Multidrug-resistant Mycobacterium Tuberculosis Strains Research Articles

Novel diaryl ether derivatives as InhA inhibitors: Design, synthesis and antimycobacterial activity

A new series of triclosan (TCL)-mimicking diaryl ether derivatives 7–25 were synthesized and evaluated as inhibitors of enoyl acyl carrier protein reductase InhA enzyme. In addition, these derivatives were screened as inhibitors of drug-susceptible (DS), multidrug-resistant (MDR), and extensive drug-resistant (XDR) Mycobacterium tuberculosis (MTB) strains. Most compounds exihibted superior anti-TB activities and improved ClogP compared to TCL as a standard drug. The present work has led to the identification of compounds 14, 19 and 24 which possess remarkable activities against DS, MDR and XDR MTB strains with MIC values of 1.95, 3.9 and 15.63 µg/ml, respectively for compound 14, 1.95, 3.9 and 7.81 µg/ml, respectively for compound 19 and 0.98, 1.95 and 3.9 µg/ml, respectively for compound 24. Most compounds did not exhibit toxicity to HePG2 normal cell line. Compounds 14, 19 and 24, presenting the best MIC values, were further evaluated as inhibitors of InhA enzyme. They showed high binding affinities in the micromolar range with IC50 values of 1.33, 0.6, and 0.29 µM for compounds 14, 19, and 24, respectively. Furthermore, molecular docking approach was utilized to understand the difference in bioactivities between the new compounds. In particular, the results revealed strong binding interactions and high docking scores of compounds 14, 19 and 24, which could correlate with their high activities. Mainly, the molecular modelling study of compound 24 provides an excellent platform for understanding the molecular mechanism regarding InhA inhibition. Thus, compound 24 could be a lead compound for future development of new antitubercular drugs.

Decreasing trend of drug-resistant TB in Italy

BACKGROUND: TB caused by rifampicin-resistant (RR) and multidrug-resistant (MDR) Mycobacterium tuberculosis strains is a major concern to TB control globally. However, in the European Union, MDR-TB notifications among all bacteriologically confirmed TB cases with available drug susceptibility testing (DST) results decreased over the last years.METHODS: We conducted a retrospective analysis on DST results reported from 2011 to 2020 by 46 laboratories in 19 out of 20 regions in Italy in order to evaluate resistance trends to first- and second-line drugs in MDR/RR-TB strains isolated from Italian-born persons (IBPs) and foreign-born persons (FBPs).RESULTS: Of 23,972 M. tuberculosis strains examined (15,519 from FBPs and 8,453 from IBPs), MDR-TB decreased from 3.2% in 2011 to 2.2% in 2020. High MDR/RR-TB rates occurred mostly in FBPs from former Soviet Union countries. In 2017, a MDR/RR-TB increase was detected in FBPs from sub-Saharan Africa. MDR-TB strains showed consistent increase in resistance to pyrazinamide (PZA), slight increase in resistance to fluoroquinolones and a decrease in resistance to other drugs.CONCLUSION: While MDR/RR-TB cases slightly decreased, a worrisome increase of resistance to PZA and fluoroquinolones among MDR/RR-TB patients was seen. This implies that a fast and efficient diagnosis aligned with therapy is crucial for TB control.

RESISTANCE MECHANISMS TO FIRST LINE DRUG IN MYCOBACTERIUM TUBERCULOSIS: A REVIEW

The emergence of multidrug-resistant (MDR) Mycobacterium tuberculosis (Mtb) strains, identified as resistant to at least isoniazid and rifampin, these two drugs form the backbone of the first line drug which is used for tuberculosis (TB) treatment, unresponsive has hampered TB control. As a result of the nearly universal calculation of with half a million new cases of MDR/first line drug TB per year, it is important to keep the database up to date awareness of the processes that contribute to the emergence of MDR Mtb. This resistance is produced for a variety of reasons, including genetic, microbiological factors, non-adherence to treatment by patients and/or failures in therapy administration by some referrer medical centre for TB. This review offers a detailed summary of genetic mechanisms that lead to resistant to first line drug therapy used in management of TB as well as up-to-date information on some new aspects lead to such problem. Keywords: Mycobacterium tuberculosis, first line drug, anti-TB treatment, World Health Organization, isoniazid and rifampin Citation: Hassan JS, Al-Tamimi BJ, Abdulrahman TR, Jamal QW. Resistance mechanisms to first line drug in Mycobacterium tuberculosis: A review. Iraqi JMS. 2022; 20(1): 35-43. doi: 10.22578/IJMS.20.1.5

Antitubercular, Cytotoxicity, and Computational Target Validation of Dihydroquinazolinone Derivatives.

