Antimycobacterial Effects Research Articles

Evaluation of the Anti-Mycobacterial and Anti-Inflammatory Activities of the New Cardiotonic Steroid γ-Benzylidene Digoxin-15 in Macrophage Models of Infection

Cardiotonic steroids modulate various aspects of the inflammatory response. The synthetic cardiotonic steroid γ-benzylidene digoxin 15 (BD-15), a digoxin derivative, has emerged as a promising candidate with potential immunomodulatory effects. However, its biological activity remains largely unexplored. This study investigated the anti-mycobacterial and anti-inflammatory effects of BD-15 in an in vitro macrophage infection model with Mycobacterium spp. Unlike digoxin, which showed significant toxicity at higher concentrations, BD-15 exhibited no cytotoxicity in RAW 264.7 cells (a murine macrophage cell line). Both compounds were evaluated in Mycobacterium smegmatis-infected RAW 264.7 cells, reducing bacterial burden without direct bactericidal activity. Additionally, both modulated pro-inflammatory cytokine levels, notably by decreasing tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) levels. BD-15 specifically reduced NOD-, LRR-, and pyrin-domain-containing protein 3 (NLRP3) inflammasome expression and increased interleukin-10 (IL-10) production. Notably, BD-15 reduced colony-forming unit (CFU) counts in Mycobacterium tuberculosis-infected RAW 264.7 cells. Toxicity assays in HepG2 cells (a human liver cancer cell line) showed that BD-15 had minimal hepatotoxicity compared to digoxin, and both demonstrated negligible acute toxicity in an Artemia salina bioassay. These findings revealed the immunomodulatory effects of cardiotonic steroids in a bacterial infection model and highlighted BD-15 as a safer alternative to digoxin for therapeutic applications.

New Frontiers in Fighting Mycobacterial Infections: Venom-Derived Peptides.

Notwithstanding the indefatigable endeavors to develop effective anti-mycobacterial therapies, mycobacterial infections still present a tough problem for medicine today. The problem is further complicated by the disquieting surge of drug-resistant mycobacterial pathogens, which considerably narrows the existing therapeutic options. Thus, there is a genuine need to discover novel anti-mycobacterial drugs. Animal venoms are considered a treasure trove of structurally variable and biologically active peptides, which may hold promise for therapeutic applications. Over the past two decades, abundant evidence has been amassed regarding anti-mycobacterial effects of various peptides derived from the venoms of honeybees, wasps, scorpions, pseudoscorpions, cone snails, and snakes. This review intends to consolidate the state-of-the-art knowledge on the anti-mycobacterial peptides of animal venoms and to sketch potentially fruitful directions for future investigations. The available data indicate that micromolar concentrations of particular venom-derived peptides can effectively inhibit the in vitro growth of Mycobacterium tuberculosis and non-tuberculous mycobacteria. The proposed mechanisms of action of venom-derived peptides include reduced activity of plasma membrane ATPase, depolarization of the cell membrane, disruption of the cell wall, and increased generation of reactive oxygen species. Interestingly, administering certain peptides (≤ 2mg/kg body weight) through daily intraperitoneal injections to mice for 8consecutive days resulted in lower levels of mycobacterial infections and inflammation, hitting two targets with one arrow. Indubitably, such peptides can usher in new possibilities for the prevention and treatment of recalcitrant mycobacterial infections.

Biomolecular Interaction of Carnosine and Anti-TB Drug: Preparation of Functional Biopeptide-Based Nanocomposites and Characterization through In Vitro and In Silico Investigations.

