Multidrug-resistant Mycobacterium Tuberculosis Strains Research Articles

The expression of two-component system response regulator in multidrug-resistant Mycobacterium tuberculosis

Objective To screen out the two-component system associated with drug resistance of Mycobacterium tuberculosis by detecting the differential expression of two-component system regulator genes between multidrug resistant Mycobacterium tuberculosis strains and drug sensitive strains. Methods Total RNA of MTB was extracted from cultured MTB during the logarithmic phase in the 7H9 brook medium, and then its purity was identified. Reverse transcription was further completed. The expressing levels of TCS response regulators were quantified using SYBR Green I qRT-PCR, which aimed at finding the differential expressions between multidrug resistant strains and sensitive strains. Finally, all of differentially expressed TCS were screened out under the stress of INH, SM and LFA. Results Compared with sensitive strains,multidrug resistant strains of Rv0491, Rv3133c, Rv3143 and Rv3246c were up-regulated 1. 03, 7.11,3.48and 1.37 folds, respectively (t/t' =5. 623, -4. 196, -3. 559 and -3. 016, respectively, P ＜0. 01 ). The expressing level of other regulators had no statistical significance between muhidrug resistant strains and drug sensitive strains. Under the antibiotic pressure, the expression of Rv1027c, Rv3246c and Rv3143 showed significant changes compared with no antibiotic group. Conclusion Rv3246c and Rv3143 may be associated with MTB drug resistance and the differentially expressed genes in multi-drug resistant strains may be used as potential drug targets against drug resistant tuberculosis. Key words: Mycobacterium tuberculosis; Drug resistance, bacterial; Polymerase chain reaction; Signal transduction

Diarylquinolines, Synthesis Pathways and Quantitative Structure–Activity Relationship Studies Leading to the Discovery of TMC207

The emergence of multidrug-resistant strains of Mycobacterium tuberculosis and resistance to current anti-TB drugs call for the discovery and development of new effective anti-TB drugs. TMC207 is the lead candidate of a novel class of antimycobacterial agents, the diarylquinolines, which specifically inhibit mycobacterial ATP synthase and displays high activity against both drug-susceptible and multidrug-resistant strains of Mycobacterium tuberculosis. This article covers both synthesis pathways as well as qualitative and quantitative analyses of the structure-activity relationships of the diarylquinoline series on Mycobacterium smegmatis activity.

In vitro evaluation of the tuberculocidal property of essential oils

The re-emergence of tuberculosis on a global scale, together with the emergence and spread of Mycobacterium tuberculosis multidrug-resistant strains, is a worldwide public health problem that places a heavy burden on resource-poor countries. We investigated the antibacterial properties of certain essential oils against M. tuberculosis.

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

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 RDRio 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.

Fast track method for the identification of multi–drug resistant tuberculosis on direct clinical specimen using combined drug media

Objective To combine isoniazid (INH) and rifampicin (RIF) in a single media to detect and evaluate multi-drug resistant Mycobacterium tuberculosis (MDR-TB) strains using clinical specimens by direct and indirect drug susceptibility testing (DST). Methods Drug susceptibility testing for INH and RIF using individual and combined drug media was performed by minimum inhibitory concentration (MIC) method on direct clinical specimens. Results The combined drug media showed complete concordance with individual drug media in the detection of MDR-TB by direct DST method and 89% efficiency with indirect DST method. Susceptibility results were available by 3 weeks after the receipt of clinical specimen using direct DST on combined drug media. Conclusions Combined drug media can be used as a fast track method in large scale studies warranting detection of MDR-TB.

