Drug-resistant Strains Of Mycobacterium Tuberculosis Research Articles

Drug Susceptibility and Mutation Profiles in Mycobacterium tuberculosis Isolates from a Tertiary Care Hospital in Kerala, India.

The rising prevalence of drug-resistant Mycobacterium tuberculosis (MTB) strains poses a significant challenge to global tuberculosis (TB) control efforts. This study aimed to analyze drug resistance patterns and investigate the molecular characteristics of 193 MTB clinical isolates to shed light on the mechanisms of drug resistance. Of the 193 MTB clinical isolates, 28.5% (n = 53) exhibited mono-drug or multidrug resistance. Pyrazinamide mono-drug resistance (PZAr) was the most prevalent (17%, n = 33), followed by isoniazid mono-drug resistance (3.6%, n = 7). Rifampicin resistance was associated with mutations in the rpoB gene (D435Y, D435V, S450L, L452P). Isoniazid resistance mutations were found in the katG (S315T), inhA (C[-15] T), and ndh (R268H) genes, whereas ethambutol resistance mutations were observed in the embB gene (M306V, M306I, M306L, G406S, Q497R). Surprisingly, 94% of PZAr isolates (n = 31) showed no mutations in the pncA or rpsA genes. The presence of the R268H mutation in the ndh gene, not previously linked to PZAr, was detected in 15% of PZAr isolates (n = 5), suggesting its potential contribution to PZAr in specific cases but not as a predominant mechanism. The specific molecular mechanisms underlying PZAr in the majority of the isolates remain unknown, emphasizing the need for further research to uncover the contributing factors. These findings contribute to the understanding of drug resistance patterns and can guide future efforts in TB control and management.

Unraveling the potential of graphene quantum dots against Mycobacterium tuberculosis infection.

The emergence of drug-resistant Mycobacterium tuberculosis (Mtb) strains has underscored the urgent need for novel therapeutic approaches. Carbon-based nanomaterials, such as graphene oxide (GO), have shown potential in anti-TB activities but suffer from significant toxicity issues. This study explores the anti-TB potential of differently functionalized graphene quantum dots (GQDs) - non-functionalized, L-GQDs, aminated (NH2-GQDs), and carboxylated (COOH-GQDs) - alone and in combination with standard TB drugs (isoniazid, amikacin, and linezolid). Their effects were assessed in both axenic cultures and in vitro infection models. GQDs alone did not demonstrate direct mycobactericidal effects nor trapping activity. However, the combination of NH2-GQDs with amikacin significantly reduced CFUs in in vitro models. NH2-GQDs and COOH-GQDs also enhanced the antimicrobial activity of amikacin in infected macrophages, although L-GQDs and COOH-GQDs alone showed no significant activity. The results suggest that specific types of GQDs, particularly NH2-GQDs, can enhance the efficacy of existing anti-TB drugs. These nanoparticles might serve as effective adjuvants in anti-TB therapy by boosting drug performance and reducing bacterial counts in host cells, highlighting their potential as part of advanced drug delivery systems in tuberculosis treatment. Further investigations are needed to better understand their mechanisms and optimize their use in clinical settings.

In silico analysis and characterization of potential inhibitors of MmaA3, a methoxy mycolic acid synthase from Mycobacterium tuberculosis

The emergence of the multi-and extensively drug-resistant (MDR and XDR) strains of Mycobacterium tuberculosis (M.tb), necessitates paradigm-shifting therapeutic approaches. The impermeable waxy lipid layer, primarily composed of mycolic acids, is a key factor in conferring resistance to conventional drugs. This study introduces a novel strategy to combat drug resistance by targeting Methoxy mycolic acid synthase 3 (MmaA3), a critical enzyme in the mycolic acid biosynthesis pathway. MmaA3 is responsible for the O-methylation of hydroxymycolate precursors and emerges as a promising therapeutic target. Through homology-based modeling, we generated a three-dimensional structure of MmaA3, providing crucial insights into its structural characteristics. High throughput virtual screening was performed against the MmaA3 model, using diverse sources: knowledge-based, FDA-approved Drugbank, and Asinex-Elite libraries. Through rigorous computational analyses, including binding affinity assessments, molecular interactions analysis, and binding free energy calculations, potential inhibitors of MmaA3 have been identified. Subsequent validation studies evaluated the stability of top protein-ligand complexes, and free energy calculations using molecular dynamics simulations. The stability of complexes within the catalytic site was confirmed through RMSD and RMSF profile analyses. Furthermore, binding free energy calculations using the MM-GBSA approach revealed significant binding affinity of identified ligands for MmaA3 target protein, comparable to its substrate/cofactors. These findings underscore the potential of the proposed molecules as candidates for further experimental exploration, offering promising avenues for the development of effective inhibitors against M.tb. Overall, our research contributes to significantly advancing the formulation of progressive therapeutic strategies in combating drug-resistant tuberculosis. Communicated by Ramaswamy H. Sarma

Revealing the Interaction Mechanism between Mycobacterium tuberculosis GyrB and Novobiocin, SPR719 through Binding Thermodynamics and Dissociation Kinetics Analysis.

