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Endogenous antimicrobial peptides (AMPs) play a key role in the host defense against pathogens. AMPs attack pathogens preferentially at the site of entry to prevent invasive infection. Mycobacterium tuberculosis (Mtb) enters its host via the airways. AMPs released into the airways are therefore likely candidates to contribute to the clearance of Mtb immediately after infection. Since lysozyme is detectable in airway secretions, we evaluated its antimicrobial activity against Mtb. We demonstrate that lysozyme inhibits the growth of extracellular Mtb, including isoniazid-resistant strains. Lysozyme also inhibited the growth of non-tuberculous mycobacteria. Even though lysozyme entered Mtb-infected human macrophages and co-localized with the pathogen we did not observe antimicrobial activity. This observation was unlikely related to the large size of lysozyme (14.74 kDa) because a smaller lysozyme-derived peptide also co-localized with Mtb without affecting the viability. To evaluate whether the activity of lysozyme against extracellular Mtb could be relevant in vivo, we incubated Mtb with fractions of human serum and screened for antimicrobial activity. After several rounds of sub-fractionation, we identified a highly active fraction-component as lysozyme by mass spectrometry. In summary, our results identify lysozyme as an antimycobacterial protein that is detectable as an active compound in human serum. Our results demonstrate that the activity of AMPs against extracellular bacilli does not predict efficacy against intracellular pathogens despite co-localization within the macrophage. Ongoing experiments are designed to unravel peptide modifications that occur in the intracellular space and interfere with the deleterious activity of lysozyme in the extracellular environment. [ABSTRACT FROM AUTHOR]

Objective: Tuberculosis (TB) is a contagious disease caused by Mycobacterium tuberculosis, that poses a threat to the international scientific community. According to the WHO, 2022 report, an estimated 1.6 million people died from tuberculosis and 10.6 million have been diagnosed. In the present study, the target protein is Rv3032 protein, which belongs to the Glycosyl transferase group 1 family and is involved in the biosynthesis of the methyl glucosyl lipopolysaccharide (MGLP). Methods: The 3D structural features of the target protein were identified using computational techniques, including model building, active site prediction, and structural evaluation. Virtual screening studies were conducted at the active site using Bionet and Rare chemical ligand databases to identify the drug-like compounds. Results and Discussion: 67 ligands were selected as lead molecules based on the highest glide scores and glide energies. Out of which 6 ligands were studied, in which Carboxylate, amino, and carboxamide groups were common pharmacophore groups present in the A1 to A6 docked complexes have common binding with the ARG224, LYS229, and GLU311 amino acid residues. Conclusions: ADME parameters were found in the permissible range proving the lead-like properties of the resultant ligand molecules. [ABSTRACT FROM AUTHOR]

Purpose: Rapid, reliable identification of mycobacteria from positive cultures is essential for patient management, particularly for the differential diagnosis of Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM) species. The aim of the present study was to evaluate a new "In-Vitro-Diagnostic"-certified PCR kit, FluoroType®-Mycobacteria VER 1.0 (Hain Lifescience GmbH) for NTM and MTBC identification from cultures. Methods: Mycobacteria identification isolated from positive cultures during routine practice at the Lyon university hospital mycobacteria laboratory obtained by hsp65 amplification/sequencing were compared retrospectively and prospectively to those obtained by and the FluoroType®-Mycobacteria VER 1.0 kit. Results: The overall agreement between hsp65 amplification/sequencing and the FluoroType®-Mycobacteria VER 1.0 kit was 88.4% (84/95); 91.2% (52/57) for the retrospective period and 84.2% (32/38) for the prospective period. There were 9 (9.5%) minor discrepancies (species in the FluoroType®-Mycobacteria VER 1.0 database and identified at genus level): 4 during the retrospective period, 5 during the prospective period; and 2 (2.1%) major discrepancies (species in the FluoroType®-Mycobacteria VER 1.0 database and identified incorrectly to species level): 1 during the retrospective period (M. kumamotonense identified as M. abscessus subsp massiliense by the kit) and 1 during the prospective period (M. chimaera identified as M. smegmatis by the kit). Including concordant results at genus level and minor discrepancies, 17.9% (17/95) of strains were identified as Mycobacterium sp. by the FluoroType®-Mycobacteria-VER 1.0 kit. Conclusion: The good performance of the FluoroType®-Mycobacteria-VER 1.0 kit with few major discrepancies could enable its use for first-line identification of positive mycobacteria cultures. However, an alternative identification method at least for reference laboratories is needed owing to the non-negligible proportion of NTM strains were identified at genus level. [ABSTRACT FROM AUTHOR]

Objectives and design: As an interferon-inducible protein, Viperin has broad-spectrum antiviral effects and regulation of host immune responses. We aim to investigate how Viperin regulates interferon-γ (IFN-γ) production in macrophages to control Mycobacterium tuberculosis (Mtb) infection. Methods: We use Viperin deficient bone-marrow-derived macrophage (BMDM) to investigate the effects and machines of Viperin on Mtb infection. Results: Viperin inhibited IFN-γ production in macrophages and in the lung of mice to promote Mtb survival. Further insight into the mechanisms of Viperin-mediated regulation of IFN-γ production revealed the role of TANK-binding kinase 1 (TBK1), the TAK1-dependent inhibition of NF-kappa B kinase-epsilon (IKKε), and interferon regulatory factor 3 (IRF3). Inhibition of the TBK1-IKKε-IRF3 axis restored IFN-γ production reduced by Viperin knockout in BMDM and suppressed intracellular Mtb survival. Moreover, Viperin deficiency activated the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway, which promoted IFN-γ production and inhibited Mtb infection in BMDM. Additionally, a combination of the anti-TB drug INH treatment in the absence of Viperin resulted in further IFN-γ production and anti-TB effect. Conclusions: This study highlights the involvement of TBK1-IKKε-IRF3 axis and JAK-STAT signaling pathways in Viperin-suppressed IFN-γ production in Mtb infected macrophages, and identifies a novel mechanism of Viperin on negatively regulating host immune response to Mtb infection. [ABSTRACT FROM AUTHOR]

Increasing Arctic temperatures are facilitating the northward expansion of more southerly hosts, vectors, and pathogens, exposing naïve populations to pathogens not typical at northern latitudes. To understand such rapidly changing host–pathogen dynamics, we need sensitive and robust surveillance tools. Here, we use a novel multiplexed magnetic-capture and droplet digital PCR (ddPCR) tool to assess a sentinel Arctic species, the polar bear (Ursus maritimus; n = 68), for the presence of five zoonotic pathogens (Erysipelothrix rhusiopathiae, Francisella tularensis, Mycobacterium tuberculosis complex, Toxoplasma gondii and Trichinella spp.), and observe associations between pathogen presence and biotic and abiotic predictors. We made two novel detections: the first detection of a Mycobacterium tuberculosis complex member in Arctic wildlife and the first of E. rhusiopathiae in a polar bear. We found a prevalence of 37% for E. rhusiopathiae, 16% for F. tularensis, 29% for Mycobacterium tuberculosis complex, 18% for T. gondii, and 75% for Trichinella spp. We also identify associations with bear age (Trichinella spp.), harvest season (F. tularensis and MTBC), and human settlements (E. rhusiopathiae, F. tularensis, MTBC, and Trichinella spp.). We demonstrate that monitoring a sentinel species, the polar bear, could be a powerful tool in disease surveillance and highlight the need to better characterize pathogen distributions and diversity in the Arctic. [ABSTRACT FROM AUTHOR]