Host-directed therapies (HDTs) resolve excessive inflammation during tuberculosis (TB) disease, which leads to irreversible lung tissue damage. The peptide-based nanostructures possess intrinsic anti-inflammatory and antioxidant properties among HDTs. Native carnosine, a natural dipeptide with superior self-organization and functionalities, was chosen for nanoformulation. In the present work, multiscale self-assembly approaches of carnosine were developed using a solvent-mediated process (hexafluoro-2-propanol) and further linked with first-line anti-TB drugs. The organofluorine compound in a solvent is attributed to the self-assembling process with heteroatom acceptors in carnosine. In the carnosine-anti-TB drug nanocomposite, the functional moieties represent the involvement of hydrogen bonding and the electrostatic force of attraction. The minimum inhibitory concentration of carnosine-anti-TB drug composites represents an antimycobacterial effect on par with standard drugs. The silicon findings complemented the in vitro results through quantum chemical simulations, elucidating the respective binding pockets between putative Mtb drug targets and carnosine-anti-TB composites. These findings confirmed that the carnosine and anti-TB drug nanocomposites prepared through a solvent-mediated process act as a rational design for functional nanodelivery systems for sustainable TB therapeutics.

Furamidine-induced autophagy exerts an anti-mycobacterial effect in a SIRT1-pAMPK-FOXO3a-dependent manner by elevation of intracellular Ca2+ level expression

Mycobacterium tuberculosis (M. tb), the etiological agent of tuberculosis (TB), continues to be a major contributor to global mortality rates. To effectively combat this pandemic, TB control has to be enhanced in several areas, including point-of-care diagnostics, shorter and safer drug regimens, and preventative vaccination. The latest findings have highlighted autophagy as a host-defense mechanism that eradicates many invading bacteria, including M. tb. Thus, novel approaches like the stimulation of autophagy using various pharmaceutical drugs can be undertaken to deal with this noxious pathogen. The present study has been formulated to evaluate the anti-mycobacterial potential of Furamidine, a DNA minor groove binder (MGB). Initially, a non-cytotoxic concentration of Furamidine (10 µM) was used to assess its impact on the intracellular persistence of mycobacteria in differentiated THP-1 (dTHP-1) cells. Furamidine treatment compromised intracellular mycobacterial growth compared to control cells. Autophagy, a well-known host-defensive strategy, was investigated as a possible contributor to revealing the mechanism of action. Multiparametric approaches such as LC3-I to II conversion, protein level expression of different autophagic markers, and MDC staining were employed to study autophagic response that conclusively suggested the autophagy induction potential of Furamidine in dTHP-1 cells. Further, elevated LC3-II expression and increased autophagic vacuole accumulation under Baf-A1 treatment demonstrated the positive regulation of autophagic flux upon Furamidine treatment. Mechanistic investigations showed increased intracellular calcium (Ca2+) level expression, SIRT1, pAMPK, and FOXO3a activation upon its treatment. Inhibition of Ca2+ level expression suppressed Ca2+-mediated-FOXO3a level in Furamidine-treated cells. Furthermore, administering various inhibitors hampered the Furamidine-induced autophagy that impacted intracellular mycobacteria clearance. These results conclude that Furamidine triggered the Ca2+/pAMPK/SIRT1/FOXO3a pathway, causing less mycobacterial load in dTHP-1 cells.

Therapeutic Potential of Silver Nanoparticles (AgNPs) as an Antimycobacterial Agent: A Comprehensive Review.

The uncontrolled emergence of multidrug-resistant mycobacterial strains presents as the primary determinant of the present crisis in antimycobacterial therapeutics and underscores tuberculosis (TB) as a daunting global health concern. There is an urgent requirement for drug development for the treatment of TB. Numerous novel molecules are presently undergoing clinical investigation as part of TB drug development. However, the complex cell wall and the lifecycle of M. tuberculosis within the host pose a significant challenge to the development of new drugs and, therefore, led to a shift in research focus towards alternative antibacterial compounds, notably nanotechnology. A novel approach to TB therapy utilizing silver nanoparticles (AgNPs) holds the potential to address the medical limitations imposed by drug resistance commonly associated with currently available antibiotics. Their broad-spectrum antimicrobial activity presents the utilization of AgNPs as a promising avenue for the development of therapeutics targeting mycobacterial-induced diseases, which can effectively target Mycobacterium tuberculosis, including drug-resistant strains. AgNPs can enhance the effectiveness of traditional antibiotics, potentially leading to better treatment outcomes and a shorter duration of therapy. However, the successful implementation of this complementary strategy is contingent upon addressing several pivotal therapeutic challenges, including suboptimal delivery, variability in intra-macrophagic antimycobacterial effect, and potential toxicity. Future perspectives may involve developing targeted delivery systems that maximize therapeutic effects and minimize side effects, as well as exploring combinations with existing TB medications to enhance treatment outcomes. We have attempted to provide a comprehensive overview of the antimycobacterial activity of AgNPs, and critically analyze the advantages and limitations of employing silver nanoparticles in the treatment of TB.

