccB3 represent novel candidates not targeted by existing TB drugs, and the availability of whole-cell active inhibitors greatly increases their potential for drug discovery.

  7. Evaluation of macrolides for possible use against multidrug-resistant Mycobacterium tuberculosis

    NARCIS (Netherlands)

    van der Paardt, Anne-Fleur; Wilffert, Bob; Akkerman, Onno W.; de Lange, Wiel C. M.; van Soolingen, Dick; Sinha, Bhanu; van der Werf, Tjip S.; Kosterink, Jos G. W.; Alffenaar, Jan-Willem C.

    2015-01-01

    Multidrug-resistant tuberculosis (MDR-TB) is a major global health problem. The loss of susceptibility to an increasing number of drugs behoves us to consider the evaluation of non-traditional anti-tuberculosis drugs. Clarithromycin, a macrolide antibiotic, is defined as a group 5 anti-tuberculosis

  8. Pre-existing isoniazid resistance, but not the genotype of Mycobacterium tuberculosis drives rifampicin resistance codon preference in vitro.

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    Indra Bergval

    Full Text Available Both the probability of a mutation occurring and the ability of the mutant to persist will influence the distribution of mutants that arise in a population. We studied the interaction of these factors for the in vitro selection of rifampicin (RIF-resistant mutants of Mycobacterium tuberculosis. We characterised two series of spontaneous RIF-resistant in vitro mutants from isoniazid (INH-sensitive and -resistant laboratory strains and clinical isolates, representing various M. tuberculosis genotypes. The first series were selected from multiple parallel 1 ml cultures and the second from single 10 ml cultures. RIF-resistant mutants were screened by Multiplex Ligation-dependent Probe Amplification (MLPA or by sequencing the rpoB gene. For all strains the mutation rate for RIF resistance was determined with a fluctuation assay. The most striking observation was a shift towards rpoB-S531L (TCG→TTG mutations in a panel of laboratory-generated INH-resistant mutants selected from the 10-ml cultures (p<0.001. All tested strains showed similar mutation rates (1.33×10⁻⁸ to 2.49×10⁻⁷ except one of the laboratory-generated INH mutants with a mutation rate measured at 5.71×10⁻⁷, more than 10 times higher than that of the INH susceptible parental strain (5.46-7.44×10⁻⁸. No significant, systematic difference in the spectrum of rpoB-mutations between strains of different genotypes was observed. The dramatic shift towards rpoB-S531L in our INH-resistant laboratory mutants suggests that the relative fitness of resistant mutants can dramatically impact the distribution of (subsequent mutations that accumulate in a M. tuberculosis population, at least in vitro. We conclude that, against specific genetic backgrounds, certain resistance mutations are particularly likely to spread. Molecular screening for these (combinations of mutations in clinical isolates could rapidly identify these particular pathogenic strains. We therefore recommend that

  9. Pre-existing isoniazid resistance, but not the genotype of Mycobacterium tuberculosis drives rifampicin resistance codon preference in vitro.

    Science.gov (United States)

    Bergval, Indra; Kwok, Brian; Schuitema, Anja; Kremer, Kristin; van Soolingen, Dick; Klatser, Paul; Anthony, Richard

    2012-01-01

    Both the probability of a mutation occurring and the ability of the mutant to persist will influence the distribution of mutants that arise in a population. We studied the interaction of these factors for the in vitro selection of rifampicin (RIF)-resistant mutants of Mycobacterium tuberculosis. We characterised two series of spontaneous RIF-resistant in vitro mutants from isoniazid (INH)-sensitive and -resistant laboratory strains and clinical isolates, representing various M. tuberculosis genotypes. The first series were selected from multiple parallel 1 ml cultures and the second from single 10 ml cultures. RIF-resistant mutants were screened by Multiplex Ligation-dependent Probe Amplification (MLPA) or by sequencing the rpoB gene. For all strains the mutation rate for RIF resistance was determined with a fluctuation assay. The most striking observation was a shift towards rpoB-S531L (TCG→TTG) mutations in a panel of laboratory-generated INH-resistant mutants selected from the 10-ml cultures (pmutation rates (1.33×10⁻⁸ to 2.49×10⁻⁷) except one of the laboratory-generated INH mutants with a mutation rate measured at 5.71×10⁻⁷, more than 10 times higher than that of the INH susceptible parental strain (5.46-7.44×10⁻⁸). No significant, systematic difference in the spectrum of rpoB-mutations between strains of different genotypes was observed. The dramatic shift towards rpoB-S531L in our INH-resistant laboratory mutants suggests that the relative fitness of resistant mutants can dramatically impact the distribution of (subsequent) mutations that accumulate in a M. tuberculosis population, at least in vitro. We conclude that, against specific genetic backgrounds, certain resistance mutations are particularly likely to spread. Molecular screening for these (combinations of) mutations in clinical isolates could rapidly identify these particular pathogenic strains. We therefore recommend that isolates are screened for the distribution of resistance

  10. Tuberculosis in Australia: bacteriologically confirmed cases and drug resistance, 2007. A report of the Australian Mycobacterium Reference Laboratory Network.

    Science.gov (United States)

    Lumb, Richard; Bastion, Ivan; Carter, Robyn; Jelfs, Peter; Keehner, Terillee; Sievers, Aina

    2009-09-01

    The Australian Mycobacterium Reference Laboratory Network collects and analyses laboratory data on new cases of disease caused by the Mycobacterium tuberculosis complex. In 2007, a total of 872 cases were identified by bacteriology; an annual reporting rate of 4.1 cases per 100,000 population. Isolates were identified as M. tuberculosis (n=867), M. africanum (n=4) and M. bovis (n=1). Fifteen children aged under 10 years had bacteriologically-confirmed tuberculosis. Results of in vitro drug susceptibility testing were available for 871 of 872 isolates for isoniazid (H), rifampicin (R), ethambutol (E), and pyrazinamide (Z). A total of 98 (11.3%) isolates of M. tuberculosis were resistant to at least one of these anti-tuberculosis agents. Resistance to at least H and R (defined as multi-drug resistance, MDR) was detected in 24 (2.8%) isolates, all from overseas-born patients; 17 were from the respiratory tract (sputum n=16, endotracheal aspirate n=1). Thirteen patients with MDR-TB were from the Papua New Guinea-Torres Strait Islands zone. Of the 98 M. tuberculosis isolates resistant to at least one of the standard drugs, 54 (55.1%) were from new cases, 9 (9.2%) from previously treated cases, and no information was available on the remaining 35 cases. Seven were Australian-born, 90 were overseas- born, and the country of birth of 1 was unknown. Of the 90 overseas-born persons with drug resistant disease, 66 (73.3%) were from 5 countries: India (n=16); Papua New Guinea (n=15); the Philippines (n=12); Vietnam (n=12); and China (n=11). No XDR-TB was detected in 2007.

  11. Whole Genome Sequencing Based Characterization of Extensively Drug-Resistant Mycobacterium tuberculosis Isolates from Pakistan

    KAUST Repository

    Ali, Asho

    2015-02-26

    Improved molecular diagnostic methods for detection drug resistance in Mycobacterium tuberculosis (MTB) strains are required. Resistance to first- and second- line anti-tuberculous drugs has been associated with single nucleotide polymorphisms (SNPs) in particular genes. However, these SNPs can vary between MTB lineages therefore local data is required to describe different strain populations. We used whole genome sequencing (WGS) to characterize 37 extensively drug-resistant (XDR) MTB isolates from Pakistan and investigated 40 genes associated with drug resistance. Rifampicin resistance was attributable to SNPs in the rpoB hot-spot region. Isoniazid resistance was most commonly associated with the katG codon 315 (92%) mutation followed by inhA S94A (8%) however, one strain did not have SNPs in katG, inhA or oxyR-ahpC. All strains were pyrazimamide resistant but only 43% had pncA SNPs. Ethambutol resistant strains predominantly had embB codon 306 (62%) mutations, but additional SNPs at embB codons 406, 378 and 328 were also present. Fluoroquinolone resistance was associated with gyrA 91-94 codons in 81% of strains; four strains had only gyr B mutations, while others did not have SNPs in either gyrA or gyrB. Streptomycin resistant strains had mutations in ribosomal RNA genes; rpsL codon 43 (42%); rrs 500 region (16%), and gidB (34%) while six strains did not have mutations in any of these genes. Amikacin/kanamycin/capreomycin resistance was associated with SNPs in rrs at nt1401 (78%) and nt1484 (3%), except in seven (19%) strains. We estimate that if only the common hot-spot region targets of current commercial assays were used, the concordance between phenotypic and genotypic testing for these XDR strains would vary between rifampicin (100%), isoniazid (92%), flouroquinolones (81%), aminoglycoside (78%) and ethambutol (62%); while pncA sequencing would provide genotypic resistance in less than half the isolates. This work highlights the importance of expanded

  12. A systematic review of gyrase mutations associated with fluoroquinolone-resistant Mycobacterium tuberculosis and a proposed gyrase numbering system

    Science.gov (United States)

    Maruri, Fernanda; Sterling, Timothy R.; Kaiga, Anne W.; Blackman, Amondrea; van der Heijden, Yuri F.; Mayer, Claudine; Cambau, Emmanuelle; Aubry, Alexandra

    2012-01-01

    Fluoroquinolone resistance in Mycobacterium tuberculosis has become increasingly important. A review of mutations in DNA gyrase, the fluoroquinolone target, is needed to improve the molecular detection of resistance. We performed a systematic review of studies reporting mutations in DNA gyrase genes in clinical M. tuberculosis isolates. From 42 studies that met inclusion criteria, 1220 fluoroquinolone-resistant M. tuberculosis isolates underwent sequencing of the quinolone resistance-determining region (QRDR) of gyrA; 780 (64%) had mutations. The QRDR of gyrB was sequenced in 534 resistant isolates; 17 (3%) had mutations. Mutations at gyrA codons 90, 91 or 94 were present in 654/1220 (54%) resistant isolates. Four different GyrB numbering systems were reported, resulting in mutation location discrepancies. We propose a consensus numbering system. Most fluoroquinolone-resistant M. tuberculosis isolates had mutations in DNA gyrase, but a substantial proportion did not. The proposed consensus numbering system can improve molecular detection of resistance and identification of novel mutations. PMID:22279180

  13. Rapid identification of mycobacteria and rapid detection of drug resistance in Mycobacterium tuberculosis in cultured isolates and in respiratory specimens.

    Science.gov (United States)

    Yam, Wing-Cheong; Siu, Kit-Hang Gilman

    2013-01-01

    Recent advances in molecular biology and better understanding of the genetic basis of drug resistance have allowed rapid identification of mycobacteria and rapid detection of drug resistance of Mycobacterium tuberculosis present in cultured isolates or in respiratory specimens. In this chapter, several simple nucleic acid amplification-based techniques are introduced as molecular approach for clinical diagnosis of tuberculosis. A one-tube nested IS6110-based polymerase chain reaction (PCR) is used for M. tuberculosis complex identification; the use of a multiplex allele-specific PCR is demonstrated to detect the isoniazid resistance; PCR-sequencing assays are applied for rifampicin and ofloxacin resistance detection and 16S rDNA sequencing is utilized for identification of mycobacterial species from cultures of acid fast bacilli (AFB). Despite the high specificity and sensitivity of the molecular techniques, mycobacterial culture remains the "Gold Standard" for tuberculosis diagnosis. Negative results of molecular tests never preclude the infection or the presence of drug resistance. These technological advancements are, therefore, not intended to replace the conventional tests, but rather have major complementary roles in tuberculosis diagnosis.

  14. Comparison of rapid tests for detection of rifampicin-resistant Mycobacterium tuberculosis in Kampala, Uganda

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    McNerney Ruth

    2009-08-01

    Full Text Available Abstract Background Drug resistant tuberculosis (TB is a growing concern worldwide. Rapid detection of resistance expedites appropriate intervention to control the disease. Several technologies have recently been reported to detect rifampicin resistant Mycobacterium tuberculosis directly in sputum samples. These include phenotypic culture based methods, tests for gene mutations and tests based on bacteriophage replication. The aim of the present study was to assess the feasibility of implementing technology for rapid detection of rifampicin resistance in a high disease burden setting in Africa. Methods Sputum specimens from re-treatment TB patients presenting to the Mulago Hospital National TB Treatment Centre in Kampala, Uganda, were examined by conventional methods and simultaneously used in one of the four direct susceptibility tests, namely direct BACTEC 460, Etest, "in-house" phage test, and INNO- Rif.TB. The reference method was the BACTEC 460 indirect culture drug susceptibility testing. Test performance, cost and turn around times were assessed. Results In comparison with indirect BACTEC 460, the respective sensitivities and specificities for detecting rifampicin resistance were 100% and 100% for direct BACTEC and the Etest, 94% and 95% for the phage test, and 87% and 87% for the Inno-LiPA assay. Turn around times ranged from an average of 3 days for the INNO-LiPA and phage tests, 8 days for the direct BACTEC 460 and 20 days for the Etest. All methods were faster than the indirect BACTEC 460 which had a mean turn around time of 24 days. The cost per test, including labour ranged from $18.60 to $41.92 (USD. Conclusion All four rapid technologies were shown capable of detecting rifampicin resistance directly from sputum. The LiPA proved rapid, but was the most expensive. It was noted, however, that the LiPA test allows sterilization of samples prior to testing thereby reducing the risk of accidental laboratory transmission. In contrast the

  15. Copper Homeostasis in Mycobacterium tuberculosis

    Science.gov (United States)

    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

  16. High proportion of heteroresistance in gyrA and gyrB in fluoroquinolone-resistant Mycobacterium tuberculosis clinical isolates.

    Science.gov (United States)

    Eilertson, Brandon; Maruri, Fernanda; Blackman, Amondrea; Herrera, Miguel; Samuels, David C; Sterling, Timothy R

    2014-06-01

    Heteroresistance is the coexistence of populations with differing nucleotides at a drug resistance locus within a sample of organisms. Although Sanger sequencing is the gold standard for sequencing, it may be less sensitive than deep sequencing for detecting fluoroquinolone heteroresistance in Mycobacterium tuberculosis. Twenty-seven fluoroquinolone monoresistant and 11 fluoroquinolone-susceptible M. tuberculosis isolates were analyzed by Sanger and Illumina deep sequencing. Individual sequencing reads were analyzed to detect heteroresistance in the gyrA and gyrB genes. Heteroresistance to fluoroquinolones was identified in 10/26 (38%) phenotypically fluoroquinolone-resistant samples and 0/11 (P = 0.02) fluoroquinolone-susceptible controls. One resistant sample was excluded because of contamination with the laboratory strain M. tuberculosis H37Rv. Sanger sequencing revealed resistance-conferring mutations in 15 isolates, while deep sequencing revealed mutations in 20 isolates. Isolates with fluoroquinolone resistance-conferring mutations by Sanger sequencing all had at least those same mutations identified by deep sequencing. By deep sequencing, 10 isolates had a single fixed (defined as >95% frequency) mutation, while 10 were heteroresistant, 5 of which had a single unfixed (defined as fluoroquinolone-resistant M. tuberculosis isolates with heteroresistance than did Sanger sequencing. The heteroresistant isolates frequently demonstrated multiple mutations, but resistant isolates with fixed mutations each had only a single mutation. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  17. Putative compensatory mutations in the rpoC gene of rifampin-resistant Mycobacterium tuberculosis are associated with ongoing transmission.

    Science.gov (United States)

    de Vos, M; Müller, B; Borrell, S; Black, P A; van Helden, P D; Warren, R M; Gagneux, S; Victor, T C

    2013-02-01

    Rifampin resistance in clinical isolates of Mycobacterium tuberculosis arises primarily through the selection of bacterial variants harboring mutations in the 81-bp rifampin resistance-determining region of the rpoB gene. While these mutations were shown to infer a fitness cost in the absence of antibiotic pressure, compensatory mutations in rpoA and rpoC were identified which restore the fitness of rifampin-resistant bacteria carrying mutations in rpoB. To investigate the epidemiological relevance of these compensatory mutations, we analyzed 286 drug-resistant and 54 drug-susceptible clinical M. tuberculosis isolates from the Western Cape, South Africa, a high-incidence setting of multidrug-resistant tuberculosis. Sequencing of a portion of the RpoA-RpoC interaction region of the rpoC gene revealed that 23.5% of all rifampin-resistant isolates tested carried a nonsynonymous mutation in this region. These putative compensatory mutations in rpoC were associated with transmission, as 30.8% of all rifampin-resistant isolates with an IS6110 restriction fragment length polymorphism (RFLP) pattern belonging to a recognized RFLP cluster harbored putative rpoC mutations. Such mutations were present in only 9.4% of rifampin-resistant isolates with unique RFLP patterns (P mutations were associated with specific strain genotypes and the rpoB S531L rifampin resistance mutation. Among isolates harboring this rpoB mutation, 44.1% also harbored rpoC mutations, while only 4.1% of the isolates with other rpoB mutations exhibited mutations in rpoC (P mutations in the transmission of multidrug-resistant tuberculosis and illustrates how epistatic interactions between drug resistance-conferring mutations, compensatory mutations, and different strain genetic backgrounds might influence compensatory evolution in drug-resistant M. tuberculosis.

  18. Tuberculosis in Australia: bacteriologically confirmed cases and drug resistance, 2000: report of the Australian Mycobacterium Laboratory Reference Network.

    Science.gov (United States)

    Lumb, Richard; Bastian, Ivan; Dawson, David; Gilpin, Chris; Havekort, Frank; Howard, Peter; Sievers, Aina

    2002-01-01

    The Australian Mycobacterium Reference Laboratory Network collected and analysed laboratory data on new diagnoses of disease caused by Mycobacterium tuberculosis complex in the year 2000. A total of 765 cases were identified, representing an annual reporting rate of 4.0 cases of laboratory-confirmed tuberculosis (TB) per 100,000 population. Pulmonary disease was diagnosed in 64.9 per cent of cases with a male:female ratio of 1.5:1. Smears were positive for 209/365 (57.3%) of sputum isolates and 39/117 (33.3%) bronchoscopy isolates. Sputum from males was more likely to be smear-positive (63.3%) than from females (47.5%). Isolates from lymph node accounted for 136 (17.7%) of all cases; only 28.7 per cent were smear-positive. Eighty-four (11.0%) isolates, comprising 82 M. tuberculosis and 2 M. bovis strains, demonstrated in vitro resistance to at least one of the standard anti-TB medications. Resistance to at least isoniazid and rifampicin (defined as multidrug-resistant TB) was observed for only 8 (1.0%) strains, a rate similar to previous years. Almost all (96.3%) of patients with drug resistant strains were classified as having initial resistance. The country of birth was known for 76 (92.7%) of 82 patients with a drug resistant strain of M. tuberculosis; 6 were Australian-born and 70 (92.1%) had migrated from a total of 17 countries. Of these 70 migrants with drug-resistant disease, 68.6 per cent had migrated from one of the following countries: Vietnam (n=15), China (n=11), Philippines (n=11), India (n=6), and Indonesia (n=5).

  19. Conspicuous multidrug-resistant Mycobacterium tuberculosis cluster strains do not trespass country borders in Latin America and Spain.

    Science.gov (United States)

    Ritacco, Viviana; Iglesias, María-José; Ferrazoli, Lucilaine; Monteserin, Johana; Dalla Costa, Elis R; Cebollada, Alberto; Morcillo, Nora; Robledo, Jaime; de Waard, Jacobus H; Araya, Pamela; Aristimuño, Liselotte; Díaz, Raúl; Gavin, Patricia; Imperiale, Belen; Simonsen, Vera; Zapata, Elsa M; Jiménez, María S; Rossetti, Maria L; Martin, Carlos; Barrera, Lucía; Samper, Sofia

    2012-06-01

    Multidrug-resistant Mycobacterium tuberculosis strain diversity in Ibero-America was examined by comparing extant genotype collections in national or state tuberculosis networks. To this end, genotypes from over 1000 patients with multidrug-resistant tuberculosis diagnosed from 2004 through 2008 in Argentina, Brazil, Chile, Colombia, Venezuela and Spain were compared in a database constructed ad hoc. Most of the 116 clusters identified by IS6110 restriction fragment length polymorphism were small and restricted to individual countries. The three largest clusters, of 116, 49 and 25 patients, were found in Argentina and corresponded to previously documented locally-epidemic strains. Only 13 small clusters involved more than one country, altogether accounting for 41 patients, of whom 13 were, in turn, immigrants from Latin American countries different from those participating in the study (Peru, Ecuador and Bolivia). Most of these international clusters belonged either to the emerging RD(Rio) LAM lineage or to the Haarlem family of M. tuberculosis and four were further split by country when analyzed with spoligotyping and rifampin resistance-conferring mutations, suggesting that they did not represent ongoing transnational transmission events. The Beijing genotype accounted for 1.3% and 10.2% of patients with multidrug-resistant tuberculosis in Latin America and Spain, respectively, including one international cluster of two cases. In brief, Euro-American genotypes were widely predominant among multidrug-resistant M. tuberculosis strains in Ibero-America, reflecting closely their predominance in the general M. tuberculosis population in the region, and no evidence was found of acknowledged outbreak strains trespassing country borders. Copyright © 2011 Elsevier B.V. All rights reserved.

  20. Genomic analysis of globally diverse Mycobacterium tuberculosis strains provides insights into the emergence and spread of multidrug resistance.

    Science.gov (United States)

    Manson, Abigail L; Cohen, Keira A; Abeel, Thomas; Desjardins, Christopher A; Armstrong, Derek T; Barry, Clifton E; Brand, Jeannette; Chapman, Sinéad B; Cho, Sang-Nae; Gabrielian, Andrei; Gomez, James; Jodals, Andreea M; Joloba, Moses; Jureen, Pontus; Lee, Jong Seok; Malinga, Lesibana; Maiga, Mamoudou; Nordenberg, Dale; Noroc, Ecaterina; Romancenco, Elena; Salazar, Alex; Ssengooba, Willy; Velayati, A A; Winglee, Kathryn; Zalutskaya, Aksana; Via, Laura E; Cassell, Gail H; Dorman, Susan E; Ellner, Jerrold; Farnia, Parissa; Galagan, James E; Rosenthal, Alex; Crudu, Valeriu; Homorodean, Daniela; Hsueh, Po-Ren; Narayanan, Sujatha; Pym, Alexander S; Skrahina, Alena; Swaminathan, Soumya; Van der Walt, Martie; Alland, David; Bishai, William R; Cohen, Ted; Hoffner, Sven; Birren, Bruce W; Earl, Ashlee M

    2017-03-01

    Multidrug-resistant tuberculosis (MDR-TB), caused by drug-resistant strains of Mycobacterium tuberculosis, is an increasingly serious problem worldwide. Here we examined a data set of whole-genome sequences from 5,310 M. tuberculosis isolates from five continents. Despite the great diversity of these isolates with respect to geographical point of isolation, genetic background and drug resistance, the patterns for the emergence of drug resistance were conserved globally. We have identified harbinger mutations that often precede multidrug resistance. In particular, the katG mutation encoding p.Ser315Thr, which confers resistance to isoniazid, overwhelmingly arose before mutations that conferred rifampicin resistance across all of the lineages, geographical regions and time periods. Therefore, molecular diagnostics that include markers for rifampicin resistance alone will be insufficient to identify pre-MDR strains. Incorporating knowledge of polymorphisms that occur before the emergence of multidrug resistance, particularly katG p.Ser315Thr, into molecular diagnostics should enable targeted treatment of patients with pre-MDR-TB to prevent further development of MDR-TB.

  1. Characterisation of rpsL, rrs and embB mutations associated with streptomycin and ethambutol resistance in Mycobacterium tuberculosis.

    Science.gov (United States)

    Tracevska, Tatjana; Jansone, Inta; Nodieva, Anda; Marga, Olgerts; Skenders, Girts; Baumanis, Viesturs

    2004-12-01

    In order to characterise molecular mechanisms of first-line drug resistance in Mycobacterium tuberculosis and to evaluate the use of molecular markers of resistance (gene point mutations), we analysed 66 multi-drug-resistant (MDR) isolates from Latvian tuberculosis patients. They were all resistant to rifampin (RIF), isoniazid (INH) and streptomycin (SM), and 33 were resistant to ethambutol (EMB). Enzymatic digestion by MboII and nucleotide sequencing of the rpsL gene fragment detected a single nucleotide substitution K43R in 40 (61%) of the 66 SM-resistant M. tuberculosis isolates. Of the other 26 SM-resistant isolates, 16 (24%) had mutations at positions 513A-->C and 516C-->T of the rrs gene and 10 (15%) had the wild-type sequence. The single-stranded DNA conformation polymorphism (SSCP) method was used to detect mutations in the embB gene associated with EMB resistance. Substitutions in the embB gene were found by SSCP analysis in 15 (45%) and by sequencing in 17 (52%) of the 33 EMB-resistant isolates. Surprisingly, SSCP revealed a nucleotide mutation at codon M306 in five (15%) of 33 in vitro EMB-susceptible MDR isolates.

  2. Genetic diversity of Mycobacterium tuberculosis from Guadalajara, Mexico and identification of a rare multidrug resistant Beijing genotype.

    Science.gov (United States)

    Flores-Treviño, Samantha; Morfín-Otero, Rayo; Rodríguez-Noriega, Eduardo; González-Díaz, Esteban; Pérez-Gómez, Héctor R; Bocanegra-García, Virgilio; Vera-Cabrera, Lucio; Garza-González, Elvira

    2015-01-01

    Determining the genetic diversity of M. tuberculosis strains allows identification of the distinct Mycobacterium tuberculosis genotypes responsible for tuberculosis in different regions. Several studies have reported the genetic diversity of M. tuberculosis strains in Mexico, but little information is available from the state of Jalisco. Therefore, the aim of this study was to determine the genetic diversity of Mycobacterium tuberculosis clinical isolates from Western Mexico. Sixty-eight M. tuberculosis isolates were tested for susceptibility to first-line drugs using manual Mycobacteria Growth Indicator Tube method and genotyped using spoligotyping and IS6110-restriction fragment length polymorphism (RFLP) pattern analyses. Forty-seven (69.1%) isolates were grouped into 10 clusters and 21 isolates displayed single patterns by spoligotyping. Three of the 21 single patterns corresponded to orphan patterns in the SITVITWEB database, and 1 new type that contained 2 isolates was created. The most prevalent lineages were T (38.2%), Haarlem (17.7%), LAM (17.7%), X (7.4%), S (5.9%), EAI (1.5%) and Beijing (1.5%). Six (12.8%) of the clustered isolates were MDR, and type 406 of the Beijing family was among the MDR isolates. Seventeen (26.2%) isolates were grouped into 8 clusters and 48 isolates displayed single patterns by IS6110-RFLP. Combination of IS6110-RFLP and spoligotyping reduced the clustering rate to 20.0%. The results show that T, Haarlem, and LAM are predominant lineages among clinical isolates of M. tuberculosis in Guadalajara, Mexico. Clustering rates indicated low transmission of MDR strains. We detected a rare Beijing genotype, SIT406, which was a highly resistant strain. This is the first report of this Beijing genotype in Latin America.

  3. Quantitative proteomic analysis of ofloxacin resistant and sensitive clinical isolates of Mycobacterium tuberculosis

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    Xiang-yu HUANG

    2014-10-01

    Full Text Available Objective To identify the proteins related to ofloxacin (OFX resistance of Mycobacterium tuberculosis (MTB. Methods Standard MTB H37Rv strain, clinical isolates of OFX resistant strain (OFXR and sensitive strain (OFXS were obtained from the Chinese Center for Disease Control and Prevention, and they were cultured in Sauton's medium, and then inactivated by 60Co. Whole cellular proteins were extracted from OFXR, OFXS and H37Rv strain of MTB, respectively. The peptides were labeled, separated and identified by isobaric tags of relative and absolute quantitation (iTRAQ combined with Nano LCMS/MS technology. Results One hundred and seventy-five and 134 differential expression proteins were identified in MTB OFXR compared with MTB OFXS and H37Rv, respectively. One hundred and four common differential expression proteins were identified in MTB OFXR compared with both MTB OFXS and H37Rv. The isoelectric point and theoretic relative molecular mass of differential expression proteins were widely distributed. The majority of the common differential expression proteins were involved in intermediary metabolism, respiration, and lipid metabolism. Twelve common differential expression proteins showed significant differences (the ratio>1.2 or <0.55 in MTB OFXR, including Rv0106, Rv0895, Rv2185c, Rv3248c and Rv3841 up-regulation and Rv2524c, Rv2986c, Rv3118 and Rv3597c down-regulation. Conclusion iTRAQ has been used to identify the common differential expression proteins in MTB OFXR compared with both MTB OFXS and H37Rv, which provides a basis for further study of the mechanism of OFX-resistance. DOI: 10.11855/j.issn.0577-7402.2014.09.06

  4. Geographical profile of rpoB gene mutations in rifampicin resistant Mycobacterium tuberculosis isolates in Sri Lanka.

    Science.gov (United States)

    Adikaram, Chamila Priyangani; Perera, Jennifer; Wijesundera, Sandhya Sulochana

    2012-10-01

    The nature and frequency of mutations in the rpoB gene of rifampicin (RIF) resistant Mycobacterium tuberculosis clinical isolates varies considerably between different geographical regions. The objective of the present study was the identification of rpoB gene mutations responsible for RIF resistance in M. tuberculosis isolates in Sri Lanka. Three regions of the rpoB gene of M. tuberculosis, one corresponding to a 437-bp region, including the rifampicin resistance-determining region (RRDR) and two other regions (1395 bp and 872 bp) spanning the RRDR, were polymerase chain reaction amplified, and were subjected to DNA sequencing. The two mutations found within the RRDR in the 31 RIF resistant strains isolated in this study were at codon 526 (n=15, 48.4%) CAC (His)→TAC (Tyr) and codon 531 (n=3, 9.7%) TCG (Ser)→TTG (Leu). A significant proportion (n=15, 48.3%) showed mutations spanning the RRDR, including two novel mutations at codon 626 (n=13, 41.9%) GAC (Asp)→GAG (Glu) and 184 (n=2, 6.4%) GAC (Asp)→GAT (Asp), a silent mutation. Two isolates revealed double mutations (codons 626+526 and 626+184). The presence of a high frequency of new mutations, and the different frequencies of the universally prevailing mutations, as reported here, emphasizes the need for expanding the geographical database of mutations for effective application of an rpoB-based diagnosis of multidrug resistant tuberculosis.

  5. Proteomic analysis of streptomycin resistant and sensitive clinical isolates of Mycobacterium tuberculosis

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    Venkatesan Krishnamurthy

    2010-11-01

    Full Text Available Abstract Background Streptomycin (SM is a broad spectrum antibiotic and is an important component of any anti-tuberculosis therapy regimen. Several mechanisms have been proposed to explain the emergence of resistance but still our knowledge is inadequate. Proteins form a very complex network and drugs are countered by their modification/efflux or over expression/modification of targets. As proteins manifest most of the biological processes, these are attractive targets for developing drugs, immunodiagnostics or therapeutics. The aim of present study was to analyze and compare the protein profile of whole cell extracts from Mycobacterium tuberculosis clinical isolates susceptible and resistant to SM. Results Two-dimensional gel electrophoresis (2DE and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF mass spectrometry was employed for analyzing the protein profiles. Homology and in silico characterization for identified proteins was assessed using BLAST, InterProScan and KEGG database searches. Computational studies on the possible interactions between SM and identified proteins were carried out by a battery of online servers and softwares, namely, CLUSTALW (KEGG, I-TASSER, VMD, PatchDock and FireDock. On comparing 2DE patterns, nine proteins were found consistently overexpressed in SM resistant isolates and were identified as Rv0350, Rv0440, Rv1240, Rv3075c, Rv2971, Rv3028c, Rv2145c, Rv2031c and Rv0569. In silico docking analysis showed significant interactions of SM with essential (Rv0350, Rv0440 and Rv2971 and non essential (Rv1240, Rv3075c and Rv2031c genes. Conclusions The computational results suggest high protein binding affinity of SM and suggested many possible interactions between identified proteins and the drug. Bioinformatic analysis proves attributive for analysis of diversity of proteins identified by whole proteome analysis. In-depth study of the these proteins will give an insight into probable sites of drug

  6. Recent transmission of drug-resistant Mycobacterium tuberculosis in a prison population in southern Brazil

    Science.gov (United States)

    Reis, Ana Julia; de David, Simone Maria Martini; Nunes, Luciana de Souza; Valim, Andreia Rosane de Moura; Possuelo, Lia Gonçalves

    2016-01-01

    ABSTRACT We conducted a cross-sectional, retrospective study, characterized by classical and molecular epidemiology, involving M. tuberculosis isolates from a regional prison in southern Brazil. Between January of 2011 and August of 2014, 379 prisoners underwent sputum smear microscopy and culture; 53 (13.9%) were diagnosed with active tuberculosis. Of those, 8 (22.9%) presented with isoniazid-resistant tuberculosis. Strain genotyping was carried out by 15-locus mycobacterial interspersed repetitive unit-variable-number tandem-repeat analysis; 68.6% of the patients were distributed into five clusters, and 87.5% of the resistant cases were in the same cluster. The frequency of drug-resistant tuberculosis cases and the rate of recent transmission were high. Our data suggest the need to implement an effective tuberculosis control program within the prison system.

  7. Recent transmission of drug-resistant Mycobacterium tuberculosis in a prison population in southern Brazil

    Directory of Open Access Journals (Sweden)

    Ana Julia Reis

    Full Text Available ABSTRACT We conducted a cross-sectional, retrospective study, characterized by classical and molecular epidemiology, involving M. tuberculosis isolates from a regional prison in southern Brazil. Between January of 2011 and August of 2014, 379 prisoners underwent sputum smear microscopy and culture; 53 (13.9% were diagnosed with active tuberculosis. Of those, 8 (22.9% presented with isoniazid-resistant tuberculosis. Strain genotyping was carried out by 15-locus mycobacterial interspersed repetitive unit-variable-number tandem-repeat analysis; 68.6% of the patients were distributed into five clusters, and 87.5% of the resistant cases were in the same cluster. The frequency of drug-resistant tuberculosis cases and the rate of recent transmission were high. Our data suggest the need to implement an effective tuberculosis control program within the prison system.

  8. IS6110 fingerprinting of sensitive and resistant strains (1991-1992 of Mycobacterium tuberculosis in Colombia

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    Jorge Enrique Gómez-Marin

    2002-10-01

    Full Text Available The standardized method to study the polymorphism of IS 6110 was used to characterize 53 isolates of Mycobacterium tuberculosis obtained during 1991-1992 from 14 regions in Colombia. In Valle region cluster rate was 25% (4/16. The mean number of IS6110 band was 10 ± 3. Similarity between strains was of 60% in 81% of strains and this tended to be correlated with geographic origin. For the first time M. tuberculosis without IS6110 bands in restriction fragment length polymorphism analysis was found in Colombia. Additional studies are necessaries in order to best characterize the situation in relation to human immunodeficiency virus epidemic and recent changes in tuberculosis control program.

  9. 21 CFR 866.3370 - Mycobacterium tuberculosis immunofluorescent reagents.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Mycobacterium tuberculosis immunofluorescent... § 866.3370 Mycobacterium tuberculosis immunofluorescent reagents. (a) Identification. Mycobacterium... used to identify Mycobacterium tuberculosis directly from clinical specimens. The identification...

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

    NARCIS (Netherlands)

    Ferro, B.E.; Garcia, P.K.; Nieto, L.M.; Soolingen, D. van

    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

  11. Detection and evaluation of the mutations of embB gene in ethambutol-susceptible and resistant Mycobacterium tuberculosis isolates from China

    Institute of Scientific and Technical Information of China (English)

    WU Xue-qiong; LIANG Jian-qin; ZHANG Jun-xian; LU Yang; LI Hong-min; ZHANG Guang-yu; YAN Guo-rui; DING Bei-chuan

    2005-01-01

    @@ The situation of tuberculosis (TB) and Mycobacterium tuberculosis (M. tuberculosis) drug resistance is still very serious in China. Improper treatment for TB may lead to a prolonged course of antimicrobial therapy and spreading of drug resistant strains. Thus, rapid detection of drug resistant strains will allow prompt initiation and adjustment of effective chemotherapeutic treatment of TB. Ethambutol (EMB) is a first line anti-TB drug. embB Met306 is located in a cytoplasmic loop that forms an EMB resistance determining region,1 Lety et al2 have demonstrated that a missense mutation in the M. smegmatis embB gene confers EMB resistance. The identification of mutations in embB gene may offer a means to rapidly screen M. tuberculosis isolates for EMB resistance. Thus, in this study, we analysed 197 isolates of M. tuberculosis from Chinese people and characterized mutations in the 258 bp region of the embB gene.

  12. Molecular analysis of isoniazid-resistant Mycobacterium tuberculosis isolates in Uruguay

    NARCIS (Netherlands)

    Greif, G.; Coitinho, C.; Rivas, C.; Ingen, J. van; Robello, C.

    2012-01-01

    The incidence of tuberculosis (TB) is increasing in high-risk populations in Uruguay, possibly owing to emerging resistance. Mycobacterial interspersed repetitive units (MIRU) genotyping and katG sequence analysis of isoniazid (INH) resistance-associated mutations were performed in 45 INH-resistant

  13. Removing the bottleneck in whole genome sequencing of Mycobacterium tuberculosis for rapid drug resistance analysis: a call to action

    Directory of Open Access Journals (Sweden)

    Ruth McNerney

    2017-03-01

    Full Text Available Whole genome sequencing (WGS can provide a comprehensive analysis of Mycobacterium tuberculosis mutations that cause resistance to anti-tuberculosis drugs. With the deployment of bench-top sequencers and rapid analytical software, WGS is poised to become a useful tool to guide treatment. However, direct sequencing from clinical specimens to provide a full drug resistance profile remains a serious challenge. This article reviews current practices for extracting M. tuberculosis DNA and possible solutions for sampling sputum. Techniques under consideration include enzymatic digestion, physical disruption, chemical degradation, detergent solubilization, solvent extraction, ligand-coated magnetic beads, silica columns, and oligonucleotide pull-down baits. Selective amplification of genomic bacterial DNA in sputum prior to WGS may provide a solution, and differential lysis to reduce the levels of contaminating human DNA is also being explored. To remove this bottleneck and accelerate access to WGS for patients with suspected drug-resistant tuberculosis, it is suggested that a coordinated and collaborative approach be taken to more rapidly optimize, compare, and validate methodologies for sequencing from patient samples.

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

    DEFF Research Database (Denmark)

    Idh, Jonna; Mekonnen, Mekidim; Abate, Ebba;

    2012-01-01

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

  15. Direct sequencing for rapid detection of multidrug resistant Mycobacterium tuberculosis strains in Morocco

    Directory of Open Access Journals (Sweden)

    Zakham F

    2013-11-01

    new case. The most recorded mutation in the rpoB gene was the substitution TCG > TTG at codon 531 (Ser531 Leu, accounting for 46.15%. Significantly, the only mutation found in the katG gene was at codon 315 (AGC to ACC with a Ser315Thr amino acid change. Only one sample harbored mutation in the inhA promoter region and was a point mutation at the -15p position (C > T.Conclusion: The polymerase chain reaction sequencing approach is an accurate and rapid method for detection of drug-resistant TB in clinical specimens, and could be of great interest in the management of TB in critical cases to adjust the treatment regimen and limit the emergence of MDR and XDR strains.Keywords: Morocco, Mycobacterium tuberculosis, multidrug resistance, rpoB, katG, inhA promoter

  16. Analysis of gene mutations associated with isoniazid, rifampicin and ethambutol resistance among Mycobacterium tuberculosis isolates from Ethiopia

    Science.gov (United States)

    2012-01-01

    Background The emergence of drug resistance is one of the most important threats to tuberculosis control programs. This study was aimed to analyze the frequency of gene mutations associated with resistance to isoniazid (INH), rifampicin (RMP) and ethambutol (EMB) among Mycobacterium tuberculosis isolates from Northwest Ethiopia, and to assess the performance of the GenoType® MTBDRplus and GenoType® MTBDRsl assays as compared to the BacT/ALERT 3D system. Methods Two hundred sixty Mycobacterium tuberculosis isolates from smear positive tuberculosis patients diagnosed between March 2009 and July 2009 were included in this study. Drug susceptibility tests were performed in the Institute of Medical Microbiology and Epidemiology of Infectious Diseases, University Hospital of Leipzig, Germany. Results Of 260 isolates, mutations conferring resistance to INH, RMP, or EMB were detected in 35, 15, and 8 isolates, respectively, while multidrug resistance (MDR) was present in 13 of the isolates. Of 35 INH resistant strains, 33 had mutations in the katG gene at Ser315Thr 1 and two strains had mutation in the inhA gene at C15T. Among 15 RMP resistant isolates, 11 had rpoB gene mutation at Ser531Leu, one at His526Asp, and three strains had mutations only at the wild type probes. Of 8 EMB resistant strains, two had mutations in the embB gene at Met306Ile, one at Met306Val, and five strains had mutations only at the wild type probes. The GenoType® MTBDRplus assay had a sensitivity of 92% and specificity of 99% for INH resistance, and 100% sensitivity and specificity to detect RMP resistance and MDR. The GenoType® MTBDRsl assay had a sensitivity of 42% and specificity of 100% for EMB resistance. Conclusion The dominance of single gene mutations associated with the resistance to INH and RMP was observed in the codon 315 of the katG gene and codon 531 of the rpoB gene, respectively. The GenoType® MTBDRplus assay is a sensitive and specific tool for diagnosis of resistance to INH

  17. Analysis of gene mutations associated with isoniazid, rifampicin and ethambutol resistance among Mycobacterium tuberculosis isolates from Ethiopia

    Directory of Open Access Journals (Sweden)

    Tessema Belay

    2012-02-01

    Full Text Available Abstract Background The emergence of drug resistance is one of the most important threats to tuberculosis control programs. This study was aimed to analyze the frequency of gene mutations associated with resistance to isoniazid (INH, rifampicin (RMP and ethambutol (EMB among Mycobacterium tuberculosis isolates from Northwest Ethiopia, and to assess the performance of the GenoType® MTBDRplus and GenoType® MTBDRsl assays as compared to the BacT/ALERT 3D system. Methods Two hundred sixty Mycobacterium tuberculosis isolates from smear positive tuberculosis patients diagnosed between March 2009 and July 2009 were included in this study. Drug susceptibility tests were performed in the Institute of Medical Microbiology and Epidemiology of Infectious Diseases, University Hospital of Leipzig, Germany. Results Of 260 isolates, mutations conferring resistance to INH, RMP, or EMB were detected in 35, 15, and 8 isolates, respectively, while multidrug resistance (MDR was present in 13 of the isolates. Of 35 INH resistant strains, 33 had mutations in the katG gene at Ser315Thr 1 and two strains had mutation in the inhA gene at C15T. Among 15 RMP resistant isolates, 11 had rpoB gene mutation at Ser531Leu, one at His526Asp, and three strains had mutations only at the wild type probes. Of 8 EMB resistant strains, two had mutations in the embB gene at Met306Ile, one at Met306Val, and five strains had mutations only at the wild type probes. The GenoType® MTBDRplus assay had a sensitivity of 92% and specificity of 99% for INH resistance, and 100% sensitivity and specificity to detect RMP resistance and MDR. The GenoType® MTBDRsl assay had a sensitivity of 42% and specificity of 100% for EMB resistance. Conclusion The dominance of single gene mutations associated with the resistance to INH and RMP was observed in the codon 315 of the katG gene and codon 531 of the rpoB gene, respectively. The GenoType® MTBDRplus assay is a sensitive and specific tool for

  18. Cytokinins beyond plants: synthesis by Mycobacterium tuberculosis

    Science.gov (United States)

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

    2015-01-01

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

  19. New insights into fluoroquinolone resistance in Mycobacterium tuberculosis: functional genetic analysis of gyrA and gyrB mutations.

    Directory of Open Access Journals (Sweden)

    Seidu Malik

    Full Text Available Fluoroquinolone antibiotics are among the most potent second-line drugs used for treatment of multidrug-resistant tuberculosis (MDR TB, and resistance to this class of antibiotics is one criterion for defining extensively drug resistant tuberculosis (XDR TB. Fluoroquinolone resistance in Mycobacterium tuberculosis has been associated with modification of the quinolone resistance determining region (QRDR of gyrA. Recent studies suggest that amino acid substitutions in gyrB may also play a crucial role in resistance, but functional genetic studies of these mutations in M. tuberculosis are lacking. In this study, we examined twenty six mutations in gyrase genes gyrA (seven and gyrB (nineteen to determine the clinical relevance and role of these mutations in fluoroquinolone resistance. Transductants or clinical isolates harboring T80A, T80A+A90G, A90G, G247S and A384V gyrA mutations were susceptible to all fluoroquinolones tested. The A74S mutation conferred low-level resistance to moxifloxacin but susceptibility to ciprofloxacin, levofloxacin and ofloxacin, and the A74S+D94G double mutation conferred cross resistance to all the fluoroquinolones tested. Functional genetic analysis and structural modeling of gyrB suggest that M330I, V340L, R485C, D500A, D533A, A543T, A543V and T546M mutations are not sufficient to confer resistance as determined by agar proportion. Only three mutations, N538D, E540V and R485C+T539N, conferred resistance to all four fluoroquinolones in at least one genetic background. The D500H and D500N mutations conferred resistance only to levofloxacin and ofloxacin while N538K and E540D consistently conferred resistance to moxifloxacin only. Transductants and clinical isolates harboring T539N, T539P or N538T+T546M mutations exhibited low-level resistance to moxifloxacin only but not consistently. These findings indicate that certain mutations in gyrB confer fluoroquinolone resistance, but the level and pattern of resistance varies

  20. Mutations in the embC-embA intergenic region contribute to Mycobacterium tuberculosis resistance to ethambutol.

    Science.gov (United States)

    Cui, Zhenling; Li, Yuanyuan; Cheng, Song; Yang, Hua; Lu, Junmei; Hu, Zhongyi; Ge, Baoxue

    2014-11-01

    The rapid increase in Mycobacterium tuberculosis resistance to ethambutol (EMB) threatens the diagnosis and treatment of tuberculosis (TB). We investigated the role of mutations in the embC-embA intergenic region (IGR) in EMB-resistant clinical strains from east China. A total of 767 M. tuberculosis clinical strains were collected and analyzed for their drug susceptibility to EMB using the MGIT 960 system and MIC assay, and the embC-embA IGRs of these strains were sequenced. The transcriptional activity of the embC-embA IGR mutations was examined by reporter gene assays in recombinant Mycobacterium smegmatis strains, and the effect of IGR mutations on its binding to EmbR, a transcription regulator of embAB, was analyzed by gel mobility shift assays. Correlation coefficient analysis showed that the embC-embA IGR mutation is associated with EMB resistance. The clinical strains carrying IGR mutations had a much higher level of embA and embB mRNA as well as higher MICs to EMB. IGR mutations had higher transcriptional activity when transformed into M. smegmatis strains. Mutated IGRs bound to EmbR with much higher affinity than wild-type fragments. The sensitivity of molecular drug susceptibility testing (DST) with IGR mutations as an additional marker increased from 65.5% to 73.5%. Mutations of the embC-embA IGR enhance the binding of EmbR to the promoter region of embAB and increase the expression of embAB, thus contributing to EMB resistance. Therefore, identification of IGR mutations as markers of EMB resistance could increase the sensitivity of molecular DST.

  1. Cholesterol Analogs with Degradation-resistant Alkyl Side Chains Are Effective Mycobacterium tuberculosis Growth Inhibitors.

    Science.gov (United States)

    Frank, Daniel J; Zhao, Yan; Wong, Siew Hoon; Basudhar, Debashree; De Voss, James J; Ortiz de Montellano, Paul R

    2016-04-01

    Cholest-4-en-3-one, whether added exogenously or generated intracellularly from cholesterol, inhibits the growth ofMycobacterium tuberculosiswhen CYP125A1 and CYP142A1, the cytochrome P450 enzymes that initiate degradation of the sterol side chain, are disabled. Here we demonstrate that a 16-hydroxy derivative of cholesterol, which was previously reported to inhibit growth ofM. tuberculosis, acts by preventing the oxidation of the sterol side chain even in the presence of the relevant cytochrome P450 enzymes. The finding that (25R)-cholest-5-en-3β,16β,26-triol (1) (and its 3-keto metabolite) inhibit growth suggests that cholesterol analogs with non-degradable side chains represent a novel class of anti-mycobacterial agents. In accord with this, two cholesterol analogs with truncated, fluorinated side chains have been synthesized and shown to similarly block the growth in culture ofM. tuberculosis.

  2. Does empirical treatment of community-acquired pneumonia with fluoroquinolones delay tuberculosis treatment and result in fluoroquinolone resistance in Mycobacterium tuberculosis? Controversies and solutions.