Decaprenylphosphoryl-β-D-ribose-2′-epimerase (DprE1), a crucial enzyme in the process of arabinogalactan and lipoarabinomannan biosynthesis, has become the target of choice for anti-TB drug discovery in the recent past. The current study aims to find the potential DprE1 inhibitors through in-silico approaches. Here, we built the pharmacophore and 3D-QSAR model using the reported 40 azaindole derivatives of DprE1 inhibitors. The best pharmacophore hypothesis (ADRRR_1) was employed for the virtual screening of the chEMBL database. To identify prospective hits, molecules with good phase scores (> 2.000) were further evaluated by molecular docking studies for their ability to bind to the DprE1 enzyme (PDB: 4KW5). Based on their binding affinities (< − 9.0 kcal/mole), the best hits were subjected to the calculation of free-binding energies (Prime/MM-GBSA), pharmacokinetic, and druglikeness evaluations. The top 10 hits retrieved from these results were selected to predict their inhibitory activities via the developed 3D-QSAR model with a regression coefficient (R2) value of 0.9608 and predictive coefficient (Q2) value of 0.7313. The induced fit docking (IFD) studies and in-silico prediction of anti-TB sensitivity for these top 10 hits were also implemented. Molecular dynamics simulations (MDS) were performed for the top 5 hit molecules for 200 ns to check the stability of the hits with DprE1. Based on their conformational stability throughout the 200 ns simulation, hit 2 (chEMBL_SDF:357100) was identified as the best hit against DprE1 with an accepted safety profile. The MD results were also in accordance with the docking score, MM-GBSA value, and 3D-QSAR predicted activity. The hit 2 molecule, (N-(3-((2-(((1r,4r)-4-(dimethylamino)cyclohexyl)amino)-9-isopropyl-9H-purin-6-yl)amino)phenyl)acrylamide) could serve as a lead for the discovery of a novel DprE1 inhibiting anti-TB drug. [ABSTRACT FROM AUTHOR]

A convergent synthesis α-(1→3)-, α-(1→5), β-(1→2)-linked hexaarabinofuranoside related to the terminal fragment of lipoarabinomannan (LAM) and arabinogalactan of mycobacteria as a glycoside with 4-(3-azidopropoxy)phenyl aglycone was accomplished. 4-(3-Azidopropoxy)phenyl aglycone belongs to the class of Janus aglycones, which can be used as both a temporary protective group for the anomeric position of the carbohydrate and a (pre)spacer for the synthesis of neoglycoconjugates. The key step of the synthesis is the formation of 1,2-trans glycosidic linkage with the use of Ara-β-(1→2)-Ara p-tolylthioarabinofuranoside bearing five triisopropylsilyl groups. [ABSTRACT FROM AUTHOR]

Background: The pathogen responsible for tuberculosis is called Mycobacterium tuberculosis. Its interaction with macrophages has a significant impact on the onset and progression of the disease. Methods: The respiratory pathway allows Mycobacterium tuberculosis to enter the body's lungs where it battles immune cells before being infected latently or actively. In the progress of tuberculosis, Mycobacterium tuberculosis activates the body's immune system and creates inflammatory factors, which cause tissue inflammation to infiltrate and the creation of granulomas, which seriously harms the body. Toll-like receptors of macrophage can mediate host recognition of Mycobacterium tuberculosis, initiate immune responses, and participate in macrophage autophagy. New host-directed therapeutic approaches targeting autophagy for drug-resistant Mycobacterium tuberculosis have emerged, providing new ideas for the effective treatment of tuberculosis. Conclusions: In-depth understanding of the mechanisms by which macrophage autophagy interacts with intracellular Mycobacterium tuberculosis, as well as the study of potent and specific autophagy-regulating molecules, will lead to much-needed advances in drug discovery and vaccine design, which will improve the prevention and treatment of human tuberculosis. [ABSTRACT FROM AUTHOR]

Implementation of whole genome sequencing (WGS) for patient care is hindered by limited Mycobacterium tuberculosis (Mtb) in clinical specimens and slow Mtb growth. We evaluated droplet multiple displacement amplification (dMDA) for amplification of minute amounts of Mtb DNA to enable WGS as an alternative to other Mtb enrichment methods. Purified genomic Mtb-DNA (0.1, 0.5, 1, and 5 pg) was encapsulated and amplified using the Samplix Xdrop-instrument and sequenced alongside a control sample using standard Illumina protocols followed by MAGMA-analysis. The control and 5 pg input dMDA samples underwent nanopore sequencing followed by Nanoseq and TB-profiler analysis. dMDA generated 105-2400 ng DNA from the 0.1-5 pg input DNA, respectively. Followed by Illumina WGS, dMDA raised mean sequencing depth from 7 × for 0.1 pg input DNA to ≥ 60 × for 5 pg input and the control sample. Bioinformatic analysis revealed a high number of false positive and false negative variants when amplifying ≤ 0.5 pg input DNA. Nanopore sequencing of the 5 pg dMDA sample presented excellent coverage depth, breadth, and accurate strain characterization, albeit elevated false positive and false negative variants compared to Illumina-sequenced dMDA sample with identical Mtb DNA input. dMDA coupled with Illumina WGS for samples with ≥ 5 pg purified Mtb DNA, equating to approximately 1000 copies of the Mtb genome, offers precision for drug resistance, phylogeny, and transmission insights. [ABSTRACT FROM AUTHOR]

The Mycobacterium tuberculosis complex (MTBC) comprises nine human-adapted lineages that differ in their geographical distribution. Local adaptation of specific MTBC genotypes to the respective human host population has been invoked in this context. We aimed to assess if bacterial genetics governs MTBC pathogenesis or if local co-adaptation translates into differential susceptibility of human macrophages to infection by different MTBC genotypes. We generated macrophages from cryopreserved blood mononuclear cells of Tanzanian tuberculosis patients, from which the infecting MTBC strains had previously been phylogenetically characterized. We infected these macrophages ex vivo with a phylogenetically similar MTBC strain ("matched infection") or with strains representative of other MTBC lineages ("mismatched infection"). We found that L1 infections resulted in a significantly lower bacterial burden and that the intra-cellular replication rate of L2 strains was significantly higher compared the other MTBC lineages, irrespective of the MTBC lineage originally infecting the patients. Moreover, L4-infected macrophages released significantly greater amounts of TNF-α, IL-6, IL-10, MIP-1β, and IL-1β compared to macrophages infected by all other strains. While our results revealed no measurable effect of local adaptation, they further highlight the strong impact of MTBC phylogenetic diversity on the variable outcome of the host–pathogen interaction in human tuberculosis. [ABSTRACT FROM AUTHOR]

