Sputum culture reversion in longer treatments with bedaquiline, delamanid, and repurposed drugs for drug-resistant tuberculosis.

Sputum culture reversion after conversion is an indicator of tuberculosis (TB) treatment failure. We analyze data from the endTB multi-country prospective observational cohort (NCT03259269) to estimate the frequency (primary endpoint) among individuals receiving a longer (18-to-20 month) regimen for multidrug- or rifampicin-resistant (MDR/RR) TB who experienced culture conversion. We also conduct Cox proportional hazard regression analyses to identify factors associated with reversion, including comorbidities, previous treatment, cavitary disease at conversion, low body mass index (BMI) at conversion, time to conversion, and number of likely-effective drugs. Of 1,286 patients, 54 (4.2%) experienced reversion, a median of 173 days (97-306) after conversion. Cavitary disease, BMI < 18.5, hepatitis C, prior treatment with second-line drugs, and longer time to initial culture conversion were positively associated with reversion. Reversion was uncommon. Those with cavitary disease, low BMI, hepatitis C, prior treatment with second-line drugs, and in whom culture conversion is delayed may benefit from close monitoring following conversion.

Identification of novel single nucleotide variants in the drug resistance mechanism of Mycobacterium tuberculosis isolates by whole-genome analysis.

BACKGROUND: Tuberculosis (TB) represents a major global health challenge. Drug resistance in Mycobacterium tuberculosis (MTB) poses a substantial obstacle to effective TB treatment. Identifying genomic mutations in MTB isolates holds promise for unraveling the underlying mechanisms of drug resistance in this bacterium. METHODS: In this study, we investigated the roles of single nucleotide variants (SNVs) in MTB isolates resistant to four antibiotics (moxifloxacin, ofloxacin, amikacin, and capreomycin) through whole-genome analysis. We identified the drug-resistance-associated SNVs by comparing the genomes of MTB isolates with reference genomes using the MuMmer4 tool. RESULTS: We observed a strikingly high proportion (94.2%) of MTB isolates resistant to ofloxacin, underscoring the current prevalence of drug resistance in MTB. An average of 3529 SNVs were detected in a single ofloxacin-resistant isolate, indicating a mutation rate of approximately 0.08% under the selective pressure of ofloxacin exposure. We identified a set of 60 SNVs associated with extensively drug-resistant tuberculosis (XDR-TB), among which 42 SNVs were non-synonymous mutations located in the coding regions of nine key genes (ctpI, desA3, mce1R, moeB1, ndhA, PE_PGRS4, PPE18, rpsA, secF). Protein structure modeling revealed that SNVs of three genes (PE_PGRS4, desA3, secF) are close to the critical catalytic active sites in the three-dimensional structure of the coding proteins. CONCLUSION: This comprehensive study elucidates novel resistance mechanisms in MTB against antibiotics, paving the way for future design and development of anti-tuberculosis drugs.

Population structure and spatial distribution of Mycobacterium tuberculosis in Ethiopia.

Ethiopia is one of the countries with a high tuberculosis (TB) burden, yet little is known about the spatial distribution of Mycobacterium tuberculosis (Mtb) lineages. This study identifies the spoligotyping of 1735 archived Mtb isolates from the National Drug Resistance Survey, collected between November 2011 and June 2013, to investigate Mtb population structure and spatial distribution. Spoligotype International Types (SITs) and lineages were retrieved from online databases. The distribution of lineages was evaluated using Fisher's exact test and logistic regression models. The Global Moran's Index and Getis-Ord Gi statistic were utilized to identify hotspot areas. Our results showed that spoligotypes could be interpreted and led to 4 lineages and 283 spoligotype patterns in 91% of the isolates, including 4% of those with multidrug/rifampicin resistance (MDR/RR) TB. The identified Mtb lineages were lineage 1 (1.8%), lineage 3 (25.9%), lineage 4 (70.6%) and lineage 7 (1.6%). The proportion of lineages 3 and 4 varied by regions, with lineage 3 being significantly greater than lineage 4 in reports from Gambella (AOR = 4.37, P < 0.001) and Tigray (AOR = 3.44, P = 0.001) and lineage 4 being significantly higher in Southern Nations Nationalities and Peoples Region (AOR = 1.97, P = 0.026) than lineage 3. Hotspots for lineage 1 were located in eastern Ethiopia, while a lineage 7 hotspot was identified in northern and western Ethiopia. The five prevalent spoligotypes, which were SIT149, SIT53, SIT25, SIT37 and SIT26 account for 42.8% of all isolates under investigation, while SIT149, SIT53 and SIT21 account for 52-57.8% of drug-resistant TB cases. TB and drug resistant TB are mainly caused by lineages 3 and 4, and significant proportions of the prevalent spoligotypes also influence drug-resistant TB and the total TB burden. Regional variations in lineages may result from both local and cross-border spread.