A series of 2,3-dihydroquinazolin-4(1H)-one derivatives (3a–3m) was screened for in vitro whole-cell antitubercular activity against the tubercular strain H37Rv and multidrug-resistant (MDR) Mycobacterium tuberculosis (MTB) strains. Compounds 3l and 3m with di-substituted aryl moiety (halogens) attached to the 2-position of the scaffold showed a minimum inhibitory concentration (MIC) of 2 µg/mL against the MTB strain H37Rv. Compound 3k with an imidazole ring at the 2-position of the dihydroquinazolin-4(1H)-one also showed significant inhibitory action against both the susceptible strain H37Rv and MDR strains with MIC values of 4 and 16 µg/mL, respectively. The computational results revealed the mycobacterial pyridoxal-5′-phosphate (PLP)-dependent aminotransferase (BioA) enzyme as the potential target for the tested compounds. In vitro, ADMET calculations and cytotoxicity studies against the normal human dermal fibroblast cells indicated the safety and tolerability of the test compounds 3k–3m. Thus, compounds 3k–3m warrant further optimization to develop novel BioA inhibitors for the treatment of drug-sensitive H37Rv and drug-resistant MTB.

Determination of genetic diversity of multidrug-resistant Mycobacterium tuberculosis strains in Turkey using 15 locus MIRU-VNTR and spoligotyping methods

ABSTRACT Tuberculosis (TB) remains the leading cause of deaths from infectious disease worldwide. Nowadays, the tendency of Mycobacterium tuberculosis complex (MTBC) to spread between continents due to uncontrolled migration movements shows that TB is a global health problem. The number of studies for the detection of MTBC strains’ epidemiological features in areas with TB spread risk using molecular-based methods such as spoligotyping and Mycobacterial Interspersed Repetitive Unit (MIRU) Variable Number Tandem Repeats (VNTR) at the clonal level is insufficient. In this study, it was aimed to determine the phylogenetic relationships of MTBC strains at the species level by spoligotyping and 15 locus MIRU-VNTR (MIRU-VNTR15) molecular methods of 96 multidrug-resistant (MDR) MTBC strains isolated from sputum samples of patients with a preliminary diagnosis of pulmonary TB or suspected contact history those sent to National Tuberculosis Reference Laboratory from the centers that are members of the Tuberculosis Laboratory Surveillance Network. The phylogenetic relationship between 96 MDR-TB strains was investigated with the combination of bead-based spoligotyping and MIRU-VNTR15 methods on the MAGPIX® Milliplex Map device. In this study, it was determined that the T1 family is more common in our country and LAM7-TUR family is less common than the Beijing family unlike other studies. It was determined that the strains in the same cluster had different locus profiles, and there was no transmission from the same clone in the clonal typing we performed with spoligotyping and MIRU-VNTR15.

Design, synthesis and anti-Mycobacterium tuberculosis evaluation of new thiazolidin-4-one and thiazolo[3,2-a][1,3,5]triazine derivatives

In response to the urgent need to encounter infection diseases, and upon increasing concerns about the devastating effects of tuberculosis (TB), the promising thiazolidin-4-one scaffold was used as a starting point to design and synthesize seventeen new compounds, relying on the pharmacophoric features of different anti-Mycobacterium tuberculosis and antibacterial active compounds. Thiazolidin-4-one was elaborated to result in bi-functioning formation, and further ring fusion into a thiazolo[3,2-a][1,3,5]triazine, which was hybridized with different heterocyclic rings and sulfonamide moieties. All the newly synthesized compounds were evaluated for their activity against drug sensitive (DS), multi-drug resistant (MDR) and extensive drug resistant (XDR) Mycobacterium tuberculosis (Mtb) strains. Additionally, their anti-bacterial activity against several bronchitis causing-bacteria (ATCC) and their antifungal activity were assessed. Several compounds showed promising results regarding all of the mentioned assays without any antifungal activities. Particularly, compound 3 showed a promising activity against the three Mtb strains (DS, MDR and XDR) with MIC of 2.49, 9.91 and 39.72 µM, respectively. Furthermore, compound 7c revealed antituberculosis activity with MIC of 2.28, 18.14 and 36.31 µM against DS, MDR and XDR strains, respectively. Both of compounds 3 and 7c surpassed azithromycin on several bronchitis causing-bacteria and showed enhanced inhibitory activity against Mycobacterium tuberculosis enoyl-acyl carrier protein reductase (InhA), with IC50 of 3.90 and 2.47 µM, respectively. The enzymatic activity was augmented by the binding characteristics of 3 and 7c in the InhA active site. Further investigations confirmed their safety on normal cell lines, and promising predicted ADME characteristics.