Tuberculosis remains a large health threat, despite the availability of the tuberculosis vaccine, BCG. As BCG efficacy gradually decreases from adolescence, BCG-Prime and antigen-booster may be an efficient strategy to confer vaccine efficacy. Mycobacterial DNA-binding protein 1 (MDP1, namely Rv2986c, hupB or HU) is a major Mycobacterium tuberculosis protein that induces vaccine-efficacy by co-administration with CpG DNA. To produce MDP1 for booster-vaccine use, we have created recombinant MDP1 produced in both Escherichia coli (eMDP1) and Mycolicibacterium smegmatis (mMDP1), an avirulent rapid-growing mycobacteria. We tested their immunogenicity by checking interferon (IFN)-gamma production by stimulated peripheral blood cells derived from BCG-vaccinated individuals. Similar to native M. tuberculosis MDP1, we observed that most lysin resides in the C-terminal half of mMDP1 are highly methylated. In contrast, eMDP1 had less post-translational modifications and IFN-gamma stimulation. mMDP1 stimulated the highest amount of IFN-gamma production among the examined native M. tuberculosis proteins including immunodominant MPT32 and Antigen 85 complex. MDP1-mediated IFN-gamma production was more strongly enhanced when combined with a new type of CpG DNA G9.1 than any other tested CpG DNAs. Taken together, these results suggest that the combination of mMDP1 and G9.1 possess high potential use for human booster vaccine against tuberculosis. [ABSTRACT FROM AUTHOR]

Genetic diversity of human populations is determined by polymorphic variants of immune response genes and can be linked to the variation in responses to infectious agents. This study compared allele and genotype frequencies of polymorphic variants of the TLR1, TLR2, TLR4, TLR6, and TLR8 genes between samples from Eastern Europe and Central Asia. We included 680 unrelated individuals from Eastern Slavic (n = 308), Armenian (n = 137), Tajik (n = 138), and Kyrgyz (n = 97) samples of patients with confirmed diagnoses of HIV and tuberculosis. The international 1000 Genomes Project data—samples of Caucasians (EUR) and countries of eastern (EAS) and southern (SAS) Asia—were used for comparison. Despite the lack of genetic diversity and population differences in allele frequencies of the analyzed genes, the samples differed in a number of loci both from each other and from EAS and SAS samples. [ABSTRACT FROM AUTHOR]

The present work describes the unrevealed potential of tetrahydropyridines as anticancer and anti-tubercular agents along with DFT, ADMET, drug-likeliness and molecular docking studies synthesized by using Bronsted acidic ionic liquid viz. 1-methyl-3-(4-sulfobutyl)-1H-imidazol-3-ium chloride [MSbIM-Cl]. The synthesized derivatives have been screened for their anticancer and anti-tubercular evaluation against MCF-7 cell lines and Mycobacterium tuberculosis respectively. The compound 4e showed the highest anticancer activity while the compound 4h showed the highest anti-tubercular activity. The in vitro evaluation has been supported by computational methods such as molecular docking, density functional study and in silico ADMET and drug-likeliness prediction. [ABSTRACT FROM AUTHOR]

Contact between humans and wildlife presents a risk for both zoonotic and anthropozoonotic disease transmission. In this study we report the detection of human strains of Mycobacterium tuberculosis in sun bears and an Asiatic black bear in a wildlife rescue centre in Cambodia, confirming for the first time the susceptibility of these bear species to tuberculosis when in close contact with humans. After genotyping revealed two different strains of M. tuberculosis from cases occurring between 2009 and 2019, 100 isolates from 30 sun bear cases, a single Asiatic black bear case, and a human case were subjected to whole genome sequencing. We combined single nucleotide polymorphism analysis and exploration of mixed base calls with epidemiological data to indicate the evolution of each outbreak. Our results confirmed two concurrent yet separate tuberculosis outbreaks and established a likely transmission route in one outbreak where the human case acted as an intermediatory between bear cases. In both outbreaks, we observed high rates of transmission and progression to active disease, suggesting that sun bears are highly susceptible to tuberculosis if exposed under these conditions. Overall, our findings highlight the risk of bi-directional transmission of tuberculosis between humans and captive bears in high human tuberculosis burden regions, with implied considerations for veterinary and public health. We also demonstrate the use of standard genomic approaches to better understand disease outbreaks in captive wildlife settings and to inform control and prevention measures. [ABSTRACT FROM AUTHOR]

Background: Tuberculosis (TB) continues to be a major global health burden, and co-infection with other pathogens further complicates the diagnosis and treatment of this infectious disease. The present retrospective study aimed to evaluate the clinical utility of nanopore sequencing in identifying co-infection caused by Mycobacterium tuberculosis (M.tb) and other pathogens. Methods: Patients with M.tb co-infection from December 2021 to March 2023 at the Jiangxi Provincial Chest Hospital were retrospectively studied. Data were collected including demographics, symptoms, imaging findings, pathogen diagnosis tests, and treatment history. Pathogen tests involved culture, AFB smear, Xpert MTB/RIF, and nanopore sequencing. Results: The enrolled patients included 20 M.tb cases and three nontuberculous mycobacteria (NTM) cases co-infected with other pathogens. Common clinical symptoms included cough (47.83%), expectoration (34.78%), and asthma (17.39%). Radiological examinations showed typical features of pulmonary tuberculosis, including nodules (73.91%), cord-like shadows (34.78%), cavities (34.78%), and destroyed lung manifestations (17.39%). Nanopore sequencing identified M.tb in a significant majority of the cases (86.96%), outperforming traditional culture tests (39.13%), acid-fast bacilli (AFB) tests (27.27%), and Xpert MTB/RIF (53.84%) tests. Notably, nanopore sequencing revealed that M.tb was frequently co-infected with Candida albicans, Klebsiella pneumoniae, and Mycobacterium abscessus. Three specific cases of co-infection with distinct diagnosis and treatment characteristics were presented in detail. They illustrated the complexity of TB co-infection management and the potential of nanopore sequencing for accurate diagnosis and informing the tailored therapeutic approaches. Conclusion: Nanopore sequencing-based metagenomics method can help clinicians to identify TB co-infection patterns and formulate a rational drug regimen in time. [ABSTRACT FROM AUTHOR]

The continuing emergence of new strains of antibiotic-resistant bacteria has renewed interest in phage therapy; however, there has been limited progress in applying phage therapy to multi-drug resistant Mycobacterium tuberculosis (Mtb) infections. In this study, we show that bacteriophage strains D29 and DS6A can efficiently lyse Mtb H37Rv in 7H10 agar plates. However, only phage DS6A efficiently kills H37Rv in liquid culture and in Mtb-infected human primary macrophages. We further show in subsequent experiments that, after the humanized mice were infected with aerosolized H37Rv, then treated with DS6A intravenously, the DS6A treated mice showed increased body weight and improved pulmonary function relative to control mice. Furthermore, DS6A reduces Mtb load in mouse organs with greater efficacy in the spleen. These results demonstrate the feasibility of developing phage therapy as an effective therapeutic against Mtb infection. Bacteriophage strain DS6A efficiently eradicates intracellular Mtb in human macrophages, while humanized mice treated with DS6A show improved pulmonary function and reduced Mtb load in mouse organs with greater efficacy in the spleen. [ABSTRACT FROM AUTHOR]