Modulating mycobacterial envelope integrity for antibiotic synergy with benzothiazoles.

Developing effective tuberculosis drugs is hindered by mycobacteria's intrinsic antibiotic resistance because of their impermeable cell envelope. Using benzothiazole compounds, we aimed to increase mycobacterial cell envelope permeability and weaken the defenses of Mycobacterium marinum, serving as a model for Mycobacterium tuberculosis Initial hit, BT-08, significantly boosted ethidium bromide uptake, indicating enhanced membrane permeability. It also demonstrated efficacy in the M. marinum-zebrafish embryo infection model and M. tuberculosis-infected macrophages. Notably, BT-08 synergized with established antibiotics, including vancomycin and rifampicin. Subsequent medicinal chemistry optimization led to BT-37, a non-toxic and more potent derivative, also enhancing ethidium bromide uptake and maintaining synergy with rifampicin in infected zebrafish embryos. Mutants of M. marinum resistant to BT-37 revealed that MMAR_0407 (Rv0164) is the molecular target and that this target plays a role in the observed synergy and permeability. This study introduces novel compounds targeting a new mycobacterial vulnerability and highlights their cooperative and synergistic interactions with existing antibiotics.

Targeted protein degradation in mycobacteria uncovers antibacterial effects and potentiates antibiotic efficacy.

Proteolysis-targeting chimeras (PROTACs) represent a new therapeutic modality involving selectively directing disease-causing proteins for degradation through proteolytic systems. Our ability to exploit targeted protein degradation (TPD) for antibiotic development remains nascent due to our limited understanding of which bacterial proteins are amenable to a TPD strategy. Here, we use a genetic system to model chemically-induced proximity and degradation to screen essential proteins in Mycobacterium smegmatis (Msm), a model for the human pathogen M. tuberculosis (Mtb). By integrating experimental screening of 72 protein candidates and machine learning, we find that drug-induced proximity to the bacterial ClpC1P1P2 proteolytic complex leads to the degradation of many endogenous proteins, especially those with disordered termini. Additionally, TPD of essential Msm proteins inhibits bacterial growth and potentiates the effects of existing antimicrobial compounds. Together, our results provide biological principles to select and evaluate attractive targets for future Mtb PROTAC development, as both standalone antibiotics and potentiators of existing antibiotic efficacy.

Synthesis, molecular docking study and biological evaluation of new pyrrole scaffolds as potential antitubercular agents for dual targeting of enoyl ACP reductase and dihydrofolate reductase.

In this study, new series of N'-(2-(substitutedphenoxy)acetyl)-4-(1H-pyrrol-1-yl)benzohydrazides (3a-j) 4-(2,5-dimethyl-1H-pyrrol-1-yl)-N'-(2-(substitutedphenoxy)acetyl)benzohydrazides (5a-j) were synthesized, characterized and assessed as inhibitors of enoyl ACP reductase and DHFR. Most of the compounds exhibited dual inhibition against the enzymes enoyl ACP reductase and DHFR. Several synthesized substances also demonstrated significant antibacterial and antitubercular properties. A molecular docking analysis was conducted in order to determine the potential mechanism of action of the synthesized compounds. The results indicated that there were binding interactions seen with the active sites of dihydrofolate reductase and enoyl ACP reductase. Additionally, important structural details were identified that play a critical role in sustaining the dual inhibitory activity. These findings were useful for the development of future dual inhibitors. Therefore, this study provided strong evidence that several synthesized molecules could exert their antitubercular properties at the cellular level through multi-target inhibition. By shedding light on the mechanisms through which these compounds exert their inhibitory effects, this research opens up promising avenues for the future development of dual inhibitors with enhanced antibacterial and antitubercular properties. The study's findings underscore the importance of multi-target approaches in drug design, providing a strong foundation for the design and optimization of novel compounds that can effectively target bacterial infections at the cellular level.