Detection of novel coupled mutations in the katG gene (His276Met, Gln295His and Ser315Thr) in a multidrug-resistant Mycobacterium tuberculosis strain from Chennai, India, and insight into the molecular mechanism of isoniazid resistance using structural bioinformatics approaches

This study reports on the structural basis of drug resistance targeting the katG gene in a multidrug-resistant Mycobacterium tuberculosis (MDR-TB) strain with two novel mutations (His276Met and Gln295His) in addition to the most commonly reported mutation (Ser315Thr). A structural bioinformatics approach was used to predict the structure of the mutant KatG enzyme (MT). Subsequent molecular dynamics and docking studies were performed to explain the mechanism of isoniazid (INH) resistance. The results show significant conformational changes in the structure of MT leading to a change in INH binding residues at the active site, with a significant increase in the inhibition constant (Ki) of 5.67μm in the mutant KatG–isoniazid complex (MT-INH) compared with the wild-type KatG–isoniazid complex (WT-INH). In the case of molecular dynamics studies, root mean square deviation (RMSD) analysis of the protein backbone in simulated biological conditions revealed an unstable trajectory with higher deviations in MT throughout the simulation process (1ns). Moreover, root mean square fluctuation (RMSF) analysis revealed an overall increase in residual fluctuations in MT compared with the wild-type KatG enzyme (WT), whilst the INH binding residues of MT showed a decreased fluctuation that can be observed as peak deviations. Hence, the present study suggests that His276Met, Gln295His and Ser315Thr mutations targeting the katG gene result in decreased stability and flexibility of the protein at INH binding residues leading to impaired enzyme function.

A Two-Step Strategy for Molecular Typing of Multidrug-Resistant Mycobacterium tuberculosis Clinical Isolates from Poland

Tuberculosis (TB) continues to be one of the most challenging public health problems in the world. An important contributor to the global burden of the disease is the emergence and spread of drug-resistant and particularly multidrug-resistant Mycobacterium tuberculosis strains (MDR), defined as being resistant to at least isoniazid and rifampicin. In recent years, the introduction of different DNA-based molecular typing methods has substantially improved the knowledge of the epidemiology of TB. The purpose of this study was to employ a combination of two PCR-based genotyping methods, namely spoligotyping and IS6110-Mtb1/Mtb2 PCR to investigate the clonal relatedness of MDR M. tuberculosis clinical isolates recovered from pulmonary TB patients from Poland. Among the 50 isolates examined, 28 (56%) were clustered by spoligotyping, whereas IS6110-Mtb1/Mtb2 PCR resulted in 16 (32%) clustered isolates. The isolates that clustered in both typing methods were assumed to be clonally related. A two-step strategy consisting of spoligotyping as a first-line test, performed on the entire pool of isolates, and IS6110-Mtb1/Mtb2 PCR typing as a confirmatory subtyping method, performed only within spoligotype-defined clusters, is an efficient approach for determining clonal relatedness among M. tuberculosis clinical isolates.

Discovery of Anti-TB Agents that Target the Cell-Division Protein FtsZ

The emergence of multidrug-resistant Mycobacterium tuberculosis strains has made many of the currently available anti-tuberculosis (TB) drugs ineffective. Accordingly, there is a pressing need to identify new drug targets. Filamentous temperature-sensitive protein Z (FtsZ), a bacterial tubulin homologue, is an essential cell-division protein that polymerizes in a GTP-dependent manner, forming a highly dynamic cytokinetic ring, designated as the Z ring, at the septum site. Other cell-division proteins are recruited to the Z ring and, upon resolution of the septum, two daughter cells are produced. Since inactivation of FtsZ or alteration of FtsZ assembly results in the inhibition of Z-ring and septum formation, FtsZ is a very promising target for novel antimicrobial drug development. This review describes the function and dynamic behaviors of FtsZ and the recent development of FtsZ inhibitors as potential anti-TB agents.