Macrophages act as the first immune defense line of the host against Mycobacterium tuberculosis (Mtb). A previous study showed that circRNA_SLC8A1 was significantly upregulated in Mtb-infected macrophages, but its regulatory mechanism in anti-tuberculosis infection is unclear. Therefore, this study aimed to investigate the role of circRNA_SLC8A1 in the anti-tuberculosis activity of macrophages. We showed that circRNA_SLC8A1 was upregulated in tuberculosis patients. Moreover, the binding sites of miR-20b-5p on circRNA_SLC8A1 and Sequestosome 1 (SQSTM1/p62) mRNA were predicted by StarBase and verified by the double luciferase reporter gene assay. Next, we found that miR-20b-5p expression was decreased, while SQSTM1 protein expression was increased in a time- and dose-dependent manner in the human macrophage U937 in response to Mtb infection. Furthermore, circRNA_SLC8A1 overexpression vector (circRNA_SLC8A1) or shRNA (sh-circRNA_SLC8A1) and/or miR-20b-5p mimic or inhibitor and/or SQSTM1 overexpression vector (SQSTM1) or small interfering RNA (si-SQSTM1) or its corresponding control were transfected into Mtb-infected macrophages. Results showed that overexpression of circRNA_SLC8A1 or miR-20b-5p inhibitor promoted the secretion of pro-inflammatory factors IL-1β, IL-6, and TNF-α, increased Nitric Oxide (NO) content and inducible nitric oxide synthase (iNOS) expression, inhibited Reactive oxygen species (ROS) production. Cleaved-caspase-3 protein expression, and cell apoptosis, and promoted Mtb survival. Silencing SQSTM1 inhibited secretion of pro-inflammatory factors and activation of the NF-κB pathway. Overexpression of miR‐20b‐5p blocked the promoting of circ‐SLC8A1 on SQSTM1 protein expression. In summary, circRNA_SLC8A1 sponged miR‐20b‐5p to upregulate SQSTM1/p62 expression and promoted Mtb survival in macrophages through the NF-κB signaling pathway. [ABSTRACT FROM AUTHOR]

Fluorescent molecule-based direct labeling of amplified DNA is a sensitive method employed across diverse DNA detection and diagnostics systems. However, using pre-labeled primers only allows for the attachment of a single fluorophore to each DNA strand and any modifications of the system are less flexible, requiring new sets of primers. As an alternative, direct labeling of amplified products with modified nucleotides is available, but still poorly characterized. To address these limitations, we sought a direct and adaptable approach to label amplicons produced through Loop-mediated isothermal amplification (LAMP), using labeled nucleotides (dUTPs) rather than primers. The focus of this study was the development and examination of a direct labeling technique of specific genes, including those associated with drug resistance in Mycobacterium tuberculosis. We used 5-(3-Aminoallyl)-2′-deoxyuridine-5′triphosphate, tagged with 5/6-TAMRA (TAMRA-dUTP) for labeling LAMP amplicons during the amplification process and characterized amplification and incorporation efficiency. The optimal TAMRA-dUTP concentration was first determined based on amplification efficiency (0.5% to total dNTPs). Higher concentrations of modified nucleotides reduced or completely inhibited the amplification yield. Target size also showed to be determinant to the success of amplification, as longer sequences showed lower amplification rates, thus less TAMRA incorporated amplicons. Finally, we were able to successfully amplify all four M. tuberculosis target genes using LAMP and TAMRA-modified dUTPs. [ABSTRACT FROM AUTHOR]

Macrophages act as the first immune defense line of the host against Mycobacterium tuberculosis (Mtb). A previous study showed that circRNA_SLC8A1 was significantly upregulated in Mtb-infected macrophages, but its regulatory mechanism in anti-tuberculosis infection is unclear. Therefore, this study aimed to investigate the role of circRNA_SLC8A1 in the anti-tuberculosis activity of macrophages. We showed that circRNA_SLC8A1 was upregulated in tuberculosis patients. Moreover, the binding sites of miR-20b-5p on circRNA_SLC8A1 and Sequestosome 1 (SQSTM1/p62) mRNA were predicted by StarBase and verified by the double luciferase reporter gene assay. Next, we found that miR-20b-5p expression was decreased, while SQSTM1 protein expression was increased in a time- and dose-dependent manner in the human macrophage U937 in response to Mtb infection. Furthermore, circRNA_SLC8A1 overexpression vector (circRNA_SLC8A1) or shRNA (sh-circRNA_SLC8A1) and/or miR-20b-5p mimic or inhibitor and/or SQSTM1 overexpression vector (SQSTM1) or small interfering RNA (si-SQSTM1) or its corresponding control were transfected into Mtb-infected macrophages. Results showed that overexpression of circRNA_SLC8A1 or miR-20b-5p inhibitor promoted the secretion of pro-inflammatory factors IL-1β, IL-6, and TNF-α, increased Nitric Oxide (NO) content and inducible nitric oxide synthase (iNOS) expression, inhibited Reactive oxygen species (ROS) production. Cleaved-caspase-3 protein expression, and cell apoptosis, and promoted Mtb survival. Silencing SQSTM1 inhibited secretion of pro-inflammatory factors and activation of the NF-κB pathway. Overexpression of miR‐20b‐5p blocked the promoting of circ‐SLC8A1 on SQSTM1 protein expression. In summary, circRNA_SLC8A1 sponged miR‐20b‐5p to upregulate SQSTM1/p62 expression and promoted Mtb survival in macrophages through the NF-κB signaling pathway. [ABSTRACT FROM AUTHOR]

Whole genome sequencing (WGS) of Mycobacterium tuberculosis offers valuable insights for tuberculosis (TB) control. High throughput platforms like Illumina and Oxford Nanopore Technology (ONT) are increasingly used globally, although ONT is known for higher error rates and is less established for genomic studies. Here we present a study comparing the sequencing outputs of both Illumina and ONT platforms, analysing DNA from 59 clinical isolates in highly endemic TB regions of Thailand. The resulting sequence data were used to profile the M. tuberculosis pairs for their lineage, drug resistance and presence in transmission chains, and were compared to publicly available WGS data from Thailand (n = 1456). Our results revealed isolates that are predominantly from lineages 1 and 2, with consistent drug resistance profiles, including six multidrug-resistant strains; however, analysis of ONT data showed longer phylogenetic branches, emphasising the technologies higher error rate. An analysis incorporating the larger dataset identified fifteen of our samples within six potential transmission clusters, including a significant clade of 41 multi-drug resistant isolates. ONT's extended sequences also revealed strain-specific structural variants in pe/ppe genes (e.g. ppe50), which are candidate loci for vaccine development. Despite some limitations, our results show that ONT sequencing is a promising approach for TB genomic research, supporting precision medicine and decision-making in areas with less developed infrastructure, which is crucial for tackling the disease's significant regional burden. [ABSTRACT FROM AUTHOR]