Exploring optimal drug targets through subtractive proteomics analysis and pangenomic insights for tailored drug design in tuberculosis.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, ranks among the top causes of global human mortality, as reported by the World Health Organization's 2022 TB report. The prevalence of M. tuberculosis strains that are multiple and extensive-drug resistant represents a significant barrier to TB eradication. Fortunately, having many completely sequenced M. tuberculosis genomes available has made it possible to investigate the species pangenome, conduct a pan-phylogenetic investigation, and find potential new drug targets. The 442 complete genome dataset was used to estimate the pangenome of M. tuberculosis. This study involved phylogenomic classification and in-depth analyses. Sequential filters were applied to the conserved core genome containing 2754 proteins. These filters assessed non-human homology, virulence, essentiality, physiochemical properties, and pathway analysis. Through these intensive filtering approaches, promising broad-spectrum therapeutic targets were identified. These targets were docked with FDA-approved compounds readily available on the ZINC database. Selected highly ranked ligands with inhibitory potential include dihydroergotamine and abiraterone acetate. The effectiveness of the ligands has been supported by molecular dynamics simulation of the ligand-protein complexes, instilling optimism that the identified lead compounds may serve as a robust basis for the development of safe and efficient drugs for TB treatment, subject to further lead optimization and subsequent experimental validation.

Targeting Mycobacterium tuberculosis pH-driven adaptation.

Mycobacterium tuberculosis (Mtb) senses and adapts to host environmental cues as part of its pathogenesis. One important cue sensed by Mtb is the acidic pH of its host niche - the macrophage. Acidic pH induces widespread transcriptional and metabolic remodelling in Mtb. These adaptations to acidic pH can lead Mtb to slow its growth and promote pathogenesis and antibiotic tolerance. Mutants defective in pH-dependent adaptations exhibit reduced virulence in macrophages and animal infection models, suggesting that chemically targeting these pH-dependent pathways may have therapeutic potential. In this review, we discuss mechanisms by which Mtb regulates its growth and metabolism at acidic pH. Additionally, we consider the therapeutic potential of disrupting pH-driven adaptations in Mtb and review the growing class of compounds that exhibit pH-dependent activity or target pathways important for adaptation to acidic pH.

Prevalence, drug resistance, and genotypic diversity of the RDRio subfamily of Mycobacterium tuberculosis in Ecuador: a retrospective analysis for years 2012-2016.

Introduction: A major sublineage within the Mycobacterium tuberculosis (MTB) LAM family characterized by a new in-frame fusion gene Rv3346c/55c was discovered in Rio de Janeiro (Brazil) in 2007, called RDRio, associated to drug resistance. The few studies about prevalence of MTB RDRio strains in Latin America reported values ranging from 3% in Chile to 69.8% in Venezuela, although no information is available for countries like Ecuador. Methods: A total of 814 MTB isolates from years 2012 to 2016 were screened by multiplex PCR for RDRio identification, followed by 24-loci MIRU-VNTR and spoligotyping. Results: A total number of 17 MTB RDRio strains were identified, representing an overall prevalence of 2.09% among MTB strains in Ecuador. While 10.9% of the MTB isolates included in the study were multidrug resistance (MDR), 29.4% (5/17) of the RDRio strains were MDR. Discussion: This is the first report of the prevalence of MTB RDRio in Ecuador, where a strong association with MDR was found, but also a very low prevalence compared to other countries in Latin America. It is important to improve molecular epidemiology tools as a part of MTB surveillance programs in Latin America to track the transmission of potentially dangerous MTB stains associated to MDR TB like MTB RDRio.

Drug-resistant Mycobacterium tuberculosis among Nepalese patients at a tuberculosis referral center.

BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB), characterized by isoniazid and rifampicin resistance, is caused by chromosomal mutations that restrict treatment options and complicate tuberculosis management. This study sought to investigate the prevalence of pre-extensively drug-resistant (pre-XDR) and extensively drug-resistant (XDR) tuberculosis, as well as mutation pattern, in Nepalese patients with MDR/rifampicin-resistant (RR)-TB strains. METHODS: A cross-sectional study was conducted on MDR/RR-TB patients at the German Nepal Tuberculosis Project from June 2017 to June 2018. The MTBDRsl line probe assay identified pre-XDR-TB and XDR-TB. Pre-XDR-TB included MDR/RR-TB with resistance to any fluoroquinolone (FLQ), while XDR-TB included MDR/RR-TB with resistance to any FLQ and at least one additional group A drug. Mutation status was determined by comparing bands on reaction zones [gyrA and gyrB for FLQ resistance, rrs for SILD resistance, and eis for low-level kanamycin resistance, according to the GenoType MTBDRsl VER 2.0, Hain Lifescience GmbH, Nehren, Germany definition of pre-XDR and XDR] to the evaluation sheet. SPSS version 17.0 was used for data analysis. RESULTS: Out of a total of 171 patients with MDR/RR-TB, 160 had (93.57%) had MTBC, of whom 57 (35.63%) had pre-XDR-TB and 10 (6.25%) had XDR-TB. Among the pre-XDR-TB strains, 56 (98.25%) were FLQ resistant, while 1 (1.75%) was SLID resistant. The most frequent mutations were found at codons MUT3C (57.14%, 32/56) and MUT1 (23.21%, 13/56) of the gyrA gene. One patient had SLID resistant genotype at the MUT1 codon of the rrs gene (100%, 1/1). XDR-TB mutation bands were mostly detected on MUT1 (30%, 3/10) of the gyrA and rrs, MUT3C (30%, 3/10) of the gyrA, and MUT1 (30%, 3/10) of the rrs. CONCLUSIONS: Pre-XDR-TB had a significantly higher likelihood than XDR-TB, with different specific mutation bands present in gyrA and rrs genes.

Repurposing of anti-malarial drugs for the treatment of tuberculosis: realistic strategy or fanciful dead end?

BACKGROUND: Drug repurposing offers a strategic alternative to the development of novel compounds, leveraging the known safety and pharmacokinetic profiles of medications, such as linezolid and levofloxacin for tuberculosis (TB). Anti-malarial drugs, including quinolones and artemisinins, are already applied to other diseases and infections and could be promising for TB treatment. METHODS: This review included studies on the activity of anti-malarial drugs, specifically quinolones and artemisinins, against Mycobacterium tuberculosis complex (MTC), summarizing results from in vitro, in vivo (animal models) studies, and clinical trials. Studies on drugs not primarily developed for TB (doxycycline, sulfonamides) and any novel developed compounds were excluded. Analysis focused on in vitro activity (minimal inhibitory concentrations), synergistic effects, pre-clinical activity, and clinical trials. RESULTS: Nineteen studies, including one ongoing Phase 1 clinical trial, were analysed: primarily investigating quinolones like mefloquine and chloroquine, and, to a lesser extent, artemisinins. In vitro findings revealed high MIC values for anti-malarials versus standard TB drugs, suggesting a limited activity. Synergistic effects with anti-TB drugs were modest, with some synergy observed in combinations with isoniazid or pyrazinamide. In vivo animal studies showed limited activity of anti-malarials against MTC, except for one study of the combination of chloroquine with isoniazid. CONCLUSIONS: The repurposing of anti-malarials for TB treatment is limited by high MIC values, poor synergy, and minimal in vivo effects. Concerns about potential toxicity at effective dosages and the risk of antimicrobial resistance, especially where TB and malaria overlap, further question their repurposing. These findings suggest that focusing on novel compounds might be both more beneficial and rewarding.

Prevalence and genetic basis of Mycobacterium tuberculosis resistance to pretomanid in China.

BACKGROUND: Pretomanid is a key component of new regimens for the treatment of drug-resistant tuberculosis (TB) which are being rolled out globally. However, there is limited information on the prevalence of pre-existing resistance to the drug. METHODS: To investigate pretomanid resistance rates in China and its underlying genetic basis, as well as to generate additional minimum inhibitory concentration (MIC) data for epidemiological cutoff (ECOFF)/breakpoint setting, we performed MIC determinations in the Mycobacterial Growth Indicator Tube™ (MGIT) system, followed by WGS analysis, on 475 Mycobacterium tuberculosis (MTB) isolated from Chinese TB patients between 2013 and 2020. RESULTS: We observed a pretomanid MIC distribution with a 99% ECOFF equal to 0.5 mg/L. Of the 15 isolates with MIC values > 0.5 mg/L, one (MIC = 1 mg/L) was identified as MTB lineage 1 (L1), a genotype previously reported to be intrinsically less susceptible to pretomanid, two were borderline resistant (MIC = 2-4 mg/L) and the remaining 12 isolates were highly resistant (MIC ≥ 16 mg/L) to the drug. Five resistant isolates did not harbor mutations in the known pretomanid resistant genes. CONCLUSIONS: Our results further support a breakpoint of 0.5 mg/L for a non-L1 MTB population, which is characteristic of China. Further, our data point to an unexpected high (14/475, 3%) pre-existing pretomanid resistance rate in the country, as well as to the existence of yet-to-be-discovered pretomanid resistance genes.