Effects of Bedaquiline on Antimicrobial Activity and Cytokine Secretion of Macrophages Infected with Multidrug-Resistant Mycobacterium tuberculosis Strains.

Background Bedaquiline (Bdq) exerts bactericidal effects against drug-susceptible and drug-resistant Mycobacterium tuberculosis strains, including multidrug-resistant M. tuberculosis strains (MDR-MTBs). However, few reported investigations exist regarding Bdq effects on MDR-MTBs-infected macrophages activities and cytokine secretion. Here, Bdq bactericidal activities against MDR-MTBs and related cellular immune mechanisms were explored. Methods Macrophages infected with MDR-MTBs or H37Rv received Bdq treatments (4 h/8 h/24 h/48 h) at 1 × the minimum inhibitory concentration (1 × MIC), 10 × MIC and 20 × MIC. Intracellular colony-forming units (CFUs) and culture supernatant IL-12/23 p40, TNF-α, IL-6, and IL-10 were determined using the Luminex® 200TM system. Normally distributed continuous data (mean ± standard deviation) were analyzed using t-test or F-test (SPSS 25.0, P < 0.05 deemed statistically significant). Results (1) 100% of Bdq-treated macrophages (all doses applied over 4–48 h) survived with 0% inhibition of proliferation observed. (2) Intracellular CFUs of Bdq-treated MDR-MTBs-infected macrophages decreased over 4–48 h of treatment, were lower than preadministration and control CFUs, decreased with increasing Bdq dose, and resembled H37Rv-infected group CFUs (48 h). (3) For MDR-MTBs-infected macrophages (various Bdq doses), IL-12/23 p40 levels resembled preadministration group levels and exceeded controls (4 h); TNF-α levels exceeded preadministration group levels (24 h/48 h) and controls (24 h); IL-12/23 p40 and TNF-α levels resembled H37Rv-infected group levels (4 h/8 h/24 h/48 h); IL-6 levels exceeded preadministration and H37Rv-infected group levels (24 h/48 h) and controls (24 h); IL-10 levels resembled preadministration and H37Rv-infected group levels (4 h/8 h/24 h/48 h) and were lower than controls (24 h/48 h); IL-12/23 p40 and IL-10 levels remained unchanged as intracellular CFUs changed, with IL-12/23 p40 levels exceeding controls (4 h) and IL-10 levels remaining lower than controls (24 h/48 h); TNF-α and IL-6 levels increased as intracellular CFUs decreased (24 h/48 h) and exceed controls (24 h). Conclusion Bdq was strongly bactericidal against intracellular MDR-MTBs and H37Rv in a time-dependent, concentration-dependent manner. Bdq potentially exerted immunomodulatory effects by inducing high-level Th1 cytokine expression (IL-12/23 p40, TNF-α) and low-level Th2 cytokine expression (IL-10).

Screening Repurposed Antiviral Small Molecules as Antimycobacterial Compounds by a Lux-Based phoP Promoter-Reporter Platform

The emergence of multidrug-resistant strains and hyper-virulent strains of Mycobacterium tuberculosis are big therapeutic challenges for tuberculosis (TB) control. Repurposing bioactive small-molecule compounds has recently become a new therapeutic approach against TB. This study aimed to identify novel anti-TB agents from a library of small-molecule compounds via a rapid screening system. A total of 320 small-molecule compounds were used to screen for their ability to suppress the expression of a key virulence gene, phop, of the M. tuberculosis complex using luminescence (lux)-based promoter-reporter platforms. The minimum inhibitory and bactericidal concentrations on drug-resistant M. tuberculosis and cytotoxicity to human macrophages were determined. RNA sequencing (RNA-seq) was conducted to determine the drug mechanisms of the selected compounds as novel antibiotics or anti-virulent agents against the M. tuberculosis complex. The results showed that six compounds displayed bactericidal activity against M. bovis BCG, of which Ebselen demonstrated the lowest cytotoxicity to macrophages and was considered as a potential antibiotic for TB. Another ten compounds did not inhibit the in vitro growth of the M. tuberculosis complex and six of them downregulated the expression of phoP/R significantly. Of these, ST-193 and ST-193 (hydrochloride) showed low cytotoxicity and were suggested to be potential anti-virulence agents for M. tuberculosis.