The synthesis of (p)ppGpp alarmones plays a vital role in the regulation of metabolism suppression, growth rate control, virulence, bacterial persistence, and biofilm formation. The (p)ppGpp alarmones are synthesized by proteins of the RelA/SpoT homolog (RSH) superfamily, including long bifunctional RSH proteins and small alarmone synthetases. Here, we investigated enzyme kinetics and dose-dependent enzyme inhibition to elucidate the mechanism of 4-(4,7-dimethyl-1,2,3,4-tetrahydronaphthalen-1-yl)pentanoic acid (DMNP) action on the (p)ppGpp synthetases RelMsm and RelZ from Mycolicibacterium smegmatis and RelMtb from Mycobacterium tuberculosis. DMNP was found to inhibit the activity of RelMtb. According to the enzyme kinetics analysis, DMNP acts as a noncompetitive inhibitor of RelMsm and RelZ. Based on the results of molecular docking, the DMNP-binding site is located in the proximity of the synthetase domain active site. This study might help in the development of alarmone synthetase inhibitors, which includes relacin and its derivatives, as well as DMNP – a synthetic analog of the marine coral metabolite erogorgiaene. Unlike conventional antibiotics, alarmone synthetase inhibitors target metabolic pathways linked to the bacterial stringent response. Although these pathways are not essential for bacteria, they regulate the development of adaptation mechanisms. Combining conventional antibiotics that target actively growing cells with compounds that impede bacterial adaptation may address challenges associated with antimicrobial resistance and bacterial persistence. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) is a major public health concern that results in significant morbidity and mortality, particularly in middle- to low-income countries. Extra-pulmonary tuberculosis (EPTB) in adults is a form of TB that affects organs other than the lungs and is challenging to diagnose and treat due to a lack of accurate early diagnostic markers and inadequate knowledge of host immunity. Next-generation sequencing-based approaches have shown potential for identifying diagnostic biomarkers and host immune responses related to EPTB. This strategic review discusses on the significance using primary human cells and cell lines for in vitro transcriptomic studies on common forms of EPTB, such as lymph node TB, brain TB, bone TB, and endometrial TB to derive potential insights. While organoids have shown promise as a model system, primary cell lines still remain a valuable tool for studying host–pathogen interplay due to their conserved immune system, non-iPSC origin, and lack of heterogeneity in cell population. This review outlines a basic workflow for researchers interested in performing transcriptomics studies in EPTB, and also discusses the potential of cell-line based dual RNA-Seq technology for deciphering comprehensive transcriptomic signatures, host–pathogen interplay, and biomarkers from the host and Mycobacterium tuberculosis. Thus, emphasizing the implementation of this technique which can significantly contribute to the global anti-TB effort and advance our understanding of EPTB. [ABSTRACT FROM AUTHOR]

With the widespread use of rifampicin (RMP) and isoniazid (INH), multidrug resistance (MDR) in Mycobacterium tuberculosis (M.tb) poses a threat to the success of tuberculosis (TB) control programs. We have developed a new polycarbonate track-etched membranes (PC-TEM) based DNA bio-chip designed for rapid detection of mutations conferring MDR in M.tb culture isolates. Bio-chips were designed to contain 14 specific probes for wild type and mutated allele of selected codons within 80 bp rifampicin resistance determining region of rpoB gene, katG gene and mabA-inhA regulatory region. RMP-resistance-associated gene mutation points rpoB 516, 526, 531 and 533, and the INH-resistance-associated gene mutation points katG315 and inhA-15 were targeted. Bio-chip signal was detected using enhanced chemiluminescence. A total of 50 culture isolates that were sensitive or resistant to RMP and/or INH were analyzed by bio-chip. The results of culture-based drug susceptibility testing (DST) were used as the gold standard and gene sequencing was performed to resolve the discordance. Amongst 50 culture isolates, we have detected 18 MDR, 9 RMP mono‐resistant, 6 INH mono‐resistant, and 17 fully susceptible isolates. The developed DNA bio-chip has a sensitivity of 90% for RMP and MDR and 100% for INH resistance. The bio-chip has a specificity of 100% for RMP and MDR and 88.8% for INH detection. The identification of mutations using the DNA bio-chip was 100% concordant with the sequencing data for the probes covered by the bio-chip. The detection of rpoB, katG and inhA gene mutation points by a DNA bio-chip may be used as a rapid, accurate, and economical, clinical detection method for MDR detection in M.tb. This is very valuable for the control of TB epidemics. [ABSTRACT FROM AUTHOR]

Extrapulmonary tuberculosis with a renal involvement can be a manifestation of a disseminated infection that requires therapeutic intervention, particularly with a decrease in efficacy of conventional regimens. In the present study, we investigated the therapeutic potency of mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) in the complex anti-tuberculosis treatment (ATT). A rabbit model of renal tuberculosis (rTB) was constructed by injecting of the standard strain Mycobacterium tuberculosis H37Rv into the cortical layer of the kidney parenchyma. Isolated rabbit MSC-EVs were intravenously administered once as an addition to standard ATT (isoniazid, pyrazinamide, and ethambutol). The therapeutic efficacy was assessed by analyzing changes of blood biochemical biomarkers and levels of anti- and pro-inflammatory cytokines as well as by renal computed tomography with subsequent histological and morphometric examination. The therapeutic effect of therapy with MSC-EVs was shown by ELISA method that confirmed a statistically significant increase of the anti-inflammatory and decrease of pro-inflammatory cytokines as compared to conventional treatment. In addition, there is a positive trend in increase of ALP level, animal weigh, and normalization of ADA activity that can indicate an improvement of kidney state. A significant reduction of the area of specific and interstitial inflammation indicated positive affect of MSC-EVs that suggests a shorter duration of ATT. The number of MSC-EVs proteins (as identified by mass-spectometry analysis) with anti-microbial, anti-inflammatory and immunoregulatory functions reduced the level of the inflammatory response and the severity of kidney damage (further proved by morphometric analysis). In conclusion, MSC-EVs can be a promising tool for the complex treatment of various infectious diseases, in particularly rTB. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (Mtb) is able to transition into a dormant state, causing the latent state of tuberculosis. Dormant mycobacteria acquire resistance to all known antibacterial drugs and can survive in the human body for decades before becoming active. In the dormant forms of M. tuberculosis, the synthesis of porphyrins and its Zn-complexes significantly increased when 5-aminolevulinic acid (ALA) was added to the growth medium. Transcriptome analysis revealed an activation of 8 genes involved in the metabolism of tetrapyrroles during the Mtb transition into a dormant state, which may lead to the observed accumulation of free porphyrins. Dormant Mtb viability was reduced by more than 99.99% under illumination for 30 min (300 J/cm2) with 565 nm light that correspond for Zn–porphyrin and coproporphyrin absorptions. We did not observe any PDI effect in vitro using active bacteria grown without ALA. However, after accumulation of active cells in lung macrophages and their persistence within macrophages for several days in the presence of ALA, a significant sensitivity of active Mtb cells (ca. 99.99%) to light exposure was developed. These findings create a perspective for the treatment of latent and multidrug-resistant tuberculosis by the eradication of the pathogen in order to prevent recurrence of this disease. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (Mtb) is able to transition into a dormant state, causing the latent state of tuberculosis. Dormant mycobacteria acquire resistance to all known antibacterial drugs and can survive in the human body for decades before becoming active. In the dormant forms of M. tuberculosis, the synthesis of porphyrins and its Zn-complexes significantly increased when 5-aminolevulinic acid (ALA) was added to the growth medium. Transcriptome analysis revealed an activation of 8 genes involved in the metabolism of tetrapyrroles during the Mtb transition into a dormant state, which may lead to the observed accumulation of free porphyrins. Dormant Mtb viability was reduced by more than 99.99% under illumination for 30 min (300 J/cm2) with 565 nm light that correspond for Zn–porphyrin and coproporphyrin absorptions. We did not observe any PDI effect in vitro using active bacteria grown without ALA. However, after accumulation of active cells in lung macrophages and their persistence within macrophages for several days in the presence of ALA, a significant sensitivity of active Mtb cells (ca. 99.99%) to light exposure was developed. These findings create a perspective for the treatment of latent and multidrug-resistant tuberculosis by the eradication of the pathogen in order to prevent recurrence of this disease. [ABSTRACT FROM AUTHOR]