The Structural and Molecular Mechanisms of Mycobacterium tuberculosis Translational Elongation Factor Proteins.

Targeting translation factor proteins holds promise for developing innovative anti-tuberculosis drugs. During protein translation, many factors cause ribosomes to stall at messenger RNA (mRNA). To maintain protein homeostasis, bacteria have evolved various ribosome rescue mechanisms, including the predominant trans-translation process, to release stalled ribosomes and remove aberrant mRNAs. The rescue systems require the participation of translation elongation factor proteins (EFs) and are essential for bacterial physiology and reproduction. However, they disappear during eukaryotic evolution, which makes the essential proteins and translation elongation factors promising antimicrobial drug targets. Here, we review the structural and molecular mechanisms of the translation elongation factors EF-Tu, EF-Ts, and EF-G, which play essential roles in the normal translation and ribosome rescue mechanisms of Mycobacterium tuberculosis (Mtb). We also briefly describe the structure-based, computer-assisted study of anti-tuberculosis drugs.

Structure-Function Analysis of the Essential Mycobacterium tuberculosis P450 Drug Target, CYP121A1.

Cytochrome P450 CYP121A1 is a well-known drug target against Mycobacterium tuberculosis, the human pathogen that causes the deadly disease tuberculosis (TB). CYP121A1 is a unique P450 enzyme because it uses classical and non-classical P450 catalytic processes and has distinct structural features among P450s. However, a detailed investigation of CYP121A1 protein structures in terms of active site cavity dynamics and key amino acids interacting with bound ligands has yet to be undertaken. To address this research knowledge gap, 53 CYP121A1 crystal structures were investigated in this study. Critical amino acids required for CYP121A1's overall activity were identified and highlighted this enzyme's rigid architecture and substrate selectivity. The CYP121A1-fluconazole crystal structure revealed a novel azole drug-P450 binding mode in which azole heme coordination was facilitated by a water molecule. Fragment-based inhibitor approaches revealed that CYP121A1 can be inhibited by molecules that block the substrate channel or by directly interacting with the P450 heme. This study serves as a reference for the precise understanding of CYP121A1 interactions with different ligands and the structure-function analysis of P450 enzymes in general. Our findings provide critical information for the synthesis of more specific CYP121A1 inhibitors and their development as novel anti-TB drugs.

Mtb-Selective 5-Aminomethyl Oxazolidinone Prodrugs: Robust Potency and Potential Liabilities.

Linezolid is a drug with proven human antitubercular activity whose use is limited to highly drug-resistant patients because of its toxicity. This toxicity is related to its mechanism of action─linezolid inhibits protein synthesis in both bacteria and eukaryotic mitochondria. A highly selective and potent series of oxazolidinones, bearing a 5-aminomethyl moiety (in place of the typical 5-acetamidomethyl moiety of linezolid), was identified. Linezolid-resistant mutants were cross-resistant to these molecules but not vice versa. Resistance to the 5-aminomethyl molecules mapped to an N-acetyl transferase (Rv0133) and these mutants remained fully linezolid susceptible. Purified Rv0133 was shown to catalyze the transformation of the 5-aminomethyl oxazolidinones to their corresponding N-acetylated metabolites, and this transformation was also observed in live cells of Mycobacterium tuberculosis. Mammalian mitochondria, which lack an appropriate N-acetyltransferase to activate these prodrugs, were not susceptible to inhibition with the 5-aminomethyl analogues. Several compounds that were more potent than linezolid were taken into C3HeB/FeJ mice and were shown to be highly efficacious, and one of these (9) was additionally taken into marmosets and found to be highly active. Penetration of these 5-aminomethyl oxazolidinone prodrugs into caseum was excellent. Unfortunately, these compounds were rapidly converted into the corresponding 5-alcohols by mammalian metabolism which retained antimycobacterial activity but resulted in substantial mitotoxicity.