Linking azoles to isoniazid via hydrazone bridge: Synthesis, crystal structure determination, antitubercular evaluation and computational studies

The current emergence of drug-resistant and multidrug-resistant (MDR) Mycobacterium tuberculosis (Mtb) strains has complicated and hampered attempts to eliminate or considerably reduce the global prevalence of the often life-threatening disease tuberculosis (TB). Hence, the development of novel antitubercular agents is crucial to combat this challenge. Here, we applied the molecular hybridization approach to link isoniazid (INH), the frontline antitubercular drug, to various azole rings (pyrazole, imidazole, and triazole) through hydrazone functionality. The designed compounds were synthesized and characterized by using spectral techniques including IR, 1H NMR, 13C NMR and HRMS. Additionally, single crystal X-ray analysis was employed to resolve the proposed chemical structure of INH-T. All compounds were then extensively screened for their antitubercular activities against Mtb H37Rv, drug-resistant and MDR Mtb strains, as well as against a clinical Mtb isolate with no mutation. Notably, INH-azole hybrids presented outstanding antimycobacterial activity with negligible cytotoxicity. Computational methods based on density functional theory calculations and molecular dynamics simulations were applied to identify the characteristic reactive centers of the title compounds, predict stability towards autoxidation, understand their interactions with water molecules and predict the temperature dependence of density. Finally, molecular docking studies revealed that new INH-azole hybrids are likely to exert their antimycobacterial activity via direct inhibition of the Mtb InhA enzyme.

Achieving reinforced broad-spectrum and sustained antimicrobial efficacy by nickel-doping AlOOH nanoflower accommodated with uniform silver nanospecies

Two-dimensional materials composed of all-oriented branches assembled with active Ag nanospecies might be attractive in the microbial inhibition application because of improved Ag ion release behavior and interaction with targeted substance. Herein, we report an excellent Ag-AlNiO nanocomposite antimicrobial materials by Ni-doping AlOOH triggering better implanting of Ag nanospecies. The TEM results revealed that the formed nanoflower-like configuration of the Ni-doping AlOOH favors the formation of refined active silver nanospecies (diameter of 6–10 nm) compared to Ni-free AlOOH (Ag-AlO). The resulting Ag-AlNiO afford good biocompatibility which is evidenced by the cell viability test via cell counting kit-8 (CCK-8) assay. More importantly, Ag-AlNiO showed promotive broad-spectrum antimicrobial properties against various gram-negative bacteria, gram-positive bacteria, and fungi. We also demonstrated that corresponding MIC values against different microbes ranging from 16 to 128 μg/mL concerning Ag-AlNiO composite were at least 2-, or 4-folds lower than Ag-AlO composite. Besides, the optimal Ag-AlNiO is endowed with excellent antimicrobial effect against H37Rv, Bacillus Calmette-Guerin (BCG), and multidrug-resistant (MDR) strain of Mycobacterium tuberculosis. In comparison with Ag-AlO, Ag-AlNiO revealed a prolonged inhibitory effect on microbial growth, which indicated that abundant silver nanospecies were sustained released from the nanoflower-like Ag-AlNiO composites in addition to the direct attacks.

The Activities and Secretion of Cytokines Caused by Delamanid on Macrophages Infected by Multidrug-Resistant Mycobacterium tuberculosis Strains.