Troglitazone reduces intracellular Mycobacterium tuberculosis survival via macrophage autophagy through LKB1-AMPKα signaling.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb), results in significant morbidity and mortality worldwide. Host-directed therapy (HDT), including conventional drugs, is a promising anti-TB strategy that shows synergistic antibacterial effects when combined with anti-TB drugs. Here, the mycobactericidal effect of three anti-diabetic drugs was examined. Of these, only Troglitazone (Trog) enhanced the antimycobacterial effect in vitro and in vivo. This was due to Trog-mediated autophagy activation. Moreover, a knock-down experiment revealed that Trog activated autophagy and exhibited antimycobacterial activity through the LKB1-AMPK signaling pathway. Molecular docking and co-immunoprecipitation experiments demonstrated that Trog promoted LKB1 phosphorylation and activation by targeting STRADA. Finally, we found that Trog inhibited the intracellular survival of clinical isoniazid (INH)-resistant Mtb, and the combination of Trog and INH showed additive antibacterial effects against Mtb H37Rv. Taken together, anti-diabetic Trog may be repurposed as an HDT candidate and combined with first-line anti-TB drugs.

Bioassay-guided isolation and structure elucidation of anti-mycobacterium tuberculosis compounds from Galatella grimmii (Regel & Schmalh.) Sennikov

BackgroundGalatella is a genus in the family Asteraceae, represented by 35-45 species. Considering the high effectiveness of the ethyl acetate (EtOAc) fraction of G. grimmii against Mycobacterium tuberculosis (MIC = 0.5 µg/mL), a bioassay-directed fractionation of this extract was carried out.MethodsThe methanolic extract of the aerial parts of G. grimmii was obtained using maceration, then it was suspended in water and partitioned with petroleum ether, dichloromethane (CH2Cl2), EtOAc, and n-butanol (n-BuOH), successively. The most potent fraction (EtOAc), was selected for further isolation by Sephadex LH–20 and semi-preparative HPLC to obtain active compounds.ResultsFractionation of the EtOAc solvent fraction resulted in the characterization of five compounds, among them, compounds 1 and 2 showed the highest anti-mycobacterial effects with MICs of 0.062 and 1.00 µg/mL against H37Rv M. tuberculosis, respectively, which were higher than those of rifampin (MIC of 1.25 µg/mL) and isoniazid (MIC of 0.31 µg/mL), as positive controls. Also, compound 1 ​​inhibited all tested strains of drug-resistant Mycobacterium (MDR and XDR). Notably, the isolated compounds have been reported for the first time from G. grimmii.ConclusionDue to the potent anti-mycobacterial effect of isolated compounds from G. grimmii, this study could pave the way for developing a novel class of natural anti-tuberculosis compounds.

A comparative study of in-vivo wound healing properties of Tithonia Diversifolia. A gray crude extracts to Silver Sulphadiazine in Albino Wistar rats.

One species of flowering plant in the Asteraceae family is Tithonia diversifolia A. Gray (T. diversifolia), which grows as a shrub or weed. Significant anti-infective therapeutic characteristics, such as anti-mycobacterial, antifungal, antibacterial, anti-inflammatory, anti-malaria, and anthelmintic effects, have been discovered in the plant's extracts. Ethnic communities have been using the plant extract to cure wounds. They appear to prefer it above conventional treatments in many circumstances, to the point where their aqueous solution may be smuggled into medical institutions in order to augment the care provided. The purpose of this study was to compare T. diversifolia with silver sulphadiazine's capacity for wound healing. For 72 hours, a 70% ethanol alone and water alone was used to extract the plant. After the extracts dried out, the powder was measured and 10 mL of reconstituted volume was assessed at various concentrations for the purpose of treating wounds. Silver sulphadiazine was used as the positive control and distilled water as the negative control. According to the findings, the aqueous extract had a 48.0% healing rate after 14 days of treatment, ethanol had a 20.0% healing rate, and silver sulphadiazine had a 22.0% healing rate. It is possible to draw the conclusion that the aqueous extract concentration of 0.6 mg/10 mL demonstrated a higher healing percentage than silver sulphadiazine and the ethanol extract.