With the rapid emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb), various levels of resistance against existing anti-tuberculosis (TB) drugs have developed. Consequently, the identification of new anti-TB targets and drugs is critically urgent. DNA gyrase subunit B (GyrB) has been identified as a potential anti-TB target, with novobiocin and SPR719 proposed as inhibitors targeting GyrB. Therefore, elucidating the molecular interactions between GyrB and its inhibitors is crucial for the discovery and design of efficient GyrB inhibitors for combating multidrug-resistant TB. In this study, we revealed the detailed binding mechanisms and dissociation processes of the representative inhibitors, novobiocin and SPR719, with GyrB using classical molecular dynamics (MD) simulations, tau-random acceleration molecular dynamics (τ-RAMD) simulations, and steered molecular dynamics (SMD) simulations. Our simulation results demonstrate that both electrostatic and van der Waals interactions contribute favorably to the inhibitors' binding to GyrB, with Asn52, Asp79, Arg82, Lys108, Tyr114, and Arg141 being key residues for the inhibitors' attachment to GyrB. The τ-RAMD simulations indicate that the inhibitors primarily dissociate from the ATP channel. The SMD simulation results reveal that both inhibitors follow a similar dissociation mechanism, requiring the overcoming of hydrophobic interactions and hydrogen bonding interactions formed with the ATP active site. The binding and dissociation mechanisms of GyrB with inhibitors novobiocin and SPR719 obtained in our work will provide new insights for the development of promising GyrB inhibitors.

Molecular docking, molecular dynamics simulations and binding free energy studies of interactions between Mycobacterium tuberculosis Pks13, PknG and bioactive constituents of extremophilic bacteria

Mycobacterial pathogens present a significant challenge to disease control efforts globally due to their inherent resistance to multiple antibiotics. The rise of drug-resistant strains of Mycobacterium tuberculosis has prompted an urgent need for innovative therapeutic solutions. One promising way to discover new tuberculosis drugs is by utilizing natural products from the vast biochemical space. Multidisciplinary methods can used to harness the bioactivity of these natural products. This study aimed to evaluate the antimycobacterial efficacy of functional crude extracts from bacteria isolated from gold mine tailings in South Africa. Bacterial strains were identified using 16S rRNA sequencing. The crude extracts obtained from the bacteria were tested against Mycobacterium tuberculosis H37Rv, Mycobacterium smegmatis mc2155, and Mycobacterium aurum A+. Untargeted HPLC-qTOF and molecular networking were used to identify the functional constituents present in extracts that exhibited inhibitory activity. A virtual screening workflow (VSW) was used to filter compounds that were strong binders to Mycobacterium tuberculosis Pks13 and PknG. The ligands returned from the VSW were subjected to optimization using density functional theory (DFT) at M06-2X/6-311++ (d,p) level of theory and basis set implemented in Gaussian16 Rev.C01. The optimized ligands were re-docked against Mycobacterium tuberculosis Pks13 and PknG. Molecular dynamics simulation and molecular mechanics generalized born surface area were used to evaluate the stability of the protein–ligand complexes formed by the identified hits. The hit that showed promising binding characteristics was virtually modified through multiple synthetic routes using reaction-driven enumeration. Three bacterial isolates showed significant activity against the two strains of Mycobacterium, while only two, Bacillus subtilis and Bacillus licheniformis, exhibited activity against both Mycobacterium tuberculosis H37Rv, Mycobacterium smegmatis mc2155, and Mycobacterium aurum A+. The tentatively identified compounds from the bacterial crude extracts belonged to various classes of natural compounds associated with antimicrobial activity. Two compounds, cyclo-(L-Pro-4-OH-L-Leu) and vazabitide A, showed strong binding against PknG and Pks13, with pre-MD MM-GBSA values of − 42.8 kcal/mol and − 47.6 kcal/mol, respectively. The DFT-optimized compounds exhibited the same docking scores as the ligands optimized using the OPSL-4 force field. After modifying vazabitide A, its affinity to the Pks13 binding site increased to − 85.8 kcal/mol, as revealed by the post-MD MM-GBSA analysis. This study highlights the potential of bacteria isolates from gold mine tailings as a source of new scaffolds for designing and optimizing anti-Mycobacterium agents. These agents synthesized in-silico can be further tested in-vitro to evaluate their efficacy.