BackgroundDelamanid (Dlm) is an effective drug against drug-susceptible and drug-resistant Mycobacterium tuberculosis strains, including Multidrug-resistant Mycobacterium tuberculosis (MDR-MTB). There are few reports on the activity and secretion of cytokines caused by Dlm on macrophages infected by MDR-MTB strains. Therefore, this article aims to observe the bactericidal activity and secretion of cytokines of the macrophages infected by MDR-MTB strains after Dlm was administered, so as to provide a basis for further perfecting the mechanism of Dlm.MethodsSamples were respectively collected to count the intracellular colony-forming unit (CFU) of macrophages infected by MDR-MTB or H37Rv strains at 4, 8, 24, and 48 h after Dlm at MIC, 10MIC, and 20MIC were administered. Samples were respectively collected to detect the level of IL-12/23 p40, TNF-α, IL-6, and IL-10 in the culture supernatant of macrophages infected by MDR-MTB or H37Rv strains at 4, 24, and 48 h after Dlm at MIC were administered. The levels of four cytokines in the culture supernatant were measured using the Luminex® 200™ (Luminex, USA) according to the manufacturer’s instructions. Data were analyzed by SPSS 25.0 software. The continuous data in normal distribution were expressed as mean ± standard deviation ( ± s) and analyzed by t or F test. P<0.05 was considered statistically significant.Results(1) After Dlm was applied to macrophages infected by MDR-MTB strains:(A) The intracellular CFU gradually decreased, reached the lowest value at 48 h, and was lower than that of Dlm before administration and infection group (P<0.05). (B) The intracellular CFU was further reduced after increasing Dlm dose to 10MIC and 20MIC, and the latter was lower than that of the former (P<0.05). (C) The intracellular CFU of MDR-MTB group was higher than that of H37Rv group at 4~48 h after administration (P<0.05). (2) After Dlm at MIC dose was applied to macrophages infected by MDR-MTB strains: (A) The level of IL-12/23 p40 at any time didn’t change compared with that of Dlm before administration (P>0.05), while the level of IL-12/23 p40 at 4 h was higher than that of the infection group (P<0.05). The levels of TNF-α at 24 and 48 h were higher than that of Dlm before administration (P<0.05), but were similar to that of the infection group (P>0.05). In addition, the levels of IL-12/23 p40 and TNF-α at any time were similar to that of the H37Rv group after administration (P>0.05). (B) The levels of IL-6 at 24 and 48 h were higher than that of Dlm before administration (P<0.05), but were similar to that of H37Rv group (P>0.05) and were lower than that of infection group (P<0.05). The level of IL-10 at any time didn’t change compared with that of Dlm before administration (P>0.05), but was lower than that of the infection group at 4~48 h and was lower than that of the H37Rv group at 24 h (P<0.05). (C) The level of IL-12/23 p40 and IL-10 didn’t change with the change of intracellular CFU (P<0.05), while the level of TNF-α and IL-6 increased with the intracellular CFU decreasing, and the increase level of TNF-α was lower than that of the infection group (P<0.05).ConclusionsDlm had strong bactericidal activity against intracellular MDR-MTB, which was time-dependent and concentration-dependent. Its bactericidal activity against intracellular MDR-MTB strains was weaker than that against drug-susceptible tuberculosis strains. Dlm might have immunomodulatory effect, inducing low expression of Th2 cytokines IL-6 and IL-10 at different times after administration.

Two New Dihydrosphingosine Analogs Against Mycobacterium tuberculosis Affect gltA1, lprQ, and rpsO Expression.

The emergence of multidrug-resistant (MDR) Mycobacterium tuberculosis strains threaten the control of tuberculosis. New antitubercular dihydrosphingosine analogs, named UCIs, have been evaluated in preclinical studies but their cellular and molecular mechanisms of action against M. tuberculosis are still unknown. The aim of this study was to evaluate the effect of UCI exposure on gene expression of drug-sensitive H37Rv and MDR CIBIN:UMF:15:99 clones of M. tuberculosis which were isolated, phenotypically, and genetically characterized, cultured to log phase and treated with UCI compounds; followed by total RNA isolation, reverse transcription and hybridization assays on Affymetrix genomic microarrays. Data were validated with RT-qPCR assays. As results, UCI-05 and UCI-14 exposure increased gltA1 expression in drug-sensitive H37Rv clones. Furthermore, UCI-05 increased lprQ expression in MDR CIBIN:UMF:15:99 M. tuberculosis clones while UCI-14 reduced the expression of this gene in drug-sensitive H37Rv clones. In addition, UCI-05 reduced rpsO expression in drug-sensitive H37Rv clones. We found gene expression alterations that suggest these molecules may alter carbon and lipid metabolism as well as interfere in the protein-producing machinery in M. tuberculosis.