Phenotypic effects of mutations are highly dependent on the genetic backgrounds in which they occur, due to epistatic effects. To test how easily the loss of enzyme activity can be compensated for, we screen mutant libraries of BlaC, a β-lactamase from Mycobacterium tuberculosis, for fitness in the presence of carbenicillin and the inhibitor clavulanic acid. Using a semi-rational approach and deep sequencing, we prepare four double-site saturation libraries and determine the relative fitness effect for 1534/1540 (99.6%) of the unique library members at two temperatures. Each library comprises variants of a residue known to be relevant for clavulanic acid resistance as well as residue 105, which regulates access to the active site. Variants with greatly improved fitness were identified within each library, demonstrating that compensatory mutations for loss of activity can be readily found. In most cases, the fittest variants are a result of positive epistasis, indicating strong synergistic effects between the chosen residue pairs. Our study sheds light on a role of epistasis in the evolution of functional residues and underlines the highly adaptive potential of BlaC. Site saturation study of BlaC β-lactamase reveals that high fitness of clavulanic acid resistant double mutants is a result of positive epistasis and strong synergistic effects between chosen functional residues. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) is an infectious disease caused by the Mycobacterium tuberculosis complex (Mtbc), which develops from asymptomatic latent TB to active stages. The microbiome was purposed as a potential factor affecting TB pathogenesis, but the study was limited. The present study explored the association between gut-pharyngeal microbiome and TB stages in cynomolgus macaques using the full-length 16S rDNA amplicon sequencing based on Oxford Nanopore Technologies. The total of 71 macaques was divided into TB (−) control, TB (+) latent and TB (+) active groups. The differential abundance analysis showed that Haemophilus hemolyticus was decreased, while Prevotella species were increased in the pharyngeal microbiome of TB (+) macaques. In addition, Eubacterium coprostanoligenes in the gut was enriched in TB (+) macaques. Alteration of these bacteria might affect immune regulation and TB severity, but details of mechanisms should be further explored and validated. In summary, microbiota may be associated with host immune regulation and affect TB progression. The findings suggested the potential mechanisms of host-microbes interaction, which may improve the understanding of the role of microbiota and help develop therapeutics for TB in the future. [ABSTRACT FROM AUTHOR]

Objective: Multiple ring-enhancing lesions of the brain are enigmatic neuroimaging abnormality. In this systematic review, we evaluated the etiological spectrum of these lesions. Methods: This systematic review adhered to the PRISMA guidelines. We searched PubMed, Embase, Scopus, and Google Scholar up until 15 June 2023. We included case reports and case series. Quality evaluation of each case was based on selection, ascertainment, causality, and reporting. The extracted information included demographic characteristics, clinical features, type and number of multiple enhancing brain lesions, diagnostic procedures, final diagnoses, treatments, and patient outcomes. Protocol Registration: PROSPERO CRD42023437081. Results: We analyzed 156 records representing 161 patients, 60 of whom were immunocompromised. The mean age was 42.6 years, and 67% of patients experienced symptoms for up to 1 month. A higher proportion of immunocompromised patients (42% vs. 30%) exhibited encephalopathy. Chest or CT thorax abnormalities were reported in 27.3% of patients, while CSF abnormalities were found in 31.7%, more frequently among the immunocompromised. Definitive diagnoses were established via brain biopsy, aspiration, or autopsy in 60% of cases, and through CSF examination or other ancillary tests in 40% of cases. Immunocompromised patients had a higher incidence of Toxoplasma gondii infection and CNS lymphoma, while immunocompetent patients had a higher incidence of Mycobacterium tuberculosis infection and immune-mediated and demyelinating disorders. The improvement rate was 74% in immunocompetent patients compared to 52% in the immunocompromised group. Conclusion: Multiple ring-enhancing lesions of the brain in immunocompromised patients are more frequently caused by Toxoplasma gondii infections and CNS lymphoma. Conversely, among immunocompetent patients, Mycobacterium tuberculosis infection and immune-related demyelinating conditions are common. [ABSTRACT FROM AUTHOR]

We studied the possibility of using 4-hexylresorcinol to increase the efficiency of anti-mycobacterial chemotherapy. In an in vitro experiment, 4-hexylresorcinol increased the efficiency of rifampicin, kanamycin, and isoniazid against Mycobacterium smegmatis by 3-5 times. Experiments in sanitation of BALB/c mice infected with M. smegmatis showed the best efficacy of the isoniazid and 4-hexylresorcinol combination in comparison with isoniazid monotherapy. The growth-inhibiting activity of the combination of antibiotic rifabutin with 4-hexylresorcinol was shown on 6 strains of M. tuberculosis. A 2-fold decrease in the minimum inhibitory concentration of this antibiotic in the presence of half-minimum inhibitory concentration of 4-hexylresorcinol was demonstrated for monoresistant strain M. tuberculosis 5360/42Hr. On the mouse model of experimental tuberculosis caused by M. tuberculosis H37Rv, a 5-fold decrease in lung contamination and more rapid complete cure were achieved in animals treated with the combination of rifabutin and 4-hexylresorcinol in comparison with rifabutin monotherapy. [ABSTRACT FROM AUTHOR]

Eis (Enhanced intracellular survival) protein is an aminoglycoside acetyltransferase enzyme classified under the family – GNAT (GCN5-related family of N-acetyltransferases) secreted by Mycobacterium tuberculosis (Mtb). The enzymatic activity of Eis results in the acetylation of kanamycin, thereby impairing the drug's action. In this study, we expressed and purified recombinant Eis (rEis) to determine the enzymatic activity of Eis and its potential inhibitor. Glide-enhanced precision docking was used to perform molecular docking with chosen ligands. Quercetin was found to interact Eis with a maximum binding affinity of -8.379 kcal/mol as compared to other ligands. Quercetin shows a specific interaction between the positively charged amino acid arginine in Eis and the aromatic ring of quercetin through π-cation interaction. Further, the effect of rEis was studied on the antibiotic activity of kanamycin A in the presence and absence of quercetin. It was observed that the activity of rEis aminoglycoside acetyltransferase decreased with increasing quercetin concentration. The results from the disk diffusion assay confirmed that increasing the concentration of quercetin inhibits the rEis protein activity. In conclusion, quercetin may act as a potential Eis inhibitor. [ABSTRACT FROM AUTHOR]

Background: In this study, we conducted this population-based study to evaluate the genetic diversity and clustering rate of Mycobacterium tuberculosis (MTB) strains using the whole-genome sequencing (WGS), to better understand its transmission in Ordos. Methods: All patients with culture-positive TB notified in Ordos from January 2021 to December 2022 were recruited. WGS was performed to analyze single-nucleotide polymorphism (SNP) and to identify genotypic drug susceptibilities of MTB isolates. Results: Overall, a total of 186 patients were included in the present study, of whom 35 (18.8%) had no symptoms suggestive of active TB. Lineage 2 was the predominant MTB sublineage, accounting for 186 of isolates tested. When the pairwise SNP difference ≤ 12 was used as the cutoff for WGS-based clusters, we identified 17 genotypic clusters, and 38 isolates belonged to these 17 clusters, resulting in a clustering rate of 20.4%. The Beijing genotype was an independent factor associating with genomic-clustering (adjusted OR 4.219, 95% CI 0.962–18.502). The overall sensitivity on WGS-based resistance prediction was 85.7% for rifampicin, 73.1% for isoniazid, 60.0% for Ethambutol, 72.7% for streptomycin, and 72.7% for fluoroquinolones. Conclusion: To conclude, the present study demonstrates the extensive recent transmission of Beijing genotype strains in the community of Ordos. The failure to provide a comprehensive pattern of transmission indicated the missed diagnosis of active TB within the community. A substantial proportion of subclinical TB cases are recognized in the bacteria-positive cases, emphasizing that we must interrupt transmission by finding people with active TB before they infect others. [ABSTRACT FROM AUTHOR]