An Exploratory Review of the Potential of Lytic Proteins and Bacteriophages for the Treatment of Tuberculosis.

Tuberculosis (TB) is a highly prevalent infectious disease that causes more than 1.5 million deaths a year. More than 25% of TB deaths occur in Africa, and TB is South Africa's leading cause of death, with about 89,000 people dying of it yearly. The emergence of multidrug-resistant TB (MDR-TB) poses a significant threat to health security and could reverse the positive gains already made in the fight against TB. Antibiotic treatments are available, but side effects and the alarming increase in the prevalence of drug-resistant strains of Mycobacterium tuberculosis (Mtb) will compromise the control of the spread and treatment of the disease. A promising option is to employ specialized enzymes encoded by bacteriophages, which destroy bacterial cell membranes and walls to treat tuberculosis. Phage therapy against bacteria is a known treatment that is now reemerging with lytic proteins. These proteins provide an alternative means to treat infectious diseases where conventional antibiotic regimens do not meet the requirements. This review explores and discusses the potential of lytic protein therapy as an antimicrobial strategy against M. tuberculosis and multidrug-resistant tuberculosis.

Genetic mechanisms of co-emergence of INH-resistant Mycobacterium tuberculosis strains during the standard course of antituberculosis therapy.

The incidence of isoniazid (INH) resistant Mycobacterium tuberculosis is increasing globally. This study aimed to identify the molecular mechanisms behind the development of INH resistance in M. tuberculosis strains collected from the same patients during the standard course of treatment. Three M. tuberculosis strains were collected from a patient before and during antituberculosis (anti-TB) therapy. The strains were characterized using phenotypic drug susceptibility tests, Mycobacterial Interspersed Repeated Unit-Variable-Number Tandem Repeats (MIRU-VNTR), and whole-genome sequencing (WGS) to identify mutations associated with INH resistance. To validate the role of the novel mutations in INH resistance, the mutated katG genes were electroporated into a KatG-deleted M. tuberculosis strain (GA03). Three-dimensional structures of mutated KatG were modeled to predict their impact on INH binding. The pre-treatment strain was susceptible to INH. However, two INH-resistant strains were isolated from the patient after anti-TB therapy. MIRU-VNTR and WGS revealed that the three strains were clonally identical. A missense mutation (P232L) and a nonsense mutation (Q461Stop) were identified in the katG of the two post-treatment strains, respectively. Transformation experiments showed that katG of the pre-treatment strain restored INH susceptibility in GA03, whereas the mutated katG genes from the post-treatment strains rendered negative catalase activity and INH resistance. The protein model indicated that P232L reduced INH-KatG binding affinity while Q461Stop truncated gene transcription. Our results showed that the two katG mutations, P232L and Q461Stop, accounted for the co-emergence of INH-resistant clones during anti-TB therapy. The inclusion of these mutations in the design of molecular assays could increase the diagnostic performance.IMPORTANCEThe evolution of drug-resistant strains of Mycobacterium tuberculosis within the lung lesions of a patient has a detrimental impact on treatment outcomes. This is particularly concerning for isoniazid (INH), which is the most potent first-line antimycobacterial drug. However, the precise genetic factors responsible for drug resistance in patients have not been fully elucidated, with approximately 15% of INH-resistant strains harboring unknown genetic factors. This raises concerns about the emergence of drug-resistant clones within patients, further contributing to the global epidemic of resistance. In this study, we revealed the presence of two novel katG mutations, which emerged independently due to the stress exerted by antituberculosis (anti-TB) treatment on a parental strain. Importantly, we experimentally demonstrated the functional significance of both mutations in conferring resistance to INH. Overall, this research sheds light on the genetic mechanisms underlying the evolution of INH resistance within patients and provides valuable insights for improving diagnostic performance by targeting specific mutations.

Review for QSAR studies and drug design of selected heterocyclic nucleus of antitubercular drugs

This review article explores the pressing issue of drug-resistant strains of Mycobacterium tuberculosis, which have emerged due to the widespread and uncontrolled use of antibiotics in clinical settings over several decades. In response to this challenge, various methods have been developed for synthesizing new antitubercular compounds. Among these, the fragment-based drug discovery (FBDD) approach has shown promise as an effective strategy. One class of compounds that has exhibited significant potential in combating tuberculosis is 1,2-diazoles. The article discusses the importance of these compounds and their potential as future antitubercular drugs. Additionally, it delves into the various strategies employed in drug development, emphasizing the relevance and efficacy of FBDD. Analytical methods play a crucial role in characterizing antitubercular antibiotics, and the article highlights liquid chromatography and voltammetry as preferred techniques for determining these compounds. The redox (oxidation/reduction) properties of antituberculars make them amenable to analysis using electrochemical methods, with voltammetry being particularly suitable. Furthermore, the article underscores the significance of utilizing voltammetry for quantifying different categories of antibiotics in both dosage forms and human body fluids. The affordability of this method makes it particularly advantageous for developing countries, providing a cost-effective approach to monitor and assess the presence of antitubercular drugs. Overall, the review article offers valuable insights into the current strategies for addressing drug resistance in tuberculosis and highlights the potential of 1,2-diazoles as future antitubercular agents.