Synergetic Effect of Rifampin Loaded Mussel‐Inspired Silver Nanoparticles for Enhanced Antibacterial Activity Against Multidrug‐Resistant Strain of Mycobacterium Tuberculosis

AbstractTuberculosis (TB) is among the top ten causes of death worldwide. The emergence of multidrug‐resistant (MDR) TB has been challenging to cure TB patients and control the airborne transmission. To ensure the effectiveness of rifampin (RF) in the treatment of MDR TB infection, new and safe silver nanoparticles were used in combination. In this study, polydopamine decorated silver nanoparticles (Ag‐PDA NPs) were synthesized under mild conditions in which antituberculosis drug RF was loaded. Subsequently, the antibacterial activity of different ratios of Ag‐PDA NPs/RF against MDR strains of Mycobacterium tuberculosis (M. tuberculosis) was evaluated by minimum inhibitory concentration (MIC) tests. MIC results showed a synergistic interaction between Ag‐PDA NPs and RF, and the optimal antibacterial effect against MDR strain of M. tuberculosis was at the mass ratio of 2 Ag‐PDA NPs : 8RF. In addition, RF loaded Ag‐PDA NPs (RF@Ag‐PDA NPs) were constructed and characterized. The results indicated that RF@Ag‐PDA NPs might be considered as promising nano drugs to inhibit the growth of MDR strain of M. tuberculosis and keep the potency of RF in clinical practice.

Hierarchically structured Ag modified nanosilica constructed by micelle modification tactics delivers integrated catalytic and antibacterial activity

The complexity of environmental pollution poses a severe threat to public health, attracting considerable interest in the search for functional nanomaterials for dye removal and direct disinfection together in wastewater. Herein, we present a deft synthetic strategy for constructing Ag functionalized hierarchically structured nanosilica (Ag modified mesoporous silica/Ag-ms) using micelle termination incorporating Ag ions as a functional metallomicelle template. The results showed that multiple active Ag cores were decorated into silica nanospheres. The resulting silica has ultrahigh porous properties (1370 m2 g−1, 1.583 m3 g−1) which are much superior to those of reports, thereby could make it a superior catalyst and disinfector. The obtained nanocomposites were carefully characterized using various advanced techniques to identify their special configuration and status of silver nanospecies in the silica. In addition, the cell viability test via cell counting kit-8 (CCK-8) assay displayed good biocompatibility of obtained Ag-ms, suggesting its promising candidate possibility for biomedical applications. Furthermore, the rapid catalytic reduction of methylene blue (MB) was easily enabled by the optimized Ag ms (0.15 Ag ms) within 5 min, which exhibited great catalytic kinetic properties (4.059 min−1 mg−1). Our results also showed that 0.15 Ag-ms had superior antibacterial properties against gram-negative Pseudomonas aeruginosa, gram-positive Staphylococcus aureus, and even multidrug-resistant (MDR) strain of Mycobacterium tuberculosis compared to other counterparts (0.05 Ag-ms and 0.1 Ag ms), which affords great value for reducing antibiotic-resistant bacteria and nosocomial infections.

Discovery of 5-methylpyrimidopyridone analogues as selective antimycobacterial agents

With the emergence of multidrug-resistant strains of Mycobacterium tuberculosis (MDR-TB) and extensive drug-resistant strains (XDR-TB), there is an urgent need to develop novel drugs for the treatment of tuberculosis. Here, we designed and synthesized a series of 5-methylpyrimidopyridone analogues as potential antitubercular agents. The most potent compound 6q exhibited a MIC value of 4 μM in vitro against Mycobacterium tuberculosis. The antitubercular activities of the synthesized compounds were impacted by the amantadine and 2-chlorophenyl groups, and were enhanced by the presence of 3-methyl(4-dimethylamino)piperidinylphenyl. Molecular modeling and binding studies suggest that PknB is the potential molecular target of 5-methylpyrimidopyridone compounds. This study provides insights for the future development of new antimycobacterial agents with novel mechanisms of action.

Structural Changes in the Cap of Rv0183/mtbMGL Modulate the Shape of the Binding Pocket.