Antimycobacterial screening of traditional medicinal plants using the microplate resazurin assay

Multidrug-resistant Mycobacterium tuberculosis strains have rapidly become a global health concern. North American First Nations communities have used traditional medicines for generations to treat many pulmonary infections. In this study, we evaluated the antimycobacterial activity of 5 medicinal plants traditionally used as general therapeutics for pulmonary illnesses and specifically as treatments for tuberculosis. Aqueous extracts of Aralia nudicaulis, Symplocarpus foetidus, Heracleum maximum, Juniperus communis, and Acorus calamus were screened for antimycobacterial activity against Bacillus Calmette-Guérin, Mycobacterium avium, and M. tuberculosis H37Ra using the colorimetric microplate resazurin assay. Extracts of Acorus calamus and H. maximum root demonstrated significant antimycobacterial activity comparable to that of the rifampin control (2 microg/mL). Evaluation of the cytotoxicity of these 2 extracts using the MTT assay also showed that the extracts were less toxic to 3 human cell lines than was the DMSO positive control. This study demonstrates that aqueous extracts of the roots of H. maximum and Acorus calamus possess strong in vitro antimycobacterial activity, validates traditional knowledge, and provides potential for the development of urgently needed novel antituberculous therapeutics.

Structural enzymology of sulphur metabolism in Mycobacterium tuberculosis

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.

Combined drug medium with isoniazid and rifampicin for identification of multi-drug resistant Mycobacterium tuberculosis

A low-cost method of detecting multi-drug resistant Mycobacterium tuberculosis (MDR-TB) with the possibility of quick adoption in a resource limited setting is urgently required. We conducted a study combining isoniazid and rifampicin in a single LJ medium, to detect MDR-TB strains. Combined and individual drug media showed 100% concordance for the detection of MDR-TB and susceptible strains by proportion method. Considering the results, combined isoniazid and rifampicin containing medium could be considered for use in settings where the sole detection of MDR-TB strains is justified.

Emerging multidrug resistant Mycobacterium tuberculosis strains of the Beijing genotype circulating in Russia express a pattern of biological properties associated with enhanced virulence

The epidemiologically important Mycobacterium tuberculosis Beijing genotype strains, highly endemic in East Asia, have become an emerging infection in certain geographic areas, including Russia, because of its increasing prevalence and association with multidrug resistance (MDR). The aim was to verify whether MDR Beijing strains circulating in the emerging regions present some biological particularities that could contribute to their success in causing disease in comparison with the sporadic strains from locations with low prevalence of the Beijing genotype. We evaluated virulence-associated characteristics of the MDR Beijing strains isolated in Russia and compared them with those of the drug-resistant and susceptible Beijing strains from Brazil and reference H37Rv strain. We found that Russian MDR strains demonstrated an increased bacterial fitness and growth in THP-1 macrophage-like cells, as well as a higher capacity to induce non-protective cytokine synthesis and necrotic macrophage death. By contrast, the biological properties of the strains isolated in Brazil largely resembled those of the H37Rv strain, with the exception of the drug-resistant isolates that presented significantly reduced fitness. The data demonstrate that the emerging MDR strains of the Beijing genotype circulating in Russia do express a pattern of properties associated with the enhanced virulence favouring its clonal dissemination in this region.

Comparison of rifabutin susceptibility and rpoB mutations in multi-drug-resistant Mycobacterium tuberculosis strains by DNA sequencing and the line probe assay

We compared rifabutin susceptibility and rpoB mutations in 98 multi-drug-resistant strains of Mycobacterium tuberculosis (MDR-TB) by DNA sequencing and with a line probe assay using the commercially available INNO-LiPA Rif. TB kit (the LiPA). Our results indicated that rifabutin continues to remain active against MDR-TB strains harboring certain genetic alterations and also that the LiPA might be useful in identifying MDR-TB strains susceptible to rifabutin.

Antimycobacterial susceptibility testing methods for natural products research

The emergence of multidrug-resistant strains of Mycobacterium tuberculosis underscores the need of continuous developments on new and efficient methods to determine the susceptibility of isolates of M. tuberculosis in the search for novel antimicrobial agents. Natural products constitute an important source of new drugs, but design and implementation of antimycobacterial susceptibility testing methods are necessary for evaluate the different extracts and compounds. A number of biological assay methodologies are in current use, ranging from the classical disk diffusion and broth dilution assay format, to radiorespirometric (BACTEC), dye-based, and fluorescent/luminescence reporter assays. This review presents an analysis on the in vitro susceptibility testing methods developed for determinate antitubercular activity in natural products and related compounds (semi-synthetic natural products and natural products-derived compounds) and the criteria to select the adequate method for determination of biological activity of new natural products.

