Drugs Isoniazid Research Articles

2nd Workshop in Microbial Molecular Biology

2nd Workshop in Microbial Molecular Biology

Evaluation of the interaction between potent small molecules against the Nipah virus Glycoprotein in Malaysia and Bangladesh strains, accompanied by the human Ephrin-B2 and Ephrin-B3 receptors; a simulation approach.

Malaysia reported the first human case of Nipah virus (NiV) in late September 1998 with encephalitis and respiratory symptoms. As a result of viral genomic mutations, two main strains (NiV-Malaysia and NiV-Bangladesh) have spread around the world. There are no licensed molecular therapeutics available for this biosafety level 4 pathogen. NiV attachment glycoprotein plays a critical role in viral transmission through its human receptors (Ephrin-B2 and Ephrin-B3), so identifying small molecules that can be repurposed to inhibit them is crucial to developing anti-NiV drugs. Consequently, in this study annealing simulations, pharmacophore modeling, molecular docking, and molecular dynamics were used to evaluate seven potential drugs (Pemirolast, Nitrofurantoin, Isoniazid Pyruvate, Eriodictyol, Cepharanthine, Ergoloid, and Hypericin) against NiV-G, Ephrin-B2, and Ephrin-B3 receptors. Based on the annealing analysis, Pemirolast for efnb2 protein and Isoniazid Pyruvate for efnb3 receptor were repurposed as the most promising small molecule candidates. Furthermore, Hypericin and Cepharanthine, with notable interaction values, are the top Glycoprotein inhibitors in Malaysia and Bangladesh strains, respectively. In addition, docking calculations revealed that their binding affinity scores are related to efnb2-pem (- 7.1kcal/mol), efnb3-iso (- 5.8kcal/mol), gm-hyp (- 9.6kcal/mol), gb-ceph (- 9.2kcal/mol). Finally, our computational research minimizes the time-consuming aspects and provides options for dealing with any new variants of Nipah virus that might emerge in the future.

Harnessing Innate Immunity to Treat Mycobacterium tuberculosis Infections: Heat-Killed Caulobacter crescentus as a Novel Biotherapeutic.

Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), is a serious and devastating infectious disease worldwide. Approximately a quarter of the world population harbors latent Mtb infection without pathological consequences. Exposure of immunocompetent healthy individuals with Mtb does not result in active disease in more than 90% individuals, suggesting a defining role of host immunity to prevent and/or clear early infection. However, innate immune stimulation strategies have been relatively underexplored for the treatment of tuberculosis. In this study, we used cell culture and mouse models to examine the role of a heat-killed form of a non-pathogenic microbe, Caulobacter crescentus (HKCC), in inducing innate immunity and limiting Mtb infection. We also examined the added benefits of a distinct chemo-immunotherapeutic strategy that incorporates concurrent treatments with low doses of a first-line drug isoniazid and HKCC. This therapeutic approach resulted in highly significant reductions in disseminated Mtb in the lungs, liver, and spleen of mice compared to either agent alone. Our studies demonstrate the potential of a novel innate immunotherapeutic strategy with or without antimycobacterial drugs in controlling Mtb infection in mice and open new avenues for the treatment of tuberculosis in humans.

Adequacy of the 10 mg/kg Daily Dose of Antituberculosis Drug Isoniazid in Infants under 6 Months of Age.

In 2010, the WHO recommended an increase in the daily doses of first-line anti-tuberculosis medicines in children. We aim to characterize the pharmacokinetics of the once-daily isoniazid (INH) dose at 10 mg/kg of body weight in infants <6 months of age. We performed a multicenter pharmacokinetic study in Spain. The N-acetyltransferase 2 gene was analyzed to determine the acetylation status. Samples were analyzed using a validated UPLC-UV assay. A non-compartmental pharmacokinetic analysis was performed. Twenty-three pharmacokinetic profiles were performed in 20 infants (8 females) at a median (IQR) age of 19.0 (12.6-23.3) weeks. The acetylator statuses were homozygous fast (n = 1), heterozygous intermediate (n = 12), and homozygous slow (n = 7). INH median (IQR) Cmax and AUC0-24h values were 4.8 (3.7-6.7) mg/L and 23.5 (13.4-36.7) h*mg/L and the adult targets (>3 mg/L and 11.6-26.3 h*mg/L) were not reached in three and five cases, respectively. The age at assessment or acetylator status had no impact on Cmax values, but a larger INH AUC0-24h (p = 0.025) and trends towards a longer half-life (p = 0.055) and slower clearance (p = 0.070) were observed in homozygous slow acetylators. Treatment was well tolerated; mildly elevated alanine aminotransferase levels were observed in three cases. In our series of young infants receiving isoniazid, no major safety concerns were raised, and the target adult levels were reached in most patients.