Durlobactam, a Diazabicyclooctane ß-Lactamase Inhibitor, Inhibits BlaC and Peptidoglycan Transpeptidases of Mycobacterium tuberculosis.

Peptidoglycan synthesis is an underutilized drug target in Mycobacterium tuberculosis (Mtb). Diazabicyclooctanes (DBOs) are a class of broad-spectrum ß-lactamase inhibitors that also inhibit certain peptidoglycan transpeptidases that are important in mycobacterial cell wall synthesis. We evaluated the DBO durlobactam as an inhibitor of BlaC, the Mtb ß-lactamase, and multiple Mtb peptidoglycan transpeptidases (PonA1, LdtMt1, LdtMt2, LdtMt3, and LdtMt5). Timed electrospray ionization mass spectrometry (ESI-MS) captured acyl-enzyme complexes with BlaC and all transpeptidases except LdtMt5. Inhibition kinetics demonstrated durlobactam was a potent and efficient DBO inhibitor of BlaC (KI app 9.2 ± 0.9 µM, k2/K 5600 ± 560 M-1 s-1) and similar to clavulanate (KI app 3.3 ± 0.6 µM, k2/K 8400 ± 840 M-1 s-1); however, durlobactam had a lower turnover number (tn = kcat/kinact) than clavulanate (1 and 8, respectively). KI app values with durlobactam and clavulanate were similar for peptidoglycan transpeptidases, but ESI-MS captured durlobactam complexes at more time points. Molecular docking and simulation demonstrated several productive interactions of durlobactam in the active sites of BlaC, PonA1, and LdtMt2. Antibiotic susceptibility testing was conducted on 11 Mtb isolates with amoxicillin, ceftriaxone, meropenem, imipenem, clavulanate, and durlobactam. Durlobactam had a minimum inhibitory concentration (MIC) range of 0.5-16 µg/mL, similar to the ranges for meropenem (1-32 µg/mL) and imipenem (0.5-64 µg/mL). In ß-lactam + durlobactam combinations (1:1 mass/volume), MICs were lowered 4- to 64-fold for all isolates except one with meropenem-durlobactam. This work supports further exploration of novel ß-lactamase inhibitors that target BlaC and Mtb peptidoglycan transpeptidases.

Inhibitors of the Thioesterase Activity of Mycobacterium tuberculosis Pks13 Discovered Using DNA-Encoded Chemical Library Screening.

DNA-encoded chemical library (DEL) technology provides a time- and cost-efficient method to simultaneously screen billions of compounds for their affinity to a protein target of interest. Here we report its use to identify a novel chemical series of inhibitors of the thioesterase activity of polyketide synthase 13 (Pks13) from Mycobacterium tuberculosis (Mtb). We present three chemically distinct series of inhibitors along with their enzymatic and Mtb whole cell potency, the measure of on-target activity in cells, and the crystal structures of inhibitor-enzyme complexes illuminating their interactions with the active site of the enzyme. One of these inhibitors showed a favorable pharmacokinetic profile and demonstrated efficacy in an acute mouse model of tuberculosis (TB) infection. These findings and assay developments will aid in the advancement of TB drug discovery.

Transmission characteristics in Tuberculosis by WGS: nationwide cross-sectional surveillance in China.

China, with the third largest share of global tuberculosis cases, faces a substantial challenge in its healthcare system as a result of the high burden of multidrug-resistant and rifampicin-resistant tuberculosis (MDR/RR-TB). This study employs a genomic epidemiological approach to assess recent tuberculosis transmissions between individuals, identifying potential risk factors and discerning the role of transmitted resistant isolates in the emergence of drug-resistant tuberculosis in China. We conducted a population-based retrospective study on 5052 Mycobacterium tuberculosis (MTB) isolates from 70 surveillance sites using whole genome sequencing (WGS). Minimum spanning tree analysis identified resistance mutations, while epidemiological data analysis pinpointed transmission risk factors. Of the 5052 isolates, 23% (1160) formed 452 genomic clusters, with 85.6% (387) of the transmissions occurring within the same counties. Individuals with younger age, larger family size, new cases, smear positive, and MDR/RR were at higher odds for recent transmission, while higher education (university and above) and occupation as a non-physical workers emerged as protective factors. At least 61.4% (251/409) of MDR/RR-TB were likely a result of recent transmission of MDR/RR isolates, with previous treatment (crude OR = 2.77), smear-positive (cOR = 2.07) and larger family population (cOR = 1.13) established as risk factors. Our findings highlight that local transmission remains the predominant form of TB transmission in China. Correspondingly, drug-resistant tuberculosis is primarily driven by the transmission of resistant tuberculosis isolates. Targeted interventions for high-risk populations to interrupt transmission within the country will likely provide an opportunity to reduce the prevalence of both tuberculosis and drug-resistant tuberculosis.