Temperature-Induced Restructuring of Mycolic Acid Bilayers Modeling the Mycobacterium tuberculosis Outer Membrane: A Molecular Dynamics Study.

The emergence of new drug-resistant strains of the tuberculosis pathogen Mycobacterium tuberculosis (Mtb) is a new challenge for modern medicine. Its resistance capacity is closely related to the properties of the outer membrane of the Mtb cell wall, which is a bilayer membrane formed by mycolic acids (MAs) and their derivatives. To date, the molecular mechanisms of the response of the Mtb outer membrane to external factors and, in particular, elevated temperatures have not been sufficiently studied. In this work, we consider the temperature-induced changes in the structure, ordering, and molecular mobility of bilayer MA membranes of various chemical and conformational compositions. Using all-atom long-term molecular dynamics simulations of various MA membranes, we report the kinetic parameters of temperature-dependent changes in the MA self-diffusion coefficients and conformational compositions, including the apparent activation energies of these processes, as well as the characteristic times of ordering changes and the features of phase transitions occurring over a wide range of elevated temperatures. Understanding these effects could be useful for the prevention of drug resistance and the development of membrane-targeting pharmaceuticals, as well as in the design of membrane-based materials.

Differential distribution of linezolid in diseased and nondiseased bones in patients with spinal tuberculosis

BackgroundLinezolid exhibits antibacterial activity against sensitive and drug-resistant strains of Mycobacterium tuberculosis. Knowledge on the distribution of linezolid in different types of bones in patients with spinal tuberculosis (TB) is lacking, which limits the pharmacokinetic and pharmacodynamic studies of linezolid. This study aimed to evaluate the distribution of linezolid in diseased and nondiseased bones in patients with spinal TB.MethodsSpinal TB patients treated with linezolid-containing regimens and whose diseased and nondiseased bones were collected during surgery were enrolled retrospectively from January 2017 to February 2022. Blood, nondiseased bones, and diseased bones were collected simultaneously during the operation. Linezolid concentrations in the plasma, nondiseased bones, and diseased bones were subjected to high-performance liquid chromatography–tandem mass spectrometry.ResultsSeven eligible spinal TB patients, including one rifampicin-resistant case, were enrolled. Following a 600 mg oral administration of linezolid before surgery, the median concentrations of linezolid in plasma, nondiseased bone, and diseased bone of the seven patients were 8.23, 1.01, and 2.13 mg/L, respectively. The mean ratios of linezolid concentration in nondiseased bones/plasma, diseased bones/plasma and diseased bones/nondiseased bones reached 0.26, 0.49, and 2.27, respectively. The diseased bones/plasma presented a higher mean ratio of linezolid concentration than nondiseased bones/plasma, and the difference was statistically significant (t = 2.55, p = 0.025). Pearson’s correlation analysis showed the positively correlation of linezolid concentrations in diseased and nondiseased bones (r = 0.810, p = 0.027).ConclusionsLinezolid exhibits a higher concentration distribution in diseased bones than in nondiseased bones.

Marine-Fungi-Derived Gliotoxin Promotes Autophagy to Suppress Mycobacteria tuberculosis Infection in Macrophage.

The Mycobacterium tuberculosis (MTB) infection causes tuberculosis (TB) and has been a long-standing public-health threat. It is urgent that we discover novel antitubercular agents to manage the increased incidence of multidrug-resistant (MDR) or extensively drug-resistant (XDR) strains of MTB and tackle the adverse effects of the first- and second-line antitubercular drugs. We previously found that gliotoxin (1), 12, 13-dihydroxy-fumitremorgin C (2), and helvolic acid (3) from the cultures of a deep-sea-derived fungus, Aspergillus sp. SCSIO Ind09F01, showed direct anti-TB effects. As macrophages represent the first line of the host defense system against a mycobacteria infection, here we showed that the gliotoxin exerted potent anti-tuberculosis effects in human THP-1-derived macrophages and mouse-macrophage-leukemia cell line RAW 264.7, using CFU assay and laser confocal scanning microscope analysis. Mechanistically, gliotoxin apparently increased the ratio of LC3-II/LC3-I and Atg5 expression, but did not influence macrophage polarization, IL-1β, TNF-a, IL-10 production upon MTB infection, or ROS generation. Further study revealed that 3-MA could suppress gliotoxin-promoted autophagy and restore gliotoxin-inhibited MTB infection, indicating that gliotoxin-inhibited MTB infection can be treated through autophagy in macrophages. Therefore, we propose that marine fungi-derived gliotoxin holds the promise for the development of novel drugs for TB therapy.