Copper(II) complexes of fused ring selenosemicarbazones: Synthesis, structure elucidation, biological activity and molecular modeling

Reaction of copper(II) acetate with cyclohexanoneselenosemicarbazone (Hcysesc), 5-chloro isatinselenosemicarbazone (5-ClHIstsesc), 1-methyl isatinselenosemicarbazone (1-MeHIstsesc), 3-indole selenosemicarbazone (3-HInsesc), 3-acetylindole selenosemicarbazone (3-AcHInsesc and 2-naphthaldehyde selenosemicarbazone (2-Hnaphsesc) in 1:2 (M: L) molar ratio yielded complexes of stiochiometry, [Cu(L)2] 1–6 (L = cysesc 1; 5-ClIstsesc 2; 1-MeIstsesc 3; 3-Insesc 4; 3-AcInsesc 5; 2-naphsesc 6). All the complexes are characterized by IR, Mass and ESR spectroscopy. Two well defined g values (g‖ and g⊥) in ESR spectrum of complexes 1–4, 6 indicate axial symmetry, whereas symmetry around copper(II) metal in complex 5 is confirmed by three different g values (g1, 2.095; g2, 2.15; g3, 2.26), which supports rhombic distortion. The value of g‖ obtained is greater than g⊥, which is greater than ge, in 1–4, 6 is in well agreement in dx2-y2 ground term of square planar geometry. The value of empirical factor f for complexes 1–4, 6 lies in the range, 102–135 cm indicating a square planar geometry with small distortion, where as in complex 5 value (f = 146 cm) is in conformity of tetrahedral geometry with larger distortion. All the compounds (selenosemicabazones and their complexes) are tested for DPPH radical scavenging, ABTS radical scavenging, FRAP ferric reducing and CUPRAC cupric reducing antioxidant capacity assay and all the compounds have shown good antioxidant capacity in general. Best results are obtained for H1L with FRAP, 6.75 ± 0.01 μg TE/ml and CUPRAC, 9.52 ± 0.07 μg TE/ml. All the compounds are also tested for their anti-tubercular activity against M. tuberculosis H37RV strain ATCC27294. Ligands, H1L, H3L, H6L and complex 6 have shown highest anti-TB activity (MIC = 1.6 µg/ml) which is similar to the standard drugs (Isoniazid, Ethambutol). anti-TB activity of H2L and H5L (MIC = 12.5 µg/ml and 25 µg/ml) increased on complexation with copper(II) (MIC = 1.6 µg/ml, 2; 12.5 µg/ml, 6). Experimental results are supported by molecular modeling studies, where a considerable intermolecular interaction of these compounds with the active site of amino acid of enoyl reductase from M. tuberculosis has been observed with minimum binding energy −6.2, −7.3, −7.7, −7.4, −7.2, −8.7 Kcal/mol for H1L, H2L, H3L, H4L, H5L, and H6L and −7.2, −9.4, −8.6, −9.3, −9.9, and −10.6 Kcal/mol for copper complexes 1, 2, 3, 4, 5 and 6 exhibited respectively.

One-pot green synthesis of chitosan biguanidine nanoparticles for targeting M. tuberculosis