Third Generation Solid Dispersion-Based Formulation of Novel Anti-Tubercular Agent Exhibited Improvement in Solubility, Dissolution and Biological Activity.

The long treatment period and development of drug resistance in tuberculosis (TB) necessitates the discovery of new anti-tubercular agents. The drug discovery program of the institute leads to the development of an anti-tubercular lead (IIIM-019), which is an analogue of nitrodihydroimidazooxazole and exhibited promising anti-tubercular action. However, IIIM-019 displays poor aqueous solubility (1.2 µg/mL), which demands suitable dosage form for its efficient oral administration. In the present study, third generation solid dispersion-based formulation was developed to increase the solubility and dissolution of IIIM-019. The solubility profile of IIIM-019 using various polymeric carriers was determined and subsequently, PVP K-30 and P-407 were selected for preparation of binary and ternary solid dispersion. The third-generation ternary solid dispersion comprising PVP K-30 and P-407 revealed a remarkable enhancement in the aqueous solubility of IIIM-019. Physicochemical characterization of the developed formulations was done by employing FTIR spectroscopy, scanning electron microscopy, X-ray diffraction analysis, differential scanning calorimetry, and dynamic light scattering analysis. The dissolution study indicated an impressive release profile with the optimized formulation. The optimized formulation was further examined for cytotoxicity, cellular uptake, and hemolytic activity. The results indicated that the formulation had no apparent cytotoxicity on Caco-2 cells and was non-hemolytic in nature. Moreover, the optimized formulation showed significantly improved anti-tubercular activity compared to the native molecule. These findings showed that the developed third generation ternary solid dispersion could be a promising option for the oral delivery of investigated anti-tubercular molecule.

Uncovering the roles of Mycobacterium tuberculosis melH in redox and bioenergetic homeostasis: implications for antitubercular therapy.

Mycobacterium tuberculosis (Mtb), the pathogenic bacterium that causes tuberculosis, has evolved sophisticated defense mechanisms to counteract the cytotoxicity of reactive oxygen species (ROS) generated within host macrophages during infection. The melH gene in Mtb and Mycobacterium marinum (Mm) plays a crucial role in defense mechanisms against ROS generated during infection. We demonstrate that melH encodes an epoxide hydrolase and contributes to ROS detoxification. Deletion of melH in Mm resulted in a mutant with increased sensitivity to oxidative stress, increased accumulation of aldehyde species, and decreased production of mycothiol and ergothioneine. This heightened vulnerability is attributed to the increased expression of whiB3, a universal stress sensor. The absence of melH also resulted in reduced intracellular levels of NAD+, NADH, and ATP. Bacterial growth was impaired, even in the absence of external stressors, and the impairment was carbon source dependent. Initial MelH substrate specificity studies demonstrate a preference for epoxides with a single aromatic substituent. Taken together, these results highlight the role of melH in mycobacterial bioenergetic metabolism and provide new insights into the complex interplay between redox homeostasis and generation of reactive aldehyde species in mycobacteria. IMPORTANCE: This study unveils the pivotal role played by the melH gene in Mycobacterium tuberculosis and in Mycobacterium marinum in combatting the detrimental impact of oxidative conditions during infection. This investigation revealed notable alterations in the level of cytokinin-associated aldehyde, para-hydroxybenzaldehyde, as well as the redox buffer ergothioneine, upon deletion of melH. Moreover, changes in crucial cofactors responsible for electron transfer highlighted melH's crucial function in maintaining a delicate equilibrium of redox and bioenergetic processes. MelH prefers epoxide small substrates with a phenyl substituted substrate. These findings collectively emphasize the potential of melH as an attractive target for the development of novel antitubercular therapies that sensitize mycobacteria to host stress, offering new avenues for combating tuberculosis.