Investigating the pharmacological potential of phytol on experimental models of gastric ulcer in rats.

Phytol is a diterpene constituent of many essential oils, belonging to the group of unsaturated acyclic alcohols. Although phytol possesses antimycobacterial and anti-inflammatory effects, no reports of a gastrointestinal action are available from the literature. Due to the well-known shortcomings of classical anti-ulcer drugs (e.g. side effects or relapses), natural products may offer an attractive alternative. In this study, a potential gastroprotective activity of phytol was evaluated using acute and chronic ulcer models in rats. Phytol 12.5, 25 and 50mg/kg, administered orally 1h prior to induction of gastric lesions by absolute ethanol, inhibited the lesion area by 96, 90 and 95%, respectively. When lesions were induced by ischemia and reperfusion, phytol 12.5 and 25mg/kg per os decreased the lesion areas by 89 and 46%, respectively. In the third acute ulcer model (lesions induced by ibuprofen), phytol 12.5mg/kg reduced the lesion area by 55%. Phytol restored the decreased level of reduced glutathione, the increased levels of myeloperoxidase and malondialdehyde and the decreased levels of catalase and superoxide dismutase in rats with gastric ulcer induced by ethanol to levels obtained in vehicle group. Finally, in a chronic model in which gastric ulcer was induced by acetic acid directly instilled into the stomach, phytol administered orally over a time period of 7 days at 12.5, 25, 50 and 100mg/kg reduced lesion areas by 84, 81, 83 and 68%. Our data suggest a gastroprotective and cicatrizing effect of phytol, possibly associated with its antioxidant effect.

Antimycobacterial and healing effects of Pranlukast against MTB infection and pathogenesis in a preclinical mouse model of tuberculosis.

It is essential to understand the interactions and relationships between Mycobacterium tuberculosis (Mtb) and macrophages during the infection in order to design host-directed, immunomodulation-dependent therapeutics to control Mtb. We had reported previously that ornithine acetyltransferase (MtArgJ), a crucial enzyme of the arginine biosynthesis pathway of Mtb, is allosterically inhibited by pranlukast (PRK), which significantly reduces bacterial growth. The present investigation is centered on the immunomodulation in the host by PRK particularly the activation of the host's immune response to counteract bacterial survival and pathogenicity. Here, we show that PRK decreased the bacterial burden in the lungs by upregulating the population of pro-inflammatory interstitial macrophages (IMs) and reducing the population of Mtb susceptible alveolar macrophages (AMs), dendritic cells (DCs), and monocytes (MO). Additionally, we deduce that PRK causes the host macrophages to change their metabolic pathway from fatty acid metabolism to glycolytic metabolism around the log phage of bacterial multiplication. Further, we report that PRK reduced tissue injury by downregulating the Ly6C-positive population of monocytes. Interestingly, PRK treatment improved tissue repair and inflammation resolution by increasing the populations of arginase 1 (Arg-1) and Ym1+Ym2 (chitinase 3-like 3) positive macrophages. In summary, our study found that PRK is useful not only for reducing the tubercular burden but also for promoting the healing of the diseased tissue.