Purpose: To evaluate the performance of core genome multilocus sequence typing (cgMLST) for genotyping Mycobacterium tuberculosis (M.tuberculosis) Strains in regions where the lineage 2 strains predominate. Methods: We compared clustering by whole-genome SNP typing with cgMLST clustering in the analysis of WGS data of 6240 strains from five regions of China. Using both the receiver operating characteristic (ROC) curve and epidemiological investigation to determine the optimal threshold for defining genomic clustering by cgMLST. The performance of cgMLST was evaluated by quantifying the sensitivity, specificity and concordance of clustering between two methods. Logistic regression was used to gauge the impact of strain genetic diversity and lineage on cgMLST clustering. Results: The optimal threshold for cgMLST to define genomic clustering was determined to be ≤ 10 allelic differences between strains. The overall sensitivity and specificity of cgMLST averaged 99.6% and 96.3%, respectively; the concordance of clustering between two methods averaged 97.1%. Concordance was significantly correlated with strain genetic diversity and was 3.99 times (95% CI, 2.94–5.42) higher in regions with high genetic diversity (π > 1.55 × 10–4) compared to regions with low genetic diversity. The difference missed statistical significance, while concordance for lineage 2 strains (96.8%) was less than that for lineage 4 strains (98.3%). Conclusion : cgMLST showed a discriminatory power comparable to whole-genome SNP typing and could be used to genotype clinical M.tuberculosis strains in different regions of China. The discriminative power of cgMLST was significantly correlated with strain genetic diversity and was slightly lower with strains from regions with low genetic diversity. [ABSTRACT FROM AUTHOR]

Context: Mycobacterial membrane proteins Large 3 (MmpL3) is responsible for the transport of mycobacterial acids out of cell membrane to form cell wall, which is essential for the survival of Mycobacterium tuberculosis (Mtb) and has become a potent anti-tuberculosis target. SQ109 is an ethambutol (EMB) analogue, as a novel anti-tuberculosis drug, can effectively inhibit MmpL3, and has completed phase 2b-3 clinical trials. Drug resistance has always been the bottleneck problem in clinical treatment of tuberculosis. The S288T mutant of MmpL3 shows significant resistance to the inhibitor SQ109, while the specific action mechanism remains unclear. The results show that MmpL3 S288T mutation causes local conformational change with little effect on the global structure. With MmpL3 bound by SQ109 inhibitor, the distance between D710 and R715 increases resulting in H-bond destruction, but their interactions and proton transfer function are still restored. In addition, the rotation of Y44 in the S288T mutant leads to an obvious bend in the periplasmic domain channel and an increased number of contact residues, reducing substrate transport efficiency. This work not only provides a possible dual drug resistance mechanism of MmpL3 S288T mutant but also aids the development of novel anti-tuberculosis inhibitors. Methods: In this work, molecular dynamics (MD) and quantum mechanics (QM) simulations both were performed to compare inhibitor (i.e., SQ109) recognition, motion characteristics, and H-bond energy change of MmpL3 after S288T mutation. In addition, the WT_SQ109 complex structure was obtained by molecular docking program (Autodock 4.2); Molecular Mechanics/ Poisson Boltzmann Surface Area (MM-PBSA) and Solvated Interaction Energy (SIE) methods were used to calculate the binding free energies (∆Gbind); Geometric criteria were used to analyze the changes of hydrogen bond networks. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (Mtb) is able to transition into a dormant state, causing the latent state of tuberculosis. Dormant mycobacteria acquire resistance to all known antibacterial drugs and can survive in the human body for decades before becoming active. In the dormant forms of M. tuberculosis, the synthesis of porphyrins and its Zn-complexes significantly increased when 5-aminolevulinic acid (ALA) was added to the growth medium. Transcriptome analysis revealed an activation of 8 genes involved in the metabolism of tetrapyrroles during the Mtb transition into a dormant state, which may lead to the observed accumulation of free porphyrins. Dormant Mtb viability was reduced by more than 99.99% under illumination for 30 min (300 J/cm2) with 565 nm light that correspond for Zn–porphyrin and coproporphyrin absorptions. We did not observe any PDI effect in vitro using active bacteria grown without ALA. However, after accumulation of active cells in lung macrophages and their persistence within macrophages for several days in the presence of ALA, a significant sensitivity of active Mtb cells (ca. 99.99%) to light exposure was developed. These findings create a perspective for the treatment of latent and multidrug-resistant tuberculosis by the eradication of the pathogen in order to prevent recurrence of this disease. [ABSTRACT FROM AUTHOR]

Accurate lipid annotation is crucial for understanding the role of lipids in health and disease and identifying therapeutic targets. However, annotating the wide variety of lipid species in biological samples remains challenging in untargeted lipidomic studies. In this work, we present a lipid annotation workflow based on LC-MS and MS/MS strategies, the combination of four bioinformatic tools, and a decision tree to support the accurate annotation and semi-quantification of the lipid species present in lung tissue from control mice. The proposed workflow allowed us to generate a lipid lung-based ATLAS (LiLA), which was then employed to unveil the lipidomic signatures of the Mycobacterium tuberculosis infection at two different time points for a deeper understanding of the disease progression. This workflow, combined with manual inspection strategies of MS/MS data, can enhance the annotation process for lipidomic studies and guide the generation of sample-specific lipidome maps. LiLA serves as a freely available data resource that can be employed in future studies to address lipidomic alterations in mice lung tissue. An LC-MS and MS/MS-based workflow, integrating 4 software tools and a decision tree, enables accurate annotation and semi-quantification of lipids, providing the Lipid Lung-based ATLAS (LiLA). LiLA then revealed the lipidomic Mtb infection signature. [ABSTRACT FROM AUTHOR]

The prediction of potential microbe–drug associations is of great value for drug research and development, especially, methods, based on deep learning, have been achieved significant improvement in bio-medicine. In this manuscript, we proposed a novel computational model named NMGMDA based on the nuclear norm minimization and graph attention network to infer latent microbe–drug associations. Firstly, we created a heterogeneous microbe–drug network in NMGMDA by fusing the drug and microbe similarities with the established drug–microbe associations. After this, by using GAT and NNM to calculate the predict scores. Lastly, we created a fivefold cross validation framework to assess the new model NMGMDA's progressiveness. According to the simulation results, NMGMDA outperforms some of the most advanced methods, with a reliable AUC of 0.9946 on both MDAD and aBioflm databases. Furthermore, case studies on Ciprofloxacin, Moxifoxacin, HIV-1 and Mycobacterium tuberculosis were carried out in order to assess the effectiveness of NMGMDA even more. The experimental results demonstrated that, following the removal of known correlations from the database, 16 and 14 medications as well as 19 and 17 microbes in the top 20 predictions were validated by pertinent literature. This demonstrates the potential of our new model, NMGMDA, to reach acceptable prediction performance. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis, the etiological agent of tuberculosis, is one of the trickiest pathogens. We have only a few protective shields, like the BCG vaccine against the pathogen, which itself has poor efficacy in preventing adult tuberculosis. Even though different vaccine trials for an alternative vaccine have been conducted, those studies have not shown much promising results. In the current study, advanced computational technology was used to study the potential of a novel hypothetical mycobacterial protein, identified by subtractive hybridization, to be a vaccine candidate. NHP2 (Novel Hypothetical Protein 2), housed in the RD7 region of the clinical strains of M. tuberculosis, was studied for its physical, chemical, immunological and structural properties using different computational tools. PFAM studies and Gene ontology studies depicted NHP2 protein to be functionally active with a possible antibiotic binding domain too. Different computational tools used to assess the toxicity, allergenicity and antigenicity of the protein indicated its antigenic nature. Immune Epitope Database (IEDB) tools were used to study the T and B cell determinants of the protein. The 3D structure of the protein was designed, refined and authenticated using bioinformatics tools. The validated tertiary structure of the protein was docked against the TLR3 immune receptor to study the binding affinity and docking scores. Molecular dynamic simulation of the protein-protein complex formed were studied. NHP2 was found to activate host immune response against tubercle bacillus and could be explored as a potential vaccine in the fight against tuberculosis. [ABSTRACT FROM AUTHOR]