Emergence of bedaquiline-resistant tuberculosis and of multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis strains with rpoB Ile491Phe mutation not detected by Xpert MTB/RIF in Mozambique: a retrospective observational study

In 2021, an estimated 4800 people developed rifampicin-resistant tuberculosis in Mozambique, 75% of which went undiagnosed. Detailed molecular data on rifampicin-resistant and multidrug-resistant (MDR) tuberculosis are not available. Here, we aimed at gaining precise data on the determinants of rifampicin-resistant and MDR tuberculosis in Mozambique. In this retrospective observational study, we performed whole-genome sequencing of 704 rifampicin-resistant Mycobacterium tuberculosis complex (Mtbc) strains submitted to the National Tuberculosis Reference Laboratory (NTRL) in Maputo, Mozambique, between 2015 and 2021. Phylogenetic strain classification, genomic resistance prediction, and cluster analysis were performed. Between Jan 1, 2015, and July 31, 2021, 2606 Mtbc isolates with an isoniazid or rifampicin resistance were identified in the NTRL biobank, of which, 1483 (56·9%) were from men, 1114 (42·7%) from women, and nine (0·4%) were unknown. Genome-based drug-resistant prediction classified 704 Mtbc strains as rifampicin resistant. 628 (89%) of the 704 Mtbc strains were classified MDR; of those, 146 (23%) were pre-extensively drug resistant (pre-XDR; additional fluoroquinolone resistance), and 24 (4%) extensively drug resistant (XDR; combined fluoroquinolone and bedaquiline resistance). Overall, 61 (9%) of 704 strains revealed resistance to bedaquiline: five (7%) of 76 rifampicin resistant plus bedaquiline resistant, 32 (7%) of 458 MDR plus bedaquiline resistant, and 24 (100%) of 24 XDR. Prevalence of bedaquiline resistance increased from 3% in 2016 to 14% in 2021. The cluster rate (12 single-nucleotide polymorphism threshold) was 42% for rifampicin-resistant strains, 78% for MDR strains, 94% for pre-XDR strains, and 96% for XDR Mtbc strains. 31 (4%) of 704 Mtbc strains, belonging to a diagnostic escape outbreak strain previously described in Eswatini (group_56), had an rpoB Ile491Phe mutation which is not detected by Xpert MTB/RIF (no other rpoB mutation). Of these, 23 (74%) showed additional resistance to bedaquiline, 13 (42%) had bedaquiline and fluoroquinolone resistance, and two (6%) were bedaquiline, fluoroquinolone, and delamanid resistant. Pre-XDR resistance is highly prevalent among MDR Mtbc strains in Mozambique and so is bedaquiline resistance; and the frequency of bedaquiline resistance quadrupled over time and was found even in Mtbc strains without fluoroquinolone resistance. Importantly, strains with Ile491Phe mutation were frequent, accounting for 31% (n=10) of MDR plus bedaquiline-resistant strains and 54% (n=13) of XDR Mtbc strains. Given the current diagnostic algorithms and treatment regimens, both the emergence of rifampicin resistance due to Ile491Phe and bedaquiline resistance might jeopardise MDR tuberculosis prevention and care unless sequencing-based technology is rolled out. The potential cross border spread of diagnostic escape strains needs further investigation. The German Ministry of Health through the Seq_MDRTB-Net project, the Deutsche Forschungsgemeinschaft under Germany's Excellence Strategy Precision Medicine in Inflammation and the Research Training Group 2501 TransEvo, the Leibniz Science Campus Evolutionary Medicine of the Lung, and the German Ministry of Education and Research via the German Center for Infection Research.

Drug-resistant strains of Mycobacterium tuberculosis: cell envelope profiles and interactions with the host.

In the past few decades, drug-resistant (DR) strains of Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis (TB), have become increasingly prevalent and pose a threat to worldwide public health. These strains range from multi (MDR) to extensively (XDR) drug-resistant, making them very difficult to treat. Further, the current and future impact of the Coronavirus Disease 2019 (COVID-19) pandemic on the development of DR-TB is still unknown. Although exhaustive studies have been conducted depicting the uniqueness of the M.tb cell envelope, little is known about how its composition changes in relation to drug resistance acquisition. This knowledge is critical to understanding the capacity of DR-M.tb strains to resist anti-TB drugs, and to inform us on the future design of anti-TB drugs to combat these difficult-to-treat strains. In this review, we discuss the complexities of the M.tb cell envelope along with recent studies investigating how M.tb structurally and biochemically changes in relation to drug resistance. Further, we will describe what is currently known about the influence of M.tb drug resistance on infection outcomes, focusing on its impact on fitness, persister-bacteria, and subclinical TB.