4-(Benzyloxy)phenol-induced p53 exhibits antimycobacterial response triggering phagosome-lysosome fusion through ROS-dependent intracellular Ca2+ pathway in THP-1 cells

Drug-resistant tuberculosis (TB) outbreak has emerged as a global public health crisis. Therefore, new and innovative therapeutic options like host-directed therapies (HDTs) through novel modulators are urgently required to overcome the challenges associated with TB. In the present study, we have investigated the anti-mycobacterial effect of 4-(Benzyloxy)phenol. Cell-viability assay asserted that 50 μM of 4-(Benzyloxy)phenol was not cytotoxic to phorbol 12-myristate 13-acetate (PMA) differentiated THP-1 (dTHP-1) cells. It was observed that 4-(Benzyloxy)phenol activates p53 expression by hindering its association with KDM1A. Increased ROS, intracellular Ca2+ and phagosome-lysosome fusion, were also observed upon 4-(Benzyloxy)phenol treatment. 4-(Benzyloxy)phenol mediated killing of intracellular mycobacteria was abrogated in the presence of specific inhibitors of ROS, Ca2+ and phagosome-lysosome fusion like NAC, BAPTA-AM, and W7, respectively. We further demonstrate that 4-(Benzyloxy)phenol mediated enhanced ROS production is mediated by acetylation of p53. Blocking of p53 acetylation by Pifithrin-α (PFT- α) enhanced intracellular mycobacterial growth by blocking the mycobactericidal effect of 4-(Benzyloxy)phenol. Altogether, the results showed that 4-(Benzyloxy)phenol executed its anti-mycobacterial effect by modulating p53-mediated ROS production to regulate phagosome-lysosome fusion through Ca2+ production.

Evaluation of the anionic effect on the formation of biologically active {CuII-phenx; x = 1, 2, 3} fragments - Synthetic and structural variations, antimycobacterial and antiblastoma effects

Two cationic and molecular copper(II) complexes - mixed ligand [{Cu(phen)2benz}{Cu(phen)2Cl}]2+·2Cl-·benz-·H3O+·4·.5H2O (1), [Cu(benz)2phen] (2) (benz- = m-Cl-benzoate anion; phen = 1,10-phenantroline) and [Cu(phen)3]2+·2OTf-·2EtOH (3) (OTf = trifluoromethylsulfonate, triflate anion, CF3SO3-) were synthesized using various synthetic approaches and thoroughly characterized by spectroscopic methods and single crystal X-ray diffraction analysis. The structures of the complexes according to X-ray diffraction data are characterized by a diverse geometric environment of the complexing agent - the cationic form of 1 comprises two structurally nonequivalent moieties: the Cu (1) and Cu (2) complex-forming atoms coordinate, in chelate manner, two phen moieties each and the benz- (1)/Cl- (2) anions, respectively, thus forming different-ligand formula units, a trigonal–bipyramidal {Cu(1)N4Cl} and a pseudo octahedral one {Cu(2)N4O2}. The copper(II) ion in the complex 2 coordinates one phen ligand in a bidentate mode and two benz- anions to produce a distorted planar square environment {CuN2O2}. In complex 3, the copper(II) cation is surrounded by three phenanthroline fragments {Cu(phen)3}2+ and non-coordinated OTf- ions. The results of an EPR study 1 show that in the solid phase, intermolecular exchange interactions that cause exchange narrowing of the spectrum are efficient, whereas in solutions, resolved spectra of copper(II) ion with rhombic symmetry of the g-tensor are observed. An in vitro study of the biological properties of 1–3 toward nonpathogenic mycobacterial strain Mycolicibacterium smegmatis showed an increase in biological effectiveness depending on the structure of complexes (MIC, 1 (2 nmol/disk) > 3 (5 nmol/disk) > 2 (12.5 nmol/disk)). Complex 1 was also tested for antitumor activity against an ovarian adenocarcinoma SKOV3 demonstrated high efficiency: the IC50 value is almost 9 times higher than the activity of cisplatin.