Comparative QSAR studies of nitrofuranyl amide derivatives using theoretical structural properties

The global increase in multidrug-resistant Mycobacterium tuberculosis strains and intolerance of first-line anti-tuberculosis drugs may cause major health problems and necessitate modification of the structural therapy regimen. In an ongoing effort to develop new and potent anti-tuberculosis agents, a series of nitrofuranyl amides were subjected to quantitative structure–activity relationship (QSAR) analysis using various feature selection methods. Nitrofuranyl amide derivatives with good therapeutic indices are known to inhibit an enzyme responsible for bacterial cell-wall synthesis and act as novel mycobacterial inhibitors. Successful implementation of a predictive QSAR model largely depends on the selection of a preferred set of molecular descriptors that can signify the chemical–biological interaction. Genetic algorithm (GA), simulated annealing and stepwise regression are applied as variable selection methods for an effective comparison and model development. The results of two-dimensional QSAR showed that a combination of topological indices, hydrophobic properties and autocorrelation descriptors of different atomic properties could be explored to design potent anti-tubercular inhibitors. Further analysis using three-dimensional QSAR technique identifies a suitable model obtained by GA-partial least square method leading to anti-tubercular activity prediction. The influences of steric and electrostatic field effects generated by the contribution plot are analysed and discussed. Both two- and three-dimensional QSAR analyses of such derivatives provide important structural insights for designing potent anti-tuberculosis drugs.

Drug-susceptibility testing in TB: current status and future prospects

The rising number of resistant and multidrug-resistant Mycobacterium tuberculosis strains and the emergence of extensively drug-resistant strains substantiate the urgent demand for rapid and reliable techniques for the detection of drug-resistant TB. In recent years, a multitude of techniques for rapid drug-susceptibility testing have been designed and evaluated. Two different strategies for the assessment of drug resistance can be followed; phenotypic determination has been common practice for years, whereas more recently the genetic detection of mutations that confer for drug resistance has been established. Novel liquid culture-based drug-susceptibility testing techniques have been evaluated; several of them have proved their reliability and accuracy, while others need more evaluation or a different performance due to biosafety risks. Among the molecular tests, line-probe assays seem to be the most promising tools for a rapid and very specific and sensitive detection of multidrug-resistant M. tuberculosis.

Resistência do Mycobacterium tuberculosis à isoniazida por mutações em duas regiões diferentes do gene katG

To analyze and compare the mutations in two different regions of the katG gene, which is responsible for isoniazid (INH) resistance. We analyzed 97 multidrug-resistant Mycobacterium tuberculosis strains isolated in cultures of sputum samples obtained from the Professor Hélio Fraga Referral Center, in Brasília, Brazil. Another 6 INH-sensitive strains did not present mutations and were included as controls. We used PCR to amplify two regions of the katG gene (GenBank accession no. U06258)-region 1, (from codon 1 to codon 119) and region 2 (from codon 267 to codon 504)-which were then sequenced in order to identify mutations. Seven strains were resistant to INH and did not contain mutations in either region. Thirty strains carried mutations in region 1, which was characterized by a high number of deletions, especially at codon 4 (24 strains). Region 2 carried 83 point mutations, especially at codon 315, and there was a serine-to-threonine (AGC-to-ACC) substitution in 73 of those cases. The analysis of region 2 allowed INH resistance to be diagnosed in 81.4% of the strains. Nine strains had mutations exclusively in region 1, which allowed the proportion of INH-resistant strains identified to be increased to 90.6%. The number of mutations at codon 315 was high, which is consistent with cases described in Brazil and in other countries, and the analysis of region 1 resulted in a 9.2% increase in the rate at which mutations were identified.