In silico virtual screening for the identification of novel inhibitors against dihydrodipicolinate reductase (DapB) of Mycobacterium tuberculosis, a key enzyme of diaminopimelate pathway.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (M. tb) is one of the most devastating infectious diseases afflicting a large number of human lives. In spite of the availability of several drugs for treating TB, emergence of drug-resistant strains of the pathogen has made treatment and eradication of TB a challenging task. Hence, there is an imperative need for new intervention strategies to target M. tb. The enzymes of the diaminopimelate pathway, involved in lysine biosynthesis as well as in cell wall biosynthesis, have been considered as important anti-TB drug targets. One such enzyme is dihydrodipicolinate reductase (DapB), catalyzing the reduction of 2,3-dihydrodipicolinic acid to 2,3,4,5-tetrahydro-dipicolinic acid by utilizing NADH/NADPH as its cofactor. Here, we have generated an antisense knockdown mutant strain of dapB and demonstrated a crucial role of dapB in the in vitro and intracellular growth of M. tb. Further, in silico virtual screening was performed by employing a library of ~95,000 compounds for the identification of inhibitory molecules against DapB. The molecules with high docking scores were screened against the enzymatic activity of DapB to determine their IC50 values. Further, hit molecules that inhibited DapB were screened against the M. tb growth in broth culture and inside macrophages. The molecules exhibiting M. tb inhibition were also evaluated for their cytotoxicity against various mammalian cell lines. In summary, we have identified a lead molecule, B59, with an IC50 value of 11 µg/mL and MIC99 value of 20 µg/mL, which can be further optimized to develop potent inhibitory compounds against M. tb DapB. IMPORTANCE Non-compliance to lengthy antituberculosis (TB) treatment regimen, associated side effects, and emergence of drug-resistant strains of Mycobacterium tuberculosis (M. tb) emphasize the need to develop more effective anti-TB drugs. Here, we have evaluated the role of M. tb dihydrodipicolinate reductase (DapB), a component of the diaminopimelate pathway, which is involved in the biosynthesis of both lysine and mycobacterial cell wall. We showed that DapB is essential for the in vitro as well as intracellular growth of M. tb. We further utilized M. tb DapB, as a target for identification of inhibitors by employing in silico virtual screening, and conducted various in vitro screening assays to identify inhibitors with potential to inhibit DapB activity and in vitro and intracellular growth of M. tb with no significant cytotoxicity against various mammalian cell lines. Altogether, M. tb DapB serves as an important drug target and a hit molecule, namely, 4-(3-Phenylazoquinoxalin-2-yl) butanoic acid methyl ester has been identified as an antimycobacterial molecule in our study.

Distribution of multi-drug resistant tuberculosis in Ekiti and Ondo states, Nigeria

BackgroundTuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is one of the top infectious killer diseases in the world. The emergence of drug-resistant MTB strains has thrown challenges in controlling TB worldwide. This study investigated the prevalence of drug-resistant tuberculosis in the states of Nigeria and the risk factors that can increase the incidence of tuberculosis. MethodsThe study is a cross-sectional epidemiological research carried out in the six senatorial districts of Ekiti and Ondo states, Nigeria, between February 2019 and January 2020. A structured questionnaire was administered to 1203 respondents for socio-demographic information, and sputum samples were collected from them for TB investigation. GeneXpert technique was used to diagnose TB from the sputum samples, followed by bacterial isolation using Löweinstein-Jensen medium and antibiotic susceptibility testing. ResultsPrevalence of TB in the two states combined was 15 ​%; with 13.8 ​% for Ekiti state and 16.1 ​% for Ondo State. The distribution of TB in the senatorial districts was such that: Ondo South ​> ​Ekiti Central ​> ​Ekiti South ​> ​Ondo North ​> ​Ekiti North ​> ​Ondo Central. The risk factors identified for TB prevalence in two states were gender, male ​> ​female (OR ​= ​0.548, p ​= ​0.004); overcrowding (OR ​= ​0.733, p ​= ​0.026); room size (OR ​= ​0.580, p ​= ​0.002); smoking (OR ​= ​0.682, p ​= ​0.019) and dry and dusty season (OR ​= ​0.468, p ​= ​0.005). The prevalence of MDR-TB in Ekiti and Ondo States were 1.2 ​% and 1.3 ​% respectively. The identified risk factors for MDR were education (OR ​= ​0.739, p ​= ​0.017), age (OR ​= ​0.846, p ​= ​0.048), religion (OR ​= ​1.95, p ​= ​0.0003), family income (OR ​= ​1.76, p ​= ​0.008), previous TB treatment (OR ​= ​3.64, p ​= ​0.004), smoking (OR ​= ​1.33, p ​= ​0.035) and HIV status (OR ​= ​1.85, p ​= ​0.006). Rifampicin monoresistant was reported in 6.7 ​% of the rifampicin-resistant strains, while 93.3 ​% were rifampicin polyresistant strains. Two (13.3 ​%) of the MDR-TB strains were resistant to all the 3 first-line antimycobacterial agents. All the Rifampicin-resistant TB strains were susceptible to the aminoglycosides (Amikacin, Capreomycin and Kanamycin), also with high susceptibility to the fluoroquinilones: Moxifloxacin (100 ​%) and Levofloxacin (86.7 ​%). Sixteen (94.1 ​%) of the 17 Rifampicin-susceptible strains were susceptible to all the eight antibiotics tested, while one (5.9 ​%) was susceptible to Rifampicin and Isoniazid but resistant to the rest antibiotics. Conclusion: The study showed that there is high prevalence of TB and MDR-TB in Ekiti and Ondo States Nigeria, hence, to meet the SDG Target 3.3 of ending TB epidemic by 2030, culturing and antibiotic susceptibility testing should be carried out on every TB-positive sputum and the patients treated accordingly.