Tuberculosis (TB) is considered as one of the most fatal infectious diseases nowadays. Several traditional anti-tuberculosis drugs like isoniazid have been largely applied; however, they are associated with toxicity and poor anti-TB treatment. So, the fabrication of new alternative anti-TB drugs containing natural biopolymers for TB treatment has attracted great attention in recent years because of their remarkable features: biodegradability, biocompatibility and non-toxicity. Therefore, their medicine is very effective with low side effects compared with synthetic drugs. Our current work intends to engineer chitosan biguanidine (ChBG) nanoparticles as a new safe and high-efficient anti-TB drug using one-pot, green, cost-effective ionic gelation method. The chemical structure of as-formed materials was chemically confirmed using various analysis techniques: H-NMR, FTIR, SEM, and TEM. TEM results have proved the formation of uniformly well-distributed ChBG nanoparticles with a small particle size of ~38 nm. The inhibitory activity of these prepared nanoparticles was investigated against the growth of three different M. tuberculosis pathogens such as sensitive, MDR, and XDR, and in a comparison with the isoniazid drug as a standard anti-tuberculosis drug. The antituberculosis assay results showed that ChBG NPs attained MIC values of 0.48, 3.9, 7.81 μg/mL for inhibiting the growth of sensitive, MDR, and XDR M. tuberculosis pathogens compared to bare Ch NPs (15.63, 62.5 > 125 μg/mL) and the isoniazid drug (0.24, 0, 0 μg/mL), respectively. Moreover, cytotoxicity of the ChBG NPs was examined against normal lung cell lines (Wi38) and was found to have cell viability of 100 % with the concentration range of 0.48–7.81 μg/mL.

Large-scale genomic analysis of Mycobacterium tuberculosis reveals extent of target and compensatory mutations linked to multi-drug resistant tuberculosis

Resistance to isoniazid (INH) and rifampicin (RIF) first-line drugs in Mycobacterium tuberculosis (Mtb), together called multi-drug resistance, threatens tuberculosis control. Resistance mutations in katG (for INH) and rpoB (RIF) genes often come with fitness costs. To overcome these costs, Mtb compensatory mutations have arisen in rpoC/rpoA (RIF) and ahpC (INH) loci. By leveraging the presence of known compensatory mutations, we aimed to detect novel resistance mutations occurring in INH and RIF target genes. Across ~ 32 k Mtb isolates with whole genome sequencing (WGS) data, there were 6262 (35.7%) with INH and 5435 (30.7%) with RIF phenotypic resistance. Known mutations in katG and rpoB explained ~ 99% of resistance. However, 188 (0.6%) isolates had ahpC compensatory mutations with no known resistance mutations in katG, leading to the identification of 31 putative resistance mutations in katG, each observed in at least 3 isolates. These putative katG mutations can co-occur with other INH variants (e.g., katG-Ser315Thr, fabG1 mutations). For RIF, there were no isolates with rpoC/rpoA compensatory mutations and unknown resistance mutations. Overall, using WGS data we identified putative resistance markers for INH that could be used for genotypic drug-resistance profiling. Establishing the complete repertoire of Mtb resistance mutations will assist the clinical management of tuberculosis.

IN SILICO STUDY OF PHYTOCONSTITUENTS OF MORINGA OLEIFERA AGAINST TREHALOSE–6–PHOSPHATE PHOSPHATASE (TPP) ENZYMES

Numerous pathogenic microorganisms use the enzymes trehalose-6-phosphate phosphatase (TPP) to biosynthesize the sugar trehalose from trehalose-6-phosphate (T6P) as part of their infection and proliferation processes. In order to build new generation candidate medications to inhibit TPP using in silico approaches, the current work is being done. The majority of the 10 phytochemicals from Moringa oleifera that were used in docking studies with the 3D model of TPP had good binding affinities to the enzyme, with campesterol and stigmat-4-en-3-one showing the highest affinities (Binding energies of - 7.8 kcal/mole and -8.0 kcal/mole, respectively), when compared to the commonly used commercial drug isoniazid (Binding energy of –6.0 kcal/mole).The active site of TPP sequences, which coordinates Mg2+ and is necessary for catalysis, has been discovered to bind to phytochemical leads during docking. Binding poses and distance measurement of TPP-phytochemical complexes of 5-Hydroxymethylfurfural, Citronellol, Xylitol, 2,4-Di-tert-butylphenol, Palmitoleic acid, cis-Vaccenic acid, Phytol, Cyclopentane, 1,1-[3-(2-cyclopentylethyl)-1,5-pentanediyl]bis-, Campesterol and Stigmat-4-en-3-one reveals that the lead phytochemicals were in close proximity with most of the active site amino acids (distance range from 1.88 to 3.77 Aº). This demonstrates the close affinity between the enzyme and the leads, which may open the door to the development of new generation medications to treat diseases caused by pathogenic microorganisms that produce TPP. KEYWORDS: Trehalose–6–phosphate phosphatase, Binding affinity, In silico, Phytochemical, Moringa oleifera

Mycobacterium smegmatis: The Vanguard of Mycobacterial Research.