Tuberculosis continues to be a major threat to the human population. Global efforts to eradicate the disease are ongoing but are hampered by the increasing occurrence of multidrug-resistant strains of Mycobacterium tuberculosis. Therefore, the development of new treatment, and the exploration of new druggable targets and treatment strategies, are of high importance. Rv0183/mtbMGL, is a monoacylglycerol lipase of M. tuberculosis and it is involved in providing fatty acids and glycerol as building blocks and as an energy source. Since the lipase is expressed during the dormant and active phase of an infection, Rv0183/mtbMGL is an interesting target for inhibition. In this work, we determined the crystal structures of a surface-entropy reduced variant K74A Rv0183/mtbMGL in its free form and in complex with a substrate mimicking inhibitor. The two structures reveal conformational changes in the cap region that forms a major part of the substrate/inhibitor binding region. We present a completely closed conformation in the free form and semi-closed conformation in the ligand-bound form. These conformations differ from the previously published, completely open conformation of Rv0183/mtbMGL. Thus, this work demonstrates the high conformational plasticity of the cap from open to closed conformations and provides useful insights into changes in the substrate-binding pocket, the target of potential small-molecule inhibitors.

Evolution and emergence of multidrug-resistant Mycobacterium tuberculosis in Chisinau, Moldova.

The evolution and emergence of drug-resistant tuberculosis (TB) has been studied extensively in some contexts, but the ecological drivers of these two processes remain poorly understood. This study sought to describe the joint evolutionary and epidemiological histories of a novel multidrug-resistant Mycobacterium tuberculosis strain recently identified in the capital city of the Republic of Moldova (MDR Ural/4.2), where genomic surveillance of drug-resistant M. tuberculosis has been limited thus far. Using whole genome sequence data and Bayesian phylogenomic methods, we reconstruct the stepwise acquisition of drug resistance mutations in the MDR Ural/4.2 strain, estimate its historical bacterial population size over time, and infer the migration history of this strain between Eastern European countries. We infer that MDR Ural/4.2 likely evolved (via acquisition of rpoB S450L, which confers resistance to rifampin) in the early 1990s, during a period of social turmoil following Moldovan independence from the Soviet Union. This strain subsequently underwent substantial population size expansion in the early 2000s, at a time when national guidelines encouraged inpatient treatment of TB patients. We infer exportation of this strain and its isoniazid-resistant ancestral precursor from Moldova to neighbouring countries starting as early as 1985. Our findings suggest temporal and ecological associations between specific public health practices, including inpatient hospitalization of drug-resistant TB cases from the early 2000s until 2013, and the evolution of drug-resistant M. tuberculosis in Moldova. These findings underscore the need for regional coordination in TB control and expanded genomic surveillance efforts across Eastern Europe.

Recent Developments on the Synthesis and Bioactivity of Ilamycins/Rufomycins and Cyclomarins, Marine Cyclopeptides That Demonstrate Anti-Malaria and Anti-Tuberculosis Activity.

Ilamycins/rufomycins and cyclomarins are marine cycloheptapeptides containing unusual amino acids. Produced by Streptomyces sp., these compounds show potent activity against a range of mycobacteria, including multidrug-resistant strains of Mycobacterium tuberculosis. The cyclomarins are also very potent inhibitors of Plasmodium falciparum. Biosynthetically the cyclopeptides are obtained via a heptamodular nonribosomal peptide synthetase (NRPS) that directly incorporates some of the nonproteinogenic amino acids. A wide range of derivatives can be obtained by fermentation, while bioengineering also allows the mutasynthesis of derivatives, especially cyclomarins. Other derivatives are accessible by semisynthesis or total syntheses, reported for both natural product classes. The anti-tuberculosis (anti-TB) activity results from the binding of the peptides to the N-terminal domain (NTD) of the bacterial protease-associated unfoldase ClpC1, causing cell death by the uncontrolled proteolytic activity of this enzyme. Diadenosine triphosphate hydrolase (PfAp3Aase) was found to be the active target of the cyclomarins in Plasmodia. SAR studies with natural and synthetic derivatives on ilamycins/rufomycins and cyclomarins indicate which parts of the molecules can be simplified or otherwise modified without losing activity for either target. This review examines all aspects of the research conducted in the syntheses of these interesting cyclopeptides.