Multi-epitope vaccine against drug-resistant strains of Mycobacterium tuberculosis: a proteome-wide subtraction and immunoinformatics approach.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, one of the most deadly infections in humans. The emergence of multidrug-resistant and extensively drug-resistant Mtb strains presents a global challenge. Mtb has shown resistance to many frontline antibiotics, including rifampicin, kanamycin, isoniazid, and capreomycin. The only licensed vaccine, Bacille Calmette-Guerin, does not efficiently protect against adult pulmonary tuberculosis. Therefore, it is urgently necessary to develop new vaccines to prevent infections caused by these strains. We used a subtractive proteomics approach on 23 virulent Mtb strains and identified a conserved membrane protein (MmpL4, NP_214964.1) as both a potential drug target and vaccine candidate. MmpL4 is a non-homologous essential protein in the host and is involved in the pathogen-specific pathway. Furthermore, MmpL4 shows no homology with anti-targets and has limited homology to human gut microflora, potentially reducing the likelihood of adverse effects and cross-reactivity if therapeutics specific to this protein are developed. Subsequently, we constructed a highly soluble, safe, antigenic, and stable multi-subunit vaccine from the MmpL4 protein using immunoinformatics. Molecular dynamics simulations revealed the stability of the vaccine-bound Toll-like receptor-4 complex on a nanosecond scale, and immune simulations indicated strong primary and secondary immune responses in the host. Therefore, our study identifies a new target that could expedite the design of effective therapeutics, and the designed vaccine should be validated. Future directions include an extensive molecular interaction analysis, in silico cloning, wet-lab experiments, and evaluation and comparison of the designed candidate as both a DNA vaccine and protein vaccine.

Dual-Target Mycobacterium tuberculosis Inhibition: Insights into the Molecular Mechanism of Antifolate Drugs.

The escalating prevalence of drug-resistant strains of Mycobacterium tuberculosis has posed a significant challenge to global efforts in combating tuberculosis. To address this issue, innovative therapeutic strategies are required that target essential biochemical pathways while minimizing the potential for resistance development. The concept of dual targeting has gained prominence in drug discovery against resistance bacteria. Dual targeting recognizes the complexity of cellular processes and disrupts more than one vital pathway, simultaneously. By inhibiting more than one essential process required for bacterial growth and survival, the chances of developing resistance are substantially reduced. A previously reported study investigated the dual-targeting potential of a series of novel compounds against the folate pathway in Mycobacterium tuberculosis. Expanding on this study, we investigated the predictive pharmacokinetic profiling and the structural mechanism of inhibition of UCP1172, UCP1175, and UCP1063 on key enzymes, dihydrofolate reductase (DHFR) and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate reductase (RV2671), involved in the folate pathway. Our findings indicate that the compounds demonstrate lipophilic physiochemical properties that promote gastrointestinal absorption, and may also inhibit the drug-metabolizing enzyme, cytochrome P450 3A4, thus enhancing their biological half-life. Furthermore, key catalytic residues (Serine, Threonine, and Aspartate), conserved in both enzymes, were found to participate in vital molecular interactions with UCP1172, which demonstrated the most favorable free binding energies to both DHFR and RV2671 (-41.63 kcal/mol, -48.04 kcal/mol, respectively). The presence of characteristic loop shifts, which are similar in both enzymes, also indicates a common inhibitory mechanism by UCP1172. This elucidation advances the understanding of UCP1172's dual inhibition mechanism against Mycobacterium tuberculosis.