Antimycobacterial potential of Trachyspermum ammi seed essential oil via fume contact and determination of major compounds

Trachyspermum ammi (L.), commonly known as carrom seeds or Ajwain, has been extensively studied for its medicinal properties. In this study, anti-mycobacterial effect of AEO in liquid and fume form was investigated against Mycobacterium smegmatis and Mycobacterium tuberculosis (M. tb). Results showed that AEO inhibits the growth of M. smegmatis at 0.03 mg/mL and becomes bactericidal at 0.125 mg/mL. MICs were observed at 0.03, 0.125 and 0.06 mg/mL against M. tb (H37Rv), isoniazid- and rifampicin-resistant (RIF-R) strains. Inverted disc-fume assay revealed AEO and Thymol efficiently inhibit the growth of M. smegmatis and M. tb. Similarly, in fume contact AEO and Thymol demonstrated antibiofilm activity at a dose of 1.25 mg/mL air and 40 mg/mL air against M.smegmatis effectively. GC-MS analysis showed that Thymol was the dominant compound. These findings suggest that the use of AEO in fume form may serve as a promising strategy as an anti-mycobacterial activity against M. tb

Antimycobacterial, hepatoprotective and cytotoxicity effects of selected plant species from the Menispermaceae family

Tuberculosis, caused by Mycobacterium tuberculosis, is a worldwide disease affecting millions of people. The rise of resistant strains, coupled with toxicity of current chemotherapy, requires effective alternatives against mycobacterial infections. Some plants from the Menispermaceae family have been used to treat tuberculosis and cough related symptoms. In this study, acetone, methanol:water (4:1), dichloromethane:methanol (1:1) and hot water extracts of different plant parts of Cissampelos owariensis, Cissampelos mucronata and Tinospora fragosa were tested against M. aurum, M. bovis, M. fortuitum, M. smegmatis and M. tuberculosis using a two-fold serial microdilution assay. Cytotoxicity of the active extracts was determined against Vero and HepG2 cells. The hepatoprotective effect of the active extracts was evaluated using rifampicin and acetaminophen as toxic drugs against HepG2 cells. The hot water leaf extracts were most active with promising minimum inhibitory concentration (MIC) values of 20 and 40 µg/mL against M. smegmatis and M. fortuitum respectively. The acetone extracts of C. owariensis, C. mucronata and T. fragosa had the lowest MIC values (0.03 – 1.67 mg/mL). The root extract of C. owariensis was not toxic to Vero cells while the leaf extract was more toxic. The acetone extract of C. mucronata leaves was toxic to Vero cells but the other extracts had low toxicity. The active leaf and root extracts had protective effects on rifampicin-induced toxicity on HepG2 cells. The root extract also had a protective effect on acetaminophen-induced toxicity on HepG2 cells but the leaf extract had no protective effect. The hot water extracts of C. owariensis, C. mucronata and T. fragosa had a more protective effect on the toxin-induced cells than the acetone extracts. These results support further investigation on the bioactive compounds in these plant extracts.

Cytotoxicity and gene expression studies in understanding the mechanism on antimycobacterial properties of methanolic leaf extract of Acalypha indica Linn.

Despite the availability of effective antituberculosis medication, tuberculosis (TB) remains a serious global health concern, raising the demand for low-after effect drug alternative. This study aims to explore the anti-mycobacterial potency of Acalypha indica Linn. on Mycobacterium tuberculosis using Mycobacterium smegmatis as a model and thereby uncover its mechanism. Characterization of bioactive phytocompounds and their cumulative effects were studied through both biochemical and molecular approaches. The antimycobacterial efficacy was screened by cell viability assay and IC50. FE-SEM and qRT-PCR were used to study the effect of A. indica leaf methanolic extract (AILME) on cell integrity and mycolic acid synthesis pathway. This study reports AILME to be the most effective solvent-extract combination which showed >40 % cell inhibition from 25 µg mL−1 onwards at 24 hr. AILME showed >80 % antioxidant potential, lowest IC50 of 48.24 µg mL−1 and a MIC value of 200 µg mL−1 at 24 hr. The FE-SEM analysis of the AILME treated bacterial cells showed significant morphological damage which supports the results of antimycobacterial activity. The gene expression study revealed that AILME inhibited the expression of inhA and mtrA genes involved in mycolic acid biosynthesis pathway and cell wall integrity, respectively. The bioactive phytocompounds characterised through GCMS and HR-LCMS revealed about nineteen potent anti-TB compounds like malic acid, catechin, pyrrolidine, hexadecanoic acid, quercetin etc. that might supervise the role behind the antimycobacterial effect of the herb. This study reveals that AILME possesses strong antimycobacterial action by impeding the formation of mycolic acid biosynthesis pathway and disorganization of cell wall. Presence of anti-TB phytocompounds in abundance suggest its potential chemotherapeutic nature against M. tuberculosis.