Phenotypic and molecular characterization of pyrazinamide resistance among multidrug-resistant Mycobacterium tuberculosis isolates in Ningbo, China

BackgroundDetection of pyrazinamide (PZA) resistance in Mycobacterium tuberculosis (TB) patients is critical, especially in dealing with multidrug-resistant Mycobacterium tuberculosis (MDR-TB) case. Up to date, PZA drug susceptibility testing (DST) has not been regularly performed in China. The prevalence and molecular characteristics of PZA resistance in M.tuberculosis isolates, especially MDR-TB have not been studied in Ningbo, China. This study aimed to analyze the phenotypic and molecular characterization of PZA resistance among MDR-TB isolates in Ningbo.MethodsA total of 110 MDR-TB isolates were collected from the TB patients who were recorded at local TB dispensaries in Ningbo. All clinical isolates were examined by drug susceptibility testing and genotyping. DNA sequencing was used to detect mutations in the pncA gene associated with PZA resistance.ResultsThe prevalence of PZA resistance among MDR-TB strains in Ningbo was 59.1%. With regard to the history and the outcome of treatments among MDR-TB cases, the percentages of re-treated MDR-TB patients in the PZA-resistant group and of successful patients in PZA-susceptible group were significantly higher than the ones in the PZA-susceptible group and in the PZA-resistant group, respectively (P = 0.027, P = 0.020). The results showed that the resistance of streptomycin (67.7% vs 46.7%, P = 0.027), ethambutol (56.9% vs 33.3%, P = 0.015), ofloxacin (43.1% vs 11.1%, P = 0.000), levofloxacin (43.1% vs 11.1%, P = 0.000), pre-XDR (pre-Xtensively Drug Resistance) (38.5% vs 15.6%, P = 0.009), were more frequently adverted among PZA-resistant isolates compared with PZA-susceptible isolates. In addition, 110 MDR-TB was composed of 87 (PZA resistant, 78.5%) Beijing strains and 23 (PZA resistant, 21.5%) non-Beijing strains. Fifty-four out of 65 (83.1%) PZA-resistant MDR strains harbored a mutation located in the pncA gene and the majority (90.7%) were point mutations. Compared with the phenotypic characterization, DNA sequencing of pncA has sensitivity and specificity of 83.1 and 95.6%.ConclusionThe mutations within pncA gene was the primary mechanism of PZA resistance among MDR-TB and DNA sequencing of pncA gene could provide a rapid detection evidence in PZA drug resistance of MDR-TB in Ningbo.

Five-year microevolution of a multidrug-resistant Mycobacterium tuberculosis strain within a patient with inadequate compliance to treatment

BackgroundWhole-genome sequencing has shown that the Mycobacterium tuberculosis infection process can be more heterogeneous than previously thought. Compartmentalized infections, exogenous reinfections, and microevolution are manifestations of this clonal complexity. The analysis of the mechanisms causing the microevolution —the genetic variability of M. tuberculosis at short time scales— of a parental strain into clonal variants with a patient is a relevant issue that has not been yet completely addressed. To our knowledge, a whole genome sequence microevolution analysis in a single patient with inadequate adherence to treatment has not been previously reported.Case presentationIn this work, we applied whole genome sequencing analysis for a more in-depth analysis of the microevolution of a parental Mycobacterium tuberculosis strain into clonal variants within a patient with poor treatment compliance in Argentina. We analyzed the whole-genome sequence of 8 consecutive Mycobacterium tuberculosis isolates obtained from a patient within 57-months of intermittent therapy. Nineteen mutations (9 short-term, 10 fixed variants) emerged, most of them associated with drug resistance. The first isolate was already resistant to isoniazid, rifampicin, and streptomycin, thereafter the strain developed resistance to fluoroquinolones and pyrazinamide. Surprisingly, isolates remained susceptible to the pro-drug ethionamide after acquiring a frameshift mutation in ethA, a gene required for its activation. We also found a novel variant, (T-54G), in the 5′ untranslated region of whiB7 (T-54G), a region allegedly related to kanamycin resistance. Notably, discrepancies between canonical and phage-based susceptibility testing to kanamycin were previously found for the isolate harboring this mutation. In our patient, microevolution was mainly driven by drug selective pressure. Rare short-term mutations fixed together with resistance-conferring mutations during therapy.ConclusionsThis report highlights the relevance of whole-genome sequencing analysis in the clinic for characterization of pre-XDR and MDR resistance profile, particularly in patients with incomplete and/or intermittent treatment.