Para-amino salicylic acid (PAS) was first reported by Lehmann in 1946 and used for tuberculosis treatment. However, due to its adverse effects, it is now used only as a second line anti-tuberculosis drug for treatment of multidrug resistant or extensively drug resistant M. tuberculosis. The structure of PAS is similar to para-amino benzoic acid (pABA), an intermediate metabolite in the folate synthesis pathway. The study has identified mutations in genes in folate pathway and their intergenic regions for their possibilities in responsible for PAS resistance. Genomic DNA from 120 PAS-resistant and 49 PAS-sensitive M. tuberculosis isolated from tuberculosis patients in Thailand were studied by whole genome sequencing. Twelve genes in the folate synthesis pathway were investigated for variants associated with PAS resistance. Fifty-one SNVs were found in nine genes and their intergenic regions (pabC, pabB, folC, ribD, thyX, dfrA, thyA, folK, folP). Functional correlation test confirmed mutations in RibD, ThyX, and ThyA are responsible for PAS resistance. Detection of mutation in thyA, folC, intergenic regions of thyX, ribD, and double deletion of thyA dfrA are proposed for determination of PAS resistant M. tuberculosis. [ABSTRACT FROM AUTHOR]

A convergent synthesis of tetraarabinofuranoside β-d-Araf-(1→2)-α-d-Araf-(1→3)-α-d-Araf-(1→5)-α-d-Araf-1↑OR (R is 4-(3-azidopropoxy)phenyl) related to the terminal fragment of lipoarabinomannan (LAM) and arabinogalactan of mycobacteria was developed. The synthesized tetraarabinofuranoside represents a linear motif of a branched hexaarabinofuranoside, which is the main LAM epitope. 4-(3-Azidopropoxy)phenyl aglycone belongs to the class of Janus aglycones, which can serve as both a temporary protective group for the anomeric position of carbohydrate and a (pre)spacer for the synthesis of neoglycoconjugates useful for the development of new tuberculosis diagnostic assays. The key step of the synthesis is the formation of 1,2-cis-glycosidic bond. [ABSTRACT FROM AUTHOR]

Mycobacterium bovis (M. bovis) infection has been identified in black (Diceros bicornis) and white (Ceratotherium simum) rhinoceros populations in Kruger National Park, South Africa. However, it is unknown whether M. bovis infected rhinoceros, like humans and cattle, can shed mycobacteria in respiratory secretions. Limited studies have suggested that rhinoceros with subclinical M. bovis infection may present minimal risk for transmission. However, recent advances that have improved detection of Mycobacterium tuberculosis complex (MTBC) members in paucibacillary samples warranted further investigation of rhinoceros secretions. In this pilot study, nasal swab samples from 75 rhinoceros with defined infection status based on M. bovis antigen-specific interferon gamma release assay (IGRA) results were analysed by GeneXpert MTB/RIF Ultra, BACTEC MGIT and TiKa–MGIT culture. Following culture, speciation was done using targeted PCRs followed by Sanger sequencing for mycobacterial species identification, and a region of difference (RD) 4 PCR. Using these techniques, MTBC was detected in secretions from 14/64 IGRA positive rhinoceros, with viable M. bovis having been isolated in 11 cases, but not in any IGRA negative rhinoceros (n = 11). This finding suggests the possibility that MTBC/M. bovis-infected rhinoceros may be a source of infection for other susceptible animals sharing the environment. [ABSTRACT FROM AUTHOR]

Purpose: To describe katG and inhA mutations, clinical characteristics, treatment outcomes and clustering of drug-resistant tuberculosis (TB) in the State of São Paulo, southeast Brazil. Methods: Mycobacterium tuberculosis isolates from patients diagnosed with drug-resistant TB were screened for mutations in katG and inhA genes by line probe assay and Sanger sequencing, and typed by IS6110-restriction fragment-length polymorphism for clustering assessment. Clinical, epidemiological and demographic data were obtained from surveillance information systems for TB. Results: Among the 298 isolates studied, 127 (42.6%) were isoniazid-monoresistant, 36 (12.1%) polydrug-resistant, 93 (31.2%) MDR, 16 (5.4%) pre-extensively drug-resistant (pre-XDR), 9 (3%) extensively drug-resistant (XDR) and 17 (5.7%) susceptible after isoniazid retesting. The frequency of katG 315 mutations alone was higher in MDR isolates, while inhA promoter mutations alone were more common in isoniazid-monoresistant isolates. Twenty-six isolates phenotypically resistant to isoniazid had no mutations either in katG or inhA genes. The isolates with inhA mutations were found more frequently in clusters (75%) when compared to the isolates with katG 315 mutations (59.8%, p = 0.04). In our population, being 35–64 years old, presenting MDR-, pre-XDR- or XDR-TB and being a retreatment case were associated with unfavourable TB treatment outcomes. Conclusion: We found that katG and inhA mutations were not equally distributed between isoniazid-monoresistant and MDR isolates. In our population, clustering was higher for isolates with inhA mutations. Finally, unfavourable TB outcomes were associated with specific factors. [ABSTRACT FROM AUTHOR]

Purpose: We aimed at evaluating the diagnostic efficacy of a nucleotide matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF–MS) assay to detect drug resistance of Mycobacterium tuberculosis. Methods: Overall, 263 M. tuberculosis clinical isolates were selected to evaluate the performance of nucleic MALDI-TOF–MS for rifampin (RIF), isoniazid (INH), ethambutol (EMB), moxifloxacin (MXF), streptomycin (SM), and pyrazinamide (PZA) resistance detection. The results for RIF, INH, EMB, and MXF were compared with phenotypic microbroth dilution drug susceptibility testing (DST) and whole-genome sequencing (WGS), and the results for SM and PZA were compared with those obtained by WGS. Results: Using DST as the gold standard, the sensitivity, specificity, and kappa values of the MALDI-TOF–MS assay for the detection of resistance were 98.2%, 98.7%, and 0.97 for RIF; 92.8%, 99%, and 0.90 for INH; 82.4%, 98.0%, and 0.82 for EMB; and 92.6%, 99.5%, and 0.94 for MXF, respectively. Compared with WGS as the reference standard, the sensitivity, specificity, and kappa values of the MALDI-TOF–MS assay for the detection of resistance were 97.4%, 100.0%, and 0.98 for RIF; 98.7%, 92.9%, and 0.92 for INH; 96.3%, 100.0%, and 0.98 for EMB; 98.1%, 100.0%, and 0.99 for MXF; 98.0%, 100.0%, and 0.98 for SM; and 50.0%, 100.0%, and 0.65 for PZA. Conclusion