Discovery of novel reversible inhibitor of DprE1 based on benzomorpholine for the treatment of tuberculosis.

About a quarter of the world's population is infected with Mycobacterium tuberculosis, equivalent to about two billion people. With the emergence of multidrug-resistant tuberculosis, those existing anti-tuberculosis drugs no longer meet the demand for cure anymore; there is an urgent need for the development of new anti-tuberculosis drugs. Decaprenylphosphoryl-β-D-ribose 2´-epimerase (DprE1) has been proven to be a potential antimycobacterial target, and several inhibitors have entered clinical trial. Herein, we designed and synthesized a series of compounds based on the indole and benzomorpholine by using the strategy of scaffold hopping. The preferred compound B18 showed strong antimycobacterial activity in H37Rv and drug-resistant clinical isolates. In addition, compound B18 did not exhibit antimycobacterial efficacy against other species of strains. Subsequently, the target of B18 was identified as DprE1 by analyzing spontaneous compound-resistant mutation data, and a docking study was performed to illustrate the binding mode between B18 and DprE1. In general, compound B18 is compatible to current DprE1 inhibitors, even higher phosphodiesterase 6C selectivity and plasma protein binding rate, which represent a new type of effective reversible DprE1 inhibitor. IMPORTANCE Drug therapy remains the cornerstone of tuberculosis (TB) treatment, yet first-line anti-tuberculosis drugs are associated with significant adverse effects that can compromise patient outcomes. Moreover, prolonged and widespread use has led to an alarming rise in drug-resistant strains of Mycobacterium tuberculosis, including multidrug-resistant [MDR-tuberculosis (TB)] and extensively drug-resistant (XDR-TB) forms. Urgent action is needed to develop novel anti-tuberculosis agents capable of overcoming these challenges. We report that compound B18, a decaprenylphosphoryl-β-D-ribose 2´-epimerase inhibitor with a benzomorpholine backbone, exhibits potent activity against not only the non-pathogenic strain H37Ra, but also the pathogenic strain H37Rv and clinical MDR and XDR strains. Preliminary druggability studies indicate that B18 possesses high safety and acceptable pharmacokinetic properties, rendering it a promising candidate for further development as a novel anti-tuberculosis agent.

SK-03-92 Drug Kills Intracellular Mycobacterium tuberculosis.

Tuberculosis affects millions of people worldwide. The emergence of drug-resistant Mycobacterium tuberculosis strains has made treatment more difficult. A drug discovery project initiated to screen natural products identified a lead stilbene compound, and structure function analysis of hundreds of analogs led to the discovery of the SK-03-92 stilbene lead compound with activity against several non-tuberculoid mycobacteria. For this study, an MIC analysis and intracellular killing assay were performed to test SK-03-92 against M. tuberculosis grown in vitro as well as within murine macrophage cells. The MIC analysis showed that SK-03-92 had activity against M. tuberculosis in the range of 0.39 to 6.25 μg/mL, including activity against single-drug-resistant strains. Further, an intracellular kill assay demonstrated that the SK-03-92 lead compound killed M. tuberculosis cells within murine macrophage cells. Together, the data show the SK-03-92 lead compound can kill M. tuberculosis bacteria within mammalian macrophages.

Outcomes of bedaquiline-containing regimen in the treatment of adults with drug-resistant tuberculosis in a tertiary care center in Rajasthan.

The emergence of drug-resistant strains of Mycobacterium tuberculosis has become a significant public health problem and has led to a setback in the efforts to end tuberculosis (TB) worldwide. The longer duration, heavier pill load, and higher toxicity profile of drug-resistant TB regimens compared to those for drug-susceptible TB lead to reduced adherence and worse treatment results, including mortality. This study was conducted to estimate treatment outcomes and adverse effects in patients with drug-resistant TB on a bedaquiline-containing regimen. Patients after the pre-treatment evaluation were enrolled in a bedaquiline-containing regimen. These patients were followed up for 18 months, and the final outcome was assessed along with the adverse effects. It was found that 49 (84.4%) patients achieved culture conversion by 3 months, 54 (93.1%) achieved culture conversion by 6 months, 52 (83.81%) had favorable outcomes (cured, treatment completed), and 10 had unfavorable outcomes (died, lost to follow-up, failed). Coupled with gradually increasing trends in success rates since 2012, lesser failure rates and fewer concerns regarding grave adverse effects are a silver lining in the cloud of increasing burden and widening resistance patterns. More funding has to be directed towards ensuring adherence and finding high-risk individuals to expedite the achievement of sustainable development goals.