The genus Mycobacterium contains several slow-growing human pathogens, including Mycobacterium tuberculosis, Mycobacterium leprae, and Mycobacterium avium. Mycobacterium smegmatis is a nonpathogenic and fast growing species within this genus. In 1990, a mutant of M. smegmatis, designated mc2155, that could be transformed with episomal plasmids was isolated, elevating M. smegmatis to model status as the ideal surrogate for mycobacterial research. Classical bacterial models, such as Escherichia coli, were inadequate for mycobacteria research because they have low genetic conservation, different physiology, and lack the novel envelope structure that distinguishes the Mycobacterium genus. By contrast, M. smegmatis encodes thousands of conserved mycobacterial gene orthologs and has the same cell architecture and physiology. Dissection and characterization of conserved genes, structures, and processes in genetically tractable M. smegmatis mc2155 have since provided previously unattainable insights on these same features in its slow-growing relatives. Notably, tuberculosis (TB) drugs, including the first-line drugs isoniazid and ethambutol, are active against M. smegmatis, but not against E. coli, allowing the identification of their physiological targets. Furthermore, Bedaquiline, the first new TB drug in 40 years, was discovered through an M. smegmatis screen. M. smegmatis has become a model bacterium, not only for M. tuberculosis, but for all other Mycobacterium species and related genera. With a repertoire of bioinformatic and physical resources, including the recently established Mycobacterial Systems Resource, M. smegmatis will continue to accelerate mycobacterial research and advance the field of microbiology.

Hepatoprotective potential of diosmin against hepatotoxic effect of isoniazid and rifampin in wistar rats.

The treatment of tuberculosis with isoniazid and rifampin is associated with hepatocellular damage. Therefore, the study was designed to evaluate the hepatoprotective potential of diosmin against hepatotoxic effect of isoniazid and rifampin in Wistar rats. Hepatotoxicity was induced by administering isoniazid and rifampin (100mg/kg), whereas diosmin was given as treatment control. Markers of liver function (ALT, AST, ALP and bilirubin), inflammatory cytokines (TNFα, IL-6 and IL-1β), apoptosis (caspase-3), oxidative stress parameters (LPO, GSH, CAT and SOD) and histological changes in liver were assessed in normal, hepatotoxic control and treatment groups. The administration of isoniazid and rifampin significantly increased markers of liver dysfunction (ALT, AST, ALP and bilirubin), cytokines (TNFα, IL-6 and IL-1β) and apoptosis (caspase-3). However, daily dosing of diosmin significantly reduced these markers of liver dysfunction, inflammatory cytokines and apoptosis to near normal levels. Additionally, markers of hepatocellular oxidative stress parameters were significantly altered as evident from increased LPO level and decreased endogenous antioxidants such as GSH, SOD and CAT in isoniazid-and rifampin-treated hepatotoxic group. It was observed that diosmin treatment reduced high levels of LPO and demonstrated significant improvement in antioxidant levels. Histological studies of liver also supported our biochemical findings, which are also manifested as diosmin treatment exhibited protection against hepatocellular degeneration and inflammation. Results of the present study demonstrate hepatoprotective potential of diosmin against isoniazid-and rifampin-treated hepatotoxicity. Thus, we conclude that diosmin may be used along with anti-tubercular drugs (isoniazid and rifampin) in tuberculosis patients to overcome their hepatotoxic adverse effect.

QSAR Studies, Molecular Docking, Molecular Dynamics, Synthesis, and Biological Evaluation of Novel Quinolinone-Based Thiosemicarbazones against Mycobacterium tuberculosis.

In this study, a series of novel quinolinone-based thiosemicarbazones were designed in silico and their activities tested in vitro against Mycobacterium tuberculosis (M. tuberculosis). Quantitative structure-activity relationship (QSAR) studies were performed using quinolinone and thiosemicarbazide as pharmacophoric nuclei; the best model showed statistical parameters of R2 = 0.83; F = 47.96; s = 0.31, and was validated by several different methods. The van der Waals volume, electron density, and electronegativity model results suggested a pivotal role in antituberculosis (anti-TB) activity. Subsequently, from this model a new series of quinolinone-thiosemicarbazone 11a-e was designed and docked against two tuberculosis protein targets: enoyl-acyl carrier protein reductase (InhA) and decaprenylphosphoryl-β-D-ribose-2'-oxidase (DprE1). Molecular dynamics simulation over 200 ns showed a binding energy of -71.3 to -12.7 Kcal/mol, suggesting likely inhibition. In vitro antimycobacterial activity of quinolinone-thiosemicarbazone for 11a-e was evaluated against M. bovis, M. tuberculosis H37Rv, and six different strains of drug-resistant M. tuberculosis. All compounds exhibited good to excellent activity against all the families of M. tuberculosis. Several of the here synthesized compounds were more effective than the standard drugs (isoniazid, oxafloxacin), 11d and 11e being the most active products. The results suggest that these compounds may contribute as lead compounds in the research of new potential antimycobacterial agents.

Heteroatoms (B, N, S) doped quantum dots as potential drug delivery system for isoniazid: insight from DFT, NCI, and QTAIM

Toxicity in drug includes target toxicity, immune hypersensitivity and off target toxicity. Recently, advances in nanotechnology in the areas of drug delivery have help reduce toxicity and enhance drug solubility and deliver drugs to target sites more efficiently. In this study, we present a novel heteroatom functionalized quantum dot (QD-NBC and QD-NBS) as an effective drug delivery system for isoniazid. The said QD has been computationally modeled to assess its effectiveness in delivering isoniazid to desired target. Density functional theory (DFT) calculations were performed on the QD at the B3LYP/6−311+G(d, p) level to assess its stability through the natural bond orbital (NBO) calculations, and frontier molecular orbital (FMO) before and after interaction with isoniazid drug to understand any change in molecular behavior of the surface. Appropriate intermolecular interactions between the QD and the drug were computed through the Quantum theory of atoms in molecules (QTAIM) and Non-covalent interaction to assess the various binding mechanism and possible interactions resulting to the effective delivery of the drug target. To understand and accurately appraise the binding energy of adsorption, DFT calculations were performed with different functionals (B3LYP, CAM-B3LYP, PBEPBE, GD3BJ & WB97XD/6−311+G (d, p)). The results from DFT calculations point the functionalized QDs to be stable with appreciable energy gap suitable for delivery purposes. The adsorption energy of the drug target with the QD is in the range of −24.73 to 33.75 kcal/mol which indicates substantial interaction of the drug with the QD surface. This absorption energy is comparable with several reported literature and thus prompt the suitability of the surface for isoniazid delivery.

Discovery of a LuxR-type regulator involved in isoniazid-dependent gene regulation in Mycobacterium smegmatis

ObjectiveMost non-tuberculous mycobacteria exhibit intrinsic resistance against the anti-tuberculosis drug isoniazid (INH). We previously found that a pyrazinamidase/nicotinamidase of Mycobacterium smegmatis, named PzaA, has an enzymatic activity to hydrolyze INH, which may contribute to intrinsic resistance. Furthermore, PzaA expression is strongly induced by INH under nitrogen-depleted conditions, although the precise mechanism of this phenomenon remains unclear. Here, we aimed to reveal the mechanism underlying the INH-dependent induction of PzaA using a transcriptomic approach. MethodsRNA sequencing was performed to identify INH-inducible genes other than pzaA. 5′ rapid amplification of cDNA ends analysis was employed to identify the transcription start sites of INH-induced transcription units. The function of a LuxR-like regulator gene (MSMEI_1050) found within the gene cluster containing pzaA was confirmed by gene deletion and complementation experiments involving INH hydrolysis assay and quantitative reverse transcription PCR. ResultsRNA sequencing revealed 23 genes that INH strongly induced under conditions of nitrogen depletion, 17 of which were in a gene cluster containing pzaA. This cluster comprised at least three transcription units, including a non–INH–inducible monocistronic unit containing MSMEI_1050. Deletion of this gene deprived M. smegmatis of the ability to respond to INH, and complementation restored this ability. ConclusionsMSMEI_1050 plays a key role in INH-dependent gene regulation. The precise mechanism of action is to be determined in future studies.

Effect of teriparatide on drug treatment of tuberculous spondylitis: an experimental study

Tuberculous spondylitis often develops catastrophic bone destruction with uncontrolled inflammation. Because anti-tuberculous drugs do not have a role in bone formation, a combination drug therapy with a bone anabolic agent could help in fracture prevention and promote bone reconstruction. This study aimed to investigate the influence of teriparatide on the effect of anti-tuberculous drugs in tuberculous spondylitis treatment. We used the virulent Mycobacterium tuberculosis (Mtb) H37Rv strain. First, we investigated the interaction between teriparatide and anti-tuberculosis drugs (isoniazid and rifampin) by measuring the minimal inhibitory concentration (MIC) against H37Rv. Second, we evaluated the therapeutic effect of anti-tuberculosis drugs and teriparatide on our previously developed in vitro tuberculous spondylitis model of an Mtb-infected MG-63 osteoblastic cell line using acid-fast bacilli staining and colony-forming unit counts. Selected chemokines (interleukin [IL]-8, interferon γ-induced protein 10 kDa [IP-10], monocyte chemoattractant protein [MCP]-1, and regulated upon activation, normal T cell expressed and presumably secreted [RANTES]) and osteoblast proliferation (alkaline phosphatase [ALP] and alizarin red S [ARS] staining) were measured. Teriparatide did not affect the MIC of isoniazid and rifampin. In the Mtb-infected MG-63 spondylitis model, isoniazid and rifampin treatment significantly reduced Mtb growth, and cotreatment with teriparatide did not change the anti-tuberculosis effect of isoniazid (INH) and rifampin (RFP). IP-10 and RANTES levels were significantly increased by Mtb infection, whereas teriparatide did not affect all chemokine levels as inflammatory markers. ALP and ARS staining indicated that teriparatide promoted osteoblastic function even with Mtb infection. Cotreatment with teriparatide and the anti-tuberculosis drugs activated bone formation (ALP-positive area increased by 705%, P = 0.0031). Teriparatide was effective against Mtb-infected MG63 cells without the anti-tuberculosis drugs (ARS-positive area increased by 326%, P = 0.0037). Teriparatide had no effect on the efficacy of anti-tuberculosis drugs and no adverse effect on the activity of Mtb infection in osteoblasts. Furthermore, regulation of representative osteoblastic inflammatory chemokines was not changed by teriparatide treatment. In the in vitro Mtb-infected MG-63 cell model of tuberculous spondylitis, cotreatment with the anti-tuberculosis drugs and teriparatide increased osteoblastic function.

Ultrasensitive Detection of Multidrug-Resistant Mycobacterium tuberculosis Using SuperSelective Primer-Based Real-Time PCR Assays

The emergence of drug-resistant tuberculosis is a significant global health issue. The presence of heteroresistant Mycobacterium tuberculosis is critical to developing fully drug-resistant tuberculosis cases. The currently available molecular techniques may detect one copy of mutant bacterial genomic DNA in the presence of about 1-1000 copies of wild-type M. tuberculosis DNA. To improve the limit of heteroresistance detection, we developed SuperSelective primer-based real-time PCR assays, which, by their unique assay design, enable selective and exponential amplification of selected point mutations in the presence of abundant wild-type DNA. We designed SuperSelective primers to detect genetic mutations associated with M. tuberculosis resistance to the anti-tuberculosis drugs isoniazid and rifampin. We evaluated the efficiency of our assay in detecting heteroresistant M. tuberculosis strains using genomic DNA isolated from laboratory strains and clinical isolates from the sputum of tuberculosis patients. Results show that our assays detected heteroresistant mutations with a specificity of 100% in a background of up to 104 copies of wild-type M. tuberculosis genomic DNA, corresponding to a detection limit of 0.01%. Therefore, the SuperSelective primer-based RT-PCR assay is an ultrasensitive tool that can efficiently diagnose heteroresistant tuberculosis in clinical specimens and contributes to understanding the drug resistance mechanisms. This approach can improve the management of antimicrobial resistance in tuberculosis and other infectious diseases.

Green engineering of TMC-CMS nanoparticles decorated graphene sheets for targeting M. tuberculosis

Our current work intends to primarily engineer a new type of antibacterial composite by preparing a highly biocompatible graphene sheet decorated with TMC-CMS IPNs nanoparticles utilizing one-pot, green, cost-effective ultrasonication approach. The microstructure of as-formed materials was chemically confirmed using various analytical techniques such as 1H-NMR, FTIR, UV/vis, SEM, and TEM. TEM data has proved the formation of uniformly distributed TCNPs on graphene surfaces with a small particle size of ~22 nm compared with that of pure nanoparticles (~30 nm). The inhibitory activity of these developed materials was examined against the growth of three different M. tuberculosis pathogens and in a comparison with the isoniazid drug as a standard anti-tuberculosis drug. The TCNPs@GRP composite attained MIC values of 0.98, 3.9, and 7.81 μg/mL for inhibiting the growth of sensitive, MDR, and XDR M. tuberculosis pathogens compared to the bare TCNPs (7.81, 31.25, >125 μg/mL) and the isoniazid drug (0.24, 0, 0 μg/mL), respectively. This reveals a considerable synergism in the antituberculosis activity between TCNPs and graphene nanosheets. Cytotoxicity of the TCNPs@GRP was examined against normal lung cell lines (WI38) and was found to have cell viability of 100% with the concentration range of 0.98–7.81 μg/mL.

Strain diversity and gene mutations associated with presumptive multidrug-resistant Mycobacterium tuberculosis complex isolates in Northwest Ethiopia

In this study, we assessed the genetic diversity and gene mutations that confer resistance to rifampicin (RIF), isoniazid (INH), fluoroquinolone (FQ), and second-line injectable (SLI) drugs in RIF-resistant (RR)/multidrug-resistant tuberculosis (MDR-TB) isolates in Northwest Ethiopia. Spoligotyping was used to assign isolates to TB lineages (Ls), and Hain line probe assays were used to detect resistance to RIF, INH, and FQs, and SLIs. Among 130 analyzed strains, 68.5% were RR, and four major Mycobacterium tuberculosis complex lineages (L1, L3, L4, and L7) were identified with a predominance of the Euro-American L4 (72, 54.7%), while L7 genotypes were less common (3, 2.3%). Overall, the L4-T3-ETH (41, 32.0%), L3-CAS1-Delhi (29, 22.7%), and L3-CAS1-Killi (19, 14.8%) families were most common. Line probe analysis showed that among rpoB mutants, 65.2% were S450L, while 87.8% of katG mutants were S315T. Only three isolates showed mutation (c-15t) at the inhA gene, and no double mutation with katG and inhA genes was found. Six strains, two each of L1, L3, and L4, were resistant to FQs, having gyrA mutations (D94G, S91P), of which three isolates had additional resistance to SLI (rrs A1401G or C1402T mutations) including one isolate with low-level kanamycin (KAN) resistance. This study showed a predominance of L4-T3-ETH, L3-CAS1-Delhi, and L3-CAS1-Killi families, with a high rate of rpoB_S450L and katG_S315T mutations and a low proportion of gyrA and rrs mutations. L7 was less frequently observed in this study. Further investigations are, therefore, needed to understand L7 and other lineages with undefined mutations.

Comparative Analysis of the Efficacies of the GeneXpert and Solid Culture Media Techniques in the Diagnosis of Mycobacterium Tuberculosis.

Tuberculosis is one of the predominant infectious diseases causing significant deaths worldwide. Detection of Mycobacterium tuberculosis bacilli (MTB) using culture media was officially recognized by World Health Organization. However, there is a significant limitation in the authenticity of evaluation for its effectiveness on clinically important attributes. GeneXpert detects the presence of Mycobacterium tuberculosis (M. tuberculosis) based on the detection of nucleic acid and is able to identify the resistance of both isoniazid (INH) and Rifampicin (RIF) drugs. In this technique, DNA amplification is done using the GeneXpert instrument in the suspected sample with a specific reagent cartridge. Although GeneXpert is a rapid technique compared to other diagnostic tools for MTB identification due to false-negative results, the culture media technique is still considered the gold standard in detecting M. tuberculosis. The current study was designed to evaluate the comparative efficacies of GeneXpert and the solid culture media technique in identifying MTB. Sputum samples of 250 (n=250) suspected tuberculosis (TB) patients were investigated using both diagnostic techniques. The results revealed that out of the 250 suspected patients, 30 (12%) samples were positive with the culture media technique, while only 17 (6.8%) samples showed positive results with GeneXpert. Culture tests and GeneXpert are not equally efficient in detecting M. tuberculosis. The current study's findings showed that the culture-based detection method for M. tuberculosis is more efficient and reliable than GeneXpert.

Determination of the Pharmacokinetics and Pharmacodynamics of Isoniazid, Rifampicin, Pyrazinamide and Ethambutol in a Cross-Over Cynomolgus Macaque Model of Mycobacterium tuberculosis Infection.

Innovative cross-over study designs were explored in non-human primate (NHP) studies to determine the value of this approach for the evaluation of drug efficacy against tuberculosis (TB). Firstly, the pharmacokinetics (PK) of each of the drugs Isoniazid (H), Rifampicin (R), Pyrazinamide (Z) and Ethambutol (E), that are standardly used for the treatment of tuberculosis, was established in the blood of macaques after oral dosing as a monotherapy or in combination. Two studies were conducted to evaluate the pharmacokinetics and pharmacodynamics of different drug combinations using cross-over designs. The first employed a balanced, three-period Pigeon design with an extra period; this ensured that treatment by period interactions and carry-over could be detected comparing the treatments HR, HZ and HRZ using H37Rv as the challenge strain of Mycobacterium tuberculosis (M. tb). Although the design accounted for considerable variability between animals, the three regimens evaluated could not be distinguished using any of the alternative endpoints assessed. However, the degree of pathology achieved using H37Rv in the model during this study was less than expected. Based on these findings, a second experiment using a classical AB/BA design comparing HE with HRZ was conducted using the M. tb Erdman strain. More extensive pathology was observed, and differences in computerized tomography (CT) scores and bacteriology counts in the lungs were detected, although due to the small group sizes, clearer differences were not distinguished. Type 1 T helper (Th1) cell response profiles were characterized using the IFN-γ ELISPOT assay and revealed differences between drug treatments that corresponded to decreases in disease burden. Therefore, the studies performed support the utility of the NHP model for the determination of PK/PD of TB drugs, although further work is required to optimize the use of cross-over study designs.

Elucidating the function of hypothetical PE_PGRS45 protein of Mycobacterium tuberculosis as an oxido-reductase: a potential target for drug repurposing for the treatment of tuberculosis

Mycobacterium tuberculosis (Mtb) encodes a total of 67 PE_PGRS proteins and definite functions of many of them are still unknown. This study reports PE_PGRS45 (Rv2615c) protein from Mtb as NADPH dependent oxido-reductase having substrate specificity for fatty acyl Coenzyme A. Computational studies predicted PE_PGRS45 to be an integral membrane protein of Mtb. Expression of PE_PGRS45 in non-pathogenic Mycobacterium smegmatis, which does not possess PE_PGRS genes, confirmed its membrane localization. This protein was observed to have NADPH binding motif. Experimental validation confirmed its NADPH dependent oxido-reductase activity (Km value = 34.85 ± 9.478 μM, Vmax = 96.77 ± 7.184 nmol/min/mg of protein). Therefore, its potential to be targeted by first line anti-tubercular drug Isoniazid (INH) was investigated. INH was predicted to bind within the active site of PE_PGRS45 protein and experiments validated its inhibitory effect on the oxido-reductase activity of PE_PGRS45 with IC50/Ki values of 5.66 μM. Mtb is resistant to first line drugs including INH. Therefore, to address the problem of drug resistant TB, docking and Molecular Dynamics (MD) simulation studies between PE_PGRS45 and three drugs (Entacapone, Tolcapone and Verapamil) which are being used in Parkinson’s and hypertension treatment were performed. PE_PGRS45 bound the three drugs with similar or better affinity in comparison to INH. Additionally, INH and these drugs bound within the same active site of PE_PGRS45. This study discovered Mtb’s PE_PGRS45 protein to have an oxido-reductase activity and could be targeted by drugs that can be repurposed for TB treatment. Furthermore, in-vitro and in-vivo validation will aid in drug-resistant TB treatment. HIGHLIGHTS In-silico and in-vitro studies of hypothetical protein PE_PGRS45 (Rv2615c) of Mycobacterium tuberculosis (Mtb) reveals it to be an integral membrane protein PE_PGRS45 protein has substrate specificity for fatty acyl Coenzyme A (fatty acyl CoA) and possess NADPH dependent oxido-reductase activity Docking and simulation studies revealed that first line anti-tubercular drug Isoniazid (INH) and other drugs with anti-TB property have strong affinity for PE_PGRS45 protein Oxido-reductase activity of PE_PGRS45 protein is inhibited by INH PE_PGRS45 protein could be targeted by drugs that can be repurposed for TB treatment Communicated by Ramaswamy H. Sarma