New Benzotriazole Derivatives: Synthesis, Biological Assessment, In vivo Oral Toxicity Analysis, Docking Studies, Molecular Dynamics, and ADME Profiling

A new series of phenyl-2-(perchloro-1H-benzo[d] [1,2,3]triazol-1-yl)- acetamide derivatives (3a-k) was synthesized and assessed for antimycobacterial, antibacterial, and antifungal effects. Compound 3c displayed potent antimycobacterial, antibacterial, and antifungal activity, comparable to the standard drug. Highly active compounds were analyzed for their binding mode in the active site using the cocrystallized structure of mycobacterium tuberculosis (PDB ID 2X22) and glucosamine-6-phosphate synthase (PDB ID 2VF5), followed by molecular dynamics. Finally, in vivo oral toxicity study confirmed the non-toxic nature of the compound 3c.. KEYWORDS :Antifungal, Antimicrobial, Antimycobacterial, Molecular docking, Molecular dynamics

The role of rifampicin within the treatment of Mycobacterium avium pulmonary disease.

Rifampicin is recommended for the treatment of Mycobacterium avium complex pulmonary disease alongside azithromycin and ethambutol. We evaluated the azithromycin-ethambutol backbone with and without rifampicin in an intracellular hollow fiber model and performed RNA sequencing to study the differences in adaptation. In an in vitro hollow fiber experiment, we simulated epithelial lining fluid pharmacokinetic profiles of the recommended 3-drug (rifampicin, ethambutol, and azithromycin) or a 2-drug (ethambutol and azithromycin) treatment. THP-1 cells infected with M. avium ATCC700898 were exposed to these regimens for 21 days. We determined intra- and extra-cellular bacterial load- and THP-1 cell densities on days 0, 3, 7, 14, and 21, alongside RNA sequencing. The emergence of macrolide resistance was studied by inoculating intra- and extra-cellular fractions of azithromycin-containing Middlebrook 7H10 agar plates. Complete pharmacokinetic profiles were determined at days 0 and 21. Both therapies maintained stasis of both intra- and extra-cellular bacterial populations for 3 days, whilst regrowth coinciding with the emergence of a macrolide-resistant subpopulation was seen after 7 days. THP-1 cell density remained static. Similar transcriptional profiles were observed for both therapies that were minimally influenced by exposure duration. Transcriptional response was slightly larger during 2-drug treatment. Rifampicin did not add to the antimycobacterial effect to the 2-drug therapy or suppression of emergence resistance. RNA transcription was not greatly altered by the addition of rifampicin, which may be due to strong transcriptional influence of azithromycin and host cells. This questions the role of rifampicin in the currently recommended therapy. These findings should be confirmed in clinical trials.

Annual short-burst mass anthelmintic administration reduces tuberculosis severity but not prevalence in a wildlife reservoir

IntroductionTuberculosis (TB), caused by the Mycobacterium tuberculosis complex (MTC), is an important disease in both human and animal systems. Helminths are commonly found in coinfection with MTC and TB is often exacerbated in such coinfections. Long-term anthelmintic administration, to control helminths, can improve a host’s ability to control MTC infection. Mass drug administration programmes, in which anthelmintics are given only once or twice a year, leaving periods where helminth reinfection can occur, are common in both human and domestic animal populations. To date, the effect of such intermittent control programmes on MTC infection and severity has not been explored.MethodsHere we investigate the consequences of a ten-day, annual, mass ivermectin administration on TB prevalence and severity in free-ranging juvenile and yearling (<2 years) wild boar (Sus scrofa).ResultsThis single annual anthelmintic treatment administered over six years reduced TB severity. Further, the proportion of wild boar with severe TB continued to decrease with successive treatments. TB prevalence, however, did not decrease significantly over the course of the study.DiscussionWhile ivermectin has direct anti-mycobacterial effects in vitro, the short duration of treatment means that the reduction in TB severity we observe in wild boar is unlikely to be accounted for by such a direct mechanism. Disruption of the helminth community and subsequent modification or enhancement of the host immune response is a potential mechanism. Future work should examine the consequences of annual anthelmintic drug administration on helminth community composition and structure and on the host immunological responses through time.