Abdominal tuberculosis: experience with three cases from an Austrian centre

BACKGROUND: Tuberculosis has remained an important infectious threat in the third world, but is relatively rare in industrialized countries. Peritoneal tuberculosis is a diagnostic challenge since its clinical presentation and radiological findings may mimic peritoneal carcinosis associated with gynaecological or gastrointestinal cancer. METHODS: Two patients with peritoneal and one with gastrointestinal tuberculosis are presented. RESULTS: Three women with unspecific abdominal symptoms were admitted to the hospital. In two patients the suspected initial diagnoses based on radiological findings were gastric and ovarian cancer with peritoneal spread. The correct diagnoses were made upon laproscopy and subsequent histopathological examination. The third patient presented with abdominal pain and bloody diarrhea six years after renal transplantation and intestinal tuberculosis was diagnosed on colonoscopy. All three cases were successfully managed with combination anti-microbial therapy. In one patient, multidrug-resistant Mycobacterium tuberculosis strain was involved. CONCLUSIONS: Abdominal tuberculosis can present with clinical and radiological signs of gastrointestinal or gynaecological cancer. Laparoscopy and endoscopy with tissue biopsies can readily establish diagnosis.

Drug resistant Mycobacterium tuberculosis in Mexico

Tuberculosis (TB) remains a serious public health problem, worsened by an increased frequency of multidrug-resistant (MDR) Mycobacterium tuberculosis strains. The World Health Organization (WHO) and the International Union Against Tuberculosis and Lung Disease (IUATLD) launched the Global Project on Anti-Tuberculosis Drug Resistance Surveillance to measure the prevalence of drug resistance. Data from the global reports on resistance to anti-tuberculosis (anti-TB) drugs have shown that drug resistance still presents worldwide and that MDR-TB is present in almost all the world. Though the Global Project (WHO) has been operating since 1994, very few countries and states have reported new information. Data from repeated surveys employing comparable methodologies over several years are essential to determine with any certainty in which direction the prevalence of drug resistance is moving. Drug-resistant tuberculosis and MDR-TB have been identified in Mexico, even with the existence of a National Tuberculosis Program based on Directly Observed Treatment, Short-course (DOTS). This review discusses available surveillance data on drug susceptibility data for TB in different states of Mexico.

Sudden Increase in Multidrug Resistant Mycobacterium tuberculosis Strains Investigated in South-Eastern Free State, South Africa

Background: During the past decade the tuberculosis (TB) incidence escalated with new cases for the Free State reported as 793/100 000 in 2006 and approximately 60% co-infected with HIV. MDR-TB has risen to 6.9% of previously treated patients and sporadic reports of XDR-TB stress the importance of molecular surveillance and rapid testing. From October 2006 in the Mohokare district there was a sudden alarming rise to 21% of retreatment cases being MDR. The aim of the study was to investigate the possibility of a MDR-TB outbreak in the Mohokare district. Method: Specimens could be collected from 9 of 11 MDRTB patients and one contact in the Mohokare district, Free State. We used a commercially available spoligotyping kit to detect 43 known spacer sequences in the direct repeat (DR) loci of MTB to determine strain similarities and a genotype strip assay for detection of MDR and confirmed results by bidirectional cycle sequencing. Spoligotyping data was entered into an Excel spreadsheet in a binary format and compared to other strains in the world captured in the spolDB3.0 database using SPOTCLUST. Results: 5/9 of the MDR-TB cases were still positive; three of these and an additional contact’s strain was analysed. Spoligotyping suggested that all four strains belonged to different lineages namely LAM9, T1, S and T2. The LAM9 strain had the 526 (GAC3GTC/Asp3Val) rpoB and the 315 (TCG15ACC) katG mutations. The T1 and S strain had the same rpoB 531 (TCG3TTG/Ser3Leu) and 315 (TCG15ACC) katG mutation. The strain from a contact belonged to spoligotype family T2 and was susceptible. Results clearly indicate that the sudden increase in MDR cases is not due to an outbreak. Enormous problems exist in this district with non adherence to treatment, inadequate therapy and absconding of patients from Moroka MDR hospital leading to increased MDR-TB cases. Urgent attention needs to be given to solve this alarming problem.