    Science.gov (United States)

    Shen, Gwan-Han; Tsao, Thomas Chang-Yao; Kao, Shang-Jyh; Lee, Jen-Jyh; Chen, Yen-Hsu; Hsieh, Wei-Chung; Hsu, Gwo-Jong; Hsu, Yen-Tao; Huang, Ching-Tai; Lau, Yeu-Jun; Tsao, Shih-Ming; Hsueh, Po-Ren

    2012-03-01

    The role of fluoroquinolones (FQs) as empirical therapy for community-acquired pneumonia (CAP) remains controversial in countries with high tuberculosis (TB) endemicity owing to the possibility of delayed TB diagnosis and treatment and the emergence of FQ resistance in Mycobacterium tuberculosis. Although the rates of macrolide-resistant Streptococcus pneumoniae and amoxicillin/clavulanic acid-resistant Haemophilus influenzae have risen to alarming levels, the rates of respiratory FQ (RFQ) resistance amongst these isolates remain relatively low. It is reported that ca. 1-7% of CAP cases are re-diagnosed as pulmonary TB in Asian countries. A longer duration (≥ 7 days) of symptoms, a history of night sweats, lack of fever (> 38 °C), infection involving the upper lobe, presence of cavitary infiltrates, opacity in the lower lung without the presence of air, low total white blood cell count and the presence of lymphopenia are predictive of pulmonary TB. Amongst patients with CAP who reside in TB-endemic countries who are suspected of having TB, imaging studies as well as aggressive microbiological investigations need to be performed early on. Previous exposure to a FQ for >10 days in patients with TB is associated with the emergence of FQ-resistant M. tuberculosis isolates. However, rates of M. tuberculosis isolates with FQ resistance are significantly higher amongst multidrug-resistant M. tuberculosis isolates than amongst susceptible isolates. Consequently, in Taiwan and also in other countries with TB endemicity, a short-course (5-day) regimen of a RFQ is still recommended for empirical therapy for CAP patients if the patient is at low risk for TB. Copyright © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

  3. Molecular epidemiology of Mycobacterium tuberculosis, Buenos Aires, Argentina.

    Science.gov (United States)

    Gonzalo, Ximena; Ambroggi, Marta; Cordova, Ezequiel; Brown, Tim; Poggi, Susana; Drobniewski, Francis

    2011-03-01

    To analyze the molecular epidemiology of Mycobacterium tuberculosis strains at a hospital in Buenos Aires, Argentina, and mutations related to multidrug-resistant and extensively drug-resistant tuberculosis, we conducted a prospective case-control study. Our findings reinforce the value of incorporating already standardized molecular methods for rapidly detecting resistance.

  4. Mefloquine and its oxazolidine derivative compound are active against drug-resistant Mycobacterium tuberculosis strains and in a murine model of tuberculosis infection.

    Science.gov (United States)

    Rodrigues-Junior, Valnês S; Villela, Anne D; Gonçalves, Raoni S B; Abbadi, Bruno Lopes; Trindade, Rogério Valim; López-Gavín, Alexandre; Tudó, Griselda; González-Martín, Julian; Basso, Luiz Augusto; de Souza, Marcus V N; Campos, Maria Martha; Santos, Diógenes Santiago

    2016-08-01

    Repurposing of drugs to treat tuberculosis (TB) has been considered an alternative to overcome the global TB epidemic, especially to combat drug-resistant forms of the disease. Mefloquine has been reported as a potent drug to kill drug-resistant strains of Mycobacterium tuberculosis. In addition, mefloquine-derived molecules have been synthesised and their effectiveness against mycobacteria has been assessed. In this work, we demonstrate for the first time the activities of mefloquine and its oxazolidine derivative compound 1E in a murine model of TB infection following administration of both drugs by the oral route. The effects of associations between mefloquine or 1E with the clinically used antituberculosis drugs isoniazid, rifampicin, ethambutol, moxifloxacin and streptomycin were also investigated. Importantly, combination of mefloquine with isoniazid and of 1E with streptomycin showed a two-fold decrease in their minimum inhibitory concentrations (MICs). Moreover, no tested combinations demonstrated antagonist interactions. Here we describe novel evidence on the activity of mefloquine and 1E against a series of quinolone-resistant M. tuberculosis strains. These data show MICs against quinolone-resistant strains (0.5-8 µg/mL) similar to or lower than those previously reported for multidrug-resistant strains. Taking these results together, we can suggest the use of mefloquine or 1E in combination with clinically available drugs, especially in the case of resistant forms of TB.

  5. [Drug resistance in Mycobacterium tuberculosis. A multicenter study of the Barcelona area. Grupo de Trabajo sobre Resistencias en Tuberculosis].

    Science.gov (United States)

    Martin-Casabona, N; Alcaide, F; Coll, P; González, J; Manterola, J M; Salvadó, M; Caylà, J A

    2000-10-21

    The aims of this multicenter study was to establish the level of primary and acquired drug resistance of M. Tuberculosis strains isolated in Barcelona and to identify possible risk groups using clinical data. All tuberculosis patients with isolation and identification of M. tuberculosis strains from October 1995 to September 1997 were included. Susceptibility tests isoniazid, rifampin, ethambutol, streptomycin and pyrazinamide were performed using the Bactec 460 system and the proportions method on solid medium. Logistic progression was used for statistical analysis. The total number of patients included was 1,749 (1,535 non-treated and 214 previously treated). Primary drug resistance was 5.7% (isoniazid 3.8%; rifampin 1.0%, streptomycin 2.1%, ethambutol 0.3% and pyrazinamide 1.0%). Acquired drug resistance was 20.5% (isoniazid 17.3%, rifampin 9.8%, ethambutol 1.9%, streptomycin 4.7% and pyrazinamide 6.5%). Primary drug resistance was associated with people over 60 years old and women. The low level of drug resistance enables antituberculosis treatment of non-treated patients to start with the standardised three-drug regimes except in the case of foreign people from countries with a high level of drug resistance. Susceptibility tests are recommended on all M. tuberculosis strains isolated, together with controlled studies of drug resistance surveillance.

  6. Evaluation of efflux pump gene expression among drug susceptible and drug resistant strains of Mycobacterium tuberculosis from Iran.

    Science.gov (United States)

    Kardan Yamchi, Jalil; Haeili, Mehri; Gizaw Feyisa, Seifu; Kazemian, Hossein; Hashemi Shahraki, Abdolrazagh; Zahednamazi, Fatemeh; Imani Fooladi, Abbas Ali; Feizabadi, Mohammad Mehdi

    2015-12-01

    Absence of mutations within the genes encoding drug targets in some phenotypically drug resistant strains of Mycobacterium tuberculosis suggests possible involvement of alternative mechanisms such as over-expression of efflux pumps. We investigated the expression level of Rv1410c, Rv2459, Rv1218c and Rv1273c efflux pumps gene by real-time quantitative reverse transcription PCR (qRT-PCR) in 31 clinical isolates of M. tuberculosis. Susceptibility to first-line drugs was performed using the proportion method. Twenty one isolates were characterized with drug resistance (DR), and among them 12 showed a significantly elevated level of expression (>4 fold) for at least one of the studied genes encoding for efflux pumps. Point mutations in the katG (codons 315 or 335) and rpoB (codons 456 and 441) genes were found in 42.85% and 66.6% of drug resistant isolates, respectively. Only one isolate showed mutation at position -15 of the inhA promoter region. Among the 7 isolates (33.33%) which had no mutation in the studied regions of drug target genes, 5 isolates showed over-expression for efflux pumps. Our results demonstrated that over-expression of efflux pumps can contribute to drug resistance in M. tuberculosis.

  7. Quadruple-first line drug resistance in Mycobacterium tuberculosis in Vietnam: What can we learn from genes?

    Science.gov (United States)

    Nguyen, Huy Quang; Nguyen, Nhung Viet; Contamin, Lucie; Tran, Thanh Hoa Thi; Vu, Thuong Thi; Nguyen, Hung Van; Nguyen, Ngoc Lan Thi; Nguyen, Son Thai; Dang, Anh Duc; Bañuls, Anne-Laure; Nguyen, Van Anh Thi

    2017-02-21

    In Vietnam, a country with high tuberculosis (137/100.000 population) and multidrug-resistant (MDR)-TB burdens (7.8/100.000 population), little is known about the molecular signatures of drug resistance in general and more particularly of second line drug (SLD) resistance. This study is specifically focused on Mycobacterium tuberculosis isolates resistant to four first-line drugs (FLDs) that make TB much more difficult to treat. The aim is to determine the proportion of SLD resistance in these quadruple drug resistant isolates and the genetic determinants linked to drug resistance to better understand the genetic processes leading to quadruple and extremely drug resistance (XDR). 91 quadruple (rifampicin, isoniazid, ethambutol and streptomycin) FLD resistant and 55 susceptible isolates were included. Spoligotyping and 24-locus MIRU-VNTR techniques were performed and 9 genes and promoters linked to FLD and SLD resistance were sequenced. SLD susceptibility testing was carried out on a subsample of isolates. High proportion of quadruple-FLD resistant isolates was resistant to fluoroquinolones (27%) and second-line injectable drugs (30.2%) by drug susceptibility testing. The sequencing revealed high mutation diversity with prevailing mutations at positions katG315, inhA-15, rpoB531, embB306, rrs1401, rpsL43 and gyrA94. The sensitivity and specificity were high for most drug resistances (>86%), but the sensitivity was lower for injectable drug resistances (<69%). The mutation patterns revealed 23.1% of pre-XDR and 7.7% of XDR isolates, mostly belonging to Beijing family. The genotypic diversity and the variety of mutations reflect the existence of various evolutionary paths leading to FLD and SLD resistance. Nevertheless, particular mutation patterns linked to high-level resistance and low fitness costs seem to be favored.

  8. Rifabutin and rifampin resistance levels and associated rpoB mutations in clinical isolates of Mycobacterium tuberculosis complex.

    Science.gov (United States)

    Berrada, Zenda L; Lin, Shou-Yean Grace; Rodwell, Timothy C; Nguyen, Duylinh; Schecter, Gisela F; Pham, Lucy; Janda, J Michael; Elmaraachli, Wael; Catanzaro, Antonino; Desmond, Edward

    2016-06-01

    Cross-resistance in rifamycins has been observed in rifampin (RIF)-resistant Mycobacterium tuberculosis complex isolates; some rpoB mutations do not confer broad in vitro rifamycin resistance. We examined 164 isolates, of which 102 were RIF-resistant, for differential resistance between RIF and rifabutin (RFB). A total of 42 unique single mutations or combinations of mutations were detected. The number of unique mutations identified exceeded that reported in any previous study. RFB and RIF MICs up to 8 μg/mL by MGIT 960 were studied; the cut-off values for susceptibility to RIF and RFB were 1 μg/mL and 0.5 μg/mL, respectively. We identified 31 isolates resistant to RIF but susceptible to RFB with the mutations D516V, D516F, 518 deletion, S522L, H526A, H526C, H526G, H526L, and two dual mutations (S522L + K527R and H526S + K527R). Clinical investigations using RFB to treat multidrug-resistant tuberculosis cases harboring those mutations are recommended.

  9. 2-[4-(4-Methoxyphenylcarbonyloxy)benzylidene]-6-dimethylaminomethyl cyclohexanone hydrochloride: a Mannich base which inhibits the growth of some drug-resistant strains of Mycobacterium tuberculosis.

    Science.gov (United States)

    Das, S; Das, U; Bandy, B; Gorecki, D K J; Dimmock, J R

    2010-11-01

    2-[4-(4-Methoxyphenylcarbonyloxy)benzylidene]-6-dime-thylaminomethyl cyclohexanone hydrochloride 1 has a MIC value of 0.78 microg/mL towards Mycobacterium tuberculosis H37Rv and displays similar or identical MIC figures towards various drug-resistant strains of this microorganism. The enone 1 along with a partial structure 2-dimethylaminomethylcyclohexanone hydrochloride 3 affected respiration in isolated rat liver mitochondria differently which may contribute to the variation in toxicity to both normal cells and M. tuberculosis.

  10. Whole-Genome Sequencing of Mycobacterium tuberculosis Provides Insight into the Evolution and Genetic Composition of Drug-Resistant Tuberculosis in Belarus

    Science.gov (United States)

    Wollenberg, Kurt R.; Desjardins, Christopher A.; Zalutskaya, Aksana; Slodovnikova, Vervara; Oler, Andrew J.; Quiñones, Mariam; Abeel, Thomas; Chapman, Sinead B.; Tartakovsky, Michael; Gabrielian, Andrei; Hoffner, Sven; Skrahin, Aliaksandr; Birren, Bruce W.; Rosenthal, Alexander

    2016-01-01

    ABSTRACT The emergence and spread of drug-resistant Mycobacterium tuberculosis (DR-TB) are critical global health issues. Eastern Europe has some of the highest incidences of DR-TB, particularly multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB. To better understand the genetic composition and evolution of MDR- and XDR-TB in the region, we sequenced and analyzed the genomes of 138 M. tuberculosis isolates from 97 patients sampled between 2010 and 2013 in Minsk, Belarus. MDR and XDR-TB isolates were significantly more likely to belong to the Beijing lineage than to the Euro-American lineage, and known resistance-conferring loci accounted for the majority of phenotypic resistance to first- and second-line drugs in MDR and XDR-TB. Using a phylogenomic approach, we estimated that the majority of MDR-TB was due to the recent transmission of already-resistant M. tuberculosis strains rather than repeated de novo evolution of resistance within patients, while XDR-TB was acquired through both routes. Longitudinal sampling of M. tuberculosis from 34 patients with treatment failure showed that most strains persisted genetically unchanged during treatment or acquired resistance to fluoroquinolones. HIV+ patients were significantly more likely to have multiple infections over time than HIV− patients, highlighting a specific need for careful infection control in these patients. These data provide a better understanding of the genomic composition, transmission, and evolution of MDR- and XDR-TB in Belarus and will enable improved diagnostics, treatment protocols, and prognostic decision-making. PMID:27903602

  11. A First Assessment of Mycobacterium tuberculosis Genetic Diversity and Drug-Resistance Patterns in Twelve Caribbean Territories

    Directory of Open Access Journals (Sweden)

    Julie Millet

    2014-01-01

    Full Text Available With the exception of some French-speaking islands, data on tuberculosis (TB in the Caribbean are scarce. In this study, we report a first assessment of genetic diversity of a convenience sample of Mycobacterium tuberculosis strains received from twelve Caribbean territories by spoligotyping and describe their drug-resistance patterns. Of the 480 isolates, 40 (8.3% isolates showed resistance to at least one anti-TB drug. The proportion of drug-resistant strains was significantly higher in The Bahamas (21.4%; P=0.02, and Guyana (27.5%; P<0.0001, while it was significantly lower in Jamaica (2.4%; P=0.03 than in other countries of the present study. Regarding genetic diversity, 104 distinct spoligotype patterns were observed: 49 corresponded to clustered strains (2 to 93 strains per cluster, while 55 remained unclustered among which 16 patterns were not reported previously. Combining the study results with regional data retrieved from the international SITVIT2 database underlined a connection between frequency of certain M. tuberculosis phylogenetic lineages and the language spoken, suggesting historical (colonial and ongoing links (trade, tourism, and migratory flows with European countries with which they shared a common past.

  12. Molecular and Growth-Based Drug Susceptibility Testing of Mycobacterium tuberculosis Complex for Ethambutol Resistance in the United States

    Directory of Open Access Journals (Sweden)

    Mitchell A. Yakrus

    2016-01-01

    Full Text Available Ethambutol (EMB is used as a part of drug regimens for treatment of tuberculosis (TB. Susceptibility of Mycobacterium tuberculosis complex (MTBC isolates to EMB can be discerned by DNA sequencing to detect mutations in the embB gene associated with resistance. US Public Health Laboratories (PHL primarily use growth-based drug susceptibility test (DST methods to determine EMB resistance. The Centers for Disease Control and Prevention (CDC provides a service for molecular detection of drug resistance (MDDR by DNA sequencing and concurrent growth-based DST using agar proportion. PHL and CDC test results were compared for 211 MTBC samples submitted to CDC from September 2009 through February 2011. Concordance between growth-based DST results from PHL and CDC was 88.2%. A growth-based comparison of 39 samples, where an embB mutation associated with EMB resistance was detected, revealed a higher percentage of EMB resistance by CDC (84.6% than by PHL (59.0% which was significant (P value = 0.002. Discordance between all growth-based test results from PHL and CDC was also significant (P value = 0.003. Most discordance was linked to false susceptibility using the BACTEC™ MGIT™ 960 (MGIT growth-based system. Our analysis supports coalescing growth-based and molecular results for an informed interpretation of potential EMB resistance.

  13. Identification of rifampin-resistant mycobacterium tuberculosis strains by hybridization, PCR, and ligase detaction reaction on oligonucleotide microchips.

    Energy Technology Data Exchange (ETDEWEB)

    Mikhailovich, V.; Lapa, S.; Gryadunov, D.; Sobolev, A.; Strizhkov, B.; Chernyh, N.; Skotnikova, O.; Irtuganova, O.; Moroz, A.; Litvinov, V.; Vladimirskii, M.; Perelman, M.; Chernousova, L.; Erokhin, V.; Mirzabekov, A.; Biochip Technology Center; Russian Academy of Sciences; Moscow Antituberculosis Center; Moscow Medical Academy; Russian Academy of Medical Sciences

    2001-07-01

    Three new molecular approaches were developed to identify drug-resistant strains of Mycobacterium tuberculosis using biochips with oligonucleotides immobilized in polyacrylamide gel pads. These approaches are significantly faster than traditional bacteriological methods. All three approaches -- hybridization, PCR, and ligase detection reaction -- were designed to analyze an 81-bp fragment of the gene rpoB encoding the {beta}-subunit of RNA polymerase, where most known mutations of rifampin resistance are located. The call set for hybridization analysis consisted of 42 immobilized oligonucleotides and enabled us to identify 30 mutant variants of the rpoB gene within 24 h. These variants are found in 95% of all mutants whose rifampin resistance is caused by mutations in the 81-bp fragment. Using the second approach, allele-specific on-chip PCR, it was possible to directly identify mutations in clinical samples within 1.5 h. The third approach, on-chip ligase detection reaction, was sensitive enough to reveal rifampin-resistant strains in a model mixture containing 1% of resistant and 99% of susceptible bacteria. This level of sensitivity is comparable to that from the determination of M. tuberculosis drug resistance by using standard bacteriological tests.

  14. Designing and comparison study of rapid detection methods of resistance to injectable drugs in clinical strains of Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Fatemeh Salehi

    2012-01-01

    Full Text Available Introduction: In this study, some molecular methods were designed for rapid detection of resistance to kanamycin and amikacin.Materials and methods: Among 120 clinical isolates of mycobacterium tuberculosis, 70 strains were selected for evaluation of possible mutations. A PCR-RFLP method was designed for detection of wild type (using enzyme ajii and mutant from (BstFNI enzyme of the isolates. Furthermore, allele specific method (as PCR was designed for detection mutations in codons 1401 and 1402 gene rrs. Some selected isolates were sequenced.Results: In PCR-RFLP method, among the 70 strains examined by BstFNI enzyme, could detect 17 mutant strains among 24 phenotypicaly resistant and 44 non-mutant isolates from 46 susceptible isolates. The sensitivity of this method was %70.83 and specificity was %95.65 on the other hand, 12 mutant from 20 resistant strains and 29 non-mutant strains from 32 susceptible strains were detected by AjiI enzyme. The sensitivity and specificity of this method was 60 and %90.62, respectively. In MAS PCR, 3 mutants from 6 resistant strains and 12 non-mutants from 17 resistant strains were detected. The sensitivity of this method was 50 and specificity was 70.58. Results of sequencing method confirmed the results of molecular methods.Discussion and conclusion: PCR-RFLP method by BstFNI enzyme was the best method for rapid detection of Mycobacterium tuberculosis resistant to second-line injectable drugs and was recommended for routine use.

  15. Detecting drug resistant genetic mutation among pneumoconiosis patients complicated with tuberculosis in Mycobacterium tuberculosis L-forms application of PCR-SSCP technique in Huainan mining district

    Institute of Scientific and Technical Information of China (English)

    Jun Lu; Shan Jiang; Song Ye; Chaopin Li

    2007-01-01

    Objective: To study the relationship between drug resistant genetic mutation and drug resistance in Mycobacterium tuberculosis L-form, discuss the internal relationship between drug resistances and drug-resistant related genes and explore the value of PCRSSCP to clinical application. Methods: A total of 52 clinically isolated strains of tuberculosis L-form were collected among 97pneumoconiosis patients complicated with tuberculosis. The gene mutations of katG, rpoB and rpsL were detected by PCR-SSCP,and the results were compared with those analyzed by traditional antimicrobial susceptibility test(AST). Results: The gene mutation rates of katG, rpoB and rpsL by PCR-SSCP were respectively 57.70% (30/52), 65.38% (32/52) and 40.38% (21/52). The rate of reversion was 78.85%(41/52) and the result of drug-resistant genes was invariable. The results of AST showed that there were 40 (76.92%) multi-drug resistant strains in 52 clinically isolated strains. The number for three-drug resistant strain was 21(40.38%) and that of two-drug resistant was 19(36.54%), but only 12 (23.08%) strains were one drug resistant. The rate of total drug-resistance was 100%, but there were 15 strains of allied mutation of three genes, 16 of two mutations and 6 of only one by PCR-SSCP. The coincidences were respectively 71.43%, 84.12% and 50.00%. Then there was no significant difference between the allied mutations of multi-drug resistant gene and the mutations of only one drug resistant gene (P > 0.05). Conclusion: PCRSSCP technique has a higher sensibility and specificity to detect the genes of katG, rpoB and rpsL in tuberculosis L-form among pneumoconiosis complicated with tuberculosis,and the detecting rate of two drug resistant strains and three drug resistant strains was higher. The combined application of PCR-SSCP and AST has advantages at earlier diagnosis and guidance of clinical medications.

  16. Diagnóstico de resistência do Mycobacterium tuberculosis à rifampicina utilizando-se da reação em cadeia da polimerase Diagnostic of rifampicin-resistance of Mycobacterium tuberculosis by polymerase chain reaction

    Directory of Open Access Journals (Sweden)

    Wânia da Silva Carvalho

    2007-03-01

    Full Text Available A resistência do Mycobacterium tuberculosis aos tuberculostáticos tem surgido como grande ameaça à Saúde Pública. A resistência à rifampicina pode ser considerada como um marcador para a multi-resistência a fármacos e tem sido atribuída a mudanças estruturais da RNA polimerase, produto de expressão do gene rpoB. Os pacientes portadores dessas cepas têm baixa perspectiva frente ao tratamento. Os testes convencionais de sensibilidade aos fármacos realizados em cultura do Mtb requerem várias semanas para o crescimento. Por este motivo, a Reação em Cadeia da Polimerase (PCR, método de baixo custo e que pode reduzir o tempo para o diagnóstico, representa alternativa viável e promissora. Neste artigo estão descritos os métodos mais comumente empregados na detecção de mutantes resistentes à rifampicina baseados na PCR, como análise de Polimorfismo Conformacional de Fita Simples (Single-Strand Conformation Polymorphism, SSCP, PCR Heteroduplex e INNO-LIPA. Recentemente, padronizou-se a técnica de PCR em baixa estringência, usando um único iniciador (Low Stringency Single Specific Primer, LSSP, que se mostrou um método rápido e sensível na detecção de mutações no gene rpoB.The resistance of Mycobacterium tuberculosis to tuberculostatic drugs has emerged as a major public health threat. The resistance to rifampicin which has been attributed to structural changes in RNA polymerase can be considered as a marker for multi-drug-resistance to tuberculosis (MDR-TB. Patients bearing rifampicin-resistant strains have poor diagnosis even with treatment. Conventional culture-based drug sensibility testing can require several weeks due to the growth. In this paper we describe the most common PCR-based methods for detection of mutations that lead to rifampicin resistance, such as Single-Strand Conformation Polymorphism (SSCP, PCR Heteroduplex and INNO-LIPA. Recently, by Low Stringency using a Single Specific Primer (LSSP assay, it

  17. Association between genotype and drug resistance profiles of Mycobacterium tuberculosis strains circulating in China in a national drug resistance survey

    Science.gov (United States)

    Zhou, Yang; van den Hof, Susan; Wang, Shengfen; Pang, Yu; Zhao, Bing; Xia, Hui; Anthony, Richard; Ou, Xichao; Li, Qiang; Zheng, Yang; Song, Yuanyuan; Zhao, Yanlin; van Soolingen, Dick

    2017-01-01

    We describe the population structure of a representative collection of 3,133 Mycobacterium tuberculosis isolates, collected within the framework of a national resistance survey from 2007 in China. Genotyping data indicate that the epidemic strains in China can be divided into seven major complexes, of which 92% belonged to the East Asian (mainly Beijing strains) or the Euro-American lineage. The epidemic Beijing strains in China are closely related to the Beijing B0/W148 strain earlier described in Russia and a large cluster of these strains has spread national wide. The density of Beijing strains is high in the whole of China (average 70%), but the highest prevalence was found North of the Yellow river. The Euro-American lineage consists of three sublineages (sublineage_1, 2, and 3) and is more prevalent in the South. Beijing lineage showed the highest cluster rate of 48% and a significantly higher level of resistance to rifampicin (14%, p<0.001), ethambutol (9%, p = 0.001), and ofloxacin (5%, p = 0.011). Within the Euro-American Lineage, sublineage_3 revealed the highest cluster rate (28%) and presented a significantly elevated level of resistance to streptomycin (44%, p<0.001). Our findings suggest that standardised treatment in this region may have contributed to the successful spread of certain strains: sublineage_3 in the Euro-American lineage may have thrived when streptomycin was used without rifampicin for treatment, while later under DOTS based treatment, in which rifampicin plays a key role, Beijing lineage appears to be spreading. PMID:28333978

  18. The Multifunctional PE_PGRS11 Protein from Mycobacterium tuberculosis Plays a Role in Regulating Resistance to Oxidative Stress*

    Science.gov (United States)

    Chaturvedi, Rashmi; Bansal, Kushagra; Narayana, Yeddula; Kapoor, Nisha; Sukumar, Namineni; Togarsimalemath, Shambhuprasad Kotresh; Chandra, Nagasuma; Mishra, Saurabh; Ajitkumar, Parthasarathi; Joshi, Beenu; Katoch, Vishwa Mohan; Patil, Shripad A.; Balaji, Kithiganahalli N.

    2010-01-01

    Mycobacterium tuberculosis utilizes unique strategies to survive amid the hostile environment of infected host cells. Infection-specific expression of a unique mycobacterial cell surface antigen that could modulate key signaling cascades can act as a key survival strategy in curtailing host effector responses like oxidative stress. We demonstrate here that hypothetical PE_PGRS11 ORF encodes a functional phosphoglycerate mutase. The transcriptional analysis revealed that PE_PGRS11 is a hypoxia-responsive gene, and enforced expression of PE_PGRS11 by recombinant adenovirus or Mycobacterium smegmatis imparted resistance to alveolar epithelial cells against oxidative stress. PE_PGRS11-induced resistance to oxidative stress necessitated the modulation of genetic signatures like induced expression of Bcl2 or COX-2. This modulation of specific antiapoptotic molecular signatures involved recognition of PE_PGRS11 by TLR2 and subsequent activation of the PI3K-ERK1/2-NF-κB signaling axis. Furthermore, PE_PGRS11 markedly diminished H2O2-induced p38 MAPK activation. Interestingly, PE_PGRS11 protein was exposed at the mycobacterial cell surface and was involved in survival of mycobacteria under oxidative stress. Furthermore, PE_PGRS11 displayed differential B cell responses during tuberculosis infection. Taken together, our investigation identified PE_PGRS11 as an in vivo expressed immunodominant antigen that plays a crucial role in modulating cellular life span restrictions imposed during oxidative stress by triggering TLR2-dependent expression of COX-2 and Bcl2. These observations clearly provide a mechanistic basis for the rescue of pathogenic Mycobacterium-infected lung epithelial cells from oxidative stress. PMID:20558725

  19. Recent transmission of drug-resistant Mycobacterium tuberculosis in a prison population in southern Brazil.

    Science.gov (United States)

    Reis, Ana Julia; David, Simone Maria Martini de; Nunes, Luciana de Souza; Valim, Andreia Rosane de Moura; Possuelo, Lia Gonçalves

    2016-01-01

    We conducted a cross-sectional, retrospective study, characterized by classical and molecular epidemiology, involving M. tuberculosis isolates from a regional prison in southern Brazil. Between January of 2011 and August of 2014, 379 prisoners underwent sputum smear microscopy and culture; 53 (13.9%) were diagnosed with active tuberculosis. Of those, 8 (22.9%) presented with isoniazid-resistant tuberculosis. Strain genotyping was carried out by 15-locus mycobacterial interspersed repetitive unit-variable-number tandem-repeat analysis; 68.6% of the patients were distributed into five clusters, and 87.5% of the resistant cases were in the same cluster. The frequency of drug-resistant tuberculosis cases and the rate of recent transmission were high. Our data suggest the need to implement an effective tuberculosis control program within the prison system. RESUMO Estudo transversal, retrospectivo, com isolados de M. tuberculosis de pacientes de um presídio regional no sul do Brasil, caracterizado através de epidemiologia clássica e molecular. Entre janeiro de 2011 e agosto de 2014, 379 detentos foram submetidos a baciloscopia e cultura, sendo 53 (13,9%) diagnosticados com tuberculose ativa. Desses, 8 (22,9%) apresentavam tuberculose resistente a isoniazida. A genotipagem das cepas foi realizada por 15-locus mycobacterial interspersed repetitive units-variable number of tandem repeat analysis; 68,6% dos pacientes estavam distribuídos em cinco clusters, e 87,5% dos casos resistentes estavam em um mesmo cluster. Verificou-se uma frequência elevada de casos de resistência e alta taxa de transmissão recente. Estes dados sugerem a necessidade da implantação de um programa efetivo de controle da tuberculose no sistema prisional.

  20. Mutations in the rpoB and katG genes leading to drug resistance in Mycobacterium tuberculosis in Latvia.

    Science.gov (United States)

    Tracevska, T; Jansone, I; Broka, L; Marga, O; Baumanis, V

    2002-10-01

    To characterize the genetic basis of drug resistance in Mycobacterium tuberculosis in Latvia, mutations involved in rifampin (rpoB gene) and isoniazid (katG gene) resistance in DNA from 19 drug-susceptible and 51 multidrug-resistant M. tuberculosis complex isolates were analyzed. The most frequent rpoB gene mutations found by the Line Probe assay were the S531L (14 of 34 isolates), D516V (7 of 34), H526D (4 of 34), and D516Y plus P535S (4 of 34) mutations. Direct sequencing of seven isolates with unclear results from Line Probe assay showed the presence of the L533P mutation and the Q510H plus H526Y (1 of 34) and D516V plus P535S (4 of 34) double mutations, neither of which has been described previously. Single-strand conformation polymorphism analysis showed strand mobility differences between the rifampin-susceptible and -resistant samples for the D516V, H526D, and D516Y plus P535S mutations but not for the S531L mutation. Nucleotide substitution at codon 315 (AGC-->ACC) of the katG gene was found in 48 of 51 multidrug-resistant samples by sequencing. Furthermore, katG gene restriction fragment length polymorphism analysis with endonuclease AciI confirmed the nucleotide change in codon 315.

  1. Enhancement of antibiotic activity by efflux inhibitors against multidrug resistant Mycobacterium tuberculosis clinical isolates from Brazil

    Directory of Open Access Journals (Sweden)

    Tatiane eCoelho

    2015-04-01

    Full Text Available Drug resistant tuberculosis continues to increase and new approaches for its treatment are necessary. The identification of M. tuberculosis clinical isolates presenting efflux as part of their resistant phenotype has a major impact in tuberculosis treatment. In this work, we used a checkerboard procedure combined with the tetrazolium microplate-based assay (TEMA to study single combinations between antituberculosis drugs and efflux inhibitors (EIs against multidrug resistant M. tuberculosis clinical isolates using the fully susceptible strain H37Rv as reference. Efflux activity was studied on a real-time basis by a fluorometric method that uses ethidium bromide as efflux substrate. Quantification of efflux pump genes mRNA transcriptional levels were performed by RT-qPCR. The fractional inhibitory concentrations (FIC indicated synergistic activity for the interactions between isoniazid, rifampicin, amikacin, ofloxacin, and ethidium bromide plus the EIs verapamil, thioridazine and chlorpromazine. The FICs ranged from 0.25, indicating a four-fold reduction on the MICs, to 0.015, 64-fold reduction. The detection of active efflux by real-time fluorometry showed that all strains presented intrinsic efflux activity that contributes to the overall resistance which can be inhibited in the presence of the EIs. The quantification of the mRNA levels of the most important efflux pump genes on these strains shows that they are intrinsically predisposed to expel toxic compounds as the exposure to subinhibitory concentrations of antibiotics were not necessary to increase the pump mRNA levels when compared with the non-exposed counterpart. The results obtained in this study confirm that the intrinsic efflux activity contributes to the overall resistance in multidrug resistant clinical isolates of M. tuberculosis and that the inhibition of efflux pumps by the EIs can enhance the clinical effect of antibiotics that are their substrates.

  2. Evaluation of the MeltPro TB/STR assay for rapid detection of streptomycin resistance in Mycobacterium tuberculosis.

    Science.gov (United States)

    Zhang, Ting; Hu, Siyu; Li, Guoli; Li, Hui; Liu, Xiaoli; Niu, Jianjun; Wang, Feng; Wen, Huixin; Xu, Ye; Li, Qingge

    2015-03-01

    Rapid and comprehensive detection of drug-resistance is essential for the control of tuberculosis, which has facilitated the development of molecular assays for the detection of drug-resistant mutations in Mycobacterium tuberculosis. We hereby assessed the analytical and clinical performance of an assay for streptomycin-resistant mutations. MeltPro TB/STR is a closed-tube, dual-color, melting curve analysis-based, real-time PCR test designed to detect 15 streptomycin-resistant mutations in rpsL 43, rpsL 88, rrs 513, rrs 514, rrs 517, and rrs 905-908 of M. tuberculosis. Analytical studies showed that the accuracy was 100%, the limit of detection was 50-500 bacilli per reaction, the reproducibility in the form of Tm variation was within 1.0 °C, and we could detect 20% STR resistance in mixed bacterial samples. The cross-platform study demonstrated that the assay could be performed on six models of real-time PCR instruments. A multicenter clinical study was conducted using 1056 clinical isolates, which were collected from three geographically different healthcare units, including 709 STR-susceptible and 347 STR-resistant isolates characterized on Löwenstein-Jensen solid medium by traditional drug susceptibility testing. The results showed that the clinical sensitivity and specificity of the MeltPro TB/STR was 88.8% and 95.8%, respectively. Sequencing analysis confirmed the accuracy of the mutation types. Among all the 8 mutation types detected, rpsL K43R (AAG → AGG), rpsL K88R (AAG → AGG) and rrs 514 A → C accounted for more than 90%. We concluded that MeltPro TB/STR represents a rapid and reliable assay for the detection of STR resistance in clinical isolates.

  3. Mycobacterium Tuberculosis in Spinal Tuberculosis

    Science.gov (United States)

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

    2017-01-01

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

  4. Esters of Pyrazinoic Acid Are Active against Pyrazinamide-Resistant Strains of Mycobacterium tuberculosis and Other Naturally Resistant Mycobacteria In Vitro and Ex Vivo within Macrophages

    Science.gov (United States)

    Valente, Emília; Simões, Marta Filipa; Carmo, Nuno; Testa, Bernard

    2015-01-01

    Pyrazinamide (PZA) is active against major Mycobacterium tuberculosis species (M. tuberculosis, M. africanum, and M. microti) but not against M. bovis and M. avium. The latter two are mycobacterial species involved in human and cattle tuberculosis and in HIV coinfections, respectively. PZA is a first-line agent for the treatment of human tuberculosis and requires activation by a mycobacterial pyrazinamidase to form the active metabolite pyrazinoic acid (POA). As a result of this mechanism, resistance to PZA, as is often found in tuberculosis patients, is caused by point mutations in pyrazinamidase. In previous work, we have shown that POA esters and amides synthesized in our laboratory were stable in plasma (M. F. Simões, E. Valente, M. J. Gómez, E. Anes, and L. Constantino, Eur J Pharm Sci 37:257–263, 2009, http://dx.doi.org/10.1016/j.ejps.2009.02.012). Although the amides did not present significant activity, the esters were active against sensitive mycobacteria at concentrations 5- to 10-fold lower than those of PZA. Here, we report that these POA derivatives possess antibacterial efficacy in vitro and ex vivo against several species and strains of Mycobacterium with natural or acquired resistance to PZA, including M. bovis and M. avium. Our results indicate that the resistance probably was overcome by cleavage of the prodrugs into POA and a long-chain alcohol. Although it is not possible to rule out that the esters have intrinsic activity per se, we bring evidence here that long-chain fatty alcohols possess a significant antimycobacterial effect against PZA-resistant species and strains and are not mere inactive promoieties. These findings may lead to candidate dual drugs having enhanced activity against both PZA-susceptible and PZA-resistant isolates and being suitable for clinical development. PMID:26438493

  5. Esters of pyrazinoic acid are active against pyrazinamide-resistant strains of Mycobacterium tuberculosis and other naturally resistant mycobacteria in vitro and ex vivo within macrophages.

    KAUST Repository

    Pires, David

    2015-10-05

    Pyrazinamide (PZA) is active against major Mycobacterium tuberculosis species (M. tuberculosis, M. africanum, and M. microti), but not against M. bovis and M. avium. The latter two are mycobacteria species involved in human and cattle tuberculosis and in HIV co-infections, respectively. PZA is a first-line agent for the treatment of human tuberculosis and requires activation by a mycobacterial pyrazinamidase to form the active metabolite pyrazinoic acid (POA). As a result of this mechanism, resistance to PZA as often found in tuberculosis patients is caused by point mutations in pyrazinamidase. In previous work, we have shown that POA esters and amides synthesized in our laboratory were stable in plasma. Although the amides did not present significant activity, the esters were active against sensitive mycobacteria at concentrations 5-to-10 fold lower than those of PZA. Here, we report that these POA derivatives possess antibacterial efficacy in vitro and ex vivo against several species and strains of Mycobacterium with natural or acquired resistance to PZA, including M. bovis and M. avium. Our results indicate that the resistance was probably overcome by cleavage of the prodrugs into POA and a long-chain alcohol. Although it is not possible to rule out that the esters may have intrinsic activity per se, we bring evidence here that long-chain fatty alcohols possess a significant anti-mycobacterial effect against PZA-resistant species and strains and are not mere inactive promoieties. These findings may lead to candidate dual-drugs having enhanced activity against both PZA-susceptible and PZA-resistant isolates and being suitable for clinical development.

  6. Mycobacterium tuberculosis: factores de virulencia

    OpenAIRE

    Reinier Borrero; Nadine Álvarez; Fátima Reyes; María Elena Sarmiento; Armando Acosta

    2011-01-01

    Mycobacterium tuberculosis es el agente causal de la tuberculosis, una de las enfermedades infecciosas más letales en el mundo. La única vacuna disponible para su control es el BCG, sin embargo, falla en la protección contra la tuberculosis pulmonar, siendo esta la forma más frecuente y responsable de la diseminación. La identificación de factores de virulencia del microorganismo causal pudiera ayudar en el desarrollo de un nuevo candidato vacunal que sea capaz de neutralizar la acción de eso...

  7. Comparative Proteomic Analysis of Aminoglycosides Resistant and Susceptible Mycobacterium tuberculosis Clinical Isolates for Exploring Potential Drug Targets.

    Science.gov (United States)

    Sharma, Divakar; Kumar, Bhavnesh; Lata, Manju; Joshi, Beenu; Venkatesan, Krishnamurthy; Shukla, Sangeeta; Bisht, Deepa

    2015-01-01

    Aminoglycosides, amikacin (AK) and kanamycin (KM) are second line anti-tuberculosis drugs used to treat tuberculosis (TB) and resistance to them affects the treatment. Membrane and membrane associated proteins have an anticipated role in biological processes and pathogenesis and are potential targets for the development of new diagnostics/vaccine/therapeutics. In this study we compared membrane and membrane associated proteins of AK and KM resistant and susceptible Mycobacterium tuberculosis isolates by 2DE coupled with MALDI-TOF/TOF-MS and bioinformatic tools. Twelve proteins were found to have increased intensities (PDQuest Advanced Software) in resistant isolates and were identified as ATP synthase subunit alpha (Rv1308), Trigger factor (Rv2462c), Dihydrolipoyl dehydrogenase (Rv0462), Elongation factor Tu (Rv0685), Transcriptional regulator MoxR1(Rv1479), Universal stress protein (Rv2005c), 35kDa hypothetical protein (Rv2744c), Proteasome subunit alpha (Rv2109c), Putative short-chain type dehydrogenase/reductase (Rv0148), Bacterioferritin (Rv1876), Ferritin (Rv3841) and Alpha-crystallin/HspX (Rv2031c). Among these Rv2005c, Rv2744c and Rv0148 are proteins with unknown functions. Docking showed that both drugs bind to the conserved domain (Usp, PspA and SDR domain) of these hypothetical proteins and GPS-PUP predicted potential pupylation sites within them. Increased intensities of these proteins and proteasome subunit alpha might not only be neutralized/modulated the drug molecules but also involved in protein turnover to overcome the AK and KM resistance. Besides that Rv1876, Rv3841 and Rv0685 were found to be associated with iron regulation signifying the role of iron in resistance. Further research is needed to explore how these potential protein targets contribute to resistance of AK and KM.

  8. Multiplex real-time PCR melting curve assay to detect drug-resistant mutations of Mycobacterium tuberculosis.

    Science.gov (United States)

    Luo, Tao; Jiang, Lili; Sun, Weiming; Fu, G; Mei, Jian; Gao, Qian

    2011-09-01

    Early diagnosis of drug-resistant Mycobacterium tuberculosis is urgently needed to optimize treatment regimens and to prevent the transmission of resistant strains. Real-time PCR assays have been developed to detect drug resistance rapidly, but none of them have been widely applied due to their complexity, high cost, or requirement for advanced instruments. In this study, we developed a real-time PCR method based on melting curve analysis of dually labeled probes. Six probes targeting the rpoB 81-bp core region, katG315, the inhA promoter, the ahpC promoter, and embB306 were designed and validated with clinical isolates. First, 10 multidrug-resistant (MDR) strains with a wide mutation spectrum were used to analyze the melting temperature (T(m)) deviations of different mutations by single real-time PCR. All mutations can be detected by significant T(m) reductions compared to the wild type. Then, three duplex real-time PCRs, with two probes in each, were developed to detect mutations in 158 MDR isolates. Comparison of the results with the sequencing data showed that all mutations covered by the six probes were detected with 100% sensitivity and 100% specificity. Our method provided a new way to rapidly detect drug-resistant mutations in M. tuberculosis. Compared to other real-time PCR methods, we use fewer probes, which are labeled with the same fluorophore, guaranteeing that this assay can be used for detection in a single fluorescent channel or can be run on single-channel instruments. In conclusion, we have developed a widely applicable real-time PCR assay to detect drug-resistant mutations in M. tuberculosis.

  9. Underestimation of the resistance of Mycobacterium tuberculosis to second-line drugs by the new GenoType MTBDRsl test.

    Science.gov (United States)

    Jin, Jialin; Shen, Yaojie; Fan, Xiaoping; Diao, Ni; Wang, Feifei; Wang, Sen; Weng, Xinhua; Zhang, Wenhong

    2013-01-01

    The GenoType MTBDRsl is a new-generation PCR-based line-probe assay for the detection of extensively drug-resistant tuberculosis (XDR-TB). This study evaluated the performance of MTBDRsl in detecting genotypic resistance to ethambutol, kanamycin, and ofloxacin in Mycobacterium tuberculosis (MTB) strains. The drug resistance of 262 unique clinical MTB isolates from China was analyzed with MTBDRsl, traditional TB drug susceptibility testing (DST), and sequencing. Sensitivity of MTBDRsl was 62.4% (93/149; 95% CI = 54.1 to 70.2) for detection of ethambutol resistance, 57.9% (55/95; 95% CI = 47.3 to 68) for kanamycin resistance, and 81% (111/137; 95% CI = 73.4 to 87.2) for ofloxacin resistance; specificity was 76.8% (86/112; 95% CI = 67.9 to 84.2), 98.8% (164/166; 95% CI = 95.7 to 99.9), and 91.1% (113/124; 95% CI = 84.7 to 95.5), respectively. Sequencing suggested that 36.9% (55/149) of ethambutol-resistant strains had no embB306 mutation and that 26.8% (40/149) had embB497 mutation not covered by MTBDRsl. Furthermore, MTBDRsl indicated ethambutol resistance in 23.2% (26/112) of ethambutol-susceptible strains, of which 92.3% (24/26) were confirmed resistant by sequencing. This study demonstrated that genotypic resistance to ethambutol, kanamycin, and ofloxacin in MTB can be quickly determined with the MTBDRsl. As a rapid and convenient genetic method, this assay could function as a supplement to traditional DST. More relevant genetic markers are needed to improve sensitivity.

  10. Impact of the E540V amino acid substitution in GyrB of Mycobacterium tuberculosis on quinolone resistance.

    Science.gov (United States)

    Kim, Hyun; Nakajima, Chie; Yokoyama, Kazumasa; Rahim, Zeaur; Kim, Youn Uck; Oguri, Hiroki; Suzuki, Yasuhiko

    2011-08-01

    Amino acid substitutions conferring resistance to quinolones in Mycobacterium tuberculosis have generally been found within the quinolone resistance-determining regions (QRDRs) in the A subunit of DNA gyrase (GyrA) rather than the B subunit of DNA gyrase (GyrB). To clarify the contribution of an amino acid substitution, E540V, in GyrB to quinolone resistance in M. tuberculosis, we expressed recombinant DNA gyrases in Escherichia coli and characterized them in vitro. Wild-type and GyrB-E540V DNA gyrases were reconstituted in vitro by mixing recombinant GyrA and GyrB. Correlation between the amino acid substitution and quinolone resistance was assessed by the ATP-dependent DNA supercoiling assay, quinolone-inhibited supercoiling assay, and DNA cleavage assay. The 50% inhibitory concentrations of eight quinolones against DNA gyrases bearing the E540V amino acid substitution in GyrB were 2.5- to 36-fold higher than those against the wild-type enzyme. Similarly, the 25% maximum DNA cleavage concentrations were 1.5- to 14-fold higher for the E540V gyrase than for the wild-type enzyme. We further demonstrated that the E540V amino acid substitution influenced the interaction between DNA gyrase and the substituent(s) at R-7, R-8, or both in quinolone structures. This is the first detailed study of the contribution of the E540V amino acid substitution in GyrB to quinolone resistance in M. tuberculosis.

  11. Evolution of extensively drug-resistant tuberculosis over four decades revealed by whole genome sequencing of Mycobacterium tuberculosis from KwaZulu-Natal, South Africa

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    Keira A Cohen

    2015-01-01

    Full Text Available The largest global outbreak of extensively drug-resistant (XDR tuberculosis (TB was identified in Tugela Ferry, KwaZulu-Natal (KZN, South Africa in 2005. The antecedents and timing of the emergence of drug resistance in this fatal epidemic XDR outbreak are unknown, and it is unclear whether drug resistance in this region continues to be driven by clonal spread or by the development of de novo resistance. A whole genome sequencing and drug susceptibility testing (DST was performed on 337 clinical isolates of Mycobacterium tuberculosis (M.tb collected in KZN from 2008 to 2013, in addition to three historical isolates, one of which was isolated during the Tugela Ferry outbreak. Using a variety of whole genome comparative approaches, 11 drug-resistant clones of M.tb circulating from 2008 to 2013 were identified, including a 50-member clone of XDR M.tb that was highly related to the Tugela Ferry XDR outbreak strain. It was calculated that the evolutionary trajectory from first-line drug resistance to XDR in this clone spanned more than four decades and began at the start of the antibiotic era. It was also observed that frequent de novo evolution of MDR and XDR was present, with 56 and 9 independent evolutions, respectively. Thus, ongoing amplification of drug-resistance in KwaZulu-Natal is driven by both clonal spread and de novo acquisition of resistance. In drug-resistant TB, isoniazid resistance was overwhelmingly the initial resistance mutation to be acquired, which would not be detected by current rapid molecular diagnostics that assess only rifampicin resistance.

  12. Consequences of noncompliance for therapy efficacy and emergence of resistance in murine tuberculosis caused by the Beijing genotype of Mycobacterium tuberculosis.

    Science.gov (United States)

    de Steenwinkel, Jurriaan E M; ten Kate, Marian T; de Knegt, Gerjo J; Verbrugh, Henri A; Aarnoutse, Rob E; Boeree, Martin J; den Bakker, Michael A; van Soolingen, Dick; Bakker-Woudenberg, Irma A J M

    2012-09-01

    Despite great effort by health organizations worldwide in fighting tuberculosis (TB), morbidity and mortality are not declining as expected. One of the reasons is related to the evolutionary development of Mycobacterium tuberculosis, in particular the Beijing genotype strains. In a previous study, we showed the association between the Beijing genotype and an increased mutation frequency for rifampin resistance. In this study, we use a Beijing genotype strain and an East-African/Indian genotype strain to investigate with our mouse TB model whether the higher mutation frequency observed in a Beijing genotype strain is associated with treatment failure particularly during noncompliance therapy. Both genotype strains showed high virulence in comparison to that of M. tuberculosis strain H37Rv, resulting in a highly progressive infection with a rapid lethal outcome in untreated mice. Compliance treatment was effective without relapse of TB irrespective of the infecting strain, showing similar decreases in the mycobacterial load in infected organs and similar histopathological changes. Noncompliance treatment, simulated by a reduced duration and dosing frequency, resulted in a relapse of infection. Relapse rates were correlated with the level of noncompliance and were identical for Beijing infection and East African/Indian infection. However, only in Beijing-infected mice, isoniazid-resistant mutants were selected at the highest level of noncompliance. This is in line with the substantial selection of isoniazid-resistant mutants in vitro in a wide isoniazid concentration window observed for the Beijing strain and not for the EAI strain. These results suggest that genotype diversity of M. tuberculosis may be involved in emergence of resistance and indicates that genotype-tailor-made treatment should be investigated.

  13. Gyrase Mutations Are Associated with Variable Levels of Fluoroquinolone Resistance in Mycobacterium tuberculosis.

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    Farhat, Maha R; Jacobson, Karen R; Franke, Molly F; Kaur, Devinder; Sloutsky, Alex; Mitnick, Carole D; Murray, Megan

    2016-03-01

    Molecular diagnostics that rapidly and accurately predict resistance to fluoroquinolone drugs and especially later-generation agents promise to improve treatment outcomes for patients with multidrug-resistant tuberculosis and prevent the spread of disease. Mutations in the gyr genes are known to confer most fluoroquinolone resistance, but knowledge about the effects of gyr mutations on susceptibility to early- versus later-generation fluoroquinolones and about the role of mutation-mutation interactions is limited. Here, we sequenced the full gyrA and gyrB open reading frames in 240 multidrug-resistant and extensively drug-resistant tuberculosis strains and quantified their ofloxacin and moxifloxacin MIC by testing growth at six concentrations for each drug. We constructed a multivariate regression model to assess both the individual mutation effects and interactions on the drug MICs. We found that gyrB mutations contribute to fluoroquinolone resistance both individually and through interactions with gyrA mutations. These effects were statistically significant. In these clinical isolates, several gyrA and gyrB mutations conferred different levels of resistance to ofloxacin and moxifloxacin. Consideration of gyr mutation combinations during the interpretation of molecular test results may improve the accuracy of predicting the fluoroquinolone resistance phenotype. Further, the differential effects of gyr mutations on the activity of early- and later-generation fluoroquinolones requires further investigation and could inform the selection of a fluoroquinolone for treatment. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  14. Dominant incidence of multidrug and extensively drug-resistant specific Mycobacterium tuberculosis clones in Osaka Prefecture, Japan.

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    Aki Tamaru

    Full Text Available Infection and transmission of multidrug-resistant Mycobacterium tuberculosis (MDR-Mtb and extensively drug-resistant M. tuberculosis (XDR-Mtb is a serious health problem. We analyzed a total of 1,110 Mtb isolates in Osaka Prefecture and neighboring areas from April 2000 to March 2009. A total of 89 MDR-Mtb were identified, 36 (48.5% of which were determined to be XDR-Mtb. Among the 89 MDR-Mtb isolates, 24 (27.0% phylogenetically distributed into six clusters based on mycobacterial interspersed repetitive units-various number of tandem repeats (MIRU-VNTR typing. Among these six clusters, the MIRU-VNTR patterns of four (OM-V02, OM-V03, OM-V04, and OM-V06 were only found for MDR-Mtb. Further analysis revealed that all isolates belonging to OM-V02 and OM-V03, and two isolates from OM-V04 were clonal. Importantly such genotypes were not observed for drug-sensitive isolates. These suggest that few but transmissible clones can transmit after acquiring multidrug resistance and colonize even in a country with a developed, well-organized healthcare system.

  15. Development of Au-Nanoprobes Combined with Loop-Mediated Isothermal Amplification for Detection of Isoniazid Resistance in Mycobacterium tuberculosis

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    Jutturong Ckumdee

    2016-01-01

    Full Text Available Multidrug resistant tuberculosis (MDR-TB is Mycobacterium tuberculosis that does not respond to isoniazid and rifampicin, so the condition worsens continuously and creates difficulties for treatment by public health control programmes, especially in developing countries. The real time polymerase chain reaction (PCR combined with agarose gel electrophoresis or strip tests is useful molecular tools for diagnosis of MDR-TB. Novel loop-mediated isothermal amplification (LAMP can also detect drug resistance, which is a one-point mutation, by designing inner primers of 5′ end specific with the mutant. Au-nanoprobes on hybridisation with LAMP products containing target-specific sequences remain red, whereas test samples without specific sequences in the probe turn purple due to salt-induced aggregation of the Au-nanoprobes. In this study, a strategy was designed based on the LAMP of a DNA sample coupled to specific Au-nanoprobes, which showed the potential to provide a rapid and sensitive method for detecting isoniazid resistance at katG gene position 315 (G→C. 46 clinical samples were tested and showed 100% specificity and sensitivity compared with Genotype® MDR-TB Plus. This method was advantageous because it is rapid, cheap, specific, and sensitive. Further, it does not require thermal cycles for MDR-TB detection.

  16. Consequences of noncompliance for therapy efficacy and emergence of resistance in murine tuberculosis caused by the Beijing genotype of Mycobacterium tuberculosis

    NARCIS (Netherlands)

    J.E.M. de Steenwinkel (Jurriaan); M.T. ten Kate (Marian); G.J. de Knegt (Gerjo); H.A. Verbrugh (Henri); E.J. Aarnoutse (E. J.); M. Boeree (Martin); M.A. den Bakker (Michael); D. van Soolingen (Dick); I.A.J.M. Bakker-Woudenberg (Irma)

    2012-01-01

    textabstractDespite great effort by health organizations worldwide in fighting tuberculosis (TB), morbidity and mortality are not declining as expected. One of the reasons is related to the evolutionary development of Mycobacterium tuberculosis, in particular the Beijing genotype strains. In a previ

  17. Consequences of Noncompliance for Therapy Efficacy and Emergence of Resistance in Murine Tuberculosis Caused by the Beijing Genotype of Mycobacterium tuberculosis.

    NARCIS (Netherlands)

    Steenwinkel, J.E. de; Kate, M.T. Ten; Knegt, G.J. de; Verbrugh, H.A.; Aarnoutse, R.E.; Boeree, M.J.; Bakker, M.A. den; Soolingen, D. van; Bakker-Woudenberg, I.A.

    2012-01-01

    Despite great effort by health organizations worldwide in fighting tuberculosis (TB), morbidity and mortality are not declining as expected. One of the reasons is related to the evolutionary development of Mycobacterium tuberculosis, in particular the Beijing genotype strains. In a previous study, w

  18. Multidrug-resistant tuberculosis

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    McNerney Ruth

    2008-01-01

    Full Text Available Abstract Background With almost 9 million new cases each year, tuberculosis remains one of the most feared diseases on the planet. Led by the STOP-TB Partnership and WHO, recent efforts to combat the disease have made considerable progress in a number of countries. However, the emergence of mutated strains of Mycobacterium tuberculosis that are resistant to the major anti-tuberculosis drugs poses a deadly threat to control efforts. Multidrug-resistant tuberculosis (MDR-TB has been reported in all regions of the world. More recently, extensively drug resistant-tuberculosis (XDR-TB that is also resistant to second line drugs has emerged in a number of countries. To ensure that adequate resources are allocated to prevent the emergence and spread of drug resistance it is important to understand the scale of the problem. In this article we propose that current methods of describing the epidemiology of drug resistant tuberculosis are not adequate for this purpose and argue for the inclusion of population based statistics in global surveillance data. Discussion Whereas the prevalence of tuberculosis is presented as the proportion of individuals within a defined population having disease, the prevalence of drug resistant tuberculosis is usually presented as the proportion of tuberculosis cases exhibiting resistance to anti-tuberculosis drugs. Global surveillance activities have identified countries in Eastern Europe, the former Soviet Union and regions of China as having a high proportion of MDR-TB cases and international commentary has focused primarily on the urgent need to improve control in these settings. Other regions, such as sub-Saharan Africa have been observed as having a low proportion of drug resistant cases. However, if one considers the incidence of new tuberculosis cases with drug resistant disease in terms of the population then countries of sub-Saharan Africa have amongst the highest rates of transmitted MDR-TB in the world. We propose

  19. RNA expression analysis of efflux pump genes in clinical isolates of multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis in South Korea.

    Science.gov (United States)

    Oh, Tae Sang; Kim, Young Jin; Kang, Hee Yoon; Kim, Chang-Ki; Cho, Sun Young; Lee, Hee Joo

    2017-04-01

    Tuberculosis (TB), caused by infection with Mycobacterium tuberculosis, is an important communicable disease. Various mechanisms of resistance to antituberculosis drugs have been reported; these are principally mutations in target genes. However, not all M. tuberculosis resistance can be explained by mutations in such genes. Other resistance mechanisms associated with drug transport, such as efflux pumps, have also been reported. In this study, we investigated the expression levels of three putative efflux pumps and mutations in target genes associated with injectable agents and fluoroquinolones with clinical MDR and XDR-TB isolates. Thirty clinical isolates of M. tuberculosis that had been phenotypically characterized were obtained from the Korean Institute of Tuberculosis. Of these, 14 were MDR-TB isolates resistant to at least one injectable aminoglycoside (amikacin; AMK, kanamycin; KAN, and/or capreomycin; CPM) and 16 were XDR-TB isolates. M. tuberculosis H37Rv (ATCC 27249) was used as a reference strain. Five putative genes (Rv1258c, Rv2686c, Rv2687c, Rv2688c and pstB) were selected for analysis in this study. Sequencing was performed to detect mutations in rrs and eis genes. qRT-PCR was performed to investigate expression levels of five efflux pump genes. Of the 30 isolates, 25 strains had mutations in rrs associated with resistance to KAN, CPM and AMK and two strains had eis mutations, as well as mutations in rrs. pstB (Rv0933) exhibited increased expression and Rv2687c and Rv2688c exhibited decreased expression compared to the reference strain. Increased expression of pstB in clinical drug-resistant tuberculosis isolates may contribute to drug resistance in M. tuberculosis. In our case, overexpression of Rv1258c may have been associated with resistance to kanamycin. No correlation was evident between Rv2686c, Rv2687c or Rv2688c expression and fluoroquinolone resistance. To explore the details of efflux pump drug-resistance mechanisms, further studies on

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

    Science.gov (United States)

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

  1. An important role of prostanoid receptor EP2 in host resistance to Mycobacterium tuberculosis infection in mice.

    Science.gov (United States)

    Kaul, Vandana; Bhattacharya, Debapriya; Singh, Yogesh; Van Kaer, Luc; Peters-Golden, Marc; Bishai, William R; Das, Gobardhan

    2012-12-15

    Mycobacterium tuberculosis, the causative agent of tuberculosis, resides and replicates within susceptible hosts by inhibiting host antimicrobial mechanisms. Prostaglandin E(2) (PGE(2)), produced by M. tuberculosis-infected macrophages, exerts a variety of immunomodulatory functions via 4 receptors (EP1-EP4), each mediating distinct PGE(2) functions. Here, we show that M. tuberculosis infection selectively upregulates EP2 messenger RNA expression in CD4(+) T cells. We found that EP2 deficiency in mice increases susceptibility to M. tuberculosis infection, which correlated with reduced antigen-specific T-cell responses and increased levels of CD4(+)CD25(+)Foxp3(+) T-regulatory cells. These findings have revealed an important role for EP2 in host immune defense against tuberculosis. As a G protein-coupled receptor, EP2 could serve as a target for immunotherapy of tuberculosis.

  2. Directed evolution of Mycobacterium tuberculosis β-lactamase reveals gatekeeper residue that regulates antibiotic resistance and catalytic efficiency.

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    Christian Feiler

    Full Text Available Directed evolution can be a powerful tool for revealing the mutational pathways that lead to more resistant bacterial strains. In this study, we focused on the bacterium Mycobacterium tuberculosis, which is resistant to members of the β-lactam class of antibiotics and thus continues to pose a major public health threat. Resistance of this organism is the result of a chromosomally encoded, extended spectrum class A β-lactamase, BlaC, that is constitutively produced. Here, combinatorial enzyme libraries were selected on ampicillin to identify mutations that increased resistance of bacteria to β-lactams. After just a single round of mutagenesis and selection, BlaC mutants were evolved that conferred 5-fold greater antibiotic resistance to cells and enhanced the catalytic efficiency of BlaC by 3-fold compared to the wild-type enzyme. All isolated mutants carried a mutation at position 105 (e.g., I105F that appears to widen access to the active site by 3.6 Å while also stabilizing the reorganized topology. In light of these findings, we propose that I105 is a 'gatekeeper' residue of the active site that regulates substrate hydrolysis by BlaC. Moreover, our results suggest that directed evolution can provide insight into the development of highly drug resistant microorganisms.

  3. Tuberculous spondylitis in Russia and prominent role of multidrug-resistant clone Mycobacterium tuberculosis Beijing B0/W148.

    Science.gov (United States)

    Vyazovaya, Anna; Mokrousov, Igor; Solovieva, Natalia; Mushkin, Alexander; Manicheva, Olga; Vishnevsky, Boris; Zhuravlev, Viacheslav; Narvskaya, Olga

    2015-04-01

    Extrapulmonary and, in particular, spinal tuberculosis (TB) constitutes a minor but significant part of the total TB incidence. In spite of this, almost no studies on the genetic diversity and drug resistance of Mycobacterium tuberculosis isolates from spinal TB patients have been published to date. Here, we report results of the first Russian and globally largest molecular study of M. tuberculosis isolates recovered from patients with tuberculous spondylitis (TBS). The majority of 107 isolates were assigned to the Beijing genotype (n = 80); the other main families were T (n = 11), Ural (n = 7), and LAM (n = 4). Multidrug resistance (MDR) was more frequently found among Beijing (90.5%) and, intriguingly, Ural (71.4%) isolates than other genotypes (5%; P mutations in Beijing strains that were similarly high in both TBS (this study) and published pulmonary TB (PTB) samples from Russia shows that TBS and PTB Beijing strains follow the same paradigm of acquisition of rifampin (RIF) and isoniazid (INH) resistance. The 24-locus mycobacterial interspersed repetitive unit-variable-number tandem-repeat (MIRU-VNTR) subtyping of 80 Beijing isolates further discriminated them into 24 types (Hunter Gaston index [HGI] = 0.83); types 100-32 and 94-32 represented the largest groups. A genotype of Russian successful clone B0/W148 was identified in 30 of 80 Beijing isolates. In conclusion, this study highlighted a crucial impact of the Beijing genotype and the especially prominent role of its MDR-associated successful clone B0/W148 cluster in the development of spinal MDR-TB in Russian patients.

  4. Mycobacterium tuberculosis Complex Genotype Diversity and Drug Resistance Profiles in a Pediatric Population in Mexico

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    Mercedes Macías Parra

    2011-01-01

    Full Text Available The aim of this study was to determine the frequency of drug resistance and the clonality of genotype patterns in M. tuberculosis clinical isolates from pediatric patients in Mexico (n=90 patients from 19 states; time period—January 2002 to December 2003. Pulmonary disease was the most frequent clinical manifestation (71%. Children with systemic tuberculosis (TB were significantly younger compared to patients with localized TB infections (mean 7.7±6.2 years versus 15±3.4 years P=0.001. Resistance to any anti-TB drug was detected in 24/90 (26.7% of the isolates; 21/90 (23.3% and 10/90 (11.1% were resistant to Isoniazid and Rifampicin, respectively, and 10/90 (11.1% strains were multidrug-resistant (MDR. Spoligotyping produced a total of 55 different patterns; 12/55 corresponded to clustered isolates (n=47, clustering rate of 52.2%, and 43/55 to unclustered isolates (19 patterns were designated as orphan by the SITVIT2 database. Database comparison led to designation of 36 shared types (SITs; 32 SITs (n=65 isolates matched a preexisting shared type in SITVIT2, whereas 4 SITs (n=6 isolates were newly created. Lineage classification based on principal genetic groups (PGG revealed that 10% of the strains belonged to PGG1 (Bovis and Manu lineages. Among PGG2/3 group, the most predominant clade was the Latin-American and Mediterranean (LAM in 27.8% of isolates, followed by Haarlem and T lineages. The number of single drug-resistant (DR and multidrug-resistant (MDR-TB isolates in this study was similar to previously reported in studies from adult population with risk factors. No association between the spoligotype, age, region, or resistance pattern was observed. However, contrary to a study on M. tuberculosis spoligotyping in Acapulco city that characterized a single cluster of SIT19 corresponding to the EAI2-Manila lineage in 70 (26% of patients, not a single SIT19 isolate was found in our pediatric patient population. Neither did we find any

  5. Mycobacterium tuberculosis Complex Genotype Diversity and Drug Resistance Profiles in a Pediatric Population in Mexico

    Science.gov (United States)

    Macías Parra, Mercedes; Kumate Rodríguez, Jesús; Arredondo García, José Luís; López-Vidal, Yolanda; Castañón-Arreola, Mauricio; Balandrano, Susana; Rastogi, Nalin; Gutiérrez Castrellón, Pedro

    2011-01-01

    The aim of this study was to determine the frequency of drug resistance and the clonality of genotype patterns in M. tuberculosis clinical isolates from pediatric patients in Mexico (n = 90 patients from 19 states; time period—January 2002 to December 2003). Pulmonary disease was the most frequent clinical manifestation (71%). Children with systemic tuberculosis (TB) were significantly younger compared to patients with localized TB infections (mean 7.7 ± 6.2 years versus 15 ± 3.4 years P = 0.001). Resistance to any anti-TB drug was detected in 24/90 (26.7%) of the isolates; 21/90 (23.3%) and 10/90 (11.1%) were resistant to Isoniazid and Rifampicin, respectively, and 10/90 (11.1%) strains were multidrug-resistant (MDR). Spoligotyping produced a total of 55 different patterns; 12/55 corresponded to clustered isolates (n = 47, clustering rate of 52.2%), and 43/55 to unclustered isolates (19 patterns were designated as orphan by the SITVIT2 database). Database comparison led to designation of 36 shared types (SITs); 32 SITs (n = 65 isolates) matched a preexisting shared type in SITVIT2, whereas 4 SITs (n = 6 isolates) were newly created. Lineage classification based on principal genetic groups (PGG) revealed that 10% of the strains belonged to PGG1 (Bovis and Manu lineages). Among PGG2/3 group, the most predominant clade was the Latin-American and Mediterranean (LAM) in 27.8% of isolates, followed by Haarlem and T lineages. The number of single drug-resistant (DR) and multidrug-resistant (MDR-TB) isolates in this study was similar to previously reported in studies from adult population with risk factors. No association between the spoligotype, age, region, or resistance pattern was observed. However, contrary to a study on M. tuberculosis spoligotyping in Acapulco city that characterized a single cluster of SIT19 corresponding to the EAI2-Manila lineage in 70 (26%) of patients, not a single SIT19 isolate was found in our pediatric patient population. Neither did we

  6. Increased transmission of Mycobacterium tuberculosis Beijing genotype strains associated with resistance to streptomycin: a population-based study.

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    Tran N Buu

    Full Text Available BACKGROUND: Studies have shown that the Mycobacterium tuberculosis Beijing genotype is an emerging pathogen that is frequently associated with drug resistance. This suggests that drug resistant Beijing strains have a relatively high transmission fitness compared to other drug-resistant strains. METHODS AND FINDINGS: We studied the relative transmission fitness of the Beijing genotype in relation to anti-tuberculosis drug resistance in a population-based study of smear-positive tuberculosis patients prospectively recruited and studied over a 4-year period in rural Vietnam. Transmission fitness was analyzed by clustering of cases on basis of three DNA typing methods. Of 2531 included patients, 2207 (87% were eligible for analysis of whom 936 (42% were in a DNA fingerprint cluster. The clustering rate varied by genotype with 292/786 (37% for the Beijing genotype, 527/802 (67% for the East-African Indian (EAI genotype, and 117/619 (19% for other genotypes. Clustering was associated with the EAI compared to the Beijing genotype (adjusted odds ratio (OR(adj 3.4: 95% CI 2.8-4.4. Patients infected with streptomycin-resistant strains were less frequently clustered than patients infected with streptomycin-susceptible strains when these were of the EAI genotype (OR(adj 0.6, 95% CI 0.4-0.9, while this pattern was reversed for strains of the Beijing genotype (OR(adj 1.3, 95% CI 1.0-1.8, p for difference 0.002. The strong association between Beijing and MDR-TB (OR(adj 7.2; 95% CI 4.2-12.3 existed only if streptomycin resistance was present. CONCLUSIONS: Beijing genotype strains showed less overall transmissibility than EAI strains, but when comparisons were made within genotypes, Beijing strains showed increased transmission fitness when streptomycin-resistant, while the reverse was observed for EAI strains. The association between MDR-TB and Beijing genotype in this population was strongly dependent on resistance to streptomycin. Streptomycin resistance may

  7. High Proportion of Fluoroquinolone-Resistant Mycobacterium tuberculosis Isolates with Novel Gyrase Polymorphisms and a gyrA Region Associated with Fluoroquinolone Susceptibility

    Science.gov (United States)

    Devasia, Rose; Blackman, Amondrea; Eden, Svetlana; Li, Haijing; Maruri, Fernanda; Shintani, Ayumi; Alexander, Charles; Kaiga, Anne; Stratton, Charles W.; Warkentin, Jon; Tang, Yi-Wei

    2012-01-01

    Fluoroquinolone resistance in Mycobacterium tuberculosis can be conferred by mutations in gyrA or gyrB. The prevalence of resistance mutations outside the quinolone resistance-determining region (QRDR) of gyrA or gyrB is unclear, since such regions are rarely sequenced. M. tuberculosis isolates from 1,111 patients with newly diagnosed culture-confirmed tuberculosis diagnosed in Tennessee from 2002 to 2009 were screened for phenotypic ofloxacin resistance (>2 μg/ml). For each resistant isolate, two ofloxacin-susceptible isolates were selected: one with antecedent fluoroquinolone exposure and one without. The complete gyrA and gyrB genes were sequenced and compared with M. tuberculosis H37Rv. Of 25 ofloxacin-resistant isolates, 11 (44%) did not have previously reported resistance mutations. Of these, 10 had novel polymorphisms: 3 in the QRDR of gyrA, 1 in the QRDR of gyrB, and 6 outside the QRDR of gyrA or gyrB; 1 did not have any gyrase polymorphisms. Polymorphisms in gyrA codons 1 to 73 were more common in fluoroquinolone-susceptible than in fluoroquinolone-resistant strains (20% versus 0%; P = 0.016). In summary, almost half of fluoroquinolone-resistant M. tuberculosis isolates did not have previously described resistance mutations, which has implications for genotypic diagnostic tests. PMID:22189117

  8. The combination of sulfamethoxazole, trimethoprim, and isoniazid or rifampin is bactericidal and prevents the emergence of drug resistance in Mycobacterium tuberculosis.

    Science.gov (United States)

    Vilchèze, Catherine; Jacobs, William R

    2012-10-01

    The challenges of developing new drugs to treat tuberculosis (TB) are indicated by the relatively small number of candidates entering clinical trials in the past decade. To overcome these issues, we reexamined two FDA-approved antibacterial drugs, sulfamethoxazole (SMX) and trimethoprim (TMP), for use in TB treatment. SMX and TMP inhibit folic acid biosynthesis and are used in combination to treat infections of the respiratory, urinary, and gastrointestinal tracts. The MICs of SMX and TMP, alone and in combination, were determined for drug-susceptible, multidrug-resistant (MDR), and extensively drug-resistant Mycobacterium tuberculosis strains. While TMP alone was not effective against M. tuberculosis, the combination of TMP and SMX was bacteriostatic against M. tuberculosis. Surprisingly, the combination of SMX and TMP was also active against a subset of MDR M. tuberculosis strains. Treatment of M. tuberculosis with TMP-SMX and a first-line anti-TB drug, either isoniazid or rifampin, was bactericidal, demonstrating that the combination of TMP and SMX with isoniazid or rifampin was not antagonistic. Moreover, the addition of SMX-TMP in combination with either isoniazid or rifampin also prevented the emergence of drug resistance in vitro. In conclusion, this study further illustrates the opportunity to reevaluate the activity of TMP-SMX in vivo to prevent the emergence of drug-resistant M. tuberculosis.

  9. Mutations in the rpoB gene of rifampicin-resistant Mycobacterium tuberculosis strains isolated in Brazil and France

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    S Spindola de Miranda

    2001-02-01

    Full Text Available We evaluated the mutations in a 193bp of the rpoB gene by automated sequencing of rifampicin (RMP-resistant and susceptible Mycobacterium tuberculosis strains isolated from Brazil (25 strains and France (37 strains. In RMP-resistant strains, mutations were identified in 100% (16/16 from France and 89% (16/18 from Brazil. No mutation was detected in the 28 RMP-susceptible strains. Among RMP-resistant or RMP-susceptible strains deletion was observed. A double point mutation which had not been reported before was detected in one strain from France. Among French resistant strains mutations were found in codons 531 (31.2%, 526, 513 and 533 (18.7% each. In Brazilian strains the most common mutations were in codons 531 (72.2%, 526 (11.1% and 513 (5.5%. The heterogeneity found in French strains may be related to the fact that most of those strains were from African or Asian patients.

  10. Draft Genome Sequence of Mycobacterium tuberculosis Clinical Strain G-12-005

    OpenAIRE

    Berland, Jean-Luc; Carvalho, Fabíola Marques; de Almeida, Luiz Gonzaga Paula; Bablishvili, Nino; Gauthier, Marie; Paranhos-Baccalà, Glaucia; de Vasconcelos, Ana Tereza Ribeiro

    2014-01-01

    Infection caused by drug-resistant Mycobacterium tuberculosis is a growing concern, especially in eastern Europe. We report an annotated draft genome sequence of M. tuberculosis strain G-12-005 obtained from a patient in Georgia.

  11. Mycobacterium tuberculosis: factores de virulencia

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    Reinier Borrero

    2011-04-01

    Full Text Available Mycobacterium tuberculosis es el agente causal de la tuberculosis, una de las enfermedades infecciosas más letales en el mundo. La única vacuna disponible para su control es el BCG, sin embargo, falla en la protección contra la tuberculosis pulmonar, siendo esta la forma más frecuente y responsable de la diseminación. La identificación de factores de virulencia del microorganismo causal pudiera ayudar en el desarrollo de un nuevo candidato vacunal que sea capaz de neutralizar la acción de esos determinantes patogénicos. El empleo de diferentes modelos animales ha permitido reproducir las etapas de la enfermedad, así como medir o cuantificar la virulencia de las distintas cepas circulantes de Mycobacterium tuberculosis. Las mutaciones génicas y otras técnicas de biología molecular han posibilitado dilucidar los genes específicos involucrados en la virulencia de este microorganismo que codifican para múltiples y complejos factores de diferente naturaleza.

  12. Rapid detection of multidrug-resistant Mycobacterium tuberculosis by use of real-time PCR and high-resolution melt analysis.

    Science.gov (United States)

    Ramirez, Melissa V; Cowart, Kelley C; Campbell, Patricia J; Morlock, Glenn P; Sikes, David; Winchell, Jonas M; Posey, James E

    2010-11-01

    The current study describes the development of a unique real-time PCR assay for the detection of mutations conferring drug resistance in Mycobacterium tuberculosis. The rifampicin resistance determinant region (RRDR) of rpoB and specific regions of katG and the inhA promoter were targeted for the detection of rifampin (RIF) and isoniazid (INH) resistance, respectively. Additionally, this assay was multiplexed to discriminate Mycobacterium tuberculosis complex (MTC) strains from nontuberculous Mycobacteria (NTM) strains by targeting the IS6110 insertion element. High-resolution melting (HRM) analysis following real-time PCR was used to identify M. tuberculosis strains containing mutations at the targeted loci, and locked nucleic acid (LNA) probes were used to enhance the detection of strains containing specific single-nucleotide polymorphism (SNP) transversion mutations. This method was used to screen 252 M. tuberculosis clinical isolates, including 154 RIF-resistant strains and 174 INH-resistant strains based on the agar proportion method of drug susceptibility testing (DST). Of the 154 RIF-resistant strains, 148 were also resistant to INH and therefore classified as multidrug resistant (MDR). The assay demonstrated sensitivity and specificity of 91% and 98%, respectively, for the detection of RIF resistance and 87% and 100% for the detection of INH resistance. Overall, this assay showed a sensitivity of 85% and a specificity of 98% for the detection of MDR strains. This method provides a rapid, robust, and inexpensive way to detect the dominant mutations known to confer MDR in M. tuberculosis strains and offers several advantages over current molecular and culture-based techniques.

  13. Definition of drug resistance of Mycobacterium tuberculosis to antituberculosis drugs in patients with multidrugresistant tuberculosis and TB with extremely drug resistant depending on the case of the disease

    Directory of Open Access Journals (Sweden)

    Kryzhanovsky D.G.

    2014-11-01

    Full Text Available There was studied the profile of drug resistance to the main (I line and reserve (II line antituberculosis drugs in patients with MDR and XDR tuberculosis, depending of the case of the disease. According to the randomized retrospective research 200 patients with MDR and XDR tuberculosis, who received treatment in the clinic of hospital Municipal institution «Dnipropetrovsk rigional clinical association «Phthisiology» Dnipropetrovsk regional Council» during the period 2010 – 2012 were involved. Data about patients contained the data on a case of the disease and the results of the test of drug sensitivity to MBT. XDR – TB was revealed in 7.5% of patients with MDR tuberculosis. In patients with MDR tuberculosis as compared with patients with XDR tuberculosis «new cases» were diagnosed in 19.5% against 18.5% (p <0.05. In patients with MDR tuberculosis and with XDR tuberculosis resistance to the antituberculosis drug more commonly developed to S - 88.5%, E - 55% and Z - 24%. The presence of MDR-TB and XDR-TB prevails in patients, who underwent previous courses of treatment with anti-TB drugs in case history as compared with patients with «new cases» of treatment. The development of resistance to anti-TB drugs depends on the availability of these drugs in the previous treatment regimens.

  14. Rapid detection of Mycobacterium tuberculosis complex and rifampin resistance in smear-negative clinical samples by use of an integrated real-time PCR method.

    Science.gov (United States)

    Moure, Raquel; Muñoz, Laura; Torres, Miriam; Santin, Miguel; Martín, Rogelio; Alcaide, Fernando

    2011-03-01

    Sixty-four of 85 (75.3%) smear-negative respiratory (n = 78) and nonrespiratory (n = 7) samples with positive cultures of Mycobacterium tuberculosis complex (MTC) were detected by the GeneXpert system using the Xpert MTB/RIF assay (GX). In addition, GX found rpoB mutations in all six of the rifampin-resistant strains detected. The test was negative in 20 culture-negative and 20 nontuberculous culture-positive samples (100% specificity). GX offers high potential for the diagnosis of tuberculosis due to its capacity for direct detection of MTC, its rapidity, and its simplicity.

  15. Rapid detection of isoniazid resistance in Mycobacterium tuberculosis isolates by use of real-time-PCR-based melting curve analysis.

    Science.gov (United States)

    Hu, Siyu; Li, Guoli; Li, Hui; Liu, Xiaoli; Niu, Jianjun; Quan, Shengmao; Wang, Feng; Wen, Huixin; Xu, Ye; Li, Qingge

    2014-05-01

    The MeltPro TB/INH assay, recently approved by the Chinese Food and Drug Administration, is a closed-tube, dual-color, melting curve analysis-based, real-time PCR test specially designed to detect 30 isoniazid (INH) resistance mutations in katG position 315 (katG 315), the inhA promoter (positions -17 to -8), inhA position 94, and the ahpC promoter (positions -44 to -30 and -15 to 3) of Mycobacterium tuberculosis. Here we evaluated both the analytical performance and clinical performance of this assay. Analytical studies with corresponding panels demonstrated that the accuracy for detection of different mutation types (10 wild-type samples and 12 mutant type samples), the limit of detection (2×10(3) to 2×10(4) bacilli/ml), reproducibility (standard deviation [SD], real-time PCR machines, with the shortest running time (105 min) obtained with the LightCycler 480 II. Clinical studies enrolled 1,096 clinical isolates collected from three geographically different tuberculosis centers, including 437 INH-resistant isolates and 659 INH-susceptible isolates characterized by traditional drug susceptibility testing on Löwenstein-Jensen solid medium. The clinical sensitivity and specificity of the MeltPro TB/INH assay were 90.8% and 96.4%, respectively. DNA sequencing analysis showed that, except for the 5 mutants outside the detection range of the MeltPro assay, a concordance rate between the two methods of 99.1% (457/461) was obtained. Among the 26 mutation types detected, katG S315T (AGC→ACC), inhA -15C→T, katG S315N (AGC→AAC), and ahpC promoter -10C→T accounted for more than 90%. Overall, the MeltPro TB/INH assay represents a reliable and rapid tool for the detection of INH resistance in clinical isolates.

  16. Nonclassical transpeptidases of Mycobacterium tuberculosis alter cell size, morphology, the cytosolic matrix, protein localization, virulence, and resistance to β-lactams.

    Science.gov (United States)

    Schoonmaker, Maia K; Bishai, William R; Lamichhane, Gyanu

    2014-04-01

    Virtually all bacteria possess a peptidoglycan layer that is essential for their growth and survival. The β-lactams, the most widely used class of antibiotics in human history, inhibit D,D-transpeptidases, which catalyze the final step in peptidoglycan biosynthesis. The existence of a second class of transpeptidases, the L,D-transpeptidases, was recently reported. Mycobacterium tuberculosis, an infectious pathogen that causes tuberculosis (TB), is known to possess as many as five proteins with L,D-transpeptidase activity. Here, for the first time, we demonstrate that loss of L,D-transpeptidases 1 and 2 of M. tuberculosis (LdtMt1 and LdtMt2) alters cell surface morphology, shape, size, organization of the intracellular matrix, sorting of some low-molecular-weight proteins that are targeted to the membrane or secreted, cellular physiology, growth, virulence, and resistance of M. tuberculosis to amoxicillin-clavulanate and vancomycin.

  17. Frequency and geographic distribution of gyrA and gyrB mutations associated with fluoroquinolone resistance in clinical Mycobacterium tuberculosis isolates: a systematic review.

    Science.gov (United States)

    Avalos, Elisea; Catanzaro, Donald; Catanzaro, Antonino; Ganiats, Theodore; Brodine, Stephanie; Alcaraz, John; Rodwell, Timothy

    2015-01-01

    The detection of mutations in the gyrA and gyrB genes in the Mycobacterium tuberculosis genome that have been demonstrated to confer phenotypic resistance to fluoroquinolones is the most promising technology for rapid diagnosis of fluoroquinolone resistance. In order to characterize the diversity and frequency of gyrA and gyrB mutations and to describe the global distribution of these mutations, we conducted a systematic review, from May 1996 to April 2013, of all published studies evaluating Mycobacterium tuberculosis mutations associated with resistance to fluoroquinolones. The overall goal of the study was to determine the potential utility and reliability of these mutations as diagnostic markers to detect phenotypic fluoroquinolone resistance in Mycobacterium tuberculosis and to describe their geographic distribution. Forty-six studies, covering four continents and 18 countries, provided mutation data for 3,846 unique clinical isolates with phenotypic resistance profiles to fluoroquinolones. The gyrA mutations occurring most frequently in fluoroquinolone-resistant isolates, ranged from 21-32% for D94G and 13-20% for A90V, by drug. Eighty seven percent of all strains that were phenotypically resistant to moxifloxacin and 83% of ofloxacin resistant isolates contained mutations in gyrA. Additionally we found that 83% and 80% of moxifloxacin and ofloxacin resistant strains respectively, were observed to have mutations in the gyrA codons interrogated by the existing MTBDRsl line probe assay. In China and Russia, 83% and 84% of fluoroquinolone resistant strains respectively, were observed to have gyrA mutations in the gene regions covered by the MTBDRsl assay. Molecular diagnostics, specifically the Genotype MTBDRsl assay, focusing on codons 88-94 should have moderate to high sensitivity in most countries. While we did observe geographic differences in the frequencies of single gyrA mutations across countries, molecular diagnostics based on detection of all gyr

  18. Frequency and geographic distribution of gyrA and gyrB mutations associated with fluoroquinolone resistance in clinical Mycobacterium tuberculosis isolates: a systematic review.

    Directory of Open Access Journals (Sweden)

    Elisea Avalos

    Full Text Available The detection of mutations in the gyrA and gyrB genes in the Mycobacterium tuberculosis genome that have been demonstrated to confer phenotypic resistance to fluoroquinolones is the most promising technology for rapid diagnosis of fluoroquinolone resistance.In order to characterize the diversity and frequency of gyrA and gyrB mutations and to describe the global distribution of these mutations, we conducted a systematic review, from May 1996 to April 2013, of all published studies evaluating Mycobacterium tuberculosis mutations associated with resistance to fluoroquinolones. The overall goal of the study was to determine the potential utility and reliability of these mutations as diagnostic markers to detect phenotypic fluoroquinolone resistance in Mycobacterium tuberculosis and to describe their geographic distribution.Forty-six studies, covering four continents and 18 countries, provided mutation data for 3,846 unique clinical isolates with phenotypic resistance profiles to fluoroquinolones. The gyrA mutations occurring most frequently in fluoroquinolone-resistant isolates, ranged from 21-32% for D94G and 13-20% for A90V, by drug. Eighty seven percent of all strains that were phenotypically resistant to moxifloxacin and 83% of ofloxacin resistant isolates contained mutations in gyrA. Additionally we found that 83% and 80% of moxifloxacin and ofloxacin resistant strains respectively, were observed to have mutations in the gyrA codons interrogated by the existing MTBDRsl line probe assay. In China and Russia, 83% and 84% of fluoroquinolone resistant strains respectively, were observed to have gyrA mutations in the gene regions covered by the MTBDRsl assay.Molecular diagnostics, specifically the Genotype MTBDRsl assay, focusing on codons 88-94 should have moderate to high sensitivity in most countries. While we did observe geographic differences in the frequencies of single gyrA mutations across countries, molecular diagnostics based on detection

  19. Locked Nucleic Acid Probe-Based Real-Time PCR Assay for the Rapid Detection of Rifampin-Resistant Mycobacterium tuberculosis.

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    Yong Zhao

    Full Text Available Drug-resistant Mycobacterium tuberculosis can be rapidly diagnosed through nucleic acid amplification techniques by analyzing the variations in the associated gene sequences. In the present study, a locked nucleic acid (LNA probe-based real-time PCR assay was developed to identify the mutations in the rpoB gene associated with rifampin (RFP resistance in M. tuberculosis. Six LNA probes with the discrimination capability of one-base mismatch were designed to monitor the 23 most frequent rpoB mutations. The target mutations were identified using the probes in a "probe dropout" manner (quantification cycle = 0; thus, the proposed technique exhibited superiority in mutation detection. The LNA probe-based real-time PCR assay was developed in a two-tube format with three LNA probes and one internal amplification control probe in each tube. The assay showed excellent specificity to M. tuberculosis with or without RFP resistance by evaluating 12 strains of common non-tuberculosis mycobacteria. The limit of detection of M. tuberculosis was 10 genomic equivalents (GE/reaction by further introducing a nested PCR method. In a blind validation of 154 clinical mycobacterium isolates, 142/142 (100% were correctly detected through the assay. Of these isolates, 88/88 (100% were determined as RFP susceptible and 52/54 (96.3% were characterized as RFP resistant. Two unrecognized RFP-resistant strains were sequenced and were found to contain mutations outside the range of the 23 mutation targets. In conclusion, this study established a sensitive, accurate, and low-cost LNA probe-based assay suitable for a four-multiplexing real-time PCR instrument. The proposed method can be used to diagnose RFP-resistant tuberculosis in clinical laboratories.

  20. Locked Nucleic Acid Probe-Based Real-Time PCR Assay for the Rapid Detection of Rifampin-Resistant Mycobacterium tuberculosis.

    Science.gov (United States)

    Zhao, Yong; Li, Guilian; Sun, Chongyun; Li, Chao; Wang, Xiaochen; Liu, Haican; Zhang, Pingping; Zhao, Xiuqin; Wang, Xinrui; Jiang, Yi; Yang, Ruifu; Wan, Kanglin; Zhou, Lei

    2015-01-01

    Drug-resistant Mycobacterium tuberculosis can be rapidly diagnosed through nucleic acid amplification techniques by analyzing the variations in the associated gene sequences. In the present study, a locked nucleic acid (LNA) probe-based real-time PCR assay was developed to identify the mutations in the rpoB gene associated with rifampin (RFP) resistance in M. tuberculosis. Six LNA probes with the discrimination capability of one-base mismatch were designed to monitor the 23 most frequent rpoB mutations. The target mutations were identified using the probes in a "probe dropout" manner (quantification cycle = 0); thus, the proposed technique exhibited superiority in mutation detection. The LNA probe-based real-time PCR assay was developed in a two-tube format with three LNA probes and one internal amplification control probe in each tube. The assay showed excellent specificity to M. tuberculosis with or without RFP resistance by evaluating 12 strains of common non-tuberculosis mycobacteria. The limit of detection of M. tuberculosis was 10 genomic equivalents (GE)/reaction by further introducing a nested PCR method. In a blind validation of 154 clinical mycobacterium isolates, 142/142 (100%) were correctly detected through the assay. Of these isolates, 88/88 (100%) were determined as RFP susceptible and 52/54 (96.3%) were characterized as RFP resistant. Two unrecognized RFP-resistant strains were sequenced and were found to contain mutations outside the range of the 23 mutation targets. In conclusion, this study established a sensitive, accurate, and low-cost LNA probe-based assay suitable for a four-multiplexing real-time PCR instrument. The proposed method can be used to diagnose RFP-resistant tuberculosis in clinical laboratories.

  1. Genotypic Analysis of Genes Associated with Independent Resistance and Cross-Resistance to Isoniazid and Ethionamide in Mycobacterium tuberculosis Clinical Isolates.

    Science.gov (United States)

    Rueda, Johana; Realpe, Teresa; Mejia, Gloria Isabel; Zapata, Elsa; Rozo, Juan Carlos; Ferro, Beatriz Eugenia; Robledo, Jaime

    2015-12-01

    Ethionamide (ETH) is an antibiotic used for the treatment of multidrug-resistant (MDR) tuberculosis (TB) (MDR-TB), and its use may be limited with the emergence of resistance in the Mycobacterium tuberculosis population. ETH resistance in M. tuberculosis is phenomenon independent or cross related when accompanied with isoniazid (INH) resistance. In most cases, resistance to INH and ETH is explained by mutations in the inhA promoter and in the following genes: katG, ethA, ethR, mshA, ndh, and inhA. We sequenced the above genes in 64 M. tuberculosis isolates (n = 57 ETH-resistant MDR-TB isolates; n = 3 ETH-susceptible MDR-TB isolates; and n = 4 fully susceptible isolates). Each isolate was tested for susceptibility to first- and second-line drugs using the agar proportion method. Mutations were observed in ETH-resistant MDR-TB isolates at the following rates: 100% in katG, 72% in ethA, 45.6% in mshA, 8.7% in ndh, and 33.3% in inhA or its promoter. Of the three ETH-susceptible MDR-TB isolates, all showed mutations in katG; one had a mutation in ethA, and another, in mshA and inhA. Finally, of the four fully susceptible isolates, two showed no detectable mutation in the studied genes, and two had mutations in mshA gene unrelated to the resistance. Mutations not previously reported were found in the ethA, mshA, katG, and ndh genes. The concordance between the phenotypic susceptibility testing to INH and ETH and the sequencing was 1 and 0.45, respectively. Among isolates exhibiting INH resistance, the high frequency of independent resistance and cross-resistance with ETH in the M. tuberculosis isolates suggests the need to confirm the susceptibility to ETH before considering it in the treatment of patients with MDR-TB.

  2. Characterization of mutations causing rifampicin and isoniazid resistance of Mycobacterium tuberculosis in Syria.

    Science.gov (United States)

    Madania, Ammar; Habous, Maya; Zarzour, Hana; Ghoury, Ifad; Hebbo, Barea

    2012-01-01

    In order to characterize mutations causing rifampicin and isoniazid resistance of M. tuberculosis in Syria, 69 rifampicin resistant (Rif(r)) and 72 isoniazid resistant (Inh(r)) isolates were screened for point mutations in hot spots of the rpoB, katG and inhA genes by DNA sequencing and real time PCR. Of 69 Rif(r) isolates, 62 (90%) had mutations in the rifampin resistance determining region (RRDR) of the rpoB gene, with codons 531 (61%), 526 (13%), and 516 (8.7%) being the most commonly mutated. We found two new mutations (Asp516Thr and Ser531Gly) described for the first time in the rpoB-RRDR in association with rifampicin resistance. Only one mutation (Ile572Phe) was found outside the rpoB-RRDR. Of 72 Inh(r) strains, 30 (41.6%) had a mutation in katGcodon315 (with Ser315Thr being the predominant alteration), and 23 (32%) harbored the inhA(-15C-->T) mutation. While the general pattern of rpoB-RRDR and katG mutations reflected those found worldwide, the prevalence of the inhA(-15C-->T mutation was above the value found in most other countries, emphasizing the great importance of testing the inhA(-15C-->T) mutation for prediction of isoniazid resistance in Syria. Sensitivity of a rapid test using real time PCR and 3'-Minor groove binder (MGB) probes in detecting Rif(r) and Inh(r) isolates was 90% and 69.4%, respectively. This demonstrates that a small set of MGB-probes can be used in real time PCR in order to detect most mutations causing resistance to rifampicin and isoniazid.

  3. Analysis of embCAB mutations associated with ethambutol resistance in multidrug-resistant mycobacterium tuberculosis isolates from China.

    Science.gov (United States)

    Zhao, Li-Li; Sun, Qing; Liu, Hai-Can; Wu, Xiao-Cui; Xiao, Tong-Yang; Zhao, Xiu-Qin; Li, Gui-Lian; Jiang, Yi; Zeng, Chun-Yan; Wan, Kang-Lin

    2015-04-01

    Ethambutol (EMB) plays a pivotal role in the chemotherapy of drug-resistant tuberculosis (TB), including multidrug-resistant tuberculosis (MDR-TB). Resistance to EMB is considered to be caused by mutations in the embCAB operon (embC, embA, and embB). In this study, we analyzed the embCAB mutations among 139 MDR-TB isolates from China and found a possible association between embCAB operon mutation and EMB resistance. Our data indicate that 56.8% of MDR-TB isolates are resistant to EMB, and 82.2% of EMB-resistant isolates belong to the Beijing family. Overall, 110 (79.1%) MDR-TB isolates had at least one mutation in the embCAB operon. The majority of mutations were present in the embB gene and the embA upstream region, which also displayed significant correlations with EMB resistance. The most common mutations occurred at codon 306 in embB (embB306), followed by embB406, embA(-16), and embB497. Mutations at embB306 were associated with EMB resistance. DNA sequencing of embB306-497 was the best strategy for detecting EMB resistance, with 89.9% sensitivity, 58.3% specificity, and 76.3% accuracy. Additionally, embB306 had limited value as a candidate predictor for EMB resistance among MDR-TB infections in China.

  4. High prevalence of multidrug-resistant tuberculosis among patients with rifampicin resistance using GeneXpert Mycobacterium tuberculosis/rifampicin in Ghana

    NARCIS (Netherlands)

    Boakye-Appiah, Justice K; Steinmetz, Alexis R; Pupulampu, Peter; Ofori-Yirenkyi, Stephen; Tetteh, Ishmael; Frimpong, Michael; Oppong, Patrick; Opare-Sem, Ohene; Norman, Betty R; Stienstra, Ymkje; van der Werf, Tjip S; Wansbrough-Jones, Mark; Bonsu, Frank; Obeng-Baah, Joseph; Phillips, Richard O

    2016-01-01

    OBJECTIVE/BACKGROUND: Drug-resistant strains of tuberculosis (TB) represent a major threat to global TB control. In low- and middle-income countries, resource constraints make it difficult to identify and monitor cases of resistance using drug susceptibility testing and culture. Molecular assays suc

  5. Prevalence of Drug Resistance Mycobacterium Tuberculosis among Patients Seen in Coast Provincial General Hospital, Mombasa, Kenya

    Science.gov (United States)

    Ombura, Ida Pam; Onyango, Noel; Odera, Susan; Mutua, Florence; Nyagol, Joshua

    2016-01-01

    Background Although prevention and control of spread of multi-drug resistant tuberculosis strains is a global challenge, there is paucity of data on the prevalence of DR-TB in patients diagnosed with TB in referral hospitals in Kenya. The present study assessed patients’ characteristics and prevalence of drug resistant TB in sputa smear positive TB patients presenting to Coast Provincial General Hospital (CPGH) in Mombasa, Kenya. Methods Drug resistance was evaluated in 258 randomly selected sputa smear TB positive cases between the periods of November 2011 to February 2012 at the CPGH-Mombasa. Basic demographic data was obtained using administered questionnaires, and clinical history extracted from the files. For laboratory analyses, 2mls of sputum was obtained, decontaminated and subjected to mycobacteria DNA analyses. Detection of first line drug resistance genes was done using MDRTDR plus kit. This was followed with random selection of 83 cases for second line drug resistance genes testing using Genotype MDRTBsl probe assay kit (HAINS Lifesciences, GmbH, Germany), in which ethambutol mutation probes were included. The data was then analyzed using SPSS statistical package version 19.0. Results Male to female ratio was 1:2. Age range was 9 to 75 years, with median of 30 years. New treatment cases constituted 253(98%), among which seven turned out to be PTB negative, and further grouped as 4 (1.6%) PTB negative and 3(1.1%) NTM. 237(91.7%) new cases were fully susceptible to INH and RIF. The remaining, 8 (3.1%) and 1(0.4%) had mono- resistance to INH and RIF, respectively. All the retreatment cases were fully susceptible to the first line drugs. HIV positivity was found in 48 (18.6%) cases, of which 46(17.8%) were co-infected with TB. Of these, 44 (17.1%) showed full susceptibility to TB drugs, while 2 (0.8%) were INH resistant. For the second line drugs, one case each showed mono resistance to both and FQ. Also, one case each showed drug cross poly resistance to

  6. The population structure of drug-resistant Mycobacterium tuberculosis clinical isolates from Sichuan in China.

    Science.gov (United States)

    Zhao, Yuding; Feng, Qin; Tang, Ke; Zhang, Congcong; Sun, Honghu; Luo, Tao; Yang, Zhirong; Couvin, David; Rastogi, Nalin; Sun, Qun

    2012-06-01

    China ranks second next to India among 22 high-burden countries despite decades' effort on tuberculosis (TB) control. The Sichuan province today contains the second-largest number of TB cases among Chinese provinces, where the prevalence of drug-resistant TB, especially MDR-TB, is much higher than the average level in eastern China. In this study, the population structure and the transmission characteristics of drug-resistant TB in Sichuan province were studied by spoligotyping and 24-locus Mycobacterial interspersed repetitive units-variable number tandem DNA repeats (MIRU-VNTR), applied to a total of 306 clinical isolates. Spoligotyping-based analysis showed that Beijing family represented 69.28% of all isolates and constituted the largest group (66.24%) of MDR-TB in Sichuan. The remaining isolates, accounting for 33.76% of MDR isolates, belonged to the ill-defined T family, Manu2, H3, LAM9, and other minor unassigned clades. The discriminatory power evaluated for spoligotyping was poor (HGI=0.595), but high for 24-locus MIRU-VNTRs (HGI=0.999). The number of the most discriminatory loci (h>0.6) was 12, including locus 424, 802, 960, 1644, 1955, 2163b, 2996, 3007, 3192, 3690, 4348 and 4052. It was concluded that 24-locus MIRU-VNTRs could be a more discriminatory tool for differentiating clinical isolates from Sichuan region. The small clustering size obtained from the current population structure analysis suggested that the high prevalence of drug-resistant TB in this region might be attributed partially to the acquired resistance due to inappropriate drug use rather than active transmission of drug-resistant TB (primary resistance).

  7. Whole genome sequencing-based characterization of extensively drug resistant (XDR) strains of Mycobacterium tuberculosis from Pakistan

    KAUST Repository

    Hasan, Zahra

    2015-03-01

    Objectives: The global increase in drug resistance in Mycobacterium tuberculosis (MTB) strains increases the focus on improved molecular diagnostics for MTB. Extensively drug-resistant (XDR) - TB is caused by MTB strains resistant to rifampicin, isoniazid, fluoroquinolone and aminoglycoside antibiotics. Resistance to anti-tuberculous drugs has been associated with single nucleotide polymorphisms (SNPs), in particular MTB genes. However, there is regional variation between MTB lineages and the SNPs associated with resistance. Therefore, there is a need to identify common resistance conferring SNPs so that effective molecular-based diagnostic tests for MTB can be developed. This study investigated used whole genome sequencing (WGS) to characterize 37 XDR MTB isolates from Pakistan and investigated SNPs related to drug resistance. Methods: XDR-TB strains were selected. DNA was extracted from MTB strains, and samples underwent WGS with 76-base-paired end fragment sizes using Illumina paired end HiSeq2000 technology. Raw sequence data were mapped uniquely to H37Rv reference genome. The mappings allowed SNPs and small indels to be called using SAMtools/BCFtools. Results: This study found that in all XDR strains, rifampicin resistance was attributable to SNPs in the rpoB RDR region. Isoniazid resistance-associated mutations were primarily related to katG codon 315 followed by inhA S94A. Fluoroquinolone resistance was attributable to gyrA 91-94 codons in most strains, while one did not have SNPs in either gyrA or gyrB. Aminoglycoside resistance was mostly associated with SNPs in rrs, except in 6 strains. Ethambutol resistant strains had embB codon 306 mutations, but many strains did not have this present. The SNPs were compared with those present in commercial assays such as LiPA Hain MDRTBsl, and the sensitivity of the assays for these strains was evaluated. Conclusions: If common drug resistance associated with SNPs evaluated the concordance between phenotypic and

  8. The Inhibition of Folylpolyglutamate Synthetase (folC in the Prevention of Drug Resistance in Mycobacterium tuberculosis by Traditional Chinese Medicine

    Directory of Open Access Journals (Sweden)

    Tzu-Chieh Hung

    2014-01-01

    Full Text Available Tuberculosis (TB is an infectious disease caused by many strains of mycobacteria, but commonly Mycobacterium tuberculosis. As a possible method of reducing the drug resistance of M. tuberculosis, this research investigates the inhibition of Folylpolyglutamate synthetase, a protein transcript from the resistance association gene folC. After molecular docking to screen the traditional Chinese medicine (TCM database, the candidate TCM compounds, with Folylpolyglutamate synthetase, were selected by molecular dynamics. The 10,000 ps simulation in association with RMSD analysis and total energy and structural variation defined the protein-ligand interaction. The selected TCM compounds Saussureamine C, methyl 3-O-feruloylquinate, and Labiatic acid have been found to inhibit the activity of bacteria and viruses and to regulate immunity. We also suggest the possible pathway in protein for each ligand. Compared with the control, similar interactions and structural variations indicate that these compounds might have an effect on Folylpolyglutamate synthetase. Finally, we suggest Saussureamine C is the best candidate compound as the complex has a high score, maintains its structural composition, and has a larger variation value than the control, thus inhibiting the drug resistance ability of Mycobacterium tuberculosis.

  9. Whole genome sequencing reveals complex evolution patterns of multidrug-resistant Mycobacterium tuberculosis Beijing strains in patients.

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    Matthias Merker

    Full Text Available Multidrug-resistant (MDR Mycobacterium tuberculosis complex (MTBC strains represent a major threat for tuberculosis (TB control. Treatment of MDR-TB patients is long and less effective, resulting in a significant number of treatment failures. The development of further resistances leads to extensively drug-resistant (XDR variants. However, data on the individual reasons for treatment failure, e.g. an induced mutational burst, and on the evolution of bacteria in the patient are only sparsely available. To address this question, we investigated the intra-patient evolution of serial MTBC isolates obtained from three MDR-TB patients undergoing longitudinal treatment, finally leading to XDR-TB. Sequential isolates displayed identical IS6110 fingerprint patterns, suggesting the absence of exogenous re-infection. We utilized whole genome sequencing (WGS to screen for variations in three isolates from Patient A and four isolates from Patient B and C, respectively. Acquired polymorphisms were subsequently validated in up to 15 serial isolates by Sanger sequencing. We determined eight (Patient A and nine (Patient B polymorphisms, which occurred in a stepwise manner during the course of the therapy and were linked to resistance or a potential compensatory mechanism. For both patients, our analysis revealed the long-term co-existence of clonal subpopulations that displayed different drug resistance allele combinations. Out of these, the most resistant clone was fixed in the population. In contrast, baseline and follow-up isolates of Patient C were distinguished each by eleven unique polymorphisms, indicating an exogenous re-infection with an XDR strain not detected by IS6110 RFLP typing. Our study demonstrates that intra-patient microevolution of MDR-MTBC strains under longitudinal treatment is more complex than previously anticipated. However, a mutator phenotype was not detected. The presence of different subpopulations might confound phenotypic and

  10. Predominance of multi-drug-resistant LAM and Beijing family strains among Mycobacterium tuberculosis isolates recovered from prison inmates in Tula Region, Russia.

    Science.gov (United States)

    Ignatova, Anna; Dubiley, Svetlana; Stepanshina, Valentina; Shemyakin, Igor

    2006-10-01

    The genotypic characteristics and drug susceptibility profiles of clinical isolates of Mycobacterium tuberculosis recovered from prison hospital patients in the Tula region (central Russia) during 2001 and 2002 are reported. The emergence of multi-drug-resistant tuberculosis (TB) poses a major health risk to the population, with economic implications for TB control. Prisons serve as a continuous source of TB transmission. The results showed that members of the LAM and Beijing families are major contributors to the epidemiological picture of TB in the population studied. The two families of strains accounted for most of the drug-resistant TB in the population. The genotypic characteristics of the M. tuberculosis predominant LAM strain that was responsible for 31 % of TB cases in this setting are presented.

  11. Feasibility of the GenoType MTBDRsl assay for fluoroquinolone, amikacin-capreomycin, and ethambutol resistance testing of Mycobacterium tuberculosis strains and clinical specimens.

    Science.gov (United States)

    Hillemann, Doris; Rüsch-Gerdes, Sabine; Richter, Elvira

    2009-06-01

    The new GenoType Mycobacterium tuberculosis drug resistance second line (MTBDRsl) assay (Hain Lifescience, Nehren, Germany) was tested on 106 clinical isolates and directly on 64 sputum specimens for the ability to detect resistance to fluoroquinolones, injectable drugs (amikacin or capreomycin), and ethambutol in Mycobacterium tuberculosis strains. A total of 63 strains harboring fluoroquinolone, amikacin/capreomycin, or ethambutol resistance and 43 fully susceptible strains were comparatively analyzed with the new MTBDRsl assay, by DNA sequencing, and by conventional drug susceptibility testing in liquid and solid media. No discrepancies were obtained in comparison with the DNA sequencing results. Fluoroquinolone resistance was detected in 29 (90.6%) of 32, amikacin/capreomycin resistance was detected in 39/39 (84.8%/86.7%) of 46/45, and ethambutol resistance was detected in 36 (69.2%) of 52 resistant strains. A total of 64 sputum specimens (42 smear positive, 12 scanty, and 10 smear negative) were tested with the new MTBDRsl assay, and the results were compared with those of conventional drug susceptibility testing. Fluoroquinolone resistance was detected in 8 (88.9%) of 9, amikacin/capreomycin resistance was detected in 6/7 (75.0%/87.5%) of 8, and ethambutol resistance was detected in 10 (38.5%) of 26 resistant strains. No mutation was detected in susceptible strains. The new GenoType MTBDRsl assay represents a reliable tool for the detection of fluoroquinolone and amikacin/capreomycin resistance and to a lesser extent also ethambutol resistance. In combination with a molecular test for detection of rifampin and isoniazid resistance, the potential for the detection of extensively resistant tuberculosis within 1 to 2 days can be postulated.

  12. Cloning and nucleotide sequence of Mycobacterium tuberculosis gyrA and gyrB genes and detection of quinolone resistance mutations.

    Science.gov (United States)

    Takiff, H E; Salazar, L; Guerrero, C; Philipp, W; Huang, W M; Kreiswirth, B; Cole, S T; Jacobs, W R; Telenti, A

    1994-04-01

    The emergence of multidrug-resistant strains of Mycobacterium tuberculosis has resulted in increased interest in the fluoroquinolones (FQs) as antituberculosis agents. To investigate the frequency and mechanisms of FQ resistance in M. tuberculosis, we cloned and sequenced the wild-type gyrA and gyrB genes, which encode the A and B subunits of the DNA gyrase, respectively; DNA gyrase is the main target of the FQs. On the basis of the sequence information, we performed DNA amplification for sequencing and single-strand conformation polymorphism analysis to examine the presumed quinolone resistance regions of gyrA and gyrB from reference strains (n = 4) and clinical isolates (n = 55). Mutations in codons of gyrA analogous to those described in other FQ-resistant bacteria were identified in all isolates (n = 14) for which the ciprofloxacin MIC was > 2 micrograms/ml. In addition, we selected ciprofloxacin-resistant mutants of Mycobacterium bovis BCG and M. tuberculosis Erdman and H37ra. Spontaneously resistant mutants developed at a frequency of 1 in 10(7) to 10(8) at ciprofloxacin concentrations of 2 micrograms/ml, but no primary resistant colonies were selected at higher ciprofloxacin concentrations. Replating of those first-step mutants selected for mutants with high levels of resistance which harbored gyrA mutations similar to those found among clinical FQ-resistant isolates. The gyrA and gyrB sequence information will facilitate analysis of the mechanisms of resistance to drugs which target the gyrase and the implementation of rapid strategies for the estimation of FQ susceptibility in clinical M. tuberculosis isolates.

  13. Mycobacterium tuberculosis monoarthritis in a child

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    Rosenberg Alan M

    2008-09-01

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

  14. Mycobacterium tuberculosis of the RDRio genotype is the predominant cause of tuberculosis and associated with multidrug resistance in Porto Alegre City, South Brazil.

    Science.gov (United States)

    Dalla Costa, Elis Regina; Lazzarini, Luiz Claudio Oliveira; Perizzolo, Paulo Fernado; Díaz, Chyntia Acosta; Spies, Fernanda S; Costa, Lucas Laux; Ribeiro, Andrezza W; Barroco, Caroline; Schuh, Sandra Jungblut; da Silva Pereira, Marcia Aparecida; Dias, Claudia F; Gomes, Harrison M; Unis, Gisela; Zaha, Arnaldo; Almeida da Silva, Pedro E; Suffys, Philip N; Rossetti, Maria L R

    2013-04-01

    Spoligotyping has shown Mycobacterium tuberculosis strains to be composed of different lineages, and some of them are not just geographically restricted but also affect specific ethnic populations and are associated with outbreaks and drug resistance. We recently described a particular subtype within the Latin American-Mediterranean (LAM) family, called RD(Rio), widespread in Brazil. Moreover, recent data also indicate that RD(Rio) is present in many countries on all continents and is associated with cavitary disease and multidrug resistance (MDR). To further explore the relationship between RD(Rio) and MDR, we conducted a study in a tuberculosis (TB) reference center responsible for the care of MDR patients in Rio Grande do Sul, the southernmost Brazilian state. From a collection of 237 clinical isolates, RD(Rio) alone was responsible for one-half of all MDR cases, including one large group composed of strains with identical IS6110-restriction fragment length polymorphism (RFLP) and having the LAM5 signature. We additionally had complete data records for 96 patients and could make comparisons between the presence and absence of RD(Rio). No difference in clinical, radiological or laboratory features was observed, but a significantly greater number of cases with MDR were described in patients infected with an RD(Rio) strain (P = 0.0015). Altogether, RD(Rio) was responsible for 38% of all TB cases. These data support and confirmed previous findings that RD(Rio) is the main agent responsible for TB in Brazil and is associated with drug resistance. Considering that RD(Rio) is a globally distributed genotype, such findings raise concern about the increase in MDR in certain human populations.

  15. The genotypic study of Mycobacterium tuberculosis complex resistant to isoniazid: Galicia, Spain (2008-2013).

    Science.gov (United States)

    Pérez Del Molino, M L; Barbeito-Castiñeiras, G; Mejuto, B; Alonso, P; Fernández, A; González-Mediero, G

    2016-11-01

    Incorporation of rapid detection systems to identify mutations in M. tuberculosis complex that confer resistance to isoniazid and rifampicin has potentiated the knowledge of their distribution, given the geographical variability. We performed antibiograms of the 2,993 strains isolated in Galicia, Spain (2008-2013). In the strains resistant to isoniazid, a concentration of 0.4 mg/mL and MTBDRplus Genotype test (Hain Lifescience, Germany) were used. We found that 3.64 % of strains were resistant to isoniazid, while 0.43 % were resistant to isoniazid and rifampicin (multidrug resistant, MDR). The MTBDRplus test showed an overall sensitivity of 72.48 %, with 62.5 % sensitivity for non MDR isoniazid-resistant strains and 100 % sensitivity for MDR strains. The katG gene mutation was detected at codon 315 in 38.53 % of strains. The S315T mutation appeared in 61.54 % of MDR strains and 34.38 % of non-MDR strains. The 28.44 % had mutations in inhA, (93.55 % in C15T), and 38.46 % of MDR strains were mutated. In non-MDR strains, 37.50 % were wild-type, 35.42 % and 27.08 % had mutations in katG and inhA, respectively. The most frequent mutation in rpoβ was S531L (46.15 %). The 38.71 % and 41.9 % of strains with resistance to isoniazid and streptomycin had mutations in katG and inhA, respectively (2 strains with mutations in T8C and T8A). The distribution pattern of resistance among strains with high and low concentrations of isoniazid showed statistically significant differences in relation to the mutation in katG and wild-type. The sensitivity of the Genotype MTBDRplus test for non-MDR strains in our area was at the lower threshold described.

  16. Declining trend of resistance to first-line anti-tubercular drugs in clinical isolates of Mycobacterium tuberculosis in a tertiary care north Indian hospital after implementation of revised national Tuberculosis control programme

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    A Jain

    2014-01-01

    Full Text Available Trends showing drug-resistance pattern are needed to understand direction of tuberculosis (TB control programme. The drug-resistance pattern in state of Uttar Pradesh, India, is not documented. Here we are reporting the prevalence of multi-drug-resistant (MDR and drug-resistant TB in previously treated cases of pulmonary tuberculosis following launch of revised national TB control programme (RNTCP in whole of Uttar Pradesh. Isolates of Mycobacterium tuberculosis, from patients of pulmonary tuberculosis, who were treated with antitubercular drugs for more than 4 weeks, were tested for resistance to first-line drugs; streptomycin (S, Ethambutol (E, Rifampicin (R and isoniazid (H over a period of 4 years, 2009-2012. Total 2496 isolates of M. tuberculosis were tested, of which 1139 isolates (45.6% were pan-sensitive and 370 (14.8% were pan-resistant. Total 695 isolates (27.8% were MDR. Maximum resistance was with Isoniazid (n = 1069, 42.8% followed by streptomycin (n = 840, 33.7%, rifampicin (n = 742, 29.7%, and ethambutol (n = 613, 24.6%. A decline in number of MDR strains and individual drug resistance was seen. Total MDR strains in the year 2009, 2010, 2011 and 2012 were 35.6%, 30.8%, 26.7% and 22.8% respectively. The drug resistance pattern reported from time to time may vary substantially. The decline in drug resistance visible over last four years, after implementation of DOTS, appears promising.

  17. Detection by GenoType MTBDRsl test of complex mechanisms of resistance to second-line drugs and ethambutol in multidrug-resistant Mycobacterium tuberculosis complex isolates.

    Science.gov (United States)

    Brossier, Florence; Veziris, Nicolas; Aubry, Alexandra; Jarlier, Vincent; Sougakoff, Wladimir

    2010-05-01

    The GenoType MTBDRsl test rapidly detects resistance to ethambutol, fluoroquinolones, and second-line aminoglycosides (amikacin and kanamycin) and cyclic peptide (capreomycin) in Mycobacterium tuberculosis. A set of 41 multidrug-resistant (MDR) M. tuberculosis strains, 8 extensively drug-resistant (XDR) M. tuberculosis strains, and 3 non-MDR M. tuberculosis strains were tested by the MTBDRsl test and by DNA sequencing of the resistance-determining regions in gyrA and gyrB (fluoroquinolones [FQ]), rpsL (streptomycin), rrs and tlyA (aminoglycosides and/or cyclic peptide), and embB (ethambutol). The sensitivity and specificity of the MTBDRsl test were as follows: 87% and 96%, respectively, for fluoroquinolones; 100% for both for amikacin; 77% and 100%, respectively, for kanamycin, 80% and 98%, respectively, for capreomycin; and 57% and 92%, respectively, for ethambutol. Analysis of the discrepant results indicated that three FQ-resistant strains (including one XDR strain) with mutations in gyrB were missed by the MTBDRsl test and that one FQ-susceptible strain, identified as resistant by the MTBDRsl test, had a double mutation (T80A-A90G) in GyrA that did not confer resistance to FQ. Five strains (including two XDR strains) without mutations in rrs were monoresistant to aminoglycosides or cyclic peptide and were missed by the MTBDRsl test. Finally, 12/28 ethambutol-resistant strains had no mutation at codon 306 in embB, while 2/24 ethambutol-susceptible strains had such a mutation. In conclusion, the MTBDRsl test efficiently detects the most common mutations involved in resistance to fluoroquinolones, aminoglycosides/cyclic peptide, and ethambutol and accurately assesses susceptibility to amikacin. However, due to mutations not included in the test (particularly in gyrB) or resistance mechanisms not yet characterized (particularly those related to ethambutol resistance and to monoresistance to aminoglycosides or cyclic peptide), the wild-type results yielded by the

  18. [Resistance to first-line drugs and major genotypic lineages of Mycobacterium tuberculosis in the 3 French Department of the Americas: Profiles, evolution, and trends (1995-2011)].

    Science.gov (United States)

    Millet, J; Berchel, M; Prudenté, F; Streit, E; Bomer, A-G; Schuster, F; Vanhomwegen, J; Paasch, D; Galbert, I; Valery, E; Aga, R; Rastogi, N

    2014-05-01

    This is the first overview on resistant and multidrug resistant isolates of Mycobacterium tuberculosis circulating in the French Department of the Americas (Guadeloupe, Martinique, and French Guiana) over 17 years (January 1995-December 2011). A total of 1,239 cases were studied: 1,199 new cases (primary and multidrug resistance of 11.8 and 1.6% respectively), and 40 persistent (defined as cases with a previous history of positive culture over 6 months interval and whose spoligotypes remain unchanged), in which significantly higher proportions of resistance to at least isoniazid (22.5%, P = 0.002), rifampicin (20.0%, P French Guiana, department in which one may also observe the presence of strains of M. tuberculosis phylogeographically associated to Guiana and Suriname (SIT 131 and SIT 1340).

  19. Phenotypic and genotypic analysis of multidrug-resistant tuberculosis in Ethiopia.

    NARCIS (Netherlands)

    Agonafir, M.; Lemma, E.; Wolde-Meskel, D.; Goshu, S.; Santhanam, A.; Girmachew, F.; Demissie, D.; Getahun, M.; Gebeyehu, M.; Soolingen, D. van

    2010-01-01

    SETTING: National Tuberculosis Reference Laboratory, Addis Ababa, Ethiopia. OBJECTIVES: To determine the drug susceptibility pattern of Mycobacterium tuberculosis isolates and to genetically characterise multidrug-resistant tuberculosis (MDR-TB) isolates. DESIGN: A total of 107 M. tuberculosis isola

  20. Detecting mutation pattern of drug-resistant Mycobacterium tuberculosis isolates in Himachal Pradesh using GenoType® MTBDRplus assay

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    C Thakur

    2015-01-01

    Full Text Available Context: Tuberculosis (TB is a major public health problem in India and a principal cause of death in adults, especially among the economically productive age group. India accounts for one-fifth of the global burden of TB. It is estimated that about 40% of Indian population is infected with TB bacillus. The GenoType® MTBDRplus molecular method allows rapid diagnosis of the clinical samples and detection of the most common mutations in the genes associated with rifampicin (R and isoniazid (H resistance. Aims: To study the drug resistance and mutational patterns in multidrug-resistant (MDR suspects clinical strains using GenoType® MTBDRplus assay. Subjects and Methods: A total of 770 sputum samples of the MDR-TB suspects were included in this study, which were received at Intermediate Reference Laboratory, Government TB Sanatorium, Dharampur, Solan, Himachal Pradesh from the Designated Microscopy Centres of Himachal Pradesh for the culture and susceptibility testing. All the 521 Mycobacterium tuberculosis complex (MTBC strains were subjected to GenoType® MTBDRplus (HAIN Lifescience assay to detect molecular resistance pattern to first line anti-tubercular drugs (isoniazid and rifampicin. Results: Of 770 samples, 556 (72.20% were from male and 214 (27.80% were from female. Among the 521 MTBC strains, 19.76% were found to be MDR and mono-resistance to isoniazid and rifampicin was detected in 8.63% and 6.14% strains respectively. About 74.81%, 76.35% and 5.40% strains harboured known mutation in rpoB, katG and inhA genes respectively. Conclusions: In rpoB gene, the most common mutation is associated with S531 L region. The GenoType® MTBDRplus assay is a rapid test for the detection of the most common mutations in MDR-TB strains. In our study, unknown rpoB gene mutations were found in 25.18% strains that may further be detected by gene sequencing.

  1. Prevalence and occurrence rate of Mycobacterium tuberculosis Haarlem family multi-drug resistant in the worldwide population: A systematic review and meta-analysis

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    Rashid Ramazanzadeh

    2015-01-01

    Full Text Available Background: Transmission of Mycobacterium tuberculosis (M. tuberculosis can occur in different ways. Furthermore, drug resistant in M. tuberculosis family is a major problem that creates obstacles in treatment and control of tuberculosis (TB in the world. One of the most prevalent families of M. tuberculosis is Haarlem, and it is associated with drug resistant. Our objectives of this study were to determine the prevalence and occurrence rate of M. tuberculosis Haarlem family multi-drug resistant (MDR in the worldwide using meta-analysis based on a systematic review that performed on published articles. Materials and Methods: Data sources of this study were 78 original articles (2002-2012 that were published in the literatures in several databases including PubMed, Science Direct, Google Scholar, Biological abstracts, ISI web of knowledge and IranMedex. The articles were systematically reviewed for prevalence and rate of MDR. Data were analyzed using meta-analysis and random effects models with the software package Meta R, Version 2.13 (P < 0.10. Results: Final analysis included 28601 persons in 78 articles. The highest and lowest occurrence rate of Haarlem family in M. tuberculosis was in Hungary in 2006 (66.20% with negative MDR-TB and in China in 2010 (0.8%, respectively. From 2002 to 2012, the lowest rate of prevalence was in 2010, and the highest prevalence rate was in 2012. Also 1.076% were positive for MDR and 9.22% were negative (confidence interval: 95%.0020. Conclusion: Many articles and studies are performed in this field globally, and we only chose some of them. Further studies are needed to be done in this field. Our study showed that M. tuberculosis Haarlem family is prevalent in European countries. According to the presence of MDR that was seen in our results, effective control programs are needed to control the spread of drug-resistant strains, especially Haarlem family.

  2. Evaluation of two line probe assays for rapid detection of Mycobacterium tuberculosis, tuberculosis (TB) drug resistance, and non-TB Mycobacteria in HIV-infected individuals with suspected TB.

    Science.gov (United States)

    Luetkemeyer, Anne F; Kendall, Michelle A; Wu, Xingye; Lourenço, Maria Cristina; Jentsch, Ute; Swindells, Susan; Qasba, Sarojini S; Sanchez, Jorge; Havlir, Diane V; Grinsztejn, Beatriz; Sanne, Ian M; Firnhaber, Cynthia

    2014-04-01

    Limited performance data from line probe assays (LPAs), nucleic acid tests used for the rapid diagnosis of tuberculosis (TB), nontuberculosis mycobacteria (NTM), and Mycobacterium tuberculosis drug resistance are available for HIV-infected individuals, in whom paucibacillary TB is common. In this study, the strategy of testing sputum with GenoType MTBDRplus (MTBDR-Plus) and GenoType Direct LPA (Direct LPA) was compared to a gold standard of one mycobacterial growth indicator tube (MGIT) liquid culture. HIV-positive (HIV(+)) individuals with suspected TB from southern Africa and South America with tuberculosis culture positive, of which 276 (72.8%) were acid-fast bacillus (AFB) smear positive. MTBDR-Plus had a sensitivity of 81.0% and a specificity of 100%, with sensitivities of 44.1% in AFB smear-negative versus 94.6% in AFB smear-positive specimens. For specimens that were positive for M. tuberculosis by MTBDR-Plus, the sensitivity and specificity for rifampin resistance were 91.7% and 96.6%, respectively, and for isoniazid (INH) they were 70.6% and 99.1%. The Direct LPA had a sensitivity of 88.4% and a specificity of 94.6% for M. tuberculosis detection, with a sensitivity of 72.5% in smear-negative specimens. Ten of 639 MGIT cultures grew Mycobacterium avium complex or Mycobacterium kansasii, half of which were detected by Direct LPA. Both LPA assays performed well in specimens from HIV-infected individuals, including in AFB smear-negative specimens, with 72.5% sensitivity for M. tuberculosis identification with the Direct LPA and 44.1% sensitivity with MTBDR-Plus. LPAs have a continued role for use in settings where rapid identification of INH resistance and clinically relevant NTM are priorities.

  3. Characterization of mutations in streptomycin-resistant Mycobacterium tuberculosis isolates in Sichuan, China and the association between Beijing-lineage and dual-mutation in gidB.

    Science.gov (United States)

    Sun, Honghu; Zhang, Congcong; Xiang, Ling; Pi, Rui; Guo, Zhen; Zheng, Chao; Li, Song; Zhao, Yuding; Tang, Ke; Luo, Mei; Rastogi, Nalin; Li, Yuqing; Sun, Qun

    2016-01-01

    Mutations in rpsL, rrs, and gidB are well linked to streptomycin (STR) resistance, some of which are suggested to be potentially associated with Mycobacterium tuberculosis genotypic lineages in certain geographic regions. In this study, we aimed to investigate the mutation characteristics of streptomycin resistance and the relationship between the polymorphism of drug-resistant genes and the lineage of M. tuberculosis isolates in Sichuan, China. A total of 227 M. tuberculosis clinical isolates, including 180 STR-resistant and 47 pan-susceptible isolates, were analyzed for presence of mutations in the rpsL, rrs and gidB loci. Mutation K43R in rpsL was strongly associated with high-level streptomycin resistance (P mutations in rrs and gidB potentially contributed to low-level resistance (P genotype, however, in STR-resistant strains, Beijing genotype was significantly correlated with high-level STR resistance, as well as the rpsL mutation K43R (P genotype has an evolutionary advantage under streptomycin pressure. Notably, in all isolates of Beijing genotype, a dual mutation E92D (a276c) and A205A (a615g) in gidB was detected, suggesting a highly significant association between this dual mutation and Beijing genotype.

  4. Rapid direct detection of multiple rifampin and isoniazid resistance mutations in Mycobacterium tuberculosis in respiratory samples by real-time PCR.

    Science.gov (United States)

    Marín, Mercedes; García de Viedma, Darío; Ruíz-Serrano, María Jesús; Bouza, Emilio

    2004-11-01

    Rapid detection of resistance in Mycobacterium tuberculosis can optimize the efficacy of antituberculous therapy and control the transmission of resistant M. tuberculosis strains. Real-time PCR has minimized the time required to obtain the susceptibility pattern of M. tuberculosis strains, but little effort has been made to adapt this rapid technique to the direct detection of resistance from clinical samples. In this study, we adapted and evaluated a real-time PCR design for direct detection of resistance mutations in clinical respiratory samples. The real-time PCR was evaluated with (i) 11 clinical respiratory samples harboring bacilli resistant to isoniazid (INH) and/or rifampin (RIF), (ii) 10 culture-negative sputa spiked with a set of strains encoding 14 different resistance mutations in 10 independent codons, and (iii) 16 sputa harboring susceptible strains. The results obtained with this real-time PCR design completely agreed with DNA sequencing data. In all sputa harboring resistant M. tuberculosis strains, the mutation encoding resistance was successfully detected. No mutation was detected in any of the susceptible sputa. The test was applied only to smear-positive specimens and succeeded in detecting a bacterial load equivalent to 10(3) CFU/ml in sputum samples (10 acid-fast bacilli/line). The analytical specificity of this method was proved with a set of 14 different non-M. tuberculosis bacteria. This real-time PCR design is an adequate method for the specific and rapid detection of RIF and INH resistance in smear-positive clinical respiratory samples.

  5. Evaluation of direct colorimetric MTT assay for rapid detection of rifampicin and isoniazid resistance in Mycobacterium tuberculosis

    NARCIS (Netherlands)

    G.B. Hundie (Gadissa Bedada); Woldemeskel, D. (Dawit); Gessesse, A. (Amare)

    2016-01-01

    textabstractWith the spread of multidrug-resistant tuberculosis (MDR-TB) strains there is an increasing need for new accurate and cost-effective methods for a rapid diagnostic and drug susceptibility testing (DST), particularly in low-income countries where tuberculosis is hyperendemic. A colorimetr

  6. Minor contribution of mutations at iniA codon 501 and embC-embA intergenic region in ethambutol-resistant clinical Mycobacterium tuberculosis isolates in Kuwait

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    Ahmad Suhail

    2009-01-01

    Full Text Available Abstract Background Ethambutol (EMB is a first-line drug for the treatment of tuberculosis (TB. Resistance to EMB in Mycobacterium tuberculosis isolates is mediated by mutations in several genes involved in arabinan synthesis notably three emb (arabinosyl transferase and iniA (isoniazid-inducible genes. Most epidemiologically unrelated EMB-resistant M. tuberculosis strains contain mutations at embB codons 306, 406 and 497, embC-embA intergenic region (IGR and iniA codon 501 (iniA501. Objective To develop a more comprehensive molecular screen for EMB-resistance detectioamong epidemiologically unrelated EMB-resistant M. tuberculosis strains previously analyzed for embB codon 306, 406 and 497 mutations by including analysis of mutations at iniA501 and in embC-embA IGR. Methods Fifty consecutive and phenotypically documented EMB-resistant and 25 pansusceptible M. tuberculosis strains isolated from 75 different TB patients over a four-year period in Kuwait were analyzed. Mutations at iniA501 were detected by PCR amplification followed by restriction fragment length polymorphism (RFLP patterns generated with Hpy 99 I. Direct DNA sequencing was used to confirm RFLP results and for detecting mutations in embC-embA IGR. Results Nearly same number of EMB-resistant M. tuberculosis strains were resistant to EMB alone and EMB together with additional resistance to rifampicin and isoniazid (9 of 50, 18% and 11 of 50, 22%, respectively. All the 25 pansusceptible strains contained wild-type sequences at iniA501 and in embC-embA IGR. The analysis of 50 EMB-resistant M. tuberculosis isolates showed that only one strain contained a mutated iniA501 while no mutation was detected in embC-embA IGR in any of the isolate. Conclusion Analysis of iniA501 and embC-embA IGR in epidemiologically unrelated EMB-resistant M. tuberculosis isolates in Kuwait indicate that mutations at these locations occur very infrequently and their inclusion for the development of a

  7. A Systematic Follow-Up of Mycobacterium tuberculosis Drug-Resistance and Associated Genotypic Lineages in the French Departments of the Americas over a Seventeen-Year Period

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    Julie Millet

    2014-01-01

    Full Text Available The population of the French Departments of the Americas (FDA is highly influenced by the intense migratory flows with mainland France and surrounding countries of the Caribbean and Latin America, some of which have high incidence rates of tuberculosis (Haiti: 230/100,000; Guyana: 111/100,000; and Suriname: 145/100,000 and drug resistance. Since the development of drug resistance to conventional antituberculous drugs has a major impact on the treatment success of tuberculosis, we therefore decided to review carefully Mycobacterium tuberculosis drug resistance and associated genotypic lineages in the FDA over a seventeen-year period (January 1995–December 2011. A total of 1239 cases were studied, including 153 drug-resistant and 26 multidrug-resistant- (MDR- TB cases, representing 12.3% and 2.1% of the TB cases in our study setting. A significantly higher proportion of M. tuberculosis isolates among relapse cases showed drug resistance to isoniazid (22.5%, P=0.002, rifampicin (20.0%, P<0.001, or both (MDR-TB, 17.5%; P<0.001. Determination of spoligotyping based phylogenetic clades showed that among the five major lineages observed—T family (30.1%; Latin-American and Mediterranean (LAM, 23.7%; Haarlem (H, 22.2%; East-African Indian (EAI, 7.2%; and X family (6.5%—two lineages, X and LAM, were overrepresented in drug-resistant and MDR-TB cases, respectively. Finally, 19 predominant spoligotypes were identified for the 1239 isolates of M. tuberculosis in our study among which 4 were significantly associated with drug resistance corresponding to SIT20/LAM1, SIT64/LAM6, SIT45/H1, and SIT46/undefined lineage.

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

    Science.gov (United States)

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

  9. Osteoarticular manifestations of Mycobacterium tuberculosis infection.

    Science.gov (United States)

    Zychowicz, Michael E

    2010-01-01

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

  10. Complex multifractal nature in Mycobacterium tuberculosis genome

    Science.gov (United States)

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

    2017-04-01

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

  11. Peritoneal tuberculosis due to Mycobacterium caprae

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    T. Nebreda

    2016-01-01

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

  12. Low rate of fluoroquinolone resistance in Mycobacterium tuberculosis isolates from northern Tanzania

    NARCIS (Netherlands)

    Boogaard, J. van den; Semvua, H.H.; Ingen, J. van; Mwaigwisya, S.; Laan, T. van der; Soolingen, D. van; Kibiki, G.S.; Boeree, M.J.; Aarnoutse, R.E.

    2011-01-01

    OBJECTIVES: Fluoroquinolones are used in second-line treatment of tuberculosis (TB) and have a potential role in shortening TB treatment duration. The wide use of fluoroquinolones in the treatment of other infections, including respiratory tract infections in patients with (undiagnosed) active TB,

  13. Low rate of fluoroquinolone resistance in Mycobacterium tuberculosis isolates from northern Tanzania

    NARCIS (Netherlands)

    Boogaard, J. van den; Semvua, H.H.; Ingen, J. van; Mwaigwisya, S.; Laan, T. van der; Soolingen, D. van; Kibiki, G.S.; Boeree, M.J.; Aarnoutse, R.E.

    2011-01-01

    OBJECTIVES: Fluoroquinolones are used in second-line treatment of tuberculosis (TB) and have a potential role in shortening TB treatment duration. The wide use of fluoroquinolones in the treatment of other infections, including respiratory tract infections in patients with (undiagnosed) active TB, c

  14. 抗痨颗粒对耐多药结核分枝杆菌蛋白质组学的影响%Effect of Anti-tuberculosis Particles on Proteomics of Drug-resistant Mycobacterium Tuberculosis

    Institute of Scientific and Technical Information of China (English)

    李建国; 刘湘花; 汤红琴; 董修兵; 李宁宁

    2012-01-01

    目的:应用蛋白质组学的双向电泳技术,比较分析抗痨颗粒提取物作用前后耐多药结核分枝杆菌的全菌蛋白表达差异,揭示药物作用机制.方法:临床耐多药结核分枝杆菌接种在7H9添加OADC液体培养基中37℃培养6~8d,细菌密度为1×108/mL~2 × 108/mL.抗痨颗粒浸提物的最低抑菌浓度(MIC)为0.638 8 g·L-1.加到液体培养基中,作用20 h,终浓度为相当于生药0.255 6 g·mL-1.局部内环境与外部环境完全隔离下,采用双向凝胶电泳分离全菌总蛋白,得到差异表达的蛋白质点进行分析比对.结果:耐多药结核分枝杆菌用药前后,发现了8个差异斑点,确定其中2个蛋白质为:硫代硫酸硫转移酶( thiosulfate sulfurtransferase)和假定蛋白Rv0634A.结论:揭示了抗痨颗粒对耐多药结核分枝杆菌的翻译,结构组成,氨基酸代谢,氰化物和H2S的解毒以及硫和铁转移等各方面均有一定影响,从而破坏结核分枝杆菌的生活能力,从而达到抑制结核分枝杆菌的目的.%Objective; To reveal the mechanism of drug action to compare and analyze of the effect of anti-tuberculosis particles extract on multi-drug resistant-mycobacterium tuberculosis proteomics to reveal the mechanism of drug action. Method; The clinical mycobacterium tuberculosis with multidrug resistance was added in Middlebrook 7H9 OADC liquid medium at 37 ℃ for 6-8 d, when the bacterial density researched 1 × l08/mL-2 × l08/mL, the minimum inhibitory concentration ( MIC) of anti-tuberculosis particulate extracts was 0. 638 8 g · L-1 , the final concentration was equivalent to 0. 255 6 g · mL . Local environment and external environment was completely isolated, two-dimensional gel electrophoresis separation of whole cell total protein was used to analyze differentially expressed protein spots. Result; Before and after treatment by anti-tuberculosis particles extract, multidrug-resistant mycobacterium tuberculosis showed eight

  15. GidB mutation as a phylogenetic marker for Q1 cluster Mycobacterium tuberculosis isolates and intermediate-level streptomycin resistance determinant in Lisbon, Portugal.

    Science.gov (United States)

    Perdigão, J; Macedo, R; Machado, D; Silva, C; Jordão, L; Couto, I; Viveiros, M; Portugal, I

    2014-05-01

    Development of streptomycin resistance in Mycobacterium tuberculosis is usually associated with mutations in rpsL and rrs genes, although up to 50% of clinical streptomycin-resistant isolates may present no mutation in either of these genes. In the present report we investigate the role of gidB gene mutations in streptomycin resistance. We have analyzed 52 streptomycin-resistant and 30 streptomycin-susceptible Mycobacterium tuberculosis clinical isolates by sequencing and endonuclease analysis of the gidB and rpsL genes. All clinical isolates were genotyped by 12-loci MIRU-VNTR. The gidB gene of 18 streptomycin-resistant isolates was sequenced and four missense mutations were found: F12L (1/18), L16R (18/18), A80P (4/18) and S100F (18/18). The remaining isolates were screened by endonuclease analysis for mutations A80P in the gidB gene and K43R in the rpsL gene. Overall, mutation A80P in the gidB gene was found in eight streptomycin-resistant isolates and 11 streptomycin-susceptible multidrug-resistant isolates. Also noteworthy, is the fact that gidB mutations were only present in isolates without rpsL and rrs mutations, all from genetic cluster Q1. Streptomycin quantitative drug susceptibility testing showed that isolates carrying the gidB A80P mutation were streptomycin intermediate-level resistant and that standard drug susceptibility testing yielded inconsistent results, probably due to borderline resistance. We conclude that gidB mutations may explain the high number of streptomycin-resistant strains with no mutation in rpsL or rrs. These mutations might occasionally confer low-level streptomycin resistance that will go undetected in standard susceptibility testing.

  16. Negligible risk of inducing resistance in Mycobacterium tuberculosis with single-dose rifampicin as post-exposure prophylaxis for leprosy.

    Science.gov (United States)

    Mieras, Liesbeth; Anthony, Richard; van Brakel, Wim; Bratschi, Martin W; van den Broek, Jacques; Cambau, Emmanuelle; Cavaliero, Arielle; Kasang, Christa; Perera, Geethal; Reichman, Lee; Richardus, Jan Hendrik; Saunderson, Paul; Steinmann, Peter; Yew, Wing Wai

    2016-06-08

    Post-exposure prophylaxis (PEP) for leprosy is administered as one single dose of rifampicin (SDR) to the contacts of newly diagnosed leprosy patients. SDR reduces the risk of developing leprosy among contacts by around 60 % in the first 2-3 years after receiving SDR. In countries where SDR is currently being implemented under routine programme conditions in defined areas, questions were raised by health authorities and professional bodies about the possible risk of inducing rifampicin resistance among the M. tuberculosis strains circulating in these areas. This issue has not been addressed in scientific literature to date. To produce an authoritative consensus statement about the risk that SDR would induce rifampicin-resistant tuberculosis, a meeting was convened with tuberculosis (TB) and leprosy experts. The experts carefully reviewed and discussed the available evidence regarding the mechanisms and risk factors for the development of (multi) drug-resistance in M. tuberculosis with a view to the special situation of the use of SDR as PEP for leprosy. They concluded that SDR given to contacts of leprosy patients, in the absence of symptoms of active TB, poses a negligible risk of generating resistance in M. tuberculosis in individuals and at the population level. Thus, the benefits of SDR prophylaxis in reducing the risk of developing leprosy in contacts of new leprosy patients far outweigh the risks of generating drug resistance in M. tuberculosis.

  17. The Genotype MTBDRplus ver. 2.0 test as a quick indicator of resistance to rifampicin and isoniazid in Mycobacterium tuberculosis strains

    Directory of Open Access Journals (Sweden)

    Salvatore Nisticò

    2013-08-01

    Full Text Available Tuberculosis is still a global emergency and a major public health problem, in some cases related to the appearance of strains of multi drug resistance (MDR and extensive drug resistance (XDR Mycobacterium tuberculosis complex.The correct determination of antibiotic sensitivity profiles is therefore crucial to carry out appropriate treatment aimed to decrease the infectivity of each patient and to reduce mortality. The poor adherence to treatment by the patient or the use of therapies based on a single drug, as a result of incorrect requirements, promote the development of drug-resistance. Have some time on the market of molecular diagnostic tests that allow, quickly and directly from biological sample to search for resistance genes some key drugs of anti-TB therapy (Rifampicin and Isoniazid. One of the tests in question is the Genotype MTBDRplus ver 2.0 which can reveal the presence of genes for resistance to Isoniazid (INH and Rifampin (RMP.The loci analyzed are those corresponding to the rpoB gene for rifampicin, katG and inhA for isoniazid. Our study is based on the analysis of 83 strains of tubercular Mycobacteria identified and isolated from patients with tuberculosis disease and subjected to the tests sensitivity, searching for mutations and phenotypic susceptibility testing for Rifampicin and Isoniazid.The comparison of the results has shown that the results obtained using the Genotype MTBDRplus ver 2.0 test, were similar to the results obtained by the traditional susceptibility testing.

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

    KAUST Repository

    Phelan, Jody

    2015-06-04

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

  19. Molecular typing of Mycobacterium tuberculosis isolates from Yaound é reveals RIF resistance markers, clonal relatedness and mutation patterns

    Directory of Open Access Journals (Sweden)

    Tedom

    2012-10-01

    Full Text Available Objective: To understand the spread of drug resistance and identifying diagnostic probes among the local tuberculosis (TB strains in order to design rational control tools for tuberculosis controls. Methods: TA cloning and sequencing were used to characterize mutation associated with RIF resistance in 69 bp region of the gene, rpoB. The analysis identified two regions of mutations but no unusual insertion and deletion. No mutation was observed in RIF sensitive strains. Results: We employed Random Amplified Polymorphic DNA (RAPD analysis for typing strains of M. tuberculosis to determine whether new strains were present among M. tuberculosis isolates circulating in Yaounde. Three groups (I to III of M. tuberculosis were identified among 93 isolates randomly selected. RAPD analysis provided a rapid and easy means of identifying polymorphism in M. tuberculosis isolates, and it was found to be a valuable alternative epidemiological tool. RAPD was used to select the new site of diagnostic by PCR. Also single nucleotide polymorphisms between M. tuberculosis and M. bovis were found, suggesting that RAPD can be a useful technique for distinguishing between species. Conclusions: Molecular typing is defined as the integration of conventional epidemiological approach to track specific strains of pathogens in order to understand the distribution of disease in populations.

  20. Molecular genetics of Mycobacterium tuberculosis resistant to aminoglycosides and cyclic peptide testing by MTBDRsl in Armenia

    Directory of Open Access Journals (Sweden)

    Hasmik Margaryan

    2016-01-01

    Conclusion: Isolates with rrs structural gene mutations were cross-resistant to streptomycin, KAN, CAP, and AMK. Detection of the A1401G mutation appeared to be 100% specific for the detection of resistance to KAN and AMK. Being the first assessment, these data establish the presence of phenotypic drug-resistant and extensively drug-resistant strains using molecular profiling and are helpful in understanding aminoglycoside resistance on a molecular level.

  1. Multidrug-resistant tuberculosis in Europe, 2010-2011

    NARCIS (Netherlands)

    Gunther, G.; Leth, F. van; Alexandru, S.; Altet, N.; Avsar, K.; Bang, D.; Barbuta, R.; Bothamley, G.; Ciobanu, A.; Crudu, V.; Davilovits, M.; Dedicoat, M.; Duarte, R.; Gualano, G.; Kunst, H.; Lange, W. de; Leimane, V.; Magis-Escurra Ibanez, C.; McLaughlin, A.M.; Muylle, I.; Polcova, V.; Pontali, E.; Popa, C; Rumetshofer, R.; Skrahina, A.; Solodovnikova, V.; Spinu, V.; Tiberi, S.; Viiklepp, P.; Lange, C.

    2015-01-01

    Drug-resistant Mycobacterium tuberculosis is challenging elimination of tuberculosis (TB). We evaluated risk factors for TB and levels of second-line drug resistance in M. tuberculosis in patients in Europe with multidrug-resistant (MDR) TB. A total of 380 patients with MDR TB and 376 patients with

  2. Multidrug-Resistant Tuberculosis in Europe, 2010-2011

    NARCIS (Netherlands)

    Guenther, Gunar; van Leth, Frank; Alexandru, Sofia; Altet, Neus; Avsar, Korkut; Bang, Didi; Barbuta, Raisa; Bothamley, Graham; Ciobanu, Ana; Crudu, Valeriu; Danilovits, Manfred; Dedicoat, Martin; Duarte, Raquel; Gualano, Gina; Kunst, Heinke; de Lange, Wiel; Leimane, Vaira; Magis-Escurra, Cecile; McLaughlin, Anne-Marie; Muylle, Inge; Polcova, Veronika; Pontalli, Emanuele; Popa, Christina; Rumetshofer, Rudolf; Skrahina, Alena; Solodovnikova, Varvara; Spinu, Victor; Tiberi, Simon; Viiklepp, Piret; Lange, Christoph

    2015-01-01

    Drug-resistant Mycobacterium tuberculosis is challenging elimination of tuberculosis (TB). We evaluated risk factors for TB and levels of second-line drug resistance in M. tuberculosis in patients in Europe with multidrug-resistant (MDR) TB. A total of 380 patients with MDR TB and 376 patients with

  3. Multidrug-resistant tuberculosis in Europe, 2010-2011

    DEFF Research Database (Denmark)

    Günther, Gunar; van Leth, Frank; Alexandru, Sofia

    2015-01-01

    Drug-resistant Mycobacterium tuberculosis is challenging elimination of tuberculosis (TB). We evaluated risk factors for TB and levels of second-line drug resistance in M. tuberculosis in patients in Europe with multidrug-resistant (MDR) TB. A total of 380 patients with MDR TB and 376 patients wi...

  4. Antitubercular Activity of Disulfiram, an Antialcoholism Drug, against Multidrug- and Extensively Drug-Resistant Mycobacterium tuberculosis Isolates

    Science.gov (United States)

    Horita, Yasuhiro; Yagi, Tetsuya; Ogawa, Kenji; Fujiwara, Nagatoshi; Inagaki, Emi; Kremer, Laurent; Sato, Yasuo; Kuroishi, Ryuji; Lee, YooSa; Makino, Toshiaki; Mizukami, Hajime; Hasegawa, Tomohiro; Yamamoto, Ryuji; Onozaki, Kikuo

    2012-01-01

    The antimycobacterial activities of disulfiram (DSF) and diethyldithiocarbamate (DDC) against multidrug- and extensively drug-resistant tuberculosis (MDR/XDR-TB) clinical isolates were evaluated in vitro. Both DSF and DDC exhibited potent antitubercular activities against 42 clinical isolates of M. tuberculosis, including MDR/XDR-TB strains. Moreover, DSF showed remarkable bactericidal activity ex vivo and in vivo. Therefore, DSF might be a drug repurposed for the treatment of MDR/XDR-TB. PMID:22615274

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

    OpenAIRE

    2012-01-01

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

  6. Drug susceptibility of Mycobacterium tuberculosis Beijing genotype and association with MDR TB.

    Science.gov (United States)

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

    2012-04-01

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

  7. Population structure and circulating genotypes of drug-sensitive and drug-resistant Mycobacterium tuberculosis clinical isolates in São Paulo state, Brazil

    Science.gov (United States)

    Martins, Maria Conceição; Saraiva Giampaglia, Carmen M.; Oliveira, Rosângela S.; Simonsen, Vera; Latrilha, Fábio Oliveira; Moniz, Letícia Lisboa; Couvin, David; Rastogi, Nalin; Ferrazoli, Lucilaine

    2013-01-01

    São Paulo is the most populous Brazilian state and reports the largest number of tuberculosis cases in the country annually (over 18,500). This study included 193 isolates obtained during the 2nd Nationwide Survey on Mycobacterium tuberculosis Drug Resistance that was conducted in São Paulo state and 547 isolates from a laboratory based study of drug resistance that were analyzed by the Mycobacteria Reference Laboratory at the Institute Adolfo Lutz. Both studies were conducted from 2006 to 2008 and sought to determine the genetic diversity and pattern of drug resistance of M. tuberculosis isolates (MTC) circulating in São Paulo. The patterns obtained from the spoligotyping analysis demonstrated that 51/740 (6.9%) of the isolates corresponded to orphan patterns and that 689 (93.1%) of the isolates distributed into 144 shared types, including 119 that matched a preexisting shared type in the SITVIT2 database and 25 that were new isolates. A total of 77/144 patterns corresponded to unique isolates, while the remaining 67 corresponded to clustered patterns (n = 612 isolates clustered into groups of 2–84 isolates each). The evolutionarily ancient PGG1 lineages (Beijing, CAS1-DEL, EAI3-IND, and PINI2) were rarely detected in São Paulo and comprised only 13/740, or 1.76%, of the total isolates; all of the remaining 727/740, or 98.24%, of the MTC isolates from São Paulo state were from the recent PGG2/3 evolutionary isolates belonging to the LAM, T, S, X, and Haarlem lineages, i.e., the Euro-American group. This study provides the first overview of circulating genotypes of M. tuberculosis in São Paulo state and demonstrates that the clustered shared types containing seven or more M. tuberculosis isolates that are spread in São Paulo state included both resistant and susceptible isolates. PMID:23201043

  8. Population structure and circulating genotypes of drug-sensitive and drug-resistant Mycobacterium tuberculosis clinical isolates in São Paulo state, Brazil.

    Science.gov (United States)

    Martins, Maria Conceição; Giampaglia, Carmen M Saraiva; Oliveira, Rosângela S; Simonsen, Vera; Latrilha, Fábio Oliveira; Moniz, Letícia Lisboa; Couvin, David; Rastogi, Nalin; Ferrazoli, Lucilaine

    2013-03-01

    São Paulo is the most populous Brazilian state and reports the largest number of tuberculosis cases in the country annually (over 18,500). This study included 193 isolates obtained during the 2nd Nationwide Survey on Mycobacterium tuberculosis Drug Resistance that was conducted in São Paulo state and 547 isolates from a laboratory based study of drug resistance that were analyzed by the Mycobacteria Reference Laboratory at the Institute Adolfo Lutz. Both studies were conducted from 2006 to 2008 and sought to determine the genetic diversity and pattern of drug resistance of M. tuberculosis isolates (MTC) circulating in São Paulo. The patterns obtained from the spoligotyping analysis demonstrated that 51/740 (6.9%) of the isolates corresponded to orphan patterns and that 689 (93.1%) of the isolates distributed into 144 shared types, including 119 that matched a preexisting shared type in the SITVIT2 database and 25 that were new isolates. A total of 77/144 patterns corresponded to unique isolates, while the remaining 67 corresponded to clustered patterns (n=612 isolates clustered into groups of 2-84 isolates each). The evolutionarily ancient PGG1 lineages (Beijing, CAS1-DEL, EAI3-IND, and PINI2) were rarely detected in São Paulo and comprised only 13/740, or 1.76%, of the total isolates; all of the remaining 727/740, or 98.24%, of the MTC isolates from São Paulo state were from the recent PGG2/3 evolutionary isolates belonging to the LAM, T, S, X, and Haarlem lineages, i.e., the Euro-American group. This study provides the first overview of circulating genotypes of M. tuberculosis in São Paulo state and demonstrates that the clustered shared types containing seven or more M. tuberculosis isolates that are spread in São Paulo state included both resistant and susceptible isolates.

  9. Overexpression of the Chromosomally Encoded Aminoglycoside Acetyltransferase eis Confers Kanamycin Resistance in Mycobacterium tuberculosis

    National Research Council Canada - National Science Library

    M. Analise Zaunbrecher; R. David Sikes; Beverly Metchock; Thomas M. Shinnick; James E. Posey

    2009-01-01

    .... The aminoglycosides kanamycin and amikacin are important bactericidal drugs used to treat MDR TB, and resistance to one or both of these drugs is a defining characteristic of extensively drug-resistant TB...

  10. Primary multidrug resistant tuberculosis

    Directory of Open Access Journals (Sweden)

    Sarkar Supriya

    2007-01-01

    Full Text Available A 37-year old man presented at our institution with back pain, low-grade fever and weight-loss. X-ray of chest (postero-anterior view showed multiple opacities with erosion of right 2nd and left 6th ribs. CT-scan of thorax and CT-guided FNAC con-firmed the diagnosis of tuberculosis of ribs. Even after 5-months of treatment with four first line drugs, the patient developed a cold abscess at the back. Mycobacterial culture and drug sensitivity of material aspirated by Radiometric method from the cold abscess showed growth of Mycobacterium tuberculosis, and those bacilli were resistant to both isoniazide and rifampicin. The patient did not have anti-tubercu-lar medication in the past, and that established the diagnosis of primary multidrug resistant tuberculosis of ribs. Patient was treated successfully with 2nd line drugs at the cost of moderate degree of hearing loss. After one and half years of treatment X-ray of chest (PA view showed complete healing of rib erosions with new bone formation.

  11. 湖南长沙市结核分枝杆菌耐药情况分析%Drug resistance analysis on Mycobacterium tuberculosis in patients with pulmonary tuberculosis in Changsha area

    Institute of Scientific and Technical Information of China (English)

    潘建华; 罗丹; 石国民; 彭雪峰; 向延根

    2016-01-01

    Objective To investigate the current situation of Mycobacterium tuberculosis drug resistance in Changsha area. Methods A total of 6198 Mycobacterium tuberculosis strains were identified in Changsha Central Hospital from 2011 to 2015 and the sensitivity of Mycobacterium tuberculosis strains to 7 kinds of anti-tuberculosis drugs was performed. Results The top 5 of the drug resistance rates of 6198 strains of Mycobacterium tuberculosis complex group were INH(14. 18% ),RFP(13. 57% ),SM(12. 31% ),EMB(3. 87)and OFLX(2. 60). The o-verall rate of resistance to anti-TB drugs was 20. 56% . MDR rate was 9. 31%. PDR rate was 4. 52% and XDR rate was 0. 45% . The detection rate of drug resistant tuberculosis of male was higher than female in all age groups except the female under 20 years old in 2012. The resistance in retreated patients was higher than in initial patients. Con-clusion The prevalence of drug resistance of Mycobacterium tuberculosis in our hospital is lower than that of the fifth national tuberculosis epidemiological survey,but MDR rate is higher than the national level. Therefore,its drug re-sistance situation is still grim.%目的:了解长沙地区结核分枝杆菌耐药情况。方法分析本院2012年1月-2015年10月分离的6198株结核分枝杆菌对7种抗结核药物的耐药情况。结果6198株结核分枝杆菌复合群菌株耐药率顺位前5位依次为 INH(14.18%)、RFP(13.57%)、SM(12.31%)、EMB(3.87)、OFLX(2.60);总耐药率为20.56%,耐多药占9.31%,多耐药占4.52%,广泛耐药占0.45%;除2012年20岁以下年龄组女性耐药结核菌的检出率略高于男性外,其余年份各年龄组男性均高于女性;复治患者的耐药率(24.76%)高于初治患者(19.49%),两者差异有统计学意义(χ2=176.21,P <0.001);结论本院结核分枝杆菌耐药率总体低于全国第五次结核病流行病学调查结果,但耐多药率高于全国水平,耐药状况仍然严峻。

  12. Genotypic Detection of rpoB and katG Gene Mutations Associated with Rifampicin and Isoniazid Resistance in Mycobacterium Tuberculosis Isolates: A Local Scenario (Kelantan).

    Science.gov (United States)

    Ismail, Nurul-Ain; Ismail, Mohd Fazli; Noor, Siti Suraiya Md; Camalxaman, Siti Nazrina

    2016-01-01

    Drug resistant tuberculosis (DR-TB) remains a public health issue that is of major concern on a global scale. The characterisation of clinical isolates may provide key information regarding the underlying mechanisms of drug resistance, and helps to augment therapeutic options. This study aims to evaluate the frequency of gene mutations associated with Rifampicin (RIF) and Isoniazid (INH) resistance among nine clinical isolates. A total of nine drug resistant Mycobacterium tuberculosis clinical isolates were screened for genetic mutations in rpoB and katusing polymerase chain reaction (PCR) amplification and DNA sequencing. Genotypic analysis was performed to detect the mutations in the sequence of the target genes. Our findings reveal that 80% of the isolates possess mutations at codon 119 (His119Tyr) and 135 (Arg135Trp and Ser135Leu) within the rpoB gene; and 70% possess mutations in the katG gene at codon 238 with amino acid change (Leu238Arg). Findings from this study provide an overview of the current situation of RIF and INH resistance in a hospital Universiti Sains Malaysia (HUSM) located in Kelantan, Malaysia, which could facilitate molecular-based detection methods of drug-resistant strains. Further information regarding the molecular mechanisms involved in resistance in RR-/MDR-TB should be addressed in the near future.

  13. Inhibition of Mycobacterium tuberculosis Glutamine Synthetase as a Novel Antibiotic Strategy against Tuberculosis: Demonstration of Efficacy In Vivo

    OpenAIRE

    2003-01-01

    Tuberculosis remains one of humankind's greatest killers, and new therapeutic strategies are needed to combat the causative agent, Mycobacterium tuberculosis, which is rapidly developing resistance to conventional antibiotics. Using the highly demanding guinea pig model of pulmonary tuberculosis, we have investigated the feasibility of inhibiting M. tuberculosis glutamine synthetase (GS), an enzyme that plays a key role in both nitrogen metabolism and cell wall biosynthesis, as a novel antibi...

  14. Dormancy models for Mycobacterium tuberculosis: A minireview

    Directory of Open Access Journals (Sweden)

    Amani M. Alnimr

    2015-09-01

    Full Text Available 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.

  15. Comparative Evaluation of Sloppy Molecular Beacon and Dual-Labeled Probe Melting Temperature Assays to Identify Mutations in Mycobacterium tuberculosis Resulting in Rifampin, Fluoroquinolone and Aminoglycoside Resistance

    Science.gov (United States)

    Lee, Jong Seok; Via, Laura E.; Barry, Clifton E.; Alland, David; Chakravorty, Soumitesh

    2015-01-01

    Several molecular assays to detect resistance to Rifampin, the Fluoroquinolones, and Aminoglycosides in Mycobacterium tuberculosis (M. tuberculosis) have been recently described. A systematic approach for comparing these assays in the laboratory is needed in order to determine the relative advantage of each assay and to decide which ones should be advanced to evaluation. We performed an analytic comparison of a Sloppy Molecular Beacon (SMB) melting temperature (Tm) assay and a Dual labeled probe (DLP) Tm assay. Both assays targeted the M. tuberculosis rpoB, gyrA, rrs genes and the eis promoter region. The sensitivity and specificity to detect mutations, analytic limit of detection (LOD) and the detection of heteroresistance were tested using a panel of 56 clinical DNA samples from drug resistant M. tuberculosis strains. Both SMB and DLP assays detected 29/29 (100%) samples with rpoB RRDR mutations and 3/3 (100%) samples with eis promoter mutations correctly. The SMB assay detected all 17/17 gyrA mutants and 22/22 rrs mutants, while the DLP assay detected 16/17 (94%) gyrA mutants and 12/22 (55%) rrs mutants. Both assays showed comparable LODs for detecting rpoB and eis mutations; however, the SMB assay LODs were at least two logs better for detecting wild type and mutants in gyrA and rrs targets. The SMB assay was also moderately better at detecting heteroresistance. In summary, both assays appeared to be promising methods to detect drug resistance associated mutations in M. tuberculosis; however, the relative advantage of each assay varied under each test condition. PMID:25938476

  16. Comparative Evaluation of Sloppy Molecular Beacon and Dual-Labeled Probe Melting Temperature Assays to Identify Mutations in Mycobacterium tuberculosis Resulting in Rifampin, Fluoroquinolone and Aminoglycoside Resistance.

    Directory of Open Access Journals (Sweden)

    Sandy S Roh

    Full Text Available Several molecular assays to detect resistance to Rifampin, the Fluoroquinolones, and Aminoglycosides in Mycobacterium tuberculosis (M. tuberculosis have been recently described. A systematic approach for comparing these assays in the laboratory is needed in order to determine the relative advantage of each assay and to decide which ones should be advanced to evaluation. We performed an analytic comparison of a Sloppy Molecular Beacon (SMB melting temperature (Tm assay and a Dual labeled probe (DLP Tm assay. Both assays targeted the M. tuberculosis rpoB, gyrA, rrs genes and the eis promoter region. The sensitivity and specificity to detect mutations, analytic limit of detection (LOD and the detection of heteroresistance were tested using a panel of 56 clinical DNA samples from drug resistant M. tuberculosis strains. Both SMB and DLP assays detected 29/29 (100% samples with rpoB RRDR mutations and 3/3 (100% samples with eis promoter mutations correctly. The SMB assay detected all 17/17 gyrA mutants and 22/22 rrs mutants, while the DLP assay detected 16/17 (94% gyrA mutants and 12/22 (55% rrs mutants. Both assays showed comparable LODs for detecting rpoB and eis mutations; however, the SMB assay LODs were at least two logs better for detecting wild type and mutants in gyrA and rrs targets. The SMB assay was also moderately better at detecting heteroresistance. In summary, both assays appeared to be promising methods to detect drug resistance associated mutations in M. tuberculosis; however, the relative advantage of each assay varied under each test condition.

  17. Importance of the Genetic Diversity within the Mycobacterium tuberculosis Complex for the Development of Novel Antibiotics and Diagnostic Tests of Drug Resistance

    KAUST Repository

    Koser, C. U.

    2012-09-24

    Despite being genetically monomorphic, the limited genetic diversity within the Mycobacterium tuberculosis complex (MTBC) has practical consequences for molecular methods for drug susceptibility testing and for the use of current antibiotics and those in clinical trials. It renders some representatives of MTBC intrinsically resistant against one or multiple antibiotics and affects the spectrum and consequences of resistance mutations selected for during treatment. Moreover, neutral or silent changes within genes responsible for drug resistance can cause false-positive results with hybridization-based assays, which have been recently introduced to replace slower phenotypic methods. We discuss the consequences of these findings and propose concrete steps to rigorously assess the genetic diversity of MTBC to support ongoing clinical trials.

  18. A study on demographic characteristics of drug resistant Mycobacterium tuberculosis isolates in Belarus

    Directory of Open Access Journals (Sweden)

    L Surkova

    2012-01-01

    Conclusion: As Belarus is a high-burden MDR-TB country and treatment of drug-resistant TB is long and complicated, the findings of this study provided useful information to deliver effective community-based disease control measures and a proposed plane for the effective management of drug-resistant TB at the national level.

  19. Whole genome analysis of an MDR Beijing/W strain of Mycobacterium tuberculosis with large genomic deletions associated with resistance to isoniazid.

    Science.gov (United States)

    Zhang, Qiufen; Wan, Baoshan; Zhou, Aiping; Ni, Jinjing; Xu, Zhihong; Li, Shuxian; Tao, Jing; Yao, YuFeng

    2016-05-15

    Mycobacterium tuberculosis (M.tb) is one of the most prevalent bacterial pathogens in the world. With geographical wide spread and hypervirulence, Beijing/W family is the most successful M.tb lineage. China is a country of high tuberculosis (TB) and high multiple drug-resistant TB (MDR-TB) burden, and the Beijing/W family strains take the largest share of MDR strains. To study the genetic basis of Beijing/W family strains' virulence and drug resistance, we performed the whole genome sequencing of M.tb strain W146, a clinical Beijing/W genotype MDR isolated from Wuxi, Jiangsu province, China. Compared with genome sequence of M.tb strain H37Rv, we found that strain W146 lacks three large fragments and the missing of furA-katG operon confers isoniazid resistance. Besides the missing of furA-katG operon, strain W146 harbored almost all known drug resistance-associated mutations. Comparison analysis of single nucleotide polymorphisms (SNPs) and indels between strain W146 and Beijing/W genotype strains and non-Beijing/W genotype strains revealed that strain W146 possessed some unique mutations, which may be related to drug resistance, transmission and pathogenicity. These findings will help to understand the large sequence polymorphisms (LSPs) and the transmission and drug resistance related genetic characteristics of the Beijing/W genotype of M.tb.

  20. IN VITRO EFFECT OF VITAMIN C ON THE LABORATORY ISOLATES OF MYCOBACTERIUM TUBERCULOSIS WITH KNOWN SENSITIVITY AND RESISTANCE TO THE FIRST LINE ANTI TUBERCULAR DRUGS: AN EXPERIMENTAL PILOT STUDY

    Directory of Open Access Journals (Sweden)

    Talaulikar Nikita.S , Dsouza Delia.B , Rodrigues Savio , Kulkarni MS

    2015-04-01

    Full Text Available Background and Objectives: Globally, 3.5% of new cases of Tuberculosis (TB and 20.5% of previously treated cases are estimated to have multidrug- resistant tuberculosis, the corresponding estimates for India are 2.2%, and 15% respectively. Progress has been made in research and development of new drugs for TB over the last decade, thus fuelling the need for more innovative options. Recent in-vitro studies that claim Vitamin C to have an inhibitory effect on Mycobacterium tuberculosis could possibly prove to be a major breakthrough in Medicine. Hence this experimental study was conducted on a pilot basis with the objective of studying the in -vitro effect of the active ingredient of vitamin C on the laboratory isolates of Mycobacterium tuberculosis that were known to be sensitive and resistant to the first line anti tubercular drugs (Isoniazid, Rifampicin, Pyrazinamide and Ethambutol and to compare the dose related response of both sensitive and resistant strains of Mycobacterium tuberculosis to varying concentrations of Vitamin C. Materials and Methods: Using a Completely Randomized Design, a total of 17 viable Mycobacterium tuberculosis strains, 10 of which were sensitive to all first line anti-TB drugs (Isoniazid, Rifampicin, Pyrazinamide and Ethambutol and seven strains resistant to all first line Anti-TB drugs were experimented upon. Proportion method was used to determine drug susceptibility of Mycobacterium tuberculosis to Ascorbic acid. Data is presented in a summary table. Results: With 1mM (millimole concentration of Ascorbic acid, growth of Mycobacterium tuberculosis was observed on both drug containing as well as control media, but with higher concentration of Ascorbic acid (10 mM and 100mM, no growth was observed on Ascorbic acid containing Lowenstein Jenson media. Conclusion: Although the findings of this pilot study add to the supportive evidence of an in- vitro susceptibility of Mycobacterium tuberculosis to Vitamin C, the authors

  1. Resistência a drogas em cepas de mycobacterium tuberculosis isoladas de amostras de escarro de pacientes ambulatoriais sintomáticos: Complexo de Manguinhos, Rio de Janeiro, Brasil Drug resistance in mycobacterium tuberculosis strains isolated from sputum samples from symptomatic outpatients: Complexo de Manguinhos, Rio de Janeiro, Brazil

    Directory of Open Access Journals (Sweden)

    Joycenea Matsuda Mendes

    2007-10-01

    Full Text Available Para descrever a resistência a drogas em cepas de Mycobacterium tuberculosis isoladas de amostras de escarro de 263 pacientes suspeitos de tuberculose moradores do Complexo de Manguinhos, Rio de Janeiro, Brasil, as culturas positivas entre outubro de 2000 e dezembro de 2002 foram submetidas a teste de sensibilidade para isoniazida, rifampicina, estreptomicina, etionamida e etambutol. Resistência a qualquer das drogas foi encontrada em 21,4% (16/75 dos pacientes diagnosticados com tuberculose. Destes, 50% (8/16 eram casos novos e 50% (8/16 eram casos com tratamento anterior. A tuberculose multirresistente foi encontrada em 10,6% (8/75 do total de pacientes, estando associada a tratamento anterior em 8% (6/75 deles. Nossos resultados podem ter sido subestimados, pois M. tuberculosis não pôde ser isolado em todas as amostras positivas para bacilos álcool-ácido resistentes. Contudo, eles pelo menos revelam parte do problema.This study aimed to assess drug resistance in Mycobacterium tuberculosis strains isolated from sputum samples. To that end, sputum samples were collected from 263 patients suspected of having tuberculosis. All subjects lived in the Complexo de Manguinhos, which is located in the city of Rio de Janeiro, Brazil. Cultures testing positive between October of 2000 and December of 2002 were tested to determine strain susceptibility to isoniazid, rifampicin, streptomycin, ETHionamide, and ETHambutol. Of the 75 patients diagnosed with tuberculosis, resistance to at least one of the drugs was found in 16 (21.4%. Of those 16 patients, 8 (50% were new cases, and 8 (50% had previously been treated. Multidrug-resistant tuberculosis was identified in 8 (10.6% of the 75 patients, being associated with previous treatment in 6 (8%. The incidence of multidrug-resistant tuberculosis might have been underestimated, since M. tuberculosis was not isolated from all of the samples testing positive for acid-fast bacilli. However, at least, our

  2. Ion Channel Blockers as Antimicrobial Agents, Efflux Inhibitors, and Enhancers of Macrophage Killing Activity against Drug Resistant Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Diana Machado

    Full Text Available Given the ability of M. tuberculosis to survive as an intracellular pathogen and its propensity to develop resistance to the existing antituberculosis drugs, its treatment requires new approaches. Here the antimycobacterial properties of verapamil, thioridazine, chlorpromazine, flupenthixol and haloperidol were investigated against a panel of drug resistant M. tuberculosis strains, both in vitro and on human-infected macrophages. These compounds are efflux inhibitors that share among them the characteristic of being ion channel blockers. In vitro, all compounds exhibited synergistic inhibitory activities when combined with isoniazid and rifampicin, and were able to inhibit active efflux, demonstrating their role as efflux inhibitors. Gene expression analysis showed that M. tuberculosis efflux genes were overexpressed in response to antibiotic exposure, in vitro and within macrophages, irrespective of their resistance pattern. These compounds displayed a rapid and high killing activity against M. tuberculosis, associated with a decrease in intracellular ATP levels demonstrating that the bactericidal action of the ion channel blockers against M. tuberculosis clinical strains is associated with their interference with energy metabolism. The compounds led to a decrease in the intracellular mycobacterial load by increasing phagosome acidification and activating lysosomal hydrolases. The results presented in this study enable us to propose the following mechanism of action for these compounds: a in the bacteria, the compounds generate a cascade of events involving the inhibition of the respiratory chain complexes and energy production for efflux activity. Indirectly, this reduce the resistance level to antituberculosis drugs potentiating their activity; b on the host cell, the treatment with the ion channel blockers increases phagosome acidification and induces the expression of phagosomal hydrolases, leading to bacterial growth restriction

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

    NARCIS (Netherlands)

    Huyen, M.N.; Buu, T.N.; Tiemersma, E.; Lan, N.T.; Dung, N.H.; Kremer, K.; Soolingen, D.V.; Cobelens, F.G.

    2013-01-01

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

  4. Tuberculosis-resistant transgenic cattle

    Science.gov (United States)

    Tuberculosis is a devastating disease that affects humans and many animal species. In humans, tuberculosis (TB) is mainly caused by Mycobacterium tuberculosis, while most cases in cattle are caused by Mycobacterium bovis. However, Mb can also cause, albeit rarely, human TB. In this issue, Wu et al. ...

  5. Systematic interpretation of molecular beacon polymerase chain reaction for identifying rpoB mutations in Mycobacterium tuberculosis isolates with mixed resistant and susceptible bacteria.

    Science.gov (United States)

    Gomez, Diana I; Fisher-Hoch, Susan P; Bordt, Andrea S; Quitugua, Teresa N; Robledo, Jaime; Alvarez, Nataly; Correa, Nidia; McCormick, Joseph B; Restrepo, Blanca I

    2010-05-01

    Detection of multidrug-resistant tuberculosis (MDR-TB), a frequent cause of treatment failure, takes 2 or more weeks to identify by culture. Rifampicin (RIF) resistance is a hallmark of MDR-TB, and detection of mutations in the rpoB gene of Mycobacterium tuberculosis using molecular beacon probes with real-time quantitative polymerase chain reaction (qPCR) is a novel approach that takes Visual and mathematical approaches were used to establish whether the qPCR cycle threshold of the experimental isolate was significantly higher (mutant) compared to a reference wild-type isolate. Visual classification of the beacon qPCR required reader training for strains with a mixture of RIF-susceptible and RIF-resistant bacteria. Only then had the visual interpretation by an experienced reader had 100% sensitivity and 94.6% specificity versus RIF resistance by culture phenotype and 98.1% sensitivity and 100% specificity versus mutations based on DNA sequence. The mathematical approach was 98% sensitive and 94.5% specific versus culture and 96.2% sensitive and 100% specific versus DNA sequence. Our findings indicate the mathematical approach has advantages over the visual reading, in that it uses a Microsoft Excel template to eliminate reader bias or inexperience, and allows objective interpretation from high-throughput analyses even in the presence of a mixture of RIF-resistant and RIF-susceptible isolates without the need for reader training.

  6. Drug-resistant tuberculosis: time for visionary political leadership

    OpenAIRE

    Abubakar, I; Zignol, M; Falzon, D; Raviglione, M.; Ditiu, L; Masham, S; Adetifa, I; Ford, N.; Cox, H.; Lawn, SD; Marais, BJ; McHugh, TD; Mwaba, P.; Bates, M; M. Lipman

    2013-01-01

    Two decades ago, WHO declared tuberculosis a global emergency, and invested in the highly cost-effective directly observed treatment short-course programme to control the epidemic. At that time, most strains of Mycobacterium tuberculosis were susceptible to first-line tuberculosis drugs, and drug resistance was not a major issue. However, in 2013, tuberculosis remains a major public health concern worldwide, with prevalence of multidrug-resistant (MDR) tuberculosis rising. WHO estimates rough...

  7. Critical and independent role for SOCS3 in either myeloid or T cells in resistance to Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Berit Carow

    Full Text Available Suppressor of cytokine signalling 3 (SOCS3 negatively regulates STAT3 activation in response to several cytokines such as those in the gp130-containing IL-6 receptor family. Thus, SOCS3 may play a major role in immune responses to pathogens. In the present study, the role of SOCS3 in M. tuberculosis infection was examined. All Socs3(fl/fl LysM cre, Socs3(fl/fl lck cre (with SOCS3-deficient myeloid and lymphoid cells, respectively and gp130(F/F mice, with a mutation in gp130 that impedes binding to SOCS3, showed increased susceptibility to infection with M. tuberculosis. SOCS3 binding to gp130 in myeloid cells conveyed resistance to M. tuberculosis infection via the regulation of IL-6/STAT3 signalling. SOCS3 was redundant for mycobacterial control by macrophages in vitro. Instead, SOCS3 expression in infected macrophages and DCs prevented the IL-6-mediated inhibition of TNF and IL-12 secretion and contributed to a timely CD4+ cell-dependent IFN-γ expression in vivo. In T cells, SOCS3 expression was essential for a gp130-independent control of infection with M. tuberculosis, but was neither required for the control of infection with attenuated M. bovis BCG nor for M. tuberculosis in BCG-vaccinated mice. Socs3(fl/fl lck cre mice showed an increased frequency of γδ+ T cells in different organs and an enhanced secretion of IL-17 by γδ+ T cells in response to infection. Socs3(fl/fl lck cre γδ+ T cells impaired the control of infection with M. tuberculosis. Thus, SOCS3 expression in either lymphoid or myeloid cells is essential for resistance against M. tuberculosis via discrete mechanisms.

  8. Critical and Independent Role for SOCS3 in Either Myeloid or T Cells in Resistance to Mycobacterium tuberculosis

    Science.gov (United States)

    Carow, Berit; Reuschl, Ann-Kathrin; Gavier-Widén, Dolores; Jenkins, Brendan J.; Ernst, Matthias; Yoshimura, Akihiko; Chambers, Benedict J.; Rottenberg, Martin E.

    2013-01-01

    Suppressor of cytokine signalling 3 (SOCS3) negatively regulates STAT3 activation in response to several cytokines such as those in the gp130-containing IL-6 receptor family. Thus, SOCS3 may play a major role in immune responses to pathogens. In the present study, the role of SOCS3 in M. tuberculosis infection was examined. All Socs3fl/fl LysM cre, Socs3fl/fl lck cre (with SOCS3-deficient myeloid and lymphoid cells, respectively) and gp130F/F mice, with a mutation in gp130 that impedes binding to SOCS3, showed increased susceptibility to infection with M. tuberculosis. SOCS3 binding to gp130 in myeloid cells conveyed resistance to M. tuberculosis infection via the regulation of IL-6/STAT3 signalling. SOCS3 was redundant for mycobacterial control by macrophages in vitro. Instead, SOCS3 expression in infected macrophages and DCs prevented the IL-6-mediated inhibition of TNF and IL-12 secretion and contributed to a timely CD4+ cell-dependent IFN-γ expression in vivo. In T cells, SOCS3 expression was essential for a gp130-independent control of infection with M. tuberculosis, but was neither required for the control of infection with attenuated M. bovis BCG nor for M. tuberculosis in BCG-vaccinated mice. Socs3fl/fl lck cre mice showed an increased frequency of γδ+ T cells in different organs and an enhanced secretion of IL-17 by γδ+ T cells in response to infection. Socs3fl/fl lck cre γδ+ T cells impaired the control of infection with M. tuberculosis. Thus, SOCS3 expression in either lymphoid or myeloid cells is essential for resistance against M. tuberculosis via discrete mechanisms. PMID:23853585

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

    Directory of Open Access Journals (Sweden)

    Muna Mohammed Buzayan

    2012-09-01

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

  10. Rifamycin inhibition of WT and Rif-resistant Mycobacterium tuberculosis and Escherichia coli RNA polymerases in vitro.

    Science.gov (United States)

    Gill, Sumandeep K; Garcia, George A

    2011-09-01

    Mycobacterium tuberculosis (MTB) infects over 9 million people globally and claims approximately 2 million lives annually. Rifampin (Rif) is one of the first-line anti-tuberculosis drugs that inhibits transcription by binding to the β subunit (encoded by the rpoB gene) of the prokaryotic RNA polymerase (RNAP). A highly conserved 81 base pair core region among the β subunit of prokaryotes harbors most of the point mutations leading to rifamycin-resistant (RifR) mutations, where the majority of the clinically relevant MTB RifR mutations result from amino acid substitutions of one of the following three amino acids: βAsp435, βHis445, and βSer450 (MTB numbering). In this study, to determine the direct effect of rifamycins on the MTB RNAP, co-overexpression vectors were constructed to co-express the core subunits of wild-type and RifR mutants of MTB RNAP. The three aforementioned amino acids were each mutated to the most prevalent substitution found in the MTB clinical isolates (Asp435Val, His445Tyr, Ser450Leu) in the rpoB gene via site-directed mutagenesis. After purification via two-step column chromatography, the in vitro activity of the wild-type and RifR mutant MTB RNAPs was assessed via rolling circle transcription assay. The apparent IC(50) values for three key rifamycins (rifampin (Rif), rifabutin (Rbn), and rifaximin (Rfx)) were determined and these results indicate that the mutant RNAPs demonstrate approximately 10(3)-fold or greater loss of affinities for rifamycins relative to wild-type MTB RNAP. Along with the MTB RNAPs, rifamycin inhibition of the Escherichia coli RNAP counterparts was also assessed. Previously, it has been reported that Gram-positive bacteria (particularly mycobacteria) are more sensitive to rifamycins than Gram-negative bacteria. Under our experimental conditions, the rifamycin IC(50)s for wild-type and RifR mutants of MTB and E. coli RNAPs (wild-type and corresponding mutants) were very similar; therefore, the difference in

  11. Thr202Ala in thyA Is a Marker for the Latin American Mediterranean Lineage of the Mycobacterium tuberculosis Complex Rather than Para-Aminosalicylic Acid Resistance

    KAUST Repository

    Feuerriegel, S.

    2010-08-30

    Single nucleotide polymorphisms (SNPs) involved in the development of resistance represent powerful markers for the rapid detection of first- and second-line resistance in clinical Mycobacterium tuberculosis complex (MTBC) isolates. However, the association between particular mutations and phenotypic resistance is not always clear-cut, and phylogenetic SNPs have been misclassified as resistance markers in the past. In the present study, we investigated the utility of a specific polymorphism in thyA (Thr202Ala) as a marker for resistance to para-aminosalicyclic acid (PAS). Sixty-three PAS-susceptible MTBC strains comprising all major phylogenetic lineages, reference strain H37Rv, and 135 multidrug-resistant (MDR) strains from Germany (comprising 8 PAS-resistant isolates) were investigated for the presence of Thr202Ala. In both strain collections, the Thr202Ala SNP was found exclusively in strains of the Latin American Mediterranean (LAM) lineage irrespective of PAS resistance. Furthermore, PAS MICs (0.5 mg/liter) for selected LAM strains (all containing the SNP) and non-LAM strains (not containing the SNP), as well as the results of growth curve analyses performed in liquid 7H9 medium in the presence of increasing PAS concentrations (0 to 2.0 mg/liter), were identical. In conclusion, our data demonstrate that the Thr202Ala polymorphism in thyA is not a valid marker for PAS resistance but, instead, represents a phylogenetic marker for the LAM lineage of the M. tuberculosis complex. These findings challenge some of the previous understanding of PAS resistance and, as a consequence, warrant further in-depth investigations of the genetic variation in PAS-resistant clinical isolates and spontaneous mutants.

  12. Temperature-mediated heteroduplex analysis for the detection of drug-resistant gene mutations in clinical isolates of Mycobacterium tuberculosis by denaturing HPLC, SURVEYOR nuclease.

    Science.gov (United States)

    Shi, Ruiru; Otomo, Koji; Yamada, Hiroyuki; Tatsumi, Taiga; Sugawara, Isamu

    2006-01-01

    Denaturing high-performance liquid chromatography (DHPLC) is a relatively new technique, which utilizes heteroduplex formation between wild-type and mutated DNA strands to identify point mutations. Heteroduplex molecules are separated from homoduplex molecules by ion-pair, reverse-phase liquid chromatography on a special column matrix with partial heat denaturation of the DNA strands. In order to investigate the application of this method for point mutation detection in drug-resistant genes of Mycobacterium tuberculosis, katG, rpoB, embB, gyrA, pncA and rpsL genes, which are responsible for isoniazid, rifampicin, ethambutol, fluoroquinolone, pyrazinamide and streptomycin resistance, respectively, were detected by temperature-mediated DHPLC in 10 multidrug-resistant and 10 drug-susceptible clinical isolates. The DHPLC data were compared with those from a conventional MIC test. The results show that DHPLC is cost-effective with high capacity and accuracy, and is potentially useful for genotypic screening for mutations associated with anti-tuberculosis drug resistance.

  13. Diterpene production in Mycobacterium tuberculosis

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  15. Chemical engineering of Mycobacterium tuberculosis dodecin hybrids.

    Science.gov (United States)

    Vinzenz, Xenia; Grosse, Wolfgang; Linne, Uwe; Meissner, Britta; Essen, Lars-Oliver

    2011-10-21

    The suitability for chemical engineering of the highly symmetrical Mycobacterium tuberculosis dodecin was investigated, its inner cavity providing a large compartment shields introduced compounds from bulk solvent. Hybrids were obtained by S-alkylation of cysteine mutants and characterized by spectroscopic methods, including the crystal structures of wild type and biohybrid dodecins.

  16. [Mycobacterium tuberculosis infection following organ transplantation].

    Science.gov (United States)

    Haas, Charles; Le Jeunne, Claire

    2006-11-01

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

  17. Detecting Mutations in the Mycobacterium tuberculosis Pyrazinamidase Gene pncA to Improve Infection Control and Decrease Drug Resistance Rates in Human Immunodeficiency Virus Coinfection

    Science.gov (United States)

    Dudley, Matthew Z.; Sheen, Patricia; Gilman, Robert H.; Ticona, Eduardo; Friedland, Jon S.; Kirwan, Daniela E.; Caviedes, Luz; Rodriguez, Richard; Cabrera, Lilia Z.; Coronel, Jorge; Grandjean, Louis; Moore, David A. J.; Evans, Carlton A.; Huaroto, Luz; Chávez-Pérez, Víctor; Zimic, Mirko

    2016-01-01

    Hospital infection control measures are crucial to tuberculosis (TB) control strategies within settings caring for human immunodeficiency virus (HIV)–positive patients, as these patients are at heightened risk of developing TB. Pyrazinamide (PZA) is a potent drug that effectively sterilizes persistent Mycobacterium tuberculosis bacilli. However, PZA resistance associated with mutations in the nicotinamidase/pyrazinamidase coding gene, pncA, is increasing. A total of 794 patient isolates obtained from four sites in Lima, Peru, underwent spoligotyping and drug resistance testing. In one of these sites, the HIV unit of Hospital Dos de Mayo (HDM), an isolation ward for HIV/TB coinfected patients opened during the study as an infection control intervention: circulating genotypes and drug resistance pre- and postintervention were compared. All other sites cared for HIV-negative outpatients: genotypes and drug resistance rates from these sites were compared with those from HDM. HDM patients showed high concordance between multidrug resistance, PZA resistance according to the Wayne method, the two most common genotypes (spoligotype international type [SIT] 42 of the Latino American-Mediterranean (LAM)-9 clade and SIT 53 of the T1 clade), and the two most common pncA mutations (G145A and A403C). These associations were absent among community isolates. The infection control intervention was associated with 58–92% reductions in TB caused by SIT 42 or SIT 53 genotypes (odds ratio [OR] = 0.420, P = 0.003); multidrug-resistant TB (OR = 0.349, P < 0.001); and PZA-resistant TB (OR = 0.076, P < 0.001). In conclusion, pncA mutation typing, with resistance testing and spoligotyping, was useful in identifying a nosocomial TB outbreak and demonstrating its resolution after implementation of infection control measures. PMID:27928075

  18. Population-based resistance of Mycobacterium tuberculosis isolates to pyrazinamide and fluoroquinolones: results from a multicountry surveillance project.

    Science.gov (United States)

    Zignol, Matteo; Dean, Anna S; Alikhanova, Natavan; Andres, Sönke; Cabibbe, Andrea Maurizio; Cirillo, Daniela Maria; Dadu, Andrei; Dreyer, Andries; Driesen, Michèle; Gilpin, Christopher; Hasan, Rumina; Hasan, Zahra; Hoffner, Sven; Husain, Ashaque; Hussain, Alamdar; Ismail, Nazir; Kamal, Mostofa; Mansjö, Mikael; Mvusi, Lindiwe; Niemann, Stefan; Omar, Shaheed V; Qadeer, Ejaz; Rigouts, Leen; Ruesch-Gerdes, Sabine; Schito, Marco; Seyfaddinova, Mehriban; Skrahina, Alena; Tahseen, Sabira; Wells, William A; Mukadi, Ya Diul; Kimerling, Michael; Floyd, Katherine; Weyer, Karin; Raviglione, Mario C

    2016-10-01

    Pyrazinamide and fluoroquinolones are essential antituberculosis drugs in new rifampicin-sparing regimens. However, little information about the extent of resistance to these drugs at the population level is available. In a molecular epidemiology analysis, we used population-based surveys from Azerbaijan, Bangladesh, Belarus, Pakistan, and South Africa to investigate resistance to pyrazinamide and fluoroquinolones among patients with tuberculosis. Resistance to pyrazinamide was assessed by gene sequencing with the detection of resistance-conferring mutations in the pncA gene, and susceptibility testing to fluoroquinolones was conducted using the MGIT system. Pyrazinamide resistance was assessed in 4972 patients. Levels of resistance varied substantially in the surveyed settings (3·0-42·1%). In all settings, pyrazinamide resistance was significantly associated with rifampicin resistance. Among 5015 patients who underwent susceptibility testing to fluoroquinolones, proportions of resistance ranged from 1·0-16·6% for ofloxacin, to 0·5-12·4% for levofloxacin, and 0·9-14·6% for moxifloxacin when tested at 0·5 μg/mL. High levels of ofloxacin resistance were detected in Pakistan. Resistance to moxifloxacin and gatifloxacin when tested at 2 μg/mL was low in all countries. Although pyrazinamide resistance was significantly associated with rifampicin resistance, this drug may still be effective in 19-63% of patients with rifampicin-resistant tuberculosis. Even though the high level of resistance to ofloxacin found in Pakistan is worrisome because it might be the expression of extensive and unregulated use of fluoroquinolones in some parts of Asia, the negligible levels of resistance to fourth-generation fluoroquinolones documented in all survey sites is an encouraging finding. Rational use of this class of antibiotics should therefore be ensured to preserve its effectiveness. Bill & Melinda Gates Foundation, United States Agency for International Development

  19. Direct Application of the INNO-LiPA Rif.TB Line-Probe Assay for Rapid Identification of Mycobacterium tuberculosis Complex Strains and Detection of Rifampin Resistance in 360 Smear-Positive Respiratory Specimens from an Area of High Incidence of Multidrug-Resistant Tuberculosis

    Science.gov (United States)

    Viveiros, Miguel; Leandro, Clara; Rodrigues, Liliana; Almeida, Josefina; Bettencourt, Rosário; Couto, Isabel; Carrilho, Lurdes; Diogo, José; Fonseca, Ana; Lito, Luís; Lopes, João; Pacheco, Teresa; Pessanha, Mariana; Quirim, Judite; Sancho, Luísa; Salfinger, Max; Amaral, Leonard

    2005-01-01

    The INNO-LiPA Rif.TB assay for the identification of Mycobacterium tuberculosis complex strains and the detection of rifampin (RIF) resistance has been evaluated with 360 smear-positive respiratory specimens from an area of high incidence of multidrug-resistant tuberculosis (MDR-TB). The sensitivity when compared to conventional identification/culture methods was 82.2%, and the specificity was 66.7%; the sensitivity and specificity were 100.0% and 96.9%, respectively, for the detection of RIF resistance. This assay has the potential to provide rapid information that is essential for the effective management of MDR-TB. PMID:16145166

  20. Perfil de sensibilidade e fatores de risco associados à resistência do Mycobacterium tuberculosis, em centro de referência de doenças infecto-contagiosas de Minas Gerais Multidrug-resistant Mycobacterium tuberculosis at a referral center for infectious diseases in the state of Minas Gerais, Brazil: sensitivity profile and related risk factors

    Directory of Open Access Journals (Sweden)

    Márcia Beatriz de Souza

    2006-10-01

    Full Text Available OBJETIVO: Estudar os fatores determinantes da multirresistência do Mycobacterium tuberculosis às drogas tuberculostáticas em centro de referência de doenças infecto-contagiosas do Estado de Minas Gerais, Hospital Eduardo de Menezes. MÉTODOS: Estudo tipo caso-controle, retrospectivo, realizado de setembro de 2000 a janeiro de 2004. Nesse período, 473 culturas com crescimento de M. tuberculosis relativas a 313 pacientes foram analisadas quanto ao perfil de sensibilidade, no Laboratório Central de Minas Gerais. Foram selecionados os casos multirresistentes definidos como resistência a pelo menos rifampicina e isoniazida, depois de pareados com o grupo controle de pacientes com tuberculose sensível a todas as drogas na razão de 1:3. A associação dos dados demográficos e clínicos foi feita por análise estatística uni e multivariada. RESULTADOS: Durante o período de estudo, doze casos de tuberculose multirresistente foram identificados (3,83%. Na análise univariada, a tuberculose multirresistente foi mais comum no sexo masculino, em pacientes com baciloscopia de escarro positiva, pacientes com cavitações maiores que 4 cm de diâmetro e pacientes com um ou mais tratamentos prévios para tuberculose (p = 0,10. Após a análise multivariada somente o tratamento anterior para tuberculose permaneceu estatisticamente significativo (p = 0,0374, com odds ratio de 14,36 (1,96 - 176,46. CONCLUSÃO: O fator de risco que se mostrou independentemente associado ao desenvolvimento de tuberculose multirresistente neste estudo foi a presença de um ou mais tratamentos prévios para tuberculose.OBJECTIVE: To assess the determining factors for Mycobacterium tuberculosis multidrug resistance at a referral center for infectious diseases in the state of Minas Gerais, Brazil. METHODS: A retrospective case-control study was conducted using data collected from September of 2000 to January of 2004. During this period, 473 cultures presenting growth of M

  1. Molecular Analysis of the embCAB Locus and embR Gene Involved in Ethambutol Resistance in Clinical Isolates of Mycobacterium tuberculosis in France.

    Science.gov (United States)

    Brossier, Florence; Sougakoff, Wladimir; Bernard, Christine; Petrou, Matthieu; Adeyema, Karine; Pham, Anne; Amy de la Breteque, Diane; Vallet, Marine; Jarlier, Vincent; Sola, Christophe; Veziris, Nicolas

    2015-08-01

    Modification of codon 306 in embB is regarded as the main mechanism leading to ethambutol (ETB) resistance in clinical isolates of Mycobacterium tuberculosis. However, numerous mutations elsewhere in the embCAB locus and in embR, a putative transcriptional activator of this locus, have been reported to be involved in ETB resistance. Here, we investigated the diversity of nucleotide variations observed in embCAB and embR in M. tuberculosis complex isolates from France. These regions were sequenced in 71 ETB-resistant (ETB-R) and 60 ETB-susceptible (ETB-S) clinical isolates of known phylogenetic lineages. The 131 isolates had 12 mutations corresponding to phylogenetic markers. Among the 60 ETB-S isolates, only 3 (5%) had nonsynonymous mutations that were not phylogenetic markers. Among the 71 ETB-R isolates, 98% had mutations in embCAB that likely contribute to ETB resistance: 70% had mutations located in embB codon 306, 406, or 497; 13% had mutations located outside these three positions between codons 296 and 426; and 15% had mutations corresponding to mutations in the embC-embA intergenic region. We found a strong association between resistance to ETB and the presence of mutations in embB and the embC-embA intergenic region (P embA were not involved in ETB resistance, and no mutation was detected in embR. These results strongly suggest that the sensitivity of diagnostic assays for detecting ETB resistance based on testing of embB codon 306 can be increased by testing of the embB region between codons 296 and 497 and by including the embC-embA intergenic region between positions -8 and -21.

  2. [Evaluation of the genotype MTBDR assay for rapid detection of rifampin and isoniazid resistance in clinical Mycobacterium tuberculosis complex clinical isolates].

    Science.gov (United States)

    Aslan, Gönül; Tezcan, Seda; Emekdaş, Gürol

    2009-04-01

    Rapid identification of resistant Mycobacterium tuberculosis complex isolates is quite important for the establishment of early and appropriate therapy. The Genotype MTBDR (Hain Lifescience, Nehren, Germany) is a commercially available DNA strip assay designed for the rapid detection of rpoB and katG gene mutations in clinical isolates. This study was conducted to determine the mutation types of phenotypically drug resistant 26 M. tuberculosis complex clinical isolates [15 isoniazid (INH), 1 rifampin (RMP) and 10 INH and RMP resistant] by Genotype MTBDR (G-MTBDR) DNA strip assay and to compare the diagnostic performance of this test. Sixteen of 25 (64%) INH-resistant and 9 of 11 (81.8%) RMP-resistant clinical isolates were correctly identified with the presence of hybridization in mutation probe or lack of hybridization at least by one of the wild type probes, by G-MTBDR assay. Hybridization with mutation probes was detected in only 5 of the RMP resistant isolates. We observed rpoB MUT3 (S531L, Ser-->4Leu) mutation in 4 and rpoB MUT1 (D516V) in one of these isolates. In 56% (14/25) of the INH-resistant isolates, katG T1 (S315T1) hybridization pattern was observed at katG mutation probe. G-MTBDR assay couldn't identify two of the 11 (18.2%) RMP-resistant isolates and one of these iSolates was shown to have a mutation at codon 531 (TCG-GCG) and the other at codon 545 (CTG-->ATG), possibly not associated with resistance, by sequence analysis. In four of the eight (8/25; 32%) INH-resistant isolates not identified by G-MTBDR assay, DNA cycle sequencing revealed different nucleotide changes outside the most common mutation zone. One of these were at codon 293 (GCT-->ACT) in katG, one with dual mutation at 279 (GGC-->ACC) in katG and at 15th C-->T in inhA gene, one at 15th C-->T in inhA gene and one at 279 (GGC-->ACC) in katG gene region. DNA membrane strip assay can be a use ful tool for the rapid detection of resistant M. tuberculosis complex isolates and therefore

  3. Multicenter evaluation of crystal violet decolorization assay (CVDA) for rapid detection of isoniazid and rifampicin resistance in Mycobacterium tuberculosis

    OpenAIRE

    Coban, Ahmet Yilmaz; Akbal,Ahmet Ugur; Bicmen, Can; Albay, Ali; Ali Korhan Sig; Uzun, Meltem; Selale, Deniz Sertel; Ozkutuk, Nuri; Surucuoglu, Suheyla; Albayrak, Nurhan; Ucarman, Nilay; Ozkutuk, Aydan; ESEN, Nuran; Ceyhan,Ismail; Ozyurt, Mustafa

    2016-01-01

    The aim of this multicenter study was to evaluate the performance of the crystal violet decolorization assay (CVDA) for detection of multidrug resistant tuberculosis (MDR-TB). This study was performed in 11 centers in two phases. A total of 156 isolates were tested for INH and RIF resistance. In the phase I, 106 clinical isolates were tested in the Center 1–7. In the phase 2, 156 clinical isolates were tested in the center 1–6, center 8–11. Eighty six of 156 tested isolates were the same in p...

  4. GenoType MTBDRsl for molecular detection of second-line-drug and ethambutol resistance in Mycobacterium tuberculosis strains and clinical samples.

    Science.gov (United States)

    Lacoma, A; García-Sierra, N; Prat, C; Maldonado, J; Ruiz-Manzano, J; Haba, L; Gavin, P; Samper, S; Ausina, V; Domínguez, J

    2012-01-01

    The purpose of this study was to evaluate the GenoType MTBDRsl assay (Hain Lifescience GmbH, Nehren, Germany) for its ability to detect resistance to fluoroquinolones (FLQ), injectable second-line antibiotics [kanamycin (KM) and capreomycin (CM)], and ethambutol (EMB) in Mycobacterium tuberculosis clinical strains and directly in clinical samples. A total of 34 clinical strains were characterized with the Bactec 460 TB system. Fifty-four clinical samples from 16 patients (5 were smear negative and 49 were smear positive) were also tested directly. The corresponding isolates of the clinical specimens were also analyzed with the Bactec 460TB. When there was a discrepancy between assays, pyrosequencing was performed. The overall rates of concordance of the MTBDRsl and the Bactec 460TB for the detection of FLQ, KM/CM, and EMB susceptibility in clinical strains were 72.4% (21/29), 88.8% (24/27), and 67.6% (23/34), whereas for clinical samples, rates were 86.5% (45/52), 92.3% (48/52), and 56% (28/50), respectively. In conclusion, the GenoType MTBDRsl assay may be a useful tool for making early decisions regarding KM/CM susceptibility and to a lesser extent regarding FLQ and EMB susceptibility. The test is able to detect mutations in both clinical strains and samples with a short turnaround time. However, for correct management of patients with extensively drug-resistant tuberculosis, results must be confirmed by a phenotypical method.

  5. Primary disseminated extrapulmonary multidrug resistant tuberculosis

    Directory of Open Access Journals (Sweden)

    S K Das

    2012-01-01

    Full Text Available Disseminated tuberculosis is a common mode of presentation of tuberculosis in patients both with and without HIV/AIDS in India. However, primary multidrug resistance in disseminated tuberculosis involving only the extrapulmonary sites in an immunocompetent adult is rare. Here, we report a case of a 19-year-old man who had disseminated tuberculosis involving left pleura, pericardium, peritoneum and intraabdominal lymph nodes. He was initially taking WHO category I antituberculous drugs, but was not responding in spite of 5 months of chemotherapy. Culture of the pleural biopsy specimen grew Mycobacterium tuberculosis which was resistant to isoniazid and rifampicin. He was put on therapy for multidrug resistant tuberculosis,following 24 months of chemotherapyhe had an uneventful recovery.

  6. Detection of rifampin resistance patterns in Mycobacterium tuberculosis strains isolated in Iran by polymerase chain reaction-single-strand conformation polymorphism and direct sequencing methods

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    Bahram Nasr Isfahani

    2006-09-01

    Full Text Available Mutations in the rpoB locus confer conformational changes leading to defective binding of rifampin (RIF to rpoB and consequently resistance in Mycobacterium tuberculosis. Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP was established as a rapid screening test for the detection of mutations in the rpoB gene, and direct sequencing has been unambiguously applied to characterize mutations. A total of 37 of Iranian isolates of M. tuberculosis, 16 sensitive and 21 resistant to RIF, were used in this study. A 193-bp region of the rpoB gene was amplified and PCR-SSCP patterns were determined by electrophoresis in 10% acrylamide gel and silver staining. Also, 21 samples of 193-bp rpoB amplicons with different PCR-SSCP patterns from RIFr and 10 from RIFs were sequenced. Seven distinguishable PCR-SSCP patterns were recognized in the 21 Iranian RIFr strains, while 15 out of 16 RIFs isolates demonstrated PCR-SSCP banding patterns similar to that of sensitive standard strain H37Rv. However one of the sensitive isolates demonstrated a different pattern. There were seen six different mutations in the amplified region of rpoB gene: codon 516(GAC/GTC, 523(GGG/GGT, 526(CAC/TAC, 531(TCG/TTG, 511(CTG/TTG, and 512(AGC/TCG. This study demonstrated the high specificity (93.8% and sensitivity (95.2% of PCR-SSCP method for detection of mutation in rpoB gene; 85.7% of RIFr strains showed a single mutation and 14.3% had no mutations. Three strains showed mutations caused polymorphism. Our data support the common notion that rifampin resistance genotypes are generally present mutations in codons 531 and 526, most frequently found in M. tuberculosis populations regardless of geographic origin.

  7. Detection of rifampin resistance patterns in Mycobacterium tuberculosis strains isolated in Iran by polymerase chain reaction-single-strand conformation polymorphism and direct sequencing methods.

    Science.gov (United States)

    Isfahani, Bahram Nasr; Tavakoli, Akbar; Salehi, Mansoor; Tazhibi, Mehdi

    2006-09-01

    Mutations in the rpoB locus confer conformational changes leading to defective binding of rifampin (RIF) to rpoB and consequently resistance in Mycobacterium tuberculosis. Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) was established as a rapid screening test for the detection of mutations in the rpoB gene, and direct sequencing has been unambiguously applied to characterize mutations. A total of 37 of Iranian isolates of M. tuberculosis, 16 sensitive and 21 resistant to RIF, were used in this study. A 193-bp region of the rpoB gene was amplified and PCR-SSCP patterns were determined by electrophoresis in 10% acrylamide gel and silver staining. Also, 21 samples of 193-bp rpoB amplicons with different PCR-SSCP patterns from RIFr and 10 from RIFs were sequenced. Seven distinguishable PCR-SSCP patterns were recognized in the 21 Iranian RIFr strains, while 15 out of 16 RIFs isolates demonstrated PCR-SSCP banding patterns similar to that of sensitive standard strain H37Rv. However one of the sensitive isolates demonstrated a different pattern. There were seen six different mutations in the amplified region of rpoB gene: codon 516(GAC/GTC), 523(GGG/GGT), 526(CAC/TAC), 531(TCG/TTG), 511(CTG/TTG), and 512(AGC/TCG). This study demonstrated the high specificity (93.8%) and sensitivity (95.2%) of PCR-SSCP method for detection of mutation in rpoB gene; 85.7% of RIFr strains showed a single mutation and 14.3% had no mutations. Three strains showed mutations caused polymorphism. Our data support the common notion that rifampin resistance genotypes are generally present mutations in codons 531 and 526, most frequently found in M. tuberculosis populations regardless of geographic origin.

  8. 耐异烟肼结核分枝杆菌对耐多药结核病流行的影响%The impact of isoniazid resistant Mycobacterium tuberculosis on the epidemic of multi-drug resistant tuberculosis

    Institute of Scientific and Technical Information of China (English)

    张沥月; 胡屹; 吴琳琳; 蒋伟利; 徐飚

    2015-01-01

    Objective To investigate the impact of isoniazid (INH)-resistant Mycobacterium tuberculosis (Mtb) on the prevalence and dissemination of multi-drug resistant tuberculosis (MDR-TB).Methods A total of 251 patients diagnosed with tuberculosis in designated hospitals of Guanyun,Jiangsu and Deqing,Zhejiang from 2010 to 2011 were included in the study.The drug susceptibility tests (DST) were performed on all the Mtb isolates available from the sputum cultures.Mycobacteral interspersed repetitive units-variable number tandem repeats (MIRU-VNTR) was conducted for genotyping for all available Mtb isolates.Chi-square test,Fisher exact test,ANOVA and non-conditional Logistic regression modelling were applied for data analysis.Results Among 251 patients with Mtb isolates and DST results available,72 (28.7%) were resistant to INH,including 13 were INH mono-drug resistant.Of the remaining 59 INH-resistant Mtb,34 (13.5%) were resistant to rifampin TB and 25 were resistant to streptomycin and/or ethambutol.The clustering analysis based on MIRU-VNTR genotyping revealed 29 clustered genotypes (including 105 isolates) and 146 unique genotypes (including 119 isolates).Twentyfive clusters contained drug resistant Mtb and 16 clusters of them comprised by 37 INH-resistant isolates and 20 MDR-TB isolates,which accounted for 51.4% of the INH-resistant isolates and 58.8% of the MDR-TB isolates.Single factor analysis showed that sex,age,previous tuberculosis treatment history and sputum smear results were all related to INH-resistant tuberculosis and MDR-TB (all P < 0.05).Multiple factors analysis showed that previous tuberculosis treatment history was risk factor of MDR-TB (OR=8.40,95 %CI:3.342-21.105),while the risk factors of INH-resistant tuberculosis were previous tuberculosis treatment history (OR=3.52,95%CI:1.570-7.910),pulmonary caviry (OR=2.27,95%CI:1.075-4.799) and sputum smear results (OR=0.50,95%CI:0.275-0.892).Conclusions That INH-resistant strain may evolve to

  9. Controlling strategy of dormant Mycobacterium tuberculosis

    Institute of Scientific and Technical Information of China (English)

    Gan Yiling; Guo Shuliang

    2014-01-01

    Objective This study aimed to review the available literatures on control of latent tuberculosis (TB) infection and propose a new control strategy to shorten the course of TB chemotherapy.Data sources The data used in this review were mainly obtained from articles listed in PubMed.The search terms were "therapy (treatment) of tuberculosis," "therapy (treatment) of latent TB infection," and "vaccine of TB."Study selection Articles regarding treatment and vaccine of TB were selected and reviewed.Results The most crucial reason causing the prolonged course of TB chemotherapy is the dormant state of Mycobacterium tuberculosis (M.tuberculosis).Nevertheless,there are,to date,no effective drugs that can directly kill the dormant cells of M.tuberculosis in clinical therapy.In accordance with the growth cycle of dormant M.tuberculosis in the body,the methods for controlling dormant M.tuberculosis include direct killing with drugs,prevention of dormant M.tuberculosis resuscitation with vaccines,and resuscitating dormant M.tuberculosis with preparations or drugs and then thoroughly killing these resuscitated M.tuberculosis by using anti-TB therapy.Conclusions The comprehensive analysis of the above three methods suggests that the drugs directly killing dormant cells are in clinical trials,TMC207 is the most beneficial for controlling TB.Because the side effect of vaccines is less and their action period is long,prevention of dormant cells resuscitation with vaccines is promising.The last control method makes it probable that when a huge number of active cells of M.tuberculosis have been killed and eradicated after 1-month short chemotherapy,only a strong short-term subsequent chemotherapy can completely kill and eradicate the remaining M.tuberculosis.This control strategy is expected to significantly shorten the course of TB chemotherapy and bring a new change and breakthrough in TB treatment.

  10. Use of the GenoType® MTBDRplus assay to assess drug resistance of Mycobacterium tuberculosis isolates from patients in rural Uganda

    Directory of Open Access Journals (Sweden)

    Bwanga Fred

    2010-08-01

    Full Text Available Abstract Background Drug resistance levels and patterns among Mycobacterium tuberculosis isolates from newly diagnosed and previously treated tuberculosis patients in Mbarara Uganda were investigated. Methods We enrolled, consecutively, all newly diagnosed and previously treated smear-positive TB patients aged ≥ 18 years. Isolates were tested for drug resistance against rifampicin (RIF and isoniazid (INH using the Genotype® MDRTBplus assay and results were compared with those obtained by the indirect proportion method on Lowenstein-Jensen media. HIV testing was performed using two rapid HIV tests. Results A total of 125 isolates from 167 TB suspects with a mean age 33.7 years and HIV prevalence of 67.9% (55/81 were analysed. A majority (92.8% of the participants were newly presenting while only 7.2% were retreatment cases. Resistance mutations to either RIF or INH were detected in 6.4% of the total isolates. Multidrug resistance, INH and RIF resistance was 1.6%, 3.2% and 4.8%, respectively. The rpoβ gene mutations seen in the sample were D516V, S531L, H526Y H526 D and D516V, while one strain had a Δ1 mutation in the wild type probes. There were three strains with katG (codon 315 gene mutations while only one strain showed the inhA promoter region gene mutation. Conclusion The TB resistance rate in Mbarara is relatively low. The GenoType® MTBDRplus assay can be used for rapid screening of MDR-TB in this setting.

  11. Mycobacterium tuberculosis L-forms

    OpenAIRE

    2011-01-01

    M. tuberculosis L-forms researches done in China during the recent years were reviewed in this article. M. tuberculosis L-forms could be produced spontaneously or induced by isoniazid or D-cyclic serine, that influenced the synthesis of cell walls. Among the acid-fast organisms isolated from the patients, more L-forms were found than vegetative forms. In extrapulmonary tuberculosis, M. tuberculosis disseminated through the blood mainly in L-forms, L-forms could adhere on the surface or harbor...

  12. Role of Mutations in Dihydrofolate Reductase DfrA (Rv2763c) and Thymidylate Synthase ThyA (Rv2764c) in Mycobacterium tuberculosis Drug Resistance

    KAUST Repository

    Koser, C. U.

    2010-09-17

    We would like to comment on a number of recent reports in this journal (6, 8, 12, 18) concerning Mycobacterium tuberculosis dihydrofolate reductase (DHFR), encoded by dfrA (Rv2763c). Around 36% of phenotypically para-aminosalicylic acid (PAS)-resistant M. tuberculosis strains harbor mutations in thyA (Rv2764c), which encodes a thymidylate synthase (20). In their effort to elucidate the remaining unknown resistance mechanism(s), Mathys et al. extended their sequence analysis to a number of additional genes, including dfrA (12). It was unclear whether the three dfrA mutations they identified in the PAS-resistant strains P-693 and P-3158 could contribute to PAS resistance on their own. Nonetheless, these findings are notable for two reasons. First, isoniazid (INH) has been shown to inhibit M. tuberculosis DHFR in vitro (1). Whether the same holds true for ethionamide, which shares a number of common resistance mechanisms with INH, was not tested (J. Blanchard, personal communication). In any case, the clinical relevance of DHFR-mediated INH resistance remains enigmatic. To date, only Ho et al. have addressed this question, but they did not identify any dfrA mutations in a screen of 127 INH-resistant clinical isolates (8). Consequently, Mathys et al. remain the first to describe mutations in this target (12). However, given that isolates with mutated DHFR are members of a cluster with baseline INH resistance, the importance of these mutations with respect to INH resistance remains unclear. Irrespective of their relevance in INH resistance, these dfrA mutations are noteworthy for a second reason. Contrary to previous wisdom, Forgacs et al. recently showed that M. tuberculosis is sensitive to the drug combination trimethoprim-sulfamethoxazole (TMP-SMX) (6, 18). DHFR is competitively inhibited by TMP, and consequently, mutations therein lead to resistance in a variety of organisms (9, 16, 19). The crystal structures of the wild-type M. tuberculosis DHFR in complex with

  13. Evaluation of point mutation detection in Mycobacterium tuberculosis with isoniazid resistance using real-time PCR and TaqMan probe assay.

    Science.gov (United States)

    Riahi, F; Derakhshan, M; Mosavat, A; Soleimanpour, S; Rezaee, S A

    2015-03-01

    Rapid methods for diagnosis of Mycobacterium tuberculosis (Mtb) drug resistance and choosing appropriate antibiotic treatment are pivotal. Thirty isoniazid (INH)-resistant and 30 INH-susceptible Mtb isolates were evaluated using minimum inhibitory concentration (MIC) method followed by multiplex real-time PCR (RT-PCR). Amplification refractory mutation system (ARMS) for detection of mutation in 315 codon of katG gene and single-nucleotide polymorphism (SNP) for detection of mutation in -15 (C>T) in the regulatory zone of mabA-inhA were carried out using the TaqMan method. Primers and probe were used for IS6110 region of Mtb as an internal amplification control. The sensitivity and specificity of the RT-PCR TaqMan probe for detection of Mtb complex were 100 %. Detection of INH-resistant Mtb using the ARMS method for KatG had 69 % sensitivity and 100 % specificity. The sensitivity and specificity of SNP in mabA-inhA fragment for detection of INH-resistant Mtb were 53 and 100 %, respectively. Furthermore, considering both regions, the sensitivity of RT-PCR has increased to 75 %. This study revealed that the qPCR-TaqMan method can be used as a standard tool for diagnosis of Mtb. Moreover, ARMS and SNP RT-PCR TaqMan methods can be used as rapid screening methods for detection of INH-resistant Mtb.

  14. Draft Genome Sequence of Mycobacterium tuberculosis Clinical Strain G-12-005

    Science.gov (United States)

    de Carvalho, Fabíola Marques; de Almeida, Luiz Gonzaga Paula; Bablishvili, Nino; Gauthier, Marie; Paranhos-Baccalà, Glaucia; de Vasconcelos, Ana Tereza Ribeiro

    2014-01-01

    Infection caused by drug-resistant Mycobacterium tuberculosis is a growing concern, especially in eastern Europe. We report an annotated draft genome sequence of M. tuberculosis strain G-12-005 obtained from a patient in Georgia. PMID:24812221

  15. Mycobacterium tuberculosis effectors interfering host apoptosis signaling.

    Science.gov (United States)

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

    2015-07-01

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

  16. FIND Tuberculosis Strain Bank: a Resource for Researchers and Developers Working on Tests To Detect Mycobacterium tuberculosis and Related Drug Resistance.

    Science.gov (United States)

    Tessema, Belay; Nabeta, Pamela; Valli, Eloise; Albertini, Audrey; Collantes, Jimena; Lan, Nguyen Huu; Romancenco, Elena; Tukavdze, Nestani; Denkinger, Claudia M; Dolinger, David L

    2017-04-01

    The spread of multidrug-resistant (MDR) tuberculosis (TB) and extensively drug-resistant (XDR) TB hampers global efforts in the fight against tuberculosis. To enhance the development and evaluation of diagnostic tests quickly and efficiently, well-characterized strains and samples from drug-resistant tuberculosis patients are necessary. In this project, the Foundation for Innovative New Diagnostics (FIND) has focused on the collection, characterization, and storage of such well-characterized reference materials and making them available to researchers and developers. The collection is being conducted at multiple centers in Southeast Asia, South America, Eastern Europe, and soon the sub-Saharan Africa regions. Strains are characterized for their phenotypic resistances and MICs to first-line drugs (FLDs) and second-line drugs (SLDs) using the automated MGIT 960 system following validated procedures and WHO criteria. Analysis of resistance-associated mutations is done by whole-genome sequencing (WGS) using the Illumina NextSeq system. Mycobacterial interspersed repetitive-unit-variable-number tandem-repeat analysis and WGS are used to determine strain lineages. All strains are maintained frozen at -80°C ± 10°C as distinct mother and daughter lots. All strains are extensively quality assured. The data presented here represent an analysis of the initial part of the collection. Currently, the bank contains 118 unique strains with extracted genomic DNA and matched sputum, serum, and plasma samples and will be expanded to a minimum of 1,000 unique strains over the next 3 years. Analysis of the current strains by phenotypic resistance testing shows 102 (86.4%), 10 (8.5%), and 6 (5.1%) MDR, XDR, and mono/poly resistant strains, respectively. Two of the strains are resistant to all 11 drugs that were phenotypically tested. WGS mutation analysis revealed FLD resistance-associated mutations in the rpoB, katG, inhA, embB, embA, and pncA genes; SLD resistance in the gyrA, gyr

  17. Suitability of IS6110-RFLP and MIRU-VNTR for Differentiating Spoligotyped Drug-Resistant Mycobacterium tuberculosis Clinical Isolates from Sichuan in China

    Directory of Open Access Journals (Sweden)

    Chao Zheng

    2014-01-01

    Full Text Available Genotypes of Mycobacterium tuberculosis complex (MTBC vary with the geographic origin of the patients and can affect tuberculosis (TB transmission. This study was aimed to further differentiate spoligotype-defined clusters of drug-resistant MTBC clinical isolates split in Beijing (n=190 versus non-Beijing isolates (n=84 from Sichuan region, the second high-burden province in China, by IS6110-restriction fragment length polymorphism (RFLP and 24-locus MIRU-VNTRs. Among 274 spoligotyped isolates, the clustering ratio of Beijing family was 5.3% by 24-locus MIRU-VNTRs versus 2.1% by IS6110-RFLP, while none of the non-Beijing isolates were clustered by 24-locus MIRU-VNTRs versus 9.5% by IS6110-RFLP. Hence, neither the 24-locus MIRU-VNTR was sufficient enough to fully discriminate the Beijing family, nor the IS6110-RFLP for the non-Beijing isolates. A region adjusted scheme combining 12 highly discriminatory VNTR loci with IS6110-RFLP was a better alternative for typing Beijing strains in Sichuan than 24-locus MIRU-VNTRs alone. IS6110-RFLP was for the first time introduced to systematically genotype MTBC in Sichuan and we conclude that the region-adjusted scheme of 12 highly discriminative VNTRs might be a suitable alternative to 24-locus MIRU-VNTR scheme for non-Beijing strains, while the clusters of the Beijing isolates should be further subtyped using IS6110-RFLP for optimal discrimination.

  18. Preliminary structure-activity relationships and biological evaluation of novel antitubercular indolecarboxamide derivatives against drug-susceptible and drug-resistant Mycobacterium tuberculosis strains.

    Science.gov (United States)

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

    2013-05-23

    Tuberculosis (TB) remains one of the leading causes of mortality and morbidity worldwide, with approximately one-third of the world's population infected with latent TB. This is further aggravated by HIV coinfection and the emergence of multidrug- and extensively drug-resistant (MDR and XDR, respectively) TB; hence the quest for highly effective antitubercular drugs with novel modes of action is imperative. We report herein the discovery of an indole-2-carboxamide analogue, 3, as a highly potent antitubercular agent, and the subsequent chemical modifications aimed at establishing a preliminary body of structure-activity relationships (SARs). These efforts led to the identification of three molecules (12-14) possessing an exceptional activity in the low nanomolar range against actively replicating Mycobacterium tuberculosis , with minimum inhibitory concentration (MIC) values lower than those of the most prominent antitubercular agents currently in use. These compounds were also devoid of apparent toxicity to Vero cells. Importantly, compound 12 was found to be active against the tested XDR-TB strains and orally active in the serum inhibition titration assay.

  19. Systematic interpretation of molecular beacon PCR for identifying rpoB mutations in Mycobacterium tuberculosis isolates with mixed resistant and susceptible bacteria

    Science.gov (United States)

    Gomez, Diana I.; Fisher-Hoch, Susan P.; Bordt, Andrea S.; Quitugua, Teresa N.; Robledo, Jaime; Alvarez, Nataly; Correa, Nidia; McCormick, Joseph B.; Restrepo, Blanca I.

    2010-01-01

    Detection of multi-drug resistant tuberculosis (MDR-TB), a frequent cause of treatment failure, takes two or more weeks to identify by culture. Rifampicin (RIF) resistance is a hallmark of MDR-TB, and detection of mutations in the rpoB gene of Mycobacterium tuberculosis using molecular beacon probes with real-time quantitative PCR (qPCR) is a novel approach that takes ≤ 2 days. However, qPCR identification of resistant isolates, particularly for isolates with mixed RIF-susceptible and RIF-resistant bacteria, is reader-dependent and limits its clinical use. The aim of this study was to develop an objective, reader independent method to define rpoB mutants using beacon qPCR. This would facilitate the transition from a research protocol to the clinical setting, where high-throughput methods with objective interpretation are required. For this, DNAs from 107 M. tuberculosis clinical isolates with known susceptibility to RIF by culture-based methods were obtained from two regions where isolates have not previously been subjected to evaluation using molecular beacon qPCR: The Texas-Mexico border and Colombia. Using coded DNA specimens, mutations within an 81 bp hot-spot region of rpoB were established by qPCR with five beacons spanning this region. Visual and mathematical approaches were used to establish whether the qPCR cycle threshold (Ct) of the experimental isolate was significantly higher (mutant) compared to a reference wild-type isolate. Visual classification of the beacon qPCR required reader training for strains with a mixture of RIF-susceptible and resistant bacteria. Only then, the visual interpretation by an experienced reader had 100% sensitivity and 94.6% specificity versus RIF-resistance by culture phenotype, and 98.1% sensitivity and 100% specificity versus mutations based on DNA sequence. The mathematical approach was 98% sensitive and 94.5% specific versus culture, and 96.2% sensitive and 100% specific versus DNA sequence. Our findings indicate the

  20. High rates of ofloxacin resistance in Mycobacterium tuberculosis among both new and previously treated patients in Tamil Nadu, South India.

    Directory of Open Access Journals (Sweden)

    N Selvakumar

    Full Text Available Periodic drug resistance surveillance provides useful information on trends of drug resistance and effectiveness of tuberculosis (TB control measures. The present study determines the prevalence of drug resistance among new sputum smear positive (NSP and previously treated (PT pulmonary TB patients, diagnosed at public sector designated microscopy centers (DMCs in the state of Tamil Nadu, India. In this single-stage cluster-sampling prevalence survey, 70 of 700 DMCs were randomly selected using a probability-proportional to size method. A cluster size of 24 for NSP and a varying size of 0 to 99 for PT cases were fixed for each selected DMC. Culture and drug susceptibility testing was done on Lowenstein-Jensen medium using the economic variant of proportion sensitivity test for isoniazid (INH, rifampicin (RMP, ofloxacin (OFX and kanamycin (KAN. Human Immunodeficiency Virus (HIV status was collected from patient records. From June 2011 to August 2012, 1524 NSP and 901 PT patients were enrolled. Any RMP resistance and any INH resistance were observed in 2.6% and 15.1%, and in 10.4% and 30% respectively in NSP and PT cases. Among PT patients, multi drug resistant TB (MDR-TB was highest in the treatment failure (35% group, followed by relapse (13% and treatment after default (10% groups. Extensively drug resistant TB (XDRTB was seen in 4.3% of MDR-TB cases. Any OFX resistance was seen in 10.4% of NSP, 13.9% of PT and 29% of PT MDR-TB patients. The HIV status of the patient had no impact on drug resistance levels. RMP resistance was present in 2.6% of new and 15.1% of previously treated patients in Tamil Nadu. Rates of OFX resistance were high among NSP and PT patients, especially among those with MDR-TB, a matter of concern for development of new treatment regimens for TB.

  1. High rates of ofloxacin resistance in Mycobacterium tuberculosis among both new and previously treated patients in Tamil Nadu, South India.

    Science.gov (United States)

    Selvakumar, N; Kumar, Vanaja; Balaji, S; Prabuseenivasan, S; Radhakrishnan, R; Sekar, Gomathi; Chandrasekaran, V; Kannan, T; Thomas, Aleyamma; Arunagiri, S; Dewan, Puneet; Swaminathan, Soumya

    2015-01-01

    Periodic drug resistance surveillance provides useful information on trends of drug resistance and effectiveness of tuberculosis (TB) control measures. The present study determines the prevalence of drug resistance among new sputum smear positive (NSP) and previously treated (PT) pulmonary TB patients, diagnosed at public sector designated microscopy centers (DMCs) in the state of Tamil Nadu, India. In this single-stage cluster-sampling prevalence survey, 70 of 700 DMCs were randomly selected using a probability-proportional to size method. A cluster size of 24 for NSP and a varying size of 0 to 99 for PT cases were fixed for each selected DMC. Culture and drug susceptibility testing was done on Lowenstein-Jensen medium using the economic variant of proportion sensitivity test for isoniazid (INH), rifampicin (RMP), ofloxacin (OFX) and kanamycin (KAN). Human Immunodeficiency Virus (HIV) status was collected from patient records. From June 2011 to August 2012, 1524 NSP and 901 PT patients were enrolled. Any RMP resistance and any INH resistance were observed in 2.6% and 15.1%, and in 10.4% and 30% respectively in NSP and PT cases. Among PT patients, multi drug resistant TB (MDR-TB) was highest in the treatment failure (35%) group, followed by relapse (13%) and treatment after default (10%) groups. Extensively drug resistant TB (XDRTB) was seen in 4.3% of MDR-TB cases. Any OFX resistance was seen in 10.4% of NSP, 13.9% of PT and 29% of PT MDR-TB patients. The HIV status of the patient had no impact on drug resistance levels. RMP resistance was present in 2.6% of new and 15.1% of previously treated patients in Tamil Nadu. Rates of OFX resistance were high among NSP and PT patients, especially among those with MDR-TB, a matter of concern for development of new treatment regimens for TB.

  2. Increased in vitro fitness of multi- and extensively drug-resistant F15/LAM4/KZN strains of Mycobacterium tuberculosis.

    Science.gov (United States)

    Naidoo, C C; Pillay, M

    2014-06-01

    The role of fitness in transmission of drug-resistant strains has been explored in previous studies; but has not been established for F15/LAM4/KZN strains, which were responsible for the extensively drug-resistant tuberculosis (XDR-TB) outbreak in Tugela Ferry, South Africa. The biological fitness of 15 clinical strains representing the F15/LAM4/KZN, Beijing, F11 and F28 families was determined by growth, viability and competition assays and correlated with DNA sequencing of eight genes associated with drug resistance and putative compensatory mechanisms. Similar growth rates were observed among susceptible, multidrug-resistant (MDR) and XDR strains of the KZN and F28 genotypes. In contrast, Beijing and F11 MDR strains demonstrated significantly reduced fitness. Resistant strains exhibited heterogeneous fitness profiles in competition with different susceptible strains, suggesting strain dependence. In addition, co-culture growth rates were consistently higher than independent growth rates in 13/14 competition pairs. All 14 drug-resistant strains retained viability, at a low CFU/mL, when paired with susceptible strains. The persistence of such resistant strains could consequently support the acquisition of additional drug-resistance-conferring mutations and/or the evolution of compensatory mechanisms. Frequently occurring mutations were detected in KZN and F28 resistant strains whereas, the Beijing MDR strain harboured a less common katG mutation and the F11 MDR strain had no katG mutation. Contrary to drug-resistant Beijing and F11 strains, the successful transmission of KZN strains, particularly during the outbreak, may be attributed to the presence of drug-resistance-conferring mutations associated with little or no associated fitness costs. Amplified growth in co-culture may be suggestive of in vivo trans-complementation.

  3. Combined real-time PCR and rpoB gene pyrosequencing for rapid identification of Mycobacterium tuberculosis and determination of rifampin resistance directly in clinical specimens.

    Science.gov (United States)

    Halse, Tanya A; Edwards, Justine; Cunningham, Phyllis L; Wolfgang, William J; Dumas, Nellie B; Escuyer, Vincent E; Musser, Kimberlee A

    2010-04-01

    Our laboratory has developed a rapid, sensitive, and specific molecular approach for detection in clinical specimens, within 48 h of receipt, of both Mycobacterium tuberculosis complex (MTBC) DNA and mutations within the 81-bp core region of the rpoB gene that are associated with rifampin (RIF) resistance. This approach, which combines an initial real-time PCR with internal inhibition assessment and a pyrosequencing assay, was validated for direct use with clinical specimens. To assess the suitability of real-time PCR for use with respiratory, nonrespiratory, acid-fast bacillus (AFB)-positive and AFB-negative specimens, we evaluated specimens received in our laboratory between 11 October 2007 and 30 June 2009. With culture used as the "gold standard," the sensitivity, specificity, and positive and negative predictive values were determined for 1,316 specimens to be as follows: for respiratory specimens, 94.7%, 99.9%, 99.6%, and 98.6%, respectively; for nonrespiratory specimens, 88.5%, 100.0%, 100.0%, and 96.9%, respectively; for AFB-positive specimens, 99.6%, 100.0%, 100.0%, and 97.7%, respectively; and for AFB-negative specimens, 75.4%, 99.9%, 98.0%, and 98.4%, respectively. PCR inhibition was determined to be minimal in this assay, occurring in 0.2% of tests. The rpoB gene pyrosequencing assay was evaluated in a similar prospective study, in which 148 clinical specimens positive for MTBC DNA by real-time PCR were tested. The final results revealed that the results of direct testing of clinical specimens by the pyrosequencing assay were 98.6% concordant with the results of conventional testing for susceptibility to RIF in liquid culture and that our assay displayed adequate sensitivity for 96.6% of the clinical specimens tested. Used together, these assays provide reliable results that aid with the initial management of patients with suspected tuberculosis prior to the availability of the results for cultured material, and they also provide the ability to predict

  4. Erythema nodosum and Mycobacterium Tuberculosis

    Directory of Open Access Journals (Sweden)

    Ovgu Kul

    2016-01-01

    Full Text Available Erythema nodosum[EN] is a common form of panniculitis and inflammatory disease of the subcutaneous fat tissue.EN occurs in a variety of disorders including tuberculosis, sarcoidosis, Behcet%u2019s disease, inflammatory bowel disease and streptococcal infection but idiopathic cases account for 55%. A 14 years old patient was diagnosed as Erythema nodosum[EN] with prolonged fever and red subcutaneous nodules in pretibial location. With tuberculin skin and Quantiferon positivity and tree in bud sign in chest x ray she was diagnosed as tuberculosis. On the same dates a 11 years old patient was admitted with EN. She was also diagnosed as tuberculosis due to tuberculin skin and Quantiferon positivity and hilar lymphadenopathy in thorax CT. The other causes were excluded in both cases. We present these two adolescents cases to keep in mind the close relationship between tuberculosis and erythema nodosum.

  5. Assess drug resistance pattern and genetic profile of Mycobacterium tuberculosis clinical isolates by molecular typing methods using direct repeats and IS6110 in pulmonary tuberculosis cases

    Science.gov (United States)

    Kalo, Deepika; Kant, Surya; Srivastava, Kanchan; Sharma, Ajay K

    2017-01-01

    Background: Tuberculosis (TB), a highly contagious disease that sees no gender, age, or race is mainly a disease of lungs. According to World Health Organization, a TB patient can be completely cured with 6–9 months of anti-TB treatment under directly observed treatment short course. Objectives: The aim of this study was to check the mono, multi- and triple-drug resistance to first line drugs (FLDs) among TB patients and to access their genetic profile using DR 3074, DR 0270, DR 0642, DR 2068, and DR 4110 using molecular techniques. Material and Methods: To gain a better understanding of drug resistant TB, we characterized 121 clinical isolates recovered from 159 drug resistant pulmonary tuberculosis patients by IS6110 genotyping. MTB isolates recovered from HIV- negative, and smear positive cases of both genders, age varied from 18 to 70 years with drug resistant-TB that was refractory to chemotherapy given for > 12 months. Of a total of 159 sputum smear positive patients sum number of male and female patients was 121 (76.10%) and 38 (23.89%), respectively. Among these patients, number of literate and illiterate patients were 123 (77.3%) and 36 (22.6%). 25 (15.7%) patients had farming as their occupation, 80 (50.3%) had nonagricultural occupation and 54 (33.9%) women were housewives. Results: Mono drug resistant, multi-drug resistant, and totally drug resistant (TDR) cases of TB were calculated as 113.83%, 125.1%, and 67.9%. Isoniazid showed the highest percentage of resistance among the patients. Conclusion: Any noncompliance to TB medications, lack of knowledge, and poor management in health centers, etc., results in the emergence of deadly direct repeat forms of TB, which are further complicated and complex to treat. PMID:28360464

  6. Medical devices; immunology and microbiology devices; classification of nucleic acid-based devices for the detection of Mycobacterium tuberculosis complex and the genetic mutations associated with antibiotic resistance. Final order.

    Science.gov (United States)

    2014-10-22

    The Food and Drug Administration (FDA) is classifying nucleic acid-based in vitro diagnostic devices for the detection of Mycobacterium tuberculosis complex (MTB-complex) and the genetic mutations associated with MTB-complex antibiotic resistance in respiratory specimens devices into class II (special controls). The Agency is classifying the device into class II (special controls) because special controls, in addition to general controls, will provide a reasonable assurance of safety and effectiveness of the device.

  7. 5-Arylaminouracil Derivatives: New Inhibitors of Mycobacterium tuberculosis.

    Science.gov (United States)

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

    2015-12-01

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

  8. Parallel reaction monitoring of clinical Mycobacterium tuberculosis lineages reveals pre-existent markers of rifampicin tolerance in the emerging Beijing lineage

    NARCIS (Netherlands)

    Keijzer, J. de; Mulder, A.; Ru, A.H. de; Soolingen, D. van; Veelen, P.A. van

    2017-01-01

    The spread of multidrug resistant Mycobacterium tuberculosis is one of the major challenges in tuberculosis control. In Eurasia, the spread of multidrug resistant tuberculosis is driven by the M. tuberculosis Beijing genotype. In this study, we examined whether selective advantages are present in th

  9. Parallel reaction monitoring of clinical Mycobacterium tuberculosis lineages reveals pre-existent markers of rifampicin tolerance in the emerging Beijing lineage

    NARCIS (Netherlands)

    Keijzer, J. de; Mulder, A.; Ru, A.H. de; Soolingen, D. van; Veelen, P.A. van

    2017-01-01

    The spread of multidrug resistant Mycobacterium tuberculosis is one of the major challenges in tuberculosis control. In Eurasia, the spread of multidrug resistant tuberculosis is driven by the M. tuberculosis Beijing genotype. In this study, we examined whether selective advantages are present in

  10. MDR-TB Antibody Response (Western Blot) to Fractions of Isoniazid and Rifampicin Resistant Antigens of Mycobacterium tuberculosis.

    Science.gov (United States)

    Hadizadeh Tasbiti, Alireza; Yari, Shamsi; Ghanei, Mostafa; Shokrgozar, Mohammad Ali; Bahrmand, Ahmadreza

    2015-12-01

    Drug-resistant TB poses a major threat to control of TB worldwide. Despite progress in the detection of Multidrug-resistant TB (MDR-TB) cases, a major diagnostic gap remains: 55% of reported TB patients estimated to have MDR-TB were not detected in 2013. MDR-TB antigens were conjugated to CNBr-activated Sepharose 4B. Specific polyclonal antibodies against MDR-TB Ags were prepared in rabbits using two boosted injections of the MDR-TB antigen. The antibodies were purified and treated with susceptible TB to remove any non-specific and cross-reactive antibodies. In the present study, comparative analysis of electrophoretic pattern of different antigens of INH/RIF-resistant TB were studied for identifying protein profiles. A RIF-resistant TB antigen was shown here to have different protein profiles from INH-resistant TB isolate. The results of Western blotting analysis showed that in the RIF- and INH-resistant antigenic fractions some bands of 14.4 and 45 kDa as immunogenic were common. Moreover, four bands of RIF-resistant TB antigen fractions (16, 19, 21, and 45 KDa) and one band of INH-resistant TB (about 26 KDa) were detected as diagnostic antigens. This study suggests that the Western blot is an accurate test to survey INH- and RIF-resistant TB antigens of M. tuberculosis infection. These findings indicate that MDR-TB diagnosis (based on Ag detection) could be useful in the identification of disease stages that precede symptomatic and microbiologically positive TB, such as subclinical and incipient TB.

  11. Rapid, automated, nonradiometric susceptibility testing of Mycobacterium tuberculosis complex to four first-line antituberculous drugs used in standard short-course chemotherapy

    DEFF Research Database (Denmark)

    Johansen, Isik Somuncu; Thomsen, Vibeke Østergaard; Marjamäki, Merja

    2004-01-01

    The increasing prevalence of drug-resistant tuberculosis necessitates rapid and accurate susceptibility testing. The nonradiometric BACTEC Mycobacteria Growth Indicator Tube 960 (MGIT) system for susceptibility testing was evaluated on 222 clinical Mycobacterium tuberculosis complex isolates for ...

  12. Analysis on the Drug resistance of 322 Cases of Mycobacterium Tuberculosis in A District%某区322株结核杆菌的耐药性分析

    Institute of Scientific and Technical Information of China (English)

    韩珍; 林日文; 李静

    2013-01-01

      Objective To understand the status of drug resistance in 322 Cases of Mycobacterium tuberculosis in sputum of patients with culture-positive strains isolated in Huizhou city tuberculosis institute, and to provide evidence for clinical rational chemotherapy. Methods In our hospital, 322 Cases of Mycobacterium tuberculosis in sputum with culture-positive strains isolated as the research object, with proportion method for first-line tuberculosis drugs medication to analysis the drug resistance. Results 36 cases of 322 mycobacterium tuberculosis were resistance in four kinds of anti-TB drugs, occupied 10.84%,in which S-resistance was highest, occupied 35.8%. Conclusion The drug resistance of tuberculosis mycobacterium in Huizhou area was serious, so we should strengthen the management of the clinical treatment of tuberculosis, the development of rational chemotherapy to prevent the spread of resistant bacteria.%  目的了解惠州市结核病防治院322株痰培养阳性结核杆菌的耐药情况,为临床合理用药提供理论依据。方法以我院322株痰培养阳性的结核杆菌为研究对象,用比例法进行一线结核用药的药敏实验。结果322株结核分枝杆菌对4种抗结核药物全部耐药者36例,占10.84%;以耐链霉素为最高,占35.8%。结论惠州市结核病耐药情况仍然十分严峻,应加强结核病临床治疗的管理,制定合理化疗方案,防止耐药菌传播。

  13. Early detection of multidrug resistant (MDR) Mycobacterium tuberculosis in a single tube with in-house designed fluorescence resonance energy transfer (FRET) probes using real-time PCR.

    Science.gov (United States)

    Chauhan, Devendra Singh; Sharma, Rahul; Parashar, Deepti; Sharma, Pragya; Das, Ram; Chahar, Madhvi; Singh, Ajay Vir; Singh, Pravin Kumar; Katoch, Kiran; Katoch, Vishwa Mohan

    2016-04-01

    Rapid and correct diagnosis is crucial for the management of multidrug resistance (MDR) in Mycobacterium tuberculosis (MTB). The present study aims at rapid diagnosis for identification of multidrug resistance tuberculosis (MDR-TB) using real-time PCR. FRET hybridization probes targeting most prominent four selected codons for rpoB526 and 531 and for katG314 and 315 genes were designed and evaluated on 143 clinical MTB isolates and paired sputa for rapid detection of MDR-TB. The results of real-time PCR were compared with gold standard L-J proportion method and further validated by DNA sequencing. Of the 143 MTB positive cultures, 85 and 58 isolates were found to be 'MDR' and 'pan susceptible', respectively by proportion L-J method. The sensitivity of real-time PCR for the detection of rifampicin (RIF) and isoniazid (INH) were 85.88 and 94.11%, respectively, and the specificity of method was found to be 98.27%. DNA sequencing of 31 MTB isolates having distinct melting temperature (Tm) as compared to the standard drug susceptible H37Rv strain showed 100% concordance with real-time PCR results. DNA sequencing revealed the mutations at Ser531Leu, His526Asp of rpoB gene and Ser315Thr, Thr314Pro of katG gene in RIF and INH resistance cases. This real-time PCR assay that targets limited number of loci in a selected range ensures direct and rapid detection of MDR-TB in Indian settings. However, future studies for revalidation as well as refinement are required to break the limitations of MDR-TB detection.

  14. High Persister Mutants in Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Heather L Torrey

    Full Text Available 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.

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

    DEFF Research Database (Denmark)

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

    2011-01-01

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

  16. Alternate efflux pump mechanism may contribute to drug resistance in extensively drug-resistant isolates of Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Akbar Kanji

    2016-01-01

    Conclusion: Our data show an nsSNP in the drrA efflux pump gene that may result in upregulation of drug efflux mechanisms in MTB strains. It is therefore imperative to understand the mechanism of efflux and its role in drug resistance, which will enable the identification of new drug targets and development of new drug regimens to counteract the drug efflux mechanism of MTB.

  17. Cryopreservation of Mycobacterium tuberculosis complex cells.

    Science.gov (United States)

    Shu, Zhiquan; Weigel, Kris M; Soelberg, Scott D; Lakey, Annie; Cangelosi, Gerard A; Lee, Kyong-Hoon; Chung, Jae-Hyun; Gao, Dayong

    2012-11-01

    Successful long-term preservation of Mycobacterium tuberculosis cells is important for sample transport, research, biobanking, and the development of new drugs, vaccines, biomarkers, and diagnostics. In this report, Mycobacterium bovis bacillus Calmette-Guérin and M. tuberculosis H37Ra were used as models of M. tuberculosis complex strains to study cryopreservation of M. tuberculosis complex cells in diverse sample matrices at different cooling rates. Cells were cryopreserved in diverse sample matrices, namely, phosphate-buffered saline (PBS), Middlebrook 7H9 medium with or without added glycerol, and human sputum. The efficacy of cryopreservation was quantified by microbiological culture and microscopy with BacLight LIVE/DEAD staining. In all sample matrices examined, the microbiological culture results showed that the cooling rate was the most critical factor influencing cell viability. Slow cooling (a few degrees Celsius per minute) resulted in much higher M. tuberculosis complex recovery rates than rapid cooling (direct immersion in liquid nitrogen) (P tuberculosis complex cells in 7H9 broth for 20 h before culture on solid Middlebrook 7H10 plates did not help the recovery of the cells from cryoinjury (P = 0.14 to 0.71). The BacLight LIVE/DEAD staining kit, based on Syto 9 and propidium iodide (PI), was also applied to assess cell envelope integrity after cryopreservation. Using the kit, similar percentages of "live" cells with intact envelopes were observed for samples cryopreserved under different conditions, which was inconsistent with the microbiological culture results. This implies that suboptimal cryopreservation might not cause severe damage to the cell wall and/or membrane but instead cause intracellular injury, which leads to the loss of cell viability.

  18. Estudio de la resistencia de Mycobacterium tuberculosis a los principales tuberculostáticos (Hospital de Navarra 1996-2003 Study of the resistance of Mycobacterium tuberculosis to the principal tuberculostatics (Hospital of Navarra 1996-2003

    Directory of Open Access Journals (Sweden)

    I. Dorronsoro

    2005-04-01

    Full Text Available Introducción. El objeto de este trabajo ha sido valorar la sensibilidad a los principales tuberculostáticos de las cepas de M. tuberculosis aisladas en nuestro laboratorio y estudiar los factores relacionados con la resistencia. Material y métodos. Se han estudiado 475 cepas de M. tuberculosis correspondientes a la totalidad de los casos diagnosticados entre los años 1996-2003 ambos inclusive. En el cultivo de las muestras, se utilizó el sistema BACTEC 460TB, junto con el medio sólido de Lowenstein. Los estudios de sensibilidad se realizaron mediante el sistema BACTEC 460TB. Resultados y conclusiones. Tanto la incidencia, como el número de casos de tuberculosis resistentes, se presentaron con oscilaciones a lo largo de los 8 años del estudio. El hecho de que no exista una tendencia homogénea, obliga a mantener una vigilancia activa frente a este proceso. La resistencia global a isoniacida fue del 8%, por lo que es conveniente realizar estudios de sensibilidad en todos los casos diagnosticados. Los casos de tuberculosis multirresistentes (resistentes al menos a isoniacida y rifampicina, no superaron el 3%. El pronóstico de los pacientes con tuberculosis resistente fue sombrío en los casos de coinfección con el VIH, sin embargo, cuando las defensas inmunológicas estaban conservadas, y se pautó un tratamiento con drogas activas, se consiguió la recuperación de la mayoría de los pacientes. En la actualidad, disponemos de procedimientos sensibles y rápidos que hacen recomendable el estudio de sensibilidad en todas las cepas de M. tuberculosis aisladas, y en el caso de pacientes VIH+ o inmigrantes, resulta imprescindible.Introduction. The aim of this study was to evaluate the sensitivity to the principal tuberculostatics of the M. tuberculosis stocks isolated in our laboratory and to study the factors related to resistance. Methods. Study of 475 stocks of M. tuberculosis corresponding to all cases diagnosed in the eight year period

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

    Science.gov (United States)

    Segura, C; Salvadó, M

    1997-09-01

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

  20. AMPLIFIKASI DAN IDENTIFIKASI MUTASI REGIO PROMOTER inhA PADA ISOLAT Mycobacterium tuberculosis MULTIDRUG RESISTANCE DENGAN TEKNIK POLYMERASE CHAIN REACTION

    Directory of Open Access Journals (Sweden)

    Devita Kusdianingrum

    2014-10-01

    Full Text Available ABSTRAK: Sekitar 8-20% isolate M. tuberculosis yang resisten terhadap isoniazid diketahui telah mengalami mutasi pada posisi regio promoter inhA [1]. Untuk memperoleh titik mutasi pada regio promoter, maka amplifikasi fragmen target perlu untuk dilakukan. Tujuan dilakukannya penelitian ini adalah untuk mengamplifikasi regio promoter inhA, mengetahui ada tidaknya mutasi dan jenis mutasi pada isolat 134 MDR-TB. Tahap isolasi DNA dilakukan menggunakan metode Boom yang telah dimodifikasi. Fragmen target diamplifikasi dengan teknik PCR menggunakan sepasang primer (forward primer 5’ ACATACCTGCTGCGCAAT 3’ dan reverse primer 5’ CTCCGGTAACCAGGACT GAA 3’. Amplikon disekuensing secara satu arah menggunakan forward primer. Analisis homologi dilakukan menggunakan program online BLASTn, sementara identifikasi mutasi dilakukan menggunakan software MEGA4. Hasil penelitian menunjukkan bahwa analisis homologi isolate 134 terhadap M. tuberculosis H37Rv adalah sebesar 99%. Tahap analisis mutasi menemukan terjadinya perubahan sitosin menjadi timin (CàT pada posisi -15 isolat 134 MDR-TB ABSTRACT: Approximately 8-20% M. tuberculosis isolates that are resistant to isoniazid habe been known to have a mutation in inhA promoter region [1]. To find the mutation in inhA promoter region, it is necessary to carry out the amplification of the target fragment. The purpose of this research were to amplify the inhA promoter region and to find out if there is a mutation and type of mutation at MDR-TB isolate. DNA isolation was done by a modified Boom method. Target fragment was amplified by a pair primer (forward primer 5’ ACATACCTGCTGCGCAAT 3’ and reverse primer 5’ CTCCGGTAACCAGGACT GAA 3’ using Polymerase Chain Reaction (PCR technique. Amplicon was sequenced in one forward direction. Homology analysis was conducted by online BLASTn program, while the mutation was identified by MEGA4. The result of this research showed that homology analysis of 134 was homolog

  1. Performance of the G4 Xpert(®) MTB/RIF assay for the detection of Mycobacterium tuberculosis and rifampin resistance: a retrospective case-control study of analytical and clinical samples from high- and low-tuberculosis prevalence settings.

    Science.gov (United States)

    Dharan, Nila J; Blakemore, Robert; Sloutsky, Alex; Kaur, Devinder; Alexander, Richard C; Ghajar, Minoo; Musser, Kimberlee A; Escuyer, Vincent E; Rowlinson, Marie-Claire; Crowe, Susanne; Laniado-Laborin, Rafael; Valli, Eloise; Nabeta, Pamela; Johnson, Pamela; Alland, David

    2016-12-20

    The Xpert(®) MTB/RIF (Xpert) assay is a rapid PCR-based assay for the detection of Mycobacterium tuberculosis complex DNA (MTBc) and mutations associated with rifampin resistance (RIF). An updated version introduced in 2011, the G4 Xpert, included modifications to probe B and updated analytic software. An analytical study was performed to assess Xpert detection of mutations associated with rifampin resistance in rifampin-susceptible and -resistant isolates. A clinical study was performed in which specimens from US and non-US persons suspected of tuberculosis (TB) were tested to determine Xpert performance characteristics. All specimens underwent smear microscopy, mycobacterial culture, conventional drug-susceptibility testing and Xpert testing; DNA from isolates with discordant rifampin resistance results was sequenced. Among 191 laboratory-prepared isolates in the analytical study, Xpert sensitivity for detection of rifampin resistance associated mutations was 97.7% and specificity was 90.8%, which increased to 99.0% after DNA sequencing analysis of the discordant samples. Of the 1,096 subjects in the four clinical studies, 49% were from the US. Overall, Xpert detected MTBc in 439 of 468 culture-positive specimens for a sensitivity of 93.8% (95% confidence interval [CI]: 91.2%-95.7%) and did not detect MTBc in 620 of 628 culture-negative specimens for a specificity of 98.7% (95% CI: 97.5%-99.4%). Sensitivity was 99.7% among smear-positive cases, and 76.1% among smear-negative cases. Non-determinate MTBc detection and false-positive RIF resistance results were low (1.2 and 0.9%, respectively). The updated Xpert assay retained the high sensitivity and specificity of the previous assay versions and demonstrated low rates of non-determinate and RIF resistance false positive results.

  2. Impact of β-lactamase inhibition on the activity of ceftaroline against Mycobacterium tuberculosis and Mycobacterium abscessus.

    Science.gov (United States)

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

    2015-05-01

    The production of β-lactamases Bla(Mab) 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.

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

    Science.gov (United States)

    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.

  4. Hypervirulent Mycobacterium tuberculosis strain triggers necrotic lung pathology associated with enhanced recruitment of neutrophils in resistant C57BL/6 mice

    Science.gov (United States)

    Almeida, Fabrício M.; Ventura, Thatiana L. B.; Amaral, Eduardo P.; Ribeiro, Simone C. M.; Calixto, Sanderson D.; Manhães, Marcelle R.; Rezende, Andreza L.; Souzal, Giliane S.; de Carvalho, Igor S.; Silva, Elisangela C.; da Silva, Juliana Azevedo; Carvalho, Eulógio C. Q.; Kritski, Afranio L.

    2017-01-01

    Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb) that in most cases induces irreversible necrosis of lung tissue as a result of excessive inflammatory reactions. The murine model of TB in resistant C57BL/6 mice infected with reference Mtb strains is widely used in TB studies; however, these mice do not show a necrotic pathology, which restricts their use in studies of irreversible tissue damage. Recently, we demonstrated that necrotic lung lesions could be induced in the C57BL/6 mice by highly virulent Mtb strains belonging to the modern Beijing sublineage. However, the pathogenic mechanisms leading to necrosis in this model were not elucidated. In this study, we investigated the dynamics of lung lesions in mice infected with highly virulent Beijing Mtb strain M299, compared with those infected with laboratory Mtb strain H37Rv. The data demonstrate that necrotic lung lesions in mice infected by the strain M299 were associated with enhanced recruitment of myeloid cells, especially neutrophils, and increased levels of proinflammatory cytokines, consistent with exacerbated inflammation. High levels of IFN-γ production contributed to the control of bacterial growth. Further progression to chronic disease was associated with a reduction in the levels of inflammatory mediators in the lungs, the accumulation of foamy macrophages and partial healing of the necrotic tissue by fibrosis. At a late stage of disease, degradation of foamy cells resulted in the liberation of accumulated lipids and persisting bacilli and further activation of inflammation, which promoted lung consolidation. Overall, our studies show that C57BL/6 mice infected with highly virulent Mtb strain may serve as a TB model reproducing an exacerbated inflammatory response in a resistant host to hypervirulent mycobacteria, leading to irreversible necrotic lung lesions. PMID:28306733

  5. Solution NMR Studies of Mycobacterium tuberculosis Proteins for Antibiotic Target Discovery

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    Do-Hee Kim

    2017-08-01

    Full Text Available Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis, which triggers severe pulmonary diseases. Recently, multidrug/extensively drug-resistant tuberculosis strains have emerged and continue to threaten global health. Because of the development of drug-resistant tuberculosis, there is an urgent need for novel antibiotics to treat these drug-resistant bacteria. In light of the clinical importance of M. tuberculosis, 2067 structures of M. tuberculsosis proteins have been determined. Among them, 52 structures have been solved and studied using solution nuclear magnetic resonance (NMR. The functional details based on structural analysis of M. tuberculosis using NMR can provide essential biochemical data for the development of novel antibiotic drugs. In this review, we introduce diverse structural and biochemical studies on M. tuberculosis proteins determined using NMR spectroscopy.

  6. In-depth molecular characterization of Mycobacterium tuberculosis from New Delhi--predominance of drug resistant isolates of the 'modern' (TbD1 type.

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    Ruth Stavrum

    Full Text Available BACKGROUND: India has the highest estimated burden of tuberculosis in the world, accounting for 21% of all tuberculosis cases world-wide. However, due to lack of systematic analysis using multiple markers the available information on the genomic diversity of Mycobacterium tuberculosis in India is limited. METHODOLOGY/PRINCIPAL FINDINGS: Thus, 65 M. tuberculosis isolates from New Delhi, India were analyzed by spoligotyping, MIRU-VNTR, large deletion PCR typing and single nucleotide polymorphism analysis (SNP. The Central Asian (CAS 1 _DELHI sub-lineage was the most prevalent sub-lineage comprising 46.2% (n = 30 of all isolates, with shared-type (ST 26 being the most dominant genotype comprising 24.6% (n = 16 of all isolates. Other sub-lineages observed were: East-African Indian (EAI-5 (9.2%, n = 6, EAI6_BGD1 (6.2%, n = 4, EAI3_IND, CAS and T1 with 6.2% each (n = 4 each, Beijing (4.6%, n = 3, CAS2 (3.1%, n = 2, and X1 and X2 with 1 isolate each. Genotyping results from five isolates (7.7% did not match any existing spoligopatterns, and one isolate, ST124, belonged to an undefined lineage. Twenty-six percent of the isolates belonged to the TbD1+ PGG1 genogroup. SNP analysis of the pncA gene revealed a CAS-lineage specific silent mutation, S65S, which was observed for all CAS-lineage isolates (except two ST26 isolates and in 1 orphan. Mutations in the pncA gene, conferring resistance to pyrazinamide, were observed in 15.4% of all isolates. Collectively, mutations in the rpoB gene, the katG gene and in both rpoB and katG genes, conferring resistance to rifampicin and isoniazid, respectively, were more frequent in CAS1_DELHI isolates compared to non-CAS_DELHI isolates (OR: 3.1, CI95% [1.11, 8.70], P = 0.045. The increased frequency of drug-resistance could not be linked to the patients' history of previous anti-tuberculosis treatment (OR: 1.156, CI95% [0.40, 3.36], P = 0.79. Fifty-six percent of all new tuberculosis patients had mutations in either

  7. 结核分枝杆菌的耐药基因及其相关分子机制%The mycobacterium tuberculosis resistance genes and their molecular mechanism of drug resistance

    Institute of Scientific and Technical Information of China (English)

    伍静; 师长宏

    2011-01-01

    结核分枝杆菌原发性和继发性耐药是当前控制和治疗结核病面临的重要问题,随着分子遗传学的发展,已经阐明了结核分枝杆菌耐药的分子基础是染色体的突变,影响了药靶本身或激活了药物前体的细菌酶,造成MTB的耐药.本文主要就MTB对其常用药物的耐药机制展开讨论,以便正确认识MTB对不同药物的耐药机制,建立快速检测耐药结核分枝杆菌基因型的分子生物学方法.%The important issues of the current control and treat TB is primary and secondary drug resistance, Along with the development of molecular genetics, Have expound that the molecular basis of Mycobacterium tuberculosis drug resistance is due to the chromosome mutation And affecting the drug target itself or activated the premise of bacterial enzymes. Cause the MTB drug resistance. This paper mainly for its are commonly used drugs MTB discuss drug-resistant mechanism and correctly understanding of different drugs MTB resistance mechanism,and Establishing a rapid detection drug-resistant Mycobacteria tuberculosis genotype molecular biological detection method.

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

    Science.gov (United States)

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

    2015-02-01

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

  9. Multicenter evaluation of crystal violet decolorization assay (CVDA) for rapid detection of isoniazid and rifampicin resistance in Mycobacterium tuberculosis.

    Science.gov (United States)

    Coban, Ahmet Yilmaz; Akbal, Ahmet Ugur; Bicmen, Can; Albay, Ali; Sig, Ali Korhan; Uzun, Meltem; Selale, Deniz Sertel; Ozkutuk, Nuri; Surucuoglu, Suheyla; Albayrak, Nurhan; Ucarman, Nilay; Ozkutuk, Aydan; Esen, Nuran; Ceyhan, Ismail; Ozyurt, Mustafa; Bektore, Bayhan; Aslan, Gonul; Delialioğlu, Nuran; Alp, Alpaslan

    2016-12-16

    The aim of this multicenter study was to evaluate the performance of the crystal violet decolorization assay (CVDA) for detection of multidrug resistant tuberculosis (MDR-TB). This study was performed in 11 centers in two phases. A total of 156 isolates were tested for INH and RIF resistance. In the phase I, 106 clinical isolates were tested in the Center 1-7. In the phase 2, 156 clinical isolates were tested in the center 1-6, center 8-11. Eighty six of 156 tested isolates were the same in phase I. Agreements were 96.2-96.8% for INH and 98.1-98.7% for RIF in the phase I-II, respectively. Mean time to obtain the results in the phase I was 14.3 ± 5.4 days. In the phase II, mean time to obtain the results was 11.6 ± 3.5 days. Test results were obtained within 14days for 62.3% (66/106) of isolates in the phase I and 81.4% (127/156) of isolates in the phase II. In conclusion, CVDA is rapid, reliable, inexpensive, and easy to perform for rapid detection of MDR-TB isolates. In addition, it could be adapted for drug susceptibility testing with all drugs both in developed and developing countries.

  10. PET/CT imaging of Mycobacterium tuberculosis infection.

    NARCIS (Netherlands)

    Ankrah, Alfred; Werf, van der Tjip; de Vries, Erik; Dierckx, Rudi; Sathekge, M. M.; Glaudemans, Andor W.J.M.

    Tuberculosis has a high morbidity and mortality worldwide. Mycobacterium tuberculosis (Mtb) has a complex pathophysiology; it is an aerobic bacillus capable of surviving in anaerobic conditions in a latent state for a very long time before reactivation to active disease. In the latent tuberculosis

  11. Radiometric studies of mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Edwaldo E. Camargo

    1987-02-01

    Full Text Available An in vitro assay system that included automated radiometric quantification of 14CO2 released as a result of oxidation of 14C- substrates was applied for studying the metabolic activity of M. tuberculosis under various experimental conditions. These experiments included the study of a mtabolic pathways, b detection times for various inoculum sizes, c effect of filtration on reproducibility of results, d influence of stress environment e minimal inhibitory concentrations for isoniazid, streptomycin, ethambutol and rifampin, and f generation times of M. tuberculosis and M. bovis. These organisms were found to metabolize 14C-for-mate, (U-14C acetate, (U-14C glycerol, (1-14C palmitic acid, 1-14C lauric acid, (U-14C L-malic acid, (U-14C D-glucose, and (U-14C D-glucose, but not (1-14C L-glucose, (U-14C glycine, or (U-14C pyruvate to 14CO2. By using either 14C-for-mate, (1-14C palmitic acid, or (1-14C lauric acid, 10(7 organisms/vial could be detected within 24 48 hours and as few as 10 organisms/vial within 16-20 days. Reproducible results could be obtained without filtering the bacterial suspension, provided that the organisms were grown in liquid 7H9 medium with 0.05% polysorbate 80 and homogenized prior to the study. Drugs that block protein synthesis were found to have lower minimal inhibitory concentrations with the radiometric method when compared to the conventional agar dilution method. The mean generation time obtained for M. bovis and different strains of M. tuberculosis with various substrates was 9 ± 1 hours.

  12. Fluoroquinolone consumption and -resistance trends in Mycobacterium tuberculosis and other respiratory pathogens: Ecological antibiotic pressure and consequences in Pakistan, 2009–2015

    Directory of Open Access Journals (Sweden)

    S Shakoor

    2016-01-01

    Conclusion: We discuss the possible reasons for the decrease in resistance rates in TB, putative drivers of resistance other than volume of FQ consumption, and the possible impact of the National Tuberculosis Programme and drug regulatory activities.

  13. Predominant Mycobacterium tuberculosis Families and High Rates of Recent Transmission among New Cases Are Not Associated with Primary Multidrug Resistance in Lima, Peru.

    Science.gov (United States)

    Barletta, Francesca; Otero, Larissa; de Jong, Bouke C; Iwamoto, Tomotada; Arikawa, Kentaro; Van der Stuyft, Patrick; Niemann, Stefan; Merker, Matthias; Uwizeye, Cécile; Seas, Carlos; Rigouts, Leen

    2015-06-01

    Sputum samples from new tuberculosis (TB) cases were collected over 2 years as part of a prospective study in the northeastern part of Lima, Peru. To measure the contribution of recent transmission to the high rates of multidrug resistance (MDR) in this area, Mycobacterium tuberculosis complex (MTBc) isolates were tested for drug susceptibility to first-line drugs and were genotyped by spoligotyping and 15-locus mycobacterial interspersed repetitive-unit (MIRU-15)-variable-number tandem repeat (VNTR) analysis. MDR was found in 6.8% of 844 isolates, of which 593 (70.3%) were identified as belonging to a known MTBc lineage, whereas 198 isolates (23.5%) could not be assigned to these lineages and 12 (1.4%) represented mixed infections. Lineage 4 accounted for 54.9% (n = 463) of the isolates, most of which belonged to the Haarlem family (n = 279). MIRU-15 analysis grouped 551/791 isolates (69.7%) in 102 clusters, with sizes ranging from 2 to 46 strains. The overall high clustering rate suggests a high level of recent transmission in this population, especially among younger patients (odds ratio [OR], 1.6; P = 0.01). Haarlem strains were more prone to cluster, compared to the other families taken together (OR, 2.0; P rates did not differ between MDR and non-MDR strains (OR, 1.8; P = 0.1). Furthermore, only 16/51 MDR strains clustered with other MDR strains, suggesting that patients with primary MDR infections acquired the infections mostly from index cases outside the study population, such as retreated cases.

  14. Mycobacterium tuberculosis multi-drug-resistant strain M induces IL-17(+) IFNγ(-) CD4(+) T cell expansion through an IL-23 and TGF-β-dependent mechanism in patients with MDR-TB tuberculosis.

    Science.gov (United States)

    Basile, J I; Kviatcovsky, D; Romero, M M; Balboa, L; Monteserin, J; Ritacco, V; Lopez, B; Sabio y García, C; García, A; Vescovo, M; Montaner, P G; Palmero, D; Del Carmen Sasiain, M; de la Barrera, S

    2017-01-01

    We have reported previously that T cells from patients with multi-drug-resistant tuberculosis (MDR-TB) express high levels of interleukin (IL)-17 in response to the MDR strain M (Haarlem family) of Mycobacterium tuberculosis (M. tuberculosis). Herein, we explore the pathways involved in the induction of Th17 cells in MDR-TB patients and healthy tuberculin reactors [purified protein derivative healthy donors (PPD(+) HD)] by the M strain and the laboratory strain H37Rv. Our results show that IL-1β and IL-6 are crucial for the H37Rv and M-induced expansion of IL-17(+) interferon (IFN)-γ(-) and IL-17(+) IFN-γ(+) in CD4(+) T cells from MDR-TB and PPD(+) HD. IL-23 plays an ambiguous role in T helper type 1 (Th1) and Th17 profiles: alone, IL-23 is responsible for M. tuberculosis-induced IL-17 and IFN-γ expression in CD4(+) T cells from PPD(+) HD whereas, together with transforming growth factor (TGF-β), it promotes IL-17(+) IFN-γ(-) expansion in MDR-TB. In fact, spontaneous and M. tuberculosis-induced TGF-β secretion is increased in cells from MDR-TB, the M strain being the highest inducer. Interestingly, Toll-like receptor (TLR)-2 signalling mediates the expansion of IL-17(+) IFN-γ(-) cells and the enhancement of latency-associated protein (LAP) expression in CD14(+) and CD4(+) T cells from MDR-TB, which suggests that the M strain promotes IL-17(+) IFN-γ(-) T cells through a strong TLR-2-dependent TGF-β production by antigen-presenting cells and CD4(+) T cells. Finally, CD4(+) T cells from MDR-TB patients infected with MDR Haarlem strains show higher IL-17(+) IFN-γ(-) and lower IL-17(+) IFN-γ(+) levels than LAM-infected patients. The present findings deepen our understanding of the role of IL-17 in MDR-TB and highlight the influence of the genetic background of the infecting M. tuberculosis strain on the ex-vivo Th17 response.

  15. Mechanisms of drug action and resistance in Mycobacterium tuberculosis%抗结核药物的作用机制及结核分枝杆菌的耐药机理

    Institute of Scientific and Technical Information of China (English)

    张颖; 徐顺清; 李传友

    2004-01-01

    Tuberculosis (TB) is a global health problem that poses increasing threat with the spread of HIV infection and drug resistant strains of Mycobacterium tuberculosis. Effective control of TB remains a significant challenge despite the availability of chemotherapy and BCG vaccine. The emergence of strains of M. tuberculosis resistant to multiple anti-tuberculosis drugs is increasing due to inadequate compliance to the lengthy TB therapy and presents a significant problem for the treatment. In order to combat the threat of drug resistant tuberculosis and to more effectively control the disease, an understanding of the mechanisms underlying drug resistance is necessary. Mechanisms of action and resistance of major anti-tuberculosis drugs are reviewed in this article. This knowledge could be used for the development of molecular tests for rapid detection of drug resistant strains and for the design of new anti-tuberculosis drugs.%结核病是一个严重的全球性疾病,随着艾滋病病毒及耐药结核菌的出现及播散成为结核病控制的又一个威胁.虽然我们有结核疫苗及抗结核药物.但控制结核病仍是一件很棘手的事情.多重耐药结核菌的不断出现给结核病的治疗带来很大的困难.为有效控制耐药结核病.我们必须了解结核菌的耐药机理.本文讨论了抗结核药物的作用机制及结核菌的耐药机理.结核菌耐药机理的阐明对耐药菌的快速分子诊断及新药的开发有重要的意义.

  16. Protein export systems of Mycobacterium tuberculosis: novel targets for drug development?

    Science.gov (United States)

    Feltcher, Meghan E; Sullivan, Jonathan Tabb; Braunstein, Miriam

    2010-10-01

    Protein export is essential in all bacteria and many bacterial pathogens depend on specialized protein export systems for virulence. In Mycobacterium tuberculosis, the etiological agent of the disease tuberculosis, the conserved general secretion (Sec) and twin-arginine translocation (Tat) pathways perform the bulk of protein export and are both essential. M. tuberculosis also has specialized export pathways that transport specific subsets of proteins. One such pathway is the accessory SecA2 system, which is important for M. tuberculosis virulence. There are also specialized ESX export systems that function in virulence (ESX-1) or essential physiologic processes (ESX-3). The increasing prevalence of drug-resistant M. tuberculosis strains makes the development of novel drugs for tuberculosis an urgent priority. In this article, we discuss our current understanding of the protein export systems of M. tuberculosis and consider the potential of these pathways to be novel targets for tuberculosis drugs.

  17. Oxidative stress response genes in Mycobacterium tuberculosis: role of ahpC in resistance to peroxynitrite and stage-specific survival in macrophages.

    Science.gov (United States)

    Master, S S; Springer, B; Sander, P; Boettger, E C; Deretic, V; Timmins, G S

    2002-10-01

    The Mycobacterium tuberculosis ahpC gene, encoding the mycobacterial orthologue of alkylhydroperoxide reductase, undergoes an unusual regulatory cycle. The levels of AhpC alternate between stages of expression silencing in virulent strains grown as aerated cultures, secondary to a natural loss of the regulatory oxyR function in all strains of the tubercle bacillus, and expression activation in static bacilli by a yet undefined mechanism. The reasons for this unorthodox regulatory cycle controlling expression of an antioxidant factor are currently not known. In this work, M. tuberculosis H37Rv and Mycobacterium smegmatis mc(2)155 ahpC knockout mutants were tested for sensitivity to reactive nitrogen intermediates, in particular peroxynitrite, a highly reactive combinatorial product of reactive nitrogen and oxygen species, and sensitivity to bactericidal mechanisms in resting and activated macrophages. Both M. tuberculosis ahpC::Km(r) and M. smegmatis ahpC::Km(r) showed increased susceptibility to peroxynitrite. In contrast, inactivation of ahpC in M. tuberculosis did not cause increased sensitivity to donors of NO alone. M. tuberculosis ahpC::Km(r) also showed decreased survival in unstimulated macrophages, but the effect was no longer detectable upon IFNgamma activation. These studies establish a specific role for ahpC in antioxidant defences involving peroxynitrite and most likely additional cidal mechanisms in macrophages, with the regulatory cycle likely contributing to survival upon coming out of the stationary phase during dormancy (latent infection) or upon transmission to a new host.

  18. Rapid confirmation of drug susceptibility in Mycobacterium tuberculosis using MPT 64 Ag based test

    Directory of Open Access Journals (Sweden)

    Ernest Afu Ochang

    2013-06-01

    Full Text Available Objective: To evaluate the possible use of MPT64 based rapid test to detect multi-drug resistant Mycobacterium tuberculosis (M. tuberculosis in antibiotic broth dilution cultures. Methods: Twenty five isolates of M. tuberculosis whose susceptibility pattern had previously been identified by HAIN Genotype MTBDRplus (HainLifecience, Herhen, Germany were processed and cultured according to the microscopic-observation drug susceptibility technique. These included 20 susceptible, two multi-drug resistant M. tuberculosis and three isoniazid mono-resistant isolates. After 10-day incubation, aspirates from each well were tested with the MPT64 rapid test. Results: The rapid test correctly identified all 25 isolates and detected rifampicin and isoniazid resistance in all but one isoniazid mono-resistant isolate. Conclusions: MPT64 rapid test could be useful in detecting M. tuberculosis and drug resistance from Middlebrook 7H9 antimicrobial broth dilution in resource poor settings without an inverted microscope.

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

    Science.gov (United States)

    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.

  20. Diagnostic Plausibility of MTBDRplus and MTBDRsl Line Probe Assays for Rapid Drug Susceptibility Testing of Drug Resistant Mycobacterium tuberculosis Strains in Pakistan

    Directory of Open Access Journals (Sweden)

    Javaid

    2016-06-01

    Full Text Available Background World health organization (WHO recommends the use of line probe assays (LiPAs for rapid drug susceptibility testing (DST. However, only a limited number of studies from Pakistan have documented the performance characteristics of line probe assays in testing multi-drug resistant (MDR strains of Mycobacterium tuberculosis (MTB. Objectives The objective of this work is to evaluate the diagnostic plausibility of the LiPA tests MTBDRplus and MTBDRsl on MDR MTB isolates from Pakistan. Patients and Methods This was a cross-sectional study conducted at the Indus hospital, Karachi. LiPA testing was performed on 196 smear-positive samples using BACTEC MGIT 960 as a gold standard. Results The sensitivity of MTBDRplus for isoniazid and rifampicin was found to be 88.8% and 90.2%, respectively, while sensitivity of MTBDRsl for fluoroquinolones, amikacin/capreomycin, and ethambutol was found to be 72.9%, 81.8%, and 56.6%, respectively. Conclusions The MTBDRplus and MTBDRsl genotypic testing can serve as useful additional tools for DST in a high-burden country like Pakistan provided it is used in combination with phenotypic testing.

  1. Mycobacterium tuberculosis Multidrug-Resistant Strain M Induces Low IL-8 and Inhibits TNF-α Secretion by Bronchial Epithelial Cells Altering Neutrophil Effector Functions

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    Denise Kviatcovsky

    2017-01-01

    Full Text Available M strain, the most prevalent multidrug-resistant strain of Mycobacterium tuberculosis (Mtb in Argentina, has mounted mechanisms to evade innate immune response. The role of human bronchial epithelium in Mtb infection remains unknown as well as its crosstalk with neutrophils (PMN. In this work, we evaluate whether M and H37Rv strains invade and replicate within bronchial epithelial cell line Calu-6 and how conditioned media (CM derived from infected cells alter PMN responses. We demonstrated that M infects and survives within Calu-6 without promoting death. CM from M-infected Calu-6 (M-CM did not attract PMN in correlation with its low IL-8 content compared to H37Rv-CM. Also, PMN activation and ROS production in response to irradiated H37Rv were impaired after treatment with M-CM due to the lack of TNF-α. Interestingly, M-CM increased H37Rv replication in PMN which would allow the spreading of mycobacteria upon PMN death and sustain IL-8 release. Thus, our results indicate that even at low invasion/replication rate within Calu-6, M induces the secretion of factors altering the crosstalk between these nonphagocytic cells and PMN, representing an evasion mechanism developed by M strain to persist in the host. These data provide new insights on the role of bronchial epithelium upon M infection.

  2. Drug susceptibility patterns of the Mycobacterium tuberculosis isolated from previously treated and new cases of pulmonary tuberculosis at German-Nepal tuberculosis project laboratory, Kathmandu, Nepal

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    Gobinda Thapa

    2016-08-01

    Full Text Available Abstract Background Multidrug resistant tuberculosis (MDR-TB is a serious public health problem in Nepal. It is a major obstacle for the control of the tuberculosis. The main objectives of this study were to determine the prevalence of the multidrug resistant pulmonary tuberculosis and to evaluate the drug susceptibility patterns of Mycobacterium tuberculosis isolated from previously treated and newly diagnosed cases of pulmonary tuberculosis. Methods A cross-sectional study was conducted from March 2013 to August 2013 at German-Nepal tuberculosis project (GENETUP laboratory, Kathmandu, Nepal. For this the sputum samples from total of 153 (49 new and 104 previously treated suspected pulmonary tuberculosis patients were used. The diagnosis of the tuberculosis was performed by using fluorescent microscopy and culture, while the drug susceptibility testing of Mycobacterium tuberculosis was performed by proportion method. Lowenstein-Jensen (L-J medium was used for the culture of Mycobacterium tuberculosis and the colonies grown were identified on the basis of the colony morphology, pigment production and biochemical characteristics. Results The prevalence of MDR-TB among all the cases of culture positive pulmonary tuberculosis was 15.6 %. The rate of MDR-TB among previously treated culture positive tuberculosis patients was 19.4 % and that among newly diagnosed culture positive pulmonary tuberculosis cases was 7.1 %. The highest rate of resistance of Mycobacterium tuberculosis, was toward streptomycin (24.4 % followed by isoniazid (23 %, rifampicin (17.8 % and ethambutol (15.6 %. Among the total of MDR-TB cases among previously treated patients, highest percentage of the cases were relapse (61.1 % followed by chronic (16.7 %. Conclusions The high prevalence of DR/MDR-TB in our study reflects poor implementation of tuberculosis control program. On the basis of the drug susceptibility patterns of M. tuberculosis we found in our study, we

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

    Science.gov (United States)

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

    2015-10-01

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

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

    Science.gov (United States)

    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.

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

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    James P. Dalton

    2016-11-01

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

  6. pncA Gene Mutations Associated with Pyrazinamide Resistance in Drug-Resistant Tuberculosis, South Africa and Georgia.

    Science.gov (United States)

    Allana, Salim; Shashkina, Elena; Mathema, Barun; Bablishvili, Nino; Tukvadze, Nestani; Shah, N Sarita; Kempker, Russell R; Blumberg, Henry M; Moodley, Pravi; Mlisana, Koleka; Brust, James C M; Gandhi, Neel R

    2017-03-01

    Although pyrazinamide is commonly used for tuberculosis treatment, drug-susceptibility testing is not routinely available. We found polymorphisms in the pncA gene for 70% of multidrug-resistant and 96% of extensively drug-resistant Mycobacterium tuberculosis isolates from South Africa and Georgia. Assessment of pyrazinamide susceptibility may be prudent before using it in regimens for drug-resistant tuberculosis.

  7. Performance assessment of the GenoType MTBDRsl test and DNA sequencing for detection of second-line and ethambutol drug resistance among patients infected with multidrug-resistant Mycobacterium tuberculosis.

    Science.gov (United States)

    Huang, Wei-Lun; Chi, Ting-Lin; Wu, Mei-Hua; Jou, Ruwen

    2011-07-01

    The GenoType MTBDRsl test and DNA sequencing were used to rapidly detect second-line drug- and ethambutol (EMB)-resistant Mycobacterium tuberculosis. The ability of these two assays to detect the presence of mutations associated with resistance to fluoroquinolones (FLQ), aminoglycosides/cyclic peptide (AG/CP), and EMB in the gyrA, rrs, and embB genes (for the GenoType MTBDRsl test) and gyrA, gyrB, rrs, eis, embC, embA, embB, and embR genes (for DNA sequencing) was compared to that of conventional agar proportion drug susceptibility testing (DST). We evaluated 234 multidrug-resistant (MDR) M. tuberculosis isolates. The two molecular methods had high levels of specificity (95.8 to 100%). The sensitivities for FLQ resistance detection for both methods were 85.1%. For AG (kanamycin [KM] and amikacin [AM]) and CP (capreomycin CAP]), the sensitivities of resistance detection using the GenoType MTBDRsl test were 43.2%, 84.2%, and 71.4%, respectively, while with the inclusion of an extra gene, eis, in sequencing, the sensitivity reached 70.3% for detection of KM resistance. The sensitivities of EMB resistance detection were 56.2% and 90.7% with the GenoType MTBDRsl test and sequencing, respectively. We found that the GenoType MTBDRsl test can rapidly detect resistance to FLQ, CAP, and AM. The accuracy of the GenoType MTBDRsl test for the detection of FLQ and AM resistance was comparable to that of conventional DST; however, the test was less accurate for the detection of KM and EMB resistance and demonstrated a poor predictive value for CAP resistance. We recommend including new alleles consisting of the eis promoter and embB genes in molecular analysis. However, conventional DST is necessary to rule out false-negative results from molecular assays.

  8. 耐药结核分枝杆菌快速检测方法的研究进展%Research progress of rapid detection methods of drug-resistant Mycobacterium tuberculosis

    Institute of Scientific and Technical Information of China (English)

    崔运勇; 杨慧

    2010-01-01

    传统的耐药结核分枝杆菌的诊断依赖药物敏感试验.近年来,随着技术的进步,出现了大量的先进检测手段,在敏感性、特异性、检测周期方面皆优于传统方法.基于核酸扩增的分子生物学技术为临床耐药结核分枝杆菌的快速检测打开了一个广阔的前景.在自动测序分析的帮助下,耐药结核的确诊越来越简便、准确,对结核菌基因中耐药突变位点的快速检测还能够为临床诊疗提供早期的帮助信息,甚至能识别潜在的感染.本文对目前已有的快速检测方法的研究进展进行综述.%The diagnosis of drug-resistant Mycobacterium tuberculosis traditionally relies on drug sensitivity test. With the technology progress in recent years, there has been a large number of advanced detection methods, in which the sensitivity, specificity and test cycle are superior to those in traditional methods. The molecular biology technology based on nucleic acid amplification opens a broad prospect for rapid detection of drug-resistant Mycobacterium tuberculosis in clinic. With the help of automatic sequencing,the diagnosis of drug-resistant tuberculosis is becoming simpler and more accurate. The rapid detection of mutations in drug resistance genes of Mycobacterium tuberculosis can offer early information for clinical diagnosis and treatment, and even identify potential infections. This article reviews the research progress of rapid detection methods of drug-resistant Mycobacterium tuberculosis.

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

    Science.gov (United States)

    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.

  10. Structural enzymology of sulphur metabolism in Mycobacterium tuberculosis.

    Science.gov (United States)

    Schnell, Robert; Schneider, Gunter

    2010-05-21

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

  11. Benzothiazinones kill Mycobacterium tuberculosis by blocking arabinan synthesis.

    Science.gov (United States)

    Makarov, Vadim; Manina, Giulia; Mikusova, Katarina; Möllmann, Ute; Ryabova, Olga; Saint-Joanis, Brigitte; Dhar, Neeraj; Pasca, Maria Rosalia; Buroni, Silvia; Lucarelli, Anna Paola; Milano, Anna; De Rossi, Edda; Belanova, Martina; Bobovska, Adela; Dianiskova, Petronela; Kordulakova, Jana; Sala, Claudia; Fullam, Elizabeth; Schneider, Patricia; McKinney, John D; Brodin, Priscille; Christophe, Thierry; Waddell, Simon; Butcher, Philip; Albrethsen, Jakob; Rosenkrands, Ida; Brosch, Roland; Nandi, Vrinda; Bharath, Sowmya; Gaonkar, Sheshagiri; Shandil, Radha K; Balasubramanian, Venkataraman; Balganesh, Tanjore; Tyagi, Sandeep; Grosset, Jacques; Riccardi, Giovanna; Cole, Stewart T

    2009-05-08

    New drugs are required to counter the tuberculosis (TB) pandemic. Here, we describe the synthesis and characterization of 1,3-benzothiazin-4-ones (BTZs), a new class of antimycobacterial agents that kill Mycobacterium tuberculosis in vitro, ex vivo, and in mouse models of TB. Using genetics and biochemistry, we identified the enzyme decaprenylphosphoryl-beta-d-ribose 2'-epimerase as a major BTZ target. Inhibition of this enzymatic activity abolishes the formation of decaprenylphosphoryl arabinose, a key precursor that is required for the synthesis of the cell-wall arabinans, thus provoking cell lysis and bacterial death. The most advanced compound, BTZ043, is a candidate for inclusion in combination therapies for both drug-sensitive and extensively drug-resistant TB.

  12. Inositol monophosphate phosphatase genes of Mycobacterium tuberculosis

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    Parish Tanya

    2010-02-01

    Full Text Available Abstract Background Mycobacteria use inositol in phosphatidylinositol, for anchoring lipoarabinomannan (LAM, lipomannan (LM and phosphatidylinosotol mannosides (PIMs in the cell envelope, and for the production of mycothiol, which maintains the redox balance of the cell. Inositol is synthesized by conversion of glucose-6-phosphate to inositol-1-phosphate, followed by dephosphorylation by inositol monophosphate phosphatases (IMPases to form myo-inositol. To gain insight into how Mycobacterium tuberculosis synthesises inositol we carried out genetic analysis of the four IMPase homologues that are present in the Mycobacterium tuberculosis genome. Results Mutants lacking either impA (Rv1604 or suhB (Rv2701c were isolated in the absence of exogenous inositol, and no differences in levels of PIMs, LM, LAM or mycothiol were observed. Mutagenesis of cysQ (Rv2131c was initially unsuccessful, but was possible when a porin-like gene of Mycobacterium smegmatis was expressed, and also by gene switching in the merodiploid strain. In contrast, we could only obtain mutations in impC (Rv3137 when a second functional copy was provided in trans, even when exogenous inositol was provided. Experiments to obtain a mutant in the presence of a second copy of impC containing an active-site mutation, in the presence of porin-like gene of M. smegmatis, or in the absence of inositol 1-phosphate synthase activity, were also unsuccessful. We showed that all four genes are expressed, although at different levels, and levels of inositol phosphatase activity did not fall significantly in any of the mutants obtained. Conclusions We have shown that neither impA, suhB nor cysQ is solely responsible for inositol synthesis. In contrast, we show that impC is essential for mycobacterial growth under the conditions we used, and suggest it may be required in the early stages of mycothiol synthesis.

  13. Multicenter Evaluation of Anyplex Plus MTB/NTM MDR-TB Assay for Rapid Detection of Mycobacterium tuberculosis Complex and Multidrug-Resistant Isolates in Pulmonary and Extrapulmonary Specimens.

    Science.gov (United States)

    Sali, Michela; De Maio, Flavio; Caccuri, Francesca; Campilongo, Federica; Sanguinetti, Maurizio; Fiorentini, Simona; Delogu, Giovanni; Giagulli, Cinzia

    2016-01-01

    The rapid diagnosis of tuberculosis (TB) and the detection of drug-resistant Mycobacterium tuberculosis strains are critical for successful public health interventions. Therefore, TB diagnosis requires the availability of diagnostic tools that allow the rapid detection of M. tuberculosis and drug resistance in clinical samples. Here, we performed a multicenter study to evaluate the performance of the Seegene Anyplex MTB/NTM MDR-TB assay, a new molecular method based on a multiplex real-time PCR system, for detection of Mycobacterium tuberculosis complex (MTBC), nontuberculous mycobacteria (NTM), and genetic determinants of drug resistance. In total, the results for 755 samples (534 pulmonary and 221 extrapulmonary samples) were compared with the results of smears and cultures. For pulmonary specimens, the sensitivities of the Anyplex assay and acid-fast bacillus smear testing were 86.4% and 75.0%, respectively, and the specificities were 99% and 99.4%. For extrapulmonary specimens, the sensitivities of the Anyplex assay and acid-fast bacillus smear testing were 83.3% and 50.0%, respectively, and the specificities of both were 100%. The negative and positive predictive values of the Anyplex assay for pulmonary specimens were 97% and 100%, respectively, and those for extrapulmonary specimens were 84.6% and 100%. The sensitivities of the Anyplex assay for detecting isoniazid resistance in MTBC strains from pulmonary and extrapulmonary specimens were 83.3% and 50%, respectively, while the specificities were 100% for both specimen types. These results demonstrate that the Anyplex MTB/NTM MDR-TB assay is an efficient and rapid method for the diagnosis of pulmonary and extrapulmonary TB and the detection of isoniazid resistance.

  14. Mycobacterium tuberculosis replicates within necrotic human macrophages

    Science.gov (United States)

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

    2017-01-01

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

  15. Proteome-scale MDR-TB-antibody responses for identification of putative biomarkers for the diagnosis of drug-resistant Mycobacterium tuberculosis.

    Science.gov (United States)

    Yari, Shamsi; Hadizadeh Tasbiti, Alireza; Ghanei, Mostafa; Siadat, Seyed Davar; Yari, Fatemeh; Bahrmand, A

    2016-12-01

    Multidrug-resistant tuberculosis (MDR-TB) is caused by Mycobacterium tuberculosis strains that do not respond to isoniazid and rifampicin, the two most effective first-line anti-TB drugs. Here, we designed and produced antibodies based on biomarkers that exist only in MDR-TB. Bacilli were cultured for 4weeks at 37°C, and protein extraction was performed by sequential extraction. Bacterial cells were sonicated, centrifuged at 5000rpm for 45min, and the supernatant was collected and subjected to multiple rounds of treatment to prior to protein isolation. Protein concentration was determined using the Bradford method, and extracted proteins (50μg) from each strain (drug-sensitive- and MDR-TB isolates) were visualized on polyacrylamide gels (5-15%) with Coomassie Brilliant Blue R-250 staining. Three extracts were mixed and dialyzed against 0.1M ammonium bicarbonate (pH 8.0), followed by mass spectrometry. Specific polyclonal antibodies against purified MDR-TB proteins were purified by affinity chromatography and prepared in rabbits using three booster injections. The ELIZA test was performed for evaluation the antibody production. The antibody was treated with normal oral flora to remove any non specificity and cross reactivity. Analyses of different protein patterns (drug-sensitive- and MDR-TB) were performed by western blot. Our revealed that the MDR-TB strains contained specific antigens, and that the protein profiles of drug-sensitive TB strains differed from those of the MDR-TB isolates. Five bands from the MDR-TB fractions were detected as diagnostic antigens and were not observed in drug-sensitive-TB fractions. Western blot results showed that the MDR-TB antigenic fractions showed immunogenic bands at 50.0kDa and 70.0kDa, with the five antigenic MDR-TB-specific bands were identified as Rv3248c, Rv0350, Rv0440, Rv0475, and Rv3588c. Western blot data revealed dynamic properties of antibody responses that led to actionable findings for further research. Moreover

  16. Detection of fluoroquinolone resistance in Mycobacterium tuberculosis clinical isolates as determined by gyrA/B gene mutation by using PCR technique

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    A. Salah Eldin

    2012-10-01

    Conclusion: The incidence of FO-resistant M. tuberculosis is gradually increasing to alarming levels this may be due to wide spread use of this vital groups of drugs in community-acquired pneumonia and urinary tract infections.

  17. Molecular Detection and Drug Resistance of Mycobacterium tuberculosis Complex from Cattle at a Dairy Farm in the Nkonkobe Region of South Africa: A Pilot Study

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    Blessing Silaigwana

    2012-05-01

    Full Text Available Mycobacterium tuberculosis complex (MTBC causes tuberculosis (TB in humans and animals. We investigated the presence of MTBC in cattle milk and its drug resistance using polymerase chain reaction (PCR. Two hundred samples (100 mL each were obtained from a dairy farm in the Nkonkobe region of South Africa. The samples were processed using the modified Petroff method. DNA was isolated using a Zymo Bacterial DNA kit and amplified using Seeplex® MTB Nested ACE assay. The Genotype® Mycobacterium tuberculosis-multidrug resistantplus (MTBDRplus assay was used to perform drug susceptibility and detection of mutations conferring resistance to isoniazid (INH and rifampicin (RIF. Eleven samples tested positive for MTBC DNA using the Seeplex® MTB Nested ACE assay. The Genotype® MTBDRplus assay showed that 10/11 samples were resistant to both INH and RIF i.e., multi-drug resistant (MDR. The most and least frequent rpoB mutations detected in RIF resistant samples were H526Y (9/10 and D516V (2/10 respectively. None of the INH resistant samples harbored mutations in the katG gene. However, all of them harbored the T8A mutation in the inhA gene. These results have clinical and epidemiological significance and calls for further studies and necessary actions to delineate the situation.

  18. Multidrug resistant to extensively drug resistant tuberculosis: What is next?

    Indian Academy of Sciences (India)

    Amita Jain; Pratima Dixit

    2008-11-01

    Drug resistant tuberculosis is a man made problem. While tuberculosis is hundred percent curable, multidrug resistant tuberculosis (MDR-TB) is difficult to treat. Inadequate and incomplete treatment and poor treatment adherence has led to a newer form of drug resistance known as extensively drug resistant tuberculosis (XDR-TB). XDR-TB is defined as tuberculosis caused by Mycobacterium tuberculosis strain, which is resistant to at least rifampicin and isoniazid among the first line anti tubercular drugs (MDR-TB) in addition to resistance to any fluroquinolones and at least one of three injectable second line anti tubercular drugs i.e. amikacin, kanamycin and/or capreomycin. Mismanagement of tuberculosis paves the way to drug resistant tuberculosis. Emergence of XDR-TB is reported world wide. Reported prevalence rates of XDR-TB of total MDR cases are; 6.6% overall worldwide, 6.5% in industrialized countries, 13.6% in Russia and Eastern Europe, 1.5% in Asia, 0.6% in Africa and Middle East and 15.4% in Republic of Korea. Better management and control of tuberculosis specially drug resistant TB by experienced and qualified doctors, access to standard microbiology laboratory, co-morbitidy of HIV and tuberculosis, new anti-TB drug regimens, better diagnostic tests, international standards for second line drugs (SLD)-susceptibility testing, invention of newer anti-tubercular molecules and vaccines and knowing the real magnitude of XDR-TB are some of the important issues to be addressed for effective prevention and management of XDR-TB.

  19. Anti-mycobacterial activity of garlic (Allium sativum) against multi-drug resistant and non-multi-drug resistant mycobacterium tuberculosis.

    Science.gov (United States)

    Hannan, Abdul; Ikram Ullah, Muhammad; Usman, Muhammad; Hussain, Shahid; Absar, Muhammad; Javed, Khursheed

    2011-01-01

    Emergence of multi-drug resistant (MDR) and extensively drug resistant (XDR) TB throughout the developing world is very disturbing in the present scenario of TB management. There is a fundamental need to explore alternative anti-TB agents. Hence natural plants should be investigated to understand their antimicrobial properties and safety. Garlic (Allium sativum) is one of natural plant which possesses variety of biological properties like anti-tumor, anti-hyperlipedemic and anti-microbial etc. The present study was evaluated for anti-bacterial activity of garlic against non-MDR and MDR isolates of M. tuberculosis. A total of 20 clinical isolates of MTB including 15 MDR and 5 non-MDR were investigated. Ethanolic extract of garlic was prepared by maceration method. Minimum inhibitory concentration (MIC) was performed by using 7H9 middle brook broth dilution technique. MIC of garlic extract was ranged from 1 to 3 mg/ml; showing inhibitory effects of garlic against both non-MDR and MDR M. tuberculosis isolates. Alternate medicine practices with plant extracts including garlic should be considered to decrease the burden of drug resistance and cost in the management of diseases. The use of garlic against MDR-TB may be of great importance regarding public health.

  20. Genome Analysis of the First Extensively Drug-Resistant (XDR) Mycobacterium tuberculosis in Malaysia Provides Insights into the Genetic Basis of Its Biology and Drug Resistance.

    Science.gov (United States)

    Kuan, Chee Sian; Chan, Chai Ling; Yew, Su Mei; Toh, Yue Fen; Khoo, Jia-Shiun; Chong, Jennifer; Lee, Kok Wei; Tan, Yung-Chie; Yee, Wai-Yan; Ngeow, Yun Fong; Ng, Kee Peng

    2015-01-01

    The outbreak of extensively drug-resistant tuberculosis (XDR-TB) has become an increasing problem in many TB-burdened countries. The underlying drug resistance mechanisms, including the genetic variation favored by selective pressure in the resistant population, are partially understood. Recently, the first case of XDR-TB was reported in Malaysia. However, the detailed genotype family and mechanisms of the formation of multiple drugs resistance are unknown. We sequenced the whole genome of the UM 1072388579 strain with a 2-kb insert-size library and combined with that from previously sequenced 500-bp-insert paired-end reads to produce an improved sequence with maximal sequencing coverage across the genome. In silico spoligotyping and phylogenetic analyses demonstrated that UM 1072388579 strain belongs to an ancestral-like, non-Beijing clade of East Asia lineage. This is supported by the presence of a number of lineage-specific markers, including fadD28, embA, nuoD and pks7. Polymorphism analysis showed that the drug-susceptibility profile is correlated with the pattern of resistance mutations. Mutations in drug-efflux pumps and the cell wall biogenesis pathway such as mmpL, pks and fadD genes may play an important role in survival and adaptation of this strain to its surrounding environment. In this work, fifty-seven putative promoter SNPs were identified. Among them, we identified a novel SNP located at -4 T allele of TetR/acrR promoter as an informative marker to recognize strains of East Asian lineage. Our work indicates that the UM 1072388579 harbors both classical and uncommon SNPs that allow it to escape from inhibition by many antibiotics. This study provides a strong foundation to dissect the biology and underlying resistance mechanisms of the first reported XDR M. tuberculosis in Malaysia.

  1. Genome Analysis of the First Extensively Drug-Resistant (XDR Mycobacterium tuberculosis in Malaysia Provides Insights into the Genetic Basis of Its Biology and Drug Resistance.

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    Chee Sian Kuan

    Full Text Available The outbreak of extensively drug-resistant tuberculosis (XDR-TB has become an increasing problem in many TB-burdened countries. The underlying drug resistance mechanisms, including the genetic variation favored by selective pressure in the resistant population, are partially understood. Recently, the first case of XDR-TB was reported in Malaysia. However, the detailed genotype family and mechanisms of the formation of multiple drugs resistance are unknown. We sequenced the whole genome of the UM 1072388579 strain with a 2-kb insert-size library and combined with that from previously sequenced 500-bp-insert paired-end reads to produce an improved sequence with maximal sequencing coverage across the genome. In silico spoligotyping and phylogenetic analyses demonstrated that UM 1072388579 strain belongs to an ancestral-like, non-Beijing clade of East Asia lineage. This is supported by the presence of a number of lineage-specific markers, including fadD28, embA, nuoD and pks7. Polymorphism analysis showed that the drug-susceptibility profile is correlated with the pattern of resistance mutations. Mutations in drug-efflux pumps and the cell wall biogenesis pathway such as mmpL, pks and fadD genes may play an important role in survival and adaptation of this strain to its surrounding environment. In this work, fifty-seven putative promoter SNPs were identified. Among them, we identified a novel SNP located at -4 T allele of TetR/acrR promoter as an informative marker to recognize strains of East Asian lineage. Our work indicates that the UM 1072388579 harbors both classical and uncommon SNPs that allow it to escape from inhibition by many antibiotics. This study provides a strong foundation to dissect the biology and underlying resistance mechanisms of the first reported XDR M. tuberculosis in Malaysia.

  2. A high-throughput cidality screen for Mycobacterium tuberculosis.

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    Parvinder Kaur

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

  3. The arabinosyltransferase EmbC is inhibited by ethambutol in Mycobacterium tuberculosis.

    Science.gov (United States)

    Goude, R; Amin, A G; Chatterjee, D; Parish, T

    2009-10-01

    Ethambutol (EMB) is an antimycobacterial drug used extensively for the treatment of tuberculosis caused by Mycobacterium tuberculosis. EMB targets the biosynthesis of the cell wall, inhibiting the synthesis of both arabinogalactan and lipoarabinomannan (LAM), and is assumed to act via inhibition of three arabinosyltransferases: EmbA, EmbB, and EmbC. EmbA and EmbB are required for the synthesis of arabinogalactan, and at least one enzyme (M. tuberculosis EmbA [EmbA(Mt)]) is essential in M. tuberculosis. EmbC(Mt) is also essential for the viability of M. tuberculosis but is involved in the synthesis of LAM. We show that mutations in EmbC(Mt) that reduce its arabinosyltransferase activity result in increased sensitivity to EMB and the production of smaller LAM species in M. tuberculosis. Overexpression of EmbC(Mt) was not tolerated in M. tuberculosis, but overexpression of Mycobacterium smegmatis EmbC (EmbC(Ms)) led to EMB resistance and the production of larger LAM species in M. tuberculosis. Treatment of wild-type M. tuberculosis strains with EMB led to inhibition of LAM synthesis, resulting in the production of smaller species of LAM. In contrast, no change in LAM production was seen in EMB-resistant strains. Overexpression of EmbB(Ms) in M. tuberculosis also resulted in EMB resistance, but at a lower level than that caused by EmbC(Ms). Overexpression of EmbA(Mt) in M. tuberculosis had no effect on EMB resistance. Thus, there is a direct correlation between EmbC activity and EMB resistance, as well as between EmbC activity and the size of the LAM species produced, confirming that EmbC is one of the cellular targets of EMB action.

  4. Mycobacterium tuberculosis Beijing genotype induces differential cytokine production by peripheral blood mononuclear cells of healthy BCG vaccinated individuals.

    NARCIS (Netherlands)

    Rivera-Ordaz, A.; Gonzaga-Bernachi, J.; Serafin-Lopez, J.; Hernandez-Pando, R.; Soolingen, D. van; Estrada-Parra, S.; Estrada-Garcia, I.; Chacon-Salinas, R.

    2012-01-01

    Members of the Mycobacterium tuberculosis (Mtb) Beijing genotype are a major concern due to their high prevalence in tuberculosis patients and their high rate of multi-drug resistance. Although it has been shown that Beijing modifies macrophage behavior, little is known about how this genotype could

  5. Exposure of dentists to Mycobacterium tuberculosis, Ibadan, Nigeria

    NARCIS (Netherlands)

    Cadmus, S.I.; Okoje, V.N.; Taiwo, B.O.; Soolingen, D. van

    2010-01-01

    To determine the prevalence of Mycobacterium tuberculosis infection among dental patients and to assess dentists' risk for exposure, we conducted a study among dental patients at a large tertiary hospital in Nigeria, a country where tuberculosis is endemic. Ten (13%) of 78 sputum samples obtained we

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

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

    2017-03-01

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

  7. Benzothiazinones kill Mycobacterium tuberculosis by blocking arabinan synthesis

    DEFF Research Database (Denmark)

    Makarov, Vadim; Manina, Giulia; Mikusova, Katarina

    2009-01-01

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

  8. First evaluation of drug-resistant Mycobacterium tuberculosis clinical isolates from Congo revealed misdetection of fluoroquinolone resistance by line probe assay due to a double substitution T80A-A90G in GyrA.

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    Alexandra Aubry

    Full Text Available BACKGROUND: Tuberculosis (TB is one of the major public health problems in Congo. However, data concerning Mycobacterium tuberculosis drug resistance are lacking because of the insufficient processing capacity. So, the aim of this study was to investigate for the first time the resistance patterns and the strain lineages of a sample of M. tuberculosis complex (MTBC isolates collected in the two main cities of Congo. METHODS: Over a 9-day period, 114 smear-positive sputa isolated from 114 patients attending centers for the diagnosis and treatment of TB in Brazzaville and Pointe Noire were collected for culture and drug susceptibility testing (DST. Detection of mutations conferring drug resistance was performed by using line probe assays (GenoType MTBDRplus and MTBDRsl and DNA sequencing. Strain lineages were determined by MIRU-VNTR genotyping. RESULTS: Of the 114 sputa, 46 were culture positive for MTBC. Twenty-one (46% were resistant to one or more first-line antiTB drugs. Of these, 15 (71% were multidrug resistant (MDR. The most prevalent mutations involved in rifampin and isoniazid resistance, D516V (60% in rpoB and S315T (87% in katG respectively, were well detected by MTBDRplus assay. All the 15 MDR strains were susceptible to fluoroquinolone and injectable second-line drug. No mutation was detected in the rrs locus involved in resistance to amikacin and capreomycin by both the MTBDRsl assay and DNA sequencing. By contrast, 9 MDR strains belonging to the same cluster related to T-family were identified as being falsely resistant to fluoroquinolone by the MTBDRsl assay due to the presence of a double substitution T80A-A90G in GyrA. CONCLUSIONS: Taken together, these data revealed a possible spread of a particular MDR clone in Congo, misidentified as fluoroquinolone resistant by MTBDRsl assay. Thus, this test cannot replace gold-standard culture method and should be interpreted carefully in view of the patient's native land.

  9. Rapid detection of fluoroquinolone-resistant and heteroresistant Mycobacterium tuberculosis by use of sloppy molecular beacons and dual melting-temperature codes in a real-time PCR assay.

    Science.gov (United States)

    Chakravorty, Soumitesh; Aladegbami, Bola; Thoms, Kimberley; Lee, Jong Seok; Lee, Eun Gae; Rajan, Vignesh; Cho, Eun-Jin; Kim, Hyunchul; Kwak, Hyunkyung; Kurepina, Natalia; Cho, Sang-Nae; Kreiswirth, Barry; Via, Laura E; Barry, Clifton E; Alland, David

    2011-03-01

    Fluoroquinolones (FQ) are important second-line drugs to treat tuberculosis; however, FQ resistance is an emerging problem. Resistance has been mainly attributed to mutations in a 21-bp region of the Mycobacterium tuberculosis gyrA gene, often called the quinolone resistance-determining region (QRDR). We have developed a simple, rapid, and specific assay to detect FQ resistance-determining QRDR mutations. The assay amplifies the M. tuberculosis gyrA QRDR in an asymmetrical PCR followed by probing with two sloppy molecular beacons (SMBs) spanning the entire QRDR. Mutations are detected by melting temperature (T(m)) shifts that occur when the SMBs bind to mismatched sequences. By testing DNA targets corresponding to all known QRDR mutations, we found that one or both of the SMBs produced a T(m) shift of at least 3.6°C for each mutation, making mutation detection very robust. The assay was also able to identify mixtures of wild-type and mutant DNA, with QRDR mutants identified in samples containing as little as 5 to 10% mutant DNA. The assay was blindly validated for its ability to identify the QRDR mutations on DNA extracted from clinical M. tuberculosis strains. Fifty QRDR wild-type samples, 34 QRDR mutant samples, and 8 heteroresistant samples containing mixtures of wild-type and mutant DNA were analyzed. The results showed 100% concordance to conventional DNA sequencing, including a complete identification of all of the mixtures. This SMB T(m) shift assay will be a valuable molecular tool to rapidly detect FQ resistance and to detect the emergence of FQ heteroresistance in clinical samples from tuberculosis patients.

  10. Autophagy in Mycobacterium tuberculosis and HIV infections

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    Lucile eEspert

    2015-06-01

    Full Text Available Human Immunodeficiency Virus (HIV and Mycobacterium tuberculosis (M.tb are among the most lethal human pathogens worldwide, each being responsible for around 1.5 million deaths annually. Moreover, synergy between acquired immune deficiency syndrome (AIDS and tuberculosis (TB has turned HIV/M.tb co-infection into a major public health threat in developing countries. In the past decade, autophagy, a lysosomal catabolic process, has emerged as a major host immune defense mechanism against infectious agents like M.tb and HIV. Nevertheless, in some instances, autophagy machinery appears to be instrumental for HIV infection. Finally, there is mounting evidence that both pathogens deploy various countermeasures to thwart autophagy. This mini-review proposes an overview of the roles and regulations of autophagy in HIV and M.tb infections with an emphasis on microbial factors. We also discuss the role of autophagy manipulation in the context of HIV/M.tb co-infection. In future, a comprehensive understanding of autophagy interaction with these pathogens will be critical for development of autophagy-based prophylactic and therapeutic interventions for AIDS and TB.

  11. Human Lung Immunity against Mycobacterium tuberculosis

    Science.gov (United States)

    Schwander, Stephan; Dheda, Keertan

    2011-01-01

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

  12. SPECIFIC GYRB SEQUENCE OF MYCOBACTERIUM TUBERCULOSIS CLINICAL ISOLATED FROM SPUTUM OF PULMONARY TUBERCULOSIS PATIENTS IN INDONESIA

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    N. M. Mertaniasih

    2014-12-01

    Full Text Available Background: Indonesia have many different geographic areas which could be various on the variant strains of Mycobacterium tuberculosis. The gyrB gene codes GyrB protein as sub unit compound of Gyrase enzyme that functioning in multiplication of bacteria. Detection of gyrB gene could be a marker of active multiplication of viable bacteria in the specimen from patients; and some of the DNA sequence regions were conserved and specific in the strain of Mycobacterium tuberculosis that would be a marker for identification. This research aims to analyze the sequence of gyrB gene of Mycobacterium tuberculosis clinical isolates from sputum of pulmonary TB patients in Indonesia, and determine the specific region. Method: Mycobacterium tuberculosis clinical isolates have been collected from sputum of the patients with pulmonary TB that live in some area in Indonesia. Isolation and identification of Mycobacterium tuberculosis clinical isolates using standard culture method; sequence analysis using PCR-direct sequencing of the part bases region of gyrB. Results: this study revealed that nucleotide sequence on a fragment 764 bases of gyrB gene Mycobacterium tuberculosis strains among clinical isolates almost identically to a wild type strain Mycobacterium tuberculosis H37Rv and subspecies member of Mycobacterium tuberculosis complex (MTBC, with a little difference of SNPs; there are many difference nucleotide sequence with MOTT and Gram positive or negative bacteria, except Corynebacterium diphtheriae identically with MTBC. Conclusion: the gyrB sequence in Mycobacterium tuberculosis strains among these clinical isolates from sputum of pulmonary TB patients in Indonesia have the conserved specific DNA region that almost identically with wild type strain H37Rv and MTBC.

  13. Standartization of broth microdilution method for Mycobacterium tuberculosis

    OpenAIRE

    Clarice Queico Fujimura Leite; Ana Laura Remédio Zeni Beretta; Ivone Shizuko Anno; Maria Alice da Silva Telles

    2000-01-01

    Indirect drug susceptibility tests of Mycobacterium tuberculosis was done to investigate the accuracy and feasibility of a broth microdilution method (BMM) for determining minimal inhibitory concentrations of conventional drugs against M. tuberculosis. Test drugs included isoniazid (H), rifampicin (R), ethambutol (E), streptomycin (S) and pyrazinamide (Z). Fifty isolates of M. tuberculosis from patients who had never received drug therapy, and H37Rv strain for control, were evaluated in the s...

  14. First insight into genetic diversity of the Mycobacterium tuberculosis complex in Albania obtained by multilocus variable-number tandem-repeat analysis and spoligotyping reveals the presence of beijing multidrug-resistant isolates.

    Science.gov (United States)

    Tafaj, Silva; Zhang, Jian; Hauck, Yolande; Pourcel, Christine; Hafizi, Hasan; Zoraqi, Grigor; Sola, Christophe

    2009-05-01

    We characterized a set of 100 Mycobacterium tuberculosis complex clinical isolates from tuberculosis (TB) patients in Albania, typing them with a 24-locus variable-number tandem-repeat-spoligotyping scheme. Depending on the cluster definition, 43 to 49 patients were distributed into 15 to 16 clusters which were likely to be epidemiologically linked, indicative of a recent transmission rate of 28 to 34%. This result suggests that TB is under control in Albania. However, two multidrug-resistant (MDR) Beijing genotypes harboring the same S531A mutation on the rpoB gene were also found, suggesting a potential recent transmission of MDR TB. Three brand new genotypes, Albania-1 to Albania-3, are also described.

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

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    Viviana Ritacco

    2008-08-01

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

  16. Mechanisms of fluoroquinolone resistance in Mycobacterium tuberculosis%结核分枝杆菌耐氟喹诺酮类药物的分子机制研究进展

    Institute of Scientific and Technical Information of China (English)

    张玉娇; 李晓静; 米凯霞

    2016-01-01

    Tuberculosis, caused by the pathogen Mycobacterium tuberculosis, is one of the world’s deadliest bacterial infectious disease. It is still a global-health threat, particularly because of the drug-resistant forms. Fluoroquinolones, with target of gyrase, are among the drugs used to treat tuberculosis. However, their widespread use has led to bacterial resistance. The molecular mechanisms of fluoroquinolone resistance in mycobacterium tuberculosis have been reported, such as DNA gyrase mutations, drug efflux pumps system, bacterial cell wall thickness and pentapeptide proteins (MfpA) mediated regulation of gyrase. Mutations in gyrase conferring quinolone resistance play important roles and have been extensively studied. Recent studies have shown that the regulation of DNA gyrase affects mycobacterial drug resistance, but the mechanisms, especially by post-translational modification and regulatory proteins, are poorly understood. In this review, we summarize the fluoroquinolone drug development, and the molecular genetics of fluoroquinolone resistance in mycobacteria. Comprehensive understanding of the mechanisms of fluoroquinolone resistance in Mycobacterium tuberculosis will open a new view on understanding drug resistance in mycobacteria and lead to novel strategies to develop new accurate diagnosis methods.%结核病是由结核分枝杆菌(Mycobacterium tuberculosis)通过空气传播引起人类感染的慢性传染病,耐药结核分枝杆菌的流行是目前结核病防治的世界难题。氟喹诺酮类药物是人工合成药物,应用于耐药结核的临床治疗中,在治疗中起着核心的作用。但近年来,氟喹诺酮类药物的抗性菌株不断出现,愈发增加了结核病治疗的困难与治疗失败风险。在临床中氟喹诺酮药物的靶点比较清楚,是结核分枝杆菌的DNA旋转酶。目前发现结核分枝杆菌耐氟喹诺酮类药物的机制主要包括药物靶点DNA旋转酶的关键氨基酸改变、

  17. Assessment of the probability of introducing Mycobacterium tuberculosis into Danish cattle herds

    DEFF Research Database (Denmark)

    Foddai, Alessandro; Nielsen, Liza Rosenbaum; Krogh, Kaspar

    2015-01-01

    Tuberculosis is a zoonosis caused by Mycobacterium spp. International trade in cattle is regulated with respect to Mycobacterium bovis (M. bovis) but not Mycobacterium tuberculosis (M. tuberculosis), despite that cattle can become infected with both species. In this study we estimated the annual...

  18. Association between embB mutations and ethambutol resistance in Mycobacterium tuberculosis isolates from Cuba and the Dominican Republic: reproducible patterns and problems Asociación entre las mutaciones en embB y la resistencia a etambutol en aislamientos de Mycobacterium tuberculosis de Cuba y República Dominicana: patrones y problemas reproducibles

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    Elba Guerrero

    2013-03-01

    Full Text Available The relation of ethambutol resistance to embB mutations remains unclear, and there are no reports on ethambutol resistance from the Caribbean. We examined the sequence of embB in 57 distinct Multi-Drug Resistant (MDR and non-MDR strains of Mycobacterium tuberculosis, mostly from Cuba and the Dominican Republic. embB306 codon mutations were found exclusively in MDR-TB, but in both ethambutol sensitive and resistant strains. Valine substitutions predominated in ethambutol resistant strains, while isoleucine replacements were more common in sensitive strains. Three ethambutol resistant MDR strains without embB306 substitutions had replacements in embB406 or embB497, but these were also found in ethambutol sensitive MDR strains. The results confirm previous findings that amino acid substitutions in EmbB306, EmbB406 and EmbB497 are found only in MDR-TB strains but in both phenotypically resistant and sensitive strains. One ethambutol resistant non-MDR strain did not have any embB mutation suggesting that other undefined mutations can also confer ethambutol resistance.La relación entre resistencia a etambutol y mutaciones en embB no se ha establecido claramente y no existen comunicaciones sobre la resistencia a etambutol en cepas provenientes de países Caribeños. Se evaluó la secuencia del gen embB en 57 cepas de Mycobacterium tuberculosis multi-drogo resistentes (MDR y no-MDR, la mayoría aislada en Cuba y República Dominicana. Se encontraron mutaciones en el codón embB306 exclusivamente en cepas MDR, tanto en cepas sensibles como resistentes a etambutol. Tres cepas MDR resistentes a etambutol, sin sustituciones en embB306, tenían mutaciones en los codones embB406 o embB497, pero estos cambios se encontraron también en cepas sensibles. Los resultados confirman otros estudios, mostrando que sustituciones aminoacídicas en EmbB306, EmbB406 y EmbB497 se encuentran exclusivamente en cepas MDR, tanto resistentes como sensibles a etambutol

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

    Science.gov (United States)

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

    2012-11-01

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

  20. PolyTB: A genomic variation map for Mycobacterium tuberculosis

    KAUST Repository

    Coll, Francesc

    2014-02-15

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

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

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    Eman Aleksic

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

  2. Cutting edge: Vitamin D regulates lipid metabolism in Mycobacterium tuberculosis infection.

    Science.gov (United States)

    Salamon, Hugh; Bruiners, Natalie; Lakehal, Karim; Shi, Lanbo; Ravi, Janani; Yamaguchi, Ken D; Pine, Richard; Gennaro, Maria Laura

    2014-07-01

    Vitamin D has long been linked to resistance to tuberculosis, an infectious respiratory disease that is increasingly hard to treat because of multidrug resistance. Previous work established that vitamin D induces macrophage antimicrobial functions against Mycobacterium tuberculosis. In this article, we report a novel, metabolic role for vitamin D in tuberculosis identified through integrated transcriptome and mechanistic studies. Transcriptome analysis revealed an association between vitamin D receptor (VDR) and lipid metabolism in human tuberculosis and infected macrophages. Vitamin D treatment of infected macrophages abrogated infection-induced accumulation of lipid droplets, which are required for intracellular M. tuberculosis growth. Additional transcriptomics results showed that vitamin D downregulates the proadipogenic peroxisome proliferator-activated receptor γ (PPARγ) in infected macrophages. PPARγ agonists reversed the antiadipogenic and the antimicrobial effects of VDR, indicating a link between VDR and PPARγ signaling in regulating both vitamin D functions. These findings suggest the potential for host-based, adjunct antituberculosis therapy targeting lipid metabolism.

  3. Mycobacterium marinum: a potential immunotherapy for Mycobacterium tuberculosis infection

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    Tian WW

    2013-07-01

    Full Text Available Wei-wei Tian,1 Qian-qiu Wang,1 Wei-da Liu,2 Jian-ping Shen,1 Hong-sheng Wang11Laboratory of Mycobacterial Disease, Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs, Nanjing, Jiangsu, People’s Republic of China; 2Department of Mycology, Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs, Nanjing, Jiangsu, People’s Republic of ChinaPurpose: The aim of the present study was to investigate the immune response induced by Mycobacterium marinum infection in vitro and the potential of M. marinum as an immunotherapy for M. tuberculosis infection.Methods: The potential human immune response to certain bacillus infections was investigated in an immune cell–bacillus coculture system in vitro. As a potential novel immunotherapy, M. marinum was studied and compared with two other bacilli, Bacillus Calmette-Guérin (BCG and live attenuated M. tuberculosis. We examined the changes in both the bacilli and immune cells, especially the time course of the viability of mycobacteria in the coculture system and host immune responses including multinuclear giant cell formation by Wright–Giemsa modified staining, macrophage polarization by cell surface antigen expression, and cytokines/chemokine production by both mRNA expression and protein secretion.Results: The M. marinum stimulated coculture group showed more expression of CD209, CD68, CD80, and CD86 than the BCG and M. tuberculosis (an attenuated strain, H37Ra groups, although the differences were not statistically significant. Moreover, the M. marinum group expressed more interleukin (IL-1B and IL-12p40 on day 3 (IL-1B: P = 0.003 and 0.004, respectively; IL-12p40: P = 0.001 and 0.011, respectively, a higher level of CXCL10 on day 1 (P = 0.006 and 0.026, respectively, and

  4. Early detection of multi-drug resistance and common mutations in Mycobacterium tuberculosis isolates from Delhi using GenoType MTBDRplus assay

    OpenAIRE

    2015-01-01

    Purpose: There is scarcity of prevalence data of multi-drug-resistant tuberculosis (MDR-TB) data and common mutations responsible in North India. This study aimed to detect MDR-TB among MDR-TB suspects from Delhi and mutation patterns using GenoType MTBDRplus assay. Materials and Methods: All MDR suspects in five districts of New Delhi were referred to the laboratory from 1 st October 2011 to 31 st December 2012 as per criterion defined by Programmatic Management of Drug Resistant Tuberculosi...

  5. Direct detection of multidrug-resistant Mycobacterium tuberculosis in clinical specimens in low- and high-incidence countries by line probe assay

    DEFF Research Database (Denmark)

    Johansen, Isik Somuncu; Lundgren, Bettina; Sosnovskaja, Anaida;

    2003-01-01

    The INNO-LiPA Rif.TB assay is designed for the detection of rpoB gene mutations causing rifampin resistance in isolates. We applied the method directly to 60 Lithuanian and Danish clinical specimens to detect rifampin resistance rapidly. Results were obtained in 78.3% of clinical specimens, and a...... were concordant with those obtained by BACTEC 460. The assay could have major impact on the management of multidrug-resistant tuberculosis....

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

    Science.gov (United States)

    Mayer-Barber, Katrin D; Barber, Daniel L

    2015-07-17

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

  7. Application of genotype MTBDRplus in rapid detection of the Mycobacterium tuberculosis complex as well as its resistance to isoniazid and rifampin in a high volume laboratory in Southern China.

    Science.gov (United States)

    Zhang, Lei; Ye, Yuanxing; Duo, Lina; Wang, Tingting; Song, Xingbo; Lu, Xiaojun; Ying, Binwu; Wang, Lanlan

    2011-03-01

    The alarmingly worsening epidemics of drug-resistant tuberculosis (TB) call urgent need for a simple method for the rapid detection of drug-resistant TB in clinical settings. In an attempt to establish a rapid procedure for laboratory diagnosis of TB and investigate the local TB epidemiology, molecular line probe assay of the Genotype MTBDRplus was used to identify Mycobacterium tuberculosis complex (MTBC) and detect mutations conferring resistance to two most active first-line drugs against TB: Rifampin and Isoniazid. 96 acid-fast bacillus (AFB) smear- positive sputums and 18 PCR-positive non-sputum specimens have been determined for the MTBC and resistance to Rifampin and Isoniazid. The MTBC detection rates in two sources of specimens were 93.8% (90/96) and 77.8% (14/18) respectively. The overall drug resistance (Rifampin or Isoniazid) occurred in 34.6% (36/104). Resistance to rifampin (RMP) was 28.8% (30/104) and 25% (26/104) was to Isoniazid (INH), in which high level drug resistance accounted for 88.5% (23/26) and low level drug resistance accounted for 7.7% (2/26). Multidrug resistance (MDR), defined as resistant to both RMP and INH, was found in 19.2% (20/104) of clinical samples, which was double that of official statistics. In addition, 63.3% (19/30) RMP-resistant mutations were identified in the region of RopB 530-533 and 57.9% (11/19) were the S531L mutation. 84.6% (22/26) of resistance to INH was mediated by Kat S315T1 mutations which conferred the high-level resistance to INH. The Genotype MTBDRplus line probe assay is a suitable and applicable method for establishing the rapidness in detection of drug-resistant TB in clinical laboratory. It will be a valuable addition to the conventional TB diagnostic approaches.

  8. Whole genome sequencing-based characterization of extensively drug resistant (XDR strains of Mycobacterium tuberculosis from Pakistan

    Directory of Open Access Journals (Sweden)

    Zahra Hasan

    2015-01-01

    Conclusions: If common drug resistance associated with SNPs evaluated the concordance between phenotypic and genotypic testing, the results would be rifampicin (100%, isoniazid (89%, fluoroquinolones (95%, aminoglycoside (81% and ethambutol (61%. This work highlights the importance of expanded targets for drug resistance detection in MTB isolates.

  9. Drug-resistant tuberculosis: time for visionary political leadership.

    Science.gov (United States)

    Abubakar, Ibrahim; Zignol, Matteo; Falzon, Dennis; Raviglione, Mario; Ditiu, Lucica; Masham, Susan; Adetifa, Ifedayo; Ford, Nathan; Cox, Helen; Lawn, Stephen D; Marais, Ben J; McHugh, Timothy D; Mwaba, Peter; Bates, Matthew; Lipman, Marc; Zijenah, Lynn; Logan, Simon; McNerney, Ruth; Zumla, Adam; Sarda, Krishna; Nahid, Payam; Hoelscher, Michael; Pletschette, Michel; Memish, Ziad A; Kim, Peter; Hafner, Richard; Cole, Stewart; Migliori, Giovanni Battista; Maeurer, Markus; Schito, Marco; Zumla, Alimuddin

    2013-06-01

    Two decades ago, WHO declared tuberculosis a global emergency, and invested in the highly cost-effective directly observed treatment short-course programme to control the epidemic. At that time, most strains of Mycobacterium tuberculosis were susceptible to first-line tuberculosis drugs, and drug resistance was not a major issue. However, in 2013, tuberculosis remains a major public health concern worldwide, with prevalence of multidrug-resistant (MDR) tuberculosis rising. WHO estimates roughly 630 000 cases of MDR tuberculosis worldwide, with great variation in the frequency of MDR tuberculosis between countries. In the past 8 years, extensively drug-resistant (XDR) tuberculosis has emerged, and has been reported in 84 countries, heralding the possibility of virtually untreatable tuberculosis. Increased population movement, the continuing HIV pandemic, and the rise in MDR tuberculosis pose formidable challenges to the global control of tuberculosis. We provide an overview of the global burden of drug-resistant disease; discuss the social, health service, management, and control issues that fuel and sustain the epidemic; and suggest specific recommendations for important next steps. Visionary political leadership is needed to curb the rise of MDR and XDR tuberculosis worldwide, through sustained funding and the implementation of global and regional action plans.

  10. Escape of Mycobacterium tuberculosis from oxidative killing by neutrophils.

    Science.gov (United States)

    Corleis, Björn; Korbel, Daniel; Wilson, Robert; Bylund, Johan; Chee, Ronnie; Schaible, Ulrich E

    2012-07-01

    Neutrophils enter sites of infection, where they can eliminate pathogenic bacteria in an oxidative manner. Despite their predominance in active tuberculosis lesions, the function of neutrophils in this important human infection is still highly controversial. We observed that virulent Mycobacterium tuberculosis survived inside human neutrophils despite prompt activation of these defence cells' microbicidal effectors. Survival of M. tuberculosis was accompanied by necrotic cell death of infected neutrophils. Necrotic cell death entirely depended on radical oxygen species production since chronic granulomatous disease neutrophils were protected from M. tuberculosis-triggered necrosis. More, importantly, the M. tuberculosis ΔRD1 mutant failed to induce neutrophil necrosis rendering this strain susceptible to radical oxygen species-mediated killing. We conclude that this virulence function is instrumental for M. tuberculosis to escape killing by neutrophils and contributes to pathogenesis in tuberculosis.

  11. Cavitary Lesions and Nodular Infiltration Patterns in Multi-Drug Resistant TB and Non-Tuberculosis Mycobacterium: Comparison of Chest CT

    Directory of Open Access Journals (Sweden)

    Shahram Kahkouee

    2010-05-01

    Full Text Available Background/Objective: By increasing of HIV frequency, MDRTB has also increased. Clinical and laboratory findings of multi-drug resistant TB (MDRTB and non-tuberculosis mycobacterium (NTM are very similar. Maybe imaging findings help early differentiation before culture result. This study has compared cavitary lesions and nodular infiltration patterns in the chest CT of MDRTB and NTM."nPatients and Methods: A retrospective study was performed on 66 hospitalized patients (43 MDRTB and 23 NTM in Masih Daneshvari Hospital (from 2006-2009. Lung CT scans were evaluated by a radiologist and two radiology residents without any information about the culture result. Nodular infiltrations were classified into 6 patterns (tree in bud, scattered nodule, lobar nodular infiltration, cavitary nodule, macronodule, miliary pattern and cavitary lesions to thick and thin, single and multiple with lobar distribution and satellite nodule adjacent to cavity. Chi-square statistics analysis was performed."nResult: Respectively in NTM and MDRTB: Mean age (51.5%-44.8%, sex (34.8% M and 65.2 F, 58.1% M and 41.9% F, treatment history (56.5%,83.7%, scattered nodular infiltration (47.8%, 60.5%, lobar nodular infiltration (13%, 9.3%, TIB (47.8%, 46.5%, miliary pattern (0%, 0%, cavitary nodule (21.7%, 27.9%, macro nodule (nodule≥10mm (43.5%, 25.6%, cavity (69.6%, 76.7%, thin wall cavity (65.2%, 46.5%, thick wall cavity (26.1%, 58.1%, satellite nodule adjacent to cavity (39.1%, 9.3%, single cavity (13%, 14%, lobar distribution of single cavity: RUL (8.7%, 4.7%, RML (0%, 0%, RLL (0%, 0%, LUL (4.3%, 4.7%, lingual (0%, 0%, LLL (0%, 4.7%, multiple cavity (56.5%, 60.5%, lobar distribution of multiple cavity: RUL (26.1%, 51.2%, RML (21.7%, 9.3%, RLL (26.1%, 23.3%, LUL (47.8%, 44.2%, lingual (26.1%, 18.6%, LLL (30.4%, 16.3%."nConclusion: Treatment history (p=0.016, satellite nodule adjacent to cavity (p=0.004, thick wall cavity (p=0.013 and multiple cavity in RUL (p=0.05 are more

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

    Science.gov (United States)

    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...

  13. Structural Basis of Mycobacterium tuberculosis Transcription and Transcription Inhibition.

    Science.gov (United States)

    Lin, Wei; Mandal, Soma; Degen, David; Liu, Yu; Ebright, Yon W; Li, Shengjian; Feng, Yu; Zhang, Yu; Mandal, Sukhendu; Jiang, Yi; Liu, Shuang; Gigliotti, Matthew; Talaue, Meliza; Connell, Nancy; Das, Kalyan; Arnold, Eddy; Ebright, Richard H

    2017-04-20

    Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, which kills 1.8 million annually. Mtb RNA polymerase (RNAP) is the target of the first-line antituberculosis drug rifampin (Rif). We report crystal structures of Mtb RNAP, alone and in complex with Rif, at 3.8-4.4 Å resolution. The results identify an Mtb-specific structural module of Mtb RNAP and establish that Rif functions by a steric-occlusion mechanism that prevents extension of RNA. We also report non-Rif-related compounds-Nα-aroyl-N-aryl-phenylalaninamides (AAPs)-that potently and selectively inhibit Mtb RNAP and Mtb growth, and we report crystal structures of Mtb RNAP in complex with AAPs. AAPs bind to a different site on Mtb RNAP than Rif, exhibit no cross-resistance with Rif, function additively when co-administered with Rif, and suppress resistance emergence when co-administered with Rif. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    LENUS (Irish Health Repository)

    Ojo, Olabisi O

    2010-10-01

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

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

    Science.gov (United States)

    Ojo, Olabisi O; Sheehan, Stella; Corcoran, Daniel G; Nikolayevsky, Vladyslav; Brown, Timothy; O'Sullivan, Margaret; O'Sullivan, Kathleen; Gordon, Stephen V; Drobniewski, Francis; Prentice, Michael B

    2010-10-01

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

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

    Science.gov (United States)

    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 mutation in the rpoB gene were significantly associated with mixed infections (χ(2), 56.78; P mutation in the rpoB gene to become rifampin resistant. Further studies should focus on this lineage to clarify its relevance to mixed infections.

  17. Osteomielite esternal por Mycobacterium tuberculosis Sternal osteomyelitis caused by infection with Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Diego Michelon De Carli

    2009-07-01

    Full Text Available Descrevemos o caso de um paciente de 74 anos, masculino, com dor torácica na porção superior do esterno com um ano de evolução associada a eritema, edema e fístula com drenagem de material purulento. Paciente HIV negativo e sem história prévia de contato com TB. A TC de tórax evidenciou lesão osteolítica esternal, e o material de biópsia revelou granuloma caseoso negativo para fungos e bacilos álcool-ácido resistentes no exame microbiológico direto. O diagnóstico de osteomielite esternal por Mycobacterium tuberculosis foi realizado por PCR.We report the case of a 74-year-old male patient with a one-year history of chest pain in the suprasternal notch associated with erythema, edema and drainage of purulent material from a fistulous lesion. The patient was HIV-negative with no history of TB. A CT scan of the chest showed an osteolytic lesion in the sternum, and a biopsy revealed caseous granuloma, which, in the microbiological evaluation, was negative for fungi and acid-fast bacilli. The diagnosis of sternal osteomyelitis caused by Mycobacterium tuberculosis was confirmed using PCR.

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

    DEFF Research Database (Denmark)

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

    2002-01-01

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

  19. At Baltic crossroads: a molecular snapshot of Mycobacterium tuberculosis population diversity in Kaliningrad, Russia.

    Science.gov (United States)

    Mokrousov, Igor; Otten, Tatiana; Zozio, Thierry; Turkin, Eugeni; Nazemtseva, Vera; Sheremet, Aleksandra; Vishnevsky, Boris; Narvskaya, Olga; Rastogi, Nalin

    2009-01-01

    The Kaliningrad region is the westernmost part of the Russian Federation; it includes an enclave on the Baltic Sea inside the European Union separated from mainland Russia by Lithuania and Poland. The incidence of tuberculosis in Kaliningrad has shown a steady and dramatic increase from 83/100,000 in 2000 to 134/100,000 in 2006; the rate of multidrug-resistant tuberculosis (MDR-tuberculosis) in the Kaliningrad region was reported to be 30.5% among newly diagnosed tuberculosis patients. This study presents a first molecular snapshot of the population diversity of Mycobacterium tuberculosis in this region. A total of 90 drug-resistant and susceptible M. tuberculosis strains from Kaliningrad were subjected to spoligotyping, 12-locus MIRU typing and mutation analysis of the drug resistance genes rpoB and katG. A comparison with international databases showed that the M. tuberculosis population in this region shares a joint pool of strains with the European part of Russia, and also exhibits a certain affinity with those of its northern European neighbours, such as Poland and Germany. Comparison of the genotyping and drug resistance data emphasized that the high prevalence of the MDR Beijing genotype strains is a major cause of the adverse epidemiological situation of MDR-tuberculosis in the Kaliningrad region.

  20. Pyrosequencing for rapid detection of resistance to rifampin, isoniazid, ofloxacin and amikacin in Mycobacterium tuberculosis%焦磷酸测序技术检测结核分枝杆菌四种药物耐药性

    Institute of Scientific and Technical Information of China (English)

    郑瑞娟; 秦莲花; 周燕; 王洁; 乐军; 胡忠义

    2011-01-01

    Objective To develop an assay to determine Mycobacterium tuberculosis resistance to rifampin, isoniazid, ofloxacin and amikacin by pyrosequencing and evaluate the value of this method in clinical application. Methods Eighty-nine clinical isolates of Mycobacterium tuberculosis from tuberculosis patients were collected from Shanghai Pulmonary Hospital during 2008 to 2009. All strains were isolated from decontaminated sputum, cultured on Lowenstein-Jensen media and identified by traditional biochemical tests with standard methods. Ten Mycobacterium tuberculosis were selected from the strain bank of Shanghai Pulmonary Hospital. The optimal conditions of detection of rpoB, katG, gyrA and rrs gene by pyroseuencing were determined, using the 10 Mycobacterium tuberculosis strains whose drug susceptibility of Bactec 960 and sequence of rpoB, katG, gyrA, rrs gene were known. Then the drug susceptibility of 89 Mycobacterium tuberculosis clinical isolate strains were detected by pyrosequencing using this conditions and the results were compared with that of the Bactec 960 methods. Results The pyrosequencing program of sequence analysis was suitable for the detection of the mutations of rpoB and gyrA genes, and the program of single nucleotide polymorphism was suitable for katG and rrs genes. Among the 89 Mycobacterium tuberculosis clinical isolates,when using the drug susceptibility of Bactec 960 as the standard, the sensitivity of rifampin, isoniazid,ofloxacin and amikacin is 98.0%, 64. 1%, 79.5%, 78. 3% respectively, the specificity is 97.5%,100. 0%, 90. 0%, 100. 0% respectively, the accuracy is 97.8%, 77. 5%, 85.4%, 94. 4% respectively,tested by pyrosequencing. The results of the detection of resistance to rifampin, isoniazid, ofloxacin and amikacin in Mycobacterium tuberculosis using pyrosequencing technique were almost the same with that of Bactec 960, and Kappa ≥0. 7 in each detection. Conclusion Pyrosequencing is thus a rapid, accurate and high throughput method for

  1. Luciferase reporter phage phAE85 for rapid detection of rifampicin resistance in clinical isolates of Mycobacterium tuberculosis

    Institute of Scientific and Technical Information of China (English)

    Gomathi Sivaramakrishnan; Balaji Subramanyam; Ponnuraja C; Vanaja Kumar

    2013-01-01

    Objective:To evaluate luciferase reporter phage (LRP) phAE85 in rapid detection of rifampicin resistance in a region where TB is endemic. Methods:One hundred and ninety primary isolates on Lowenstein-Jensen medium were tested. Middlebrook 7H9 complete medium with and without rifampicin at 2μg/mL was inoculated with standard inoculum from suspensions of the clinical isolate. After incubation for 72 h, LRP was added. Following 4 h of further incubation, light output from both control and test was measured as relative light units. Strains exhibiting a reduction of less than 50%relative light units in the drug containing vial compared to control were classified as resistant. Results were compared with the conventional minimum inhibitory concentration method (MIC) of drug susceptibility testing. Results:The two methods showed high level of agreement of 97% (CI 0.94, 0.99) and P value was 0.000 1. The sensitivity and specificity of LRP assay for detection of rifampicin resistance were 91%(CI 0.75, 0.98) and 99%(CI 0.95, 1.00) respectively. Time to detection of resistance by LRP assay was 3 d in comparison with 28 d by the minimum inhibitory concentration method. Conclusions: LRP assay with phAE85 is 99%specific, 91%sensitive and is highly reproducible. Thus the assay offers a simple procedure for drug sensitivity testing, within the scope of semi-automation.

  2. Interaction of wild type, G68R and L125M isoforms of the arylamine-N-acetyltransferase from Mycobacterium tuberculosis with isoniazid: a computational study on a new possible mechanism of resistance.

    Science.gov (United States)

    Ramos, Ricardo Martins; Perez, Janaína Menezes; Baptista, Luis André; de Amorim, Hermes Luís Neubauer

    2012-09-01

    Isoniazid (INH) is a front-line drug used in the treatment of tuberculosis (TB), a disease that remains a major cause of death worldwide. Isoniazid is a prodrug, requiring activation in the mycobacterial cell by the catalase-peroxidase (CP) enzyme. Recent studies have suggested that acetylation of INH by the arylamine-N-acetyltransferase from Mycobacterium tuberculosis (TBNAT) may be a possible cause of inactivation of the drug thus resulting in resistant strains. In this study, computational techniques were applied to investigate the binding of isoniazid to three TBNAT isoforms: wild type, G68R and L125M. Since there is no experimental structure available, molecular dynamics (MD) simulations were initially used for the refinement of TBNAT homology models. Distinct conformations of the models were selected during the production stage of MD simulations for molecular docking experiments with the drug. Finally, each mode of binding was refined by new molecular MD simulations. Essential dynamics (ED) analysis and linear interaction energy calculations (LIE) were used to evaluate the impact of amino acid substitutions on the structural and binding properties of the enzymes. The results suggest that the wild type and the G68R TBNATs have a similar pattern of affinity to INH. On the other hand, the calculated enzyme-INH dissociation constant (KD) was estimated 33 times lower for L125M isoform in comparison with wild type enzyme. This last finding is consistent with the hypothesis that isolated mutations in the tbnat gene can produce M. tuberculosis strains resistant to isoniazid.

  3. The crystal structure of Rv1347c, a putative antibiotic resistance protein from Mycobacterium tuberculosis, reveals a GCN5-related fold and suggests an alternative function in siderophore biosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    Card, G L; Peterson, N A; Smith, C A; Rupp, B; Schick, B M; Baker, E N

    2005-02-15

    Mycobacterium tuberculosis, the cause of TB, is a devastating human pathogen. The emergence of multi-drug resistance in recent years has prompted a search for new drug targets and for a better understanding of mechanisms of resistance. Here we focus on the gene product of an open reading frame from M. tuberculosis, Rv1347c, which is annotated as a putative aminoglycoside N-acetyltransferase. The Rv1347c protein does not show this activity, however, and we show from its crystal structure, coupled with functional and bioinformatic data, that its most likely role is in the biosynthesis of mycobactin, the M. tuberculosis siderophore. The crystal structure of Rv1347c was determined by MAD phasing from selenomethionine-substituted protein and refined at 2.2 {angstrom} resolution (R = 0.227, R{sub free} = 0.257). The protein is monomeric, with a fold that places it in the GCN5-related N-acetyltransferase (GNAT) family of acyltransferases. Features of the structure are an acylCoA binding site that is shared with other GNAT family members, and an adjacent hydrophobic channel leading to the surface that could accommodate long-chain acyl groups. Modeling the postulated substrate, the N{sup {var_epsilon}}-hydroxylysine side chain of mycobactin, into the acceptor substrate binding groove identifies two residues at the active site, His130 and Asp168, that have putative roles in substrate binding and catalysis.

  4. Tuberculosis:an experience from Mycobacterium smears and culture analysis

    Institute of Scientific and Technical Information of China (English)

    Zeehaida M; Siti Asma H; Siti Hawa H; Zaidah AR; Norbanee TH

    2009-01-01

    Objective:Simple tests like direct smear of the acid fast bacilli (AFB)and Mycobacterium culture could assist the diagnosis of tuberculosis.This study is aimed at reviewing the outcome of smears,culture results and con-tamination rate among specimens requested for AFB smear and Mycobacterium culture.Methods:Retrospec-tive laboratory data analysis requesting for Mycobacterium culture from January 2005 till December 2006 was done in a tertiary teaching hospital of Universiti Sains Malaysia,Kubang Kerian,Kelantan,Malaysia.Re-sults:Four hundred and sixty seven (36.6%)isolates grew from 1 277 specimens.Of these isolates,314 (67.2%)grew Mycobacterium tuberculosis,23 (4.9%)grew Mycobacterium other than tuberculosis and 38 (8.1%)grew contaminants.Among the M.tuberculosis cultures,165 (52.5%)had growth of more than 100 confluent colonies,whereas 39 cultures (12.4%)had growth of less than 19 colonies.Direct smear for AFB among smear positive cases showed presence of more than 50 bacilli /line in 231 (49.5%)cases and smear negative cases accounted for 63 (13.5%).Among smear positive cases,291 (94.5%)cultures grew Myco-bacterium species and another 17 (5.5%)cultures grew contaminants.In smear negative cases,32 (62.7%) cultures grew Mycobacterium species and 19 (37.3%)cultures grew contaminants.Conclusion:The results from data analysis of the Mycobacterium cultures should be critically utilized in order to review the laboratory performance and to improve its services in the future.Some of the data is also useful to the administrators of the hospital in terms of estimating the risk of occupational hazard faced by the health care workers.

  5. Multidrug-Resistant Tuberculosis

    Centers for Disease Control (CDC) Podcasts

    2008-10-28

    In this podcast, Dr. Oeltmann discusses multidrug-resistant tuberculosis. An outbreak occurred in Thailand, which led to 45 cases in the U.S. This serious illness can take up to 2 years to treat. MDR TB is a real threat and a serious condition.  Created: 10/28/2008 by Emerging Infectious Diseases.   Date Released: 10/28/2008.

  6. Evaluation of GenoFlow DR-MTB Array Test for Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis

    Science.gov (United States)

    Molina-Moya, B.; Kazdaglis, G.; Lacoma, A.; Prat, C.; Gómez, A.; Villar-Hernández, R.; García-García, E.; Haba, L.; Maldonado, J.; Samper, S.; Ruiz-Manzano, J.; Ausina, V.

    2016-01-01

    The aim of this study was to evaluate the GenoFlow DR-MTB array test (DiagCor Bioscience, Hong Kong) on 70 cultured isolates and 50 sputum specimens. The GenoFlow array test showed good sensitivity and specificity compared to the phenotypic Bactec 460TB. This array accurately detected mutations in rpoB, katG, and inhA associated with resistance to rifampin and isoniazid. PMID:26865688

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

    Directory of Open Access Journals (Sweden)

    Edward Gildeh

    2016-01-01

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

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

    Science.gov (United States)

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

    2014-01-01

    Mycobacterium tuberculosis, the