NAD(H) homeostasis underlies host protection mediated by glycolytic myeloid cells in tuberculosis

Mycobacterium tuberculosis (Mtb) disrupts glycolytic flux in infected myeloid cells through an unclear mechanism. Flux through the glycolytic pathway in myeloid cells is inextricably linked to the availability of NAD+, which is maintained by NAD+ salvage and lactate metabolism. Using lung tissue from tuberculosis (TB) patients and myeloid deficient LDHA (LdhaLysM−/−) mice, we demonstrate that glycolysis in myeloid cells is essential for protective immunity in TB. Glycolytic myeloid cells are essential for the early recruitment of multiple classes of immune cells and IFNγ-mediated protection. We identify NAD+ depletion as central to the glycolytic inhibition caused by Mtb. Lastly, we show that the NAD+ precursor nicotinamide exerts a host-dependent, antimycobacterial effect, and that nicotinamide prophylaxis and treatment reduce Mtb lung burden in mice. These findings provide insight into how Mtb alters host metabolism through perturbation of NAD(H) homeostasis and reprogramming of glycolysis, highlighting this pathway as a potential therapeutic target.

Clofazimine nanoclusters show high efficacy in experimental TB with amelioration in paradoxical lung inflammation

The rise of tuberculosis (TB) superbugs has impeded efforts to control this infectious ailment, and new treatment options are few. Paradoxical Inflammation (PI) is another major problem associated with current anti-TB therapy, which can complicate the treatment and leads to clinical worsening of disease despite a decrease in bacterial burden in the lungs. TB infection is generally accompanied by an intense local inflammatory response which may be critical to TB pathogenesis. Clofazimine (CLF), a second-line anti-TB drug, delineated potential anti-mycobacterial effects in-vitro and in-vivo and also demonstrated anti-inflammatory potential in in-vitro experiments. However, clinical implications may be restricted owing to poor solubility and low bioavailability rendering a suboptimal drug concentration in the target organ. To unravel these issues, nanocrystals of CLF (CLF-NC) were prepared using a microfluidizer® technology, which was further processed into micro-sized CLF nano-clusters (CLF-NCLs) by spray drying technique. This particle engineering offers combined advantages of micron- and nano-scale particles where micron-size (∼5 μm) promise optimum aerodynamic parameters for the finest lung deposition, and nano-scale dimensions (∼600 nm) improve the dissolution profile of apparently insoluble clofazimine. An inhalable formulation was evaluated against virulent mycobacterium tuberculosis in in-vitro studies and in mice infected with aerosol TB infection. CLF-NCLs resulted in the significant killing of virulent TB bacteria with a MIC value of ∼0.62 μg/mL, as demonstrated by Resazurin microtiter assay (REMA). In TB-infected mice, inhaled doses of CLF-NCLs equivalent to ∼300 μg and ∼ 600 μg of CLF administered on every alternate day over 30 days significantly reduced the number of bacteria in the lung. With an inhaled dose of ∼600 μg/mice, reduction of mycobacterial colony forming units (CFU) was achieved by ∼1.95 Log10CFU times compared to CLF administered via oral gavage (∼1.18 Log10CFU). Lung histology scoring showed improved pathogenesis and inflammation in infected animals after 30 days of inhalation dosing of CLF-NCLs. The levels of pro-inflammatory mediators, including cytokines, TNF-α & IL-6, and MMP-2 in bronchoalveolar lavage fluid (BAL-F) and lung tissue homogenates, were attenuated after inhalation treatment. These pre-clinical data suggest inhalable CLF-NCLs are well tolerated, show significant anti-TB activity and apparently able to tackle the challenge of paradoxical chronic lung inflammation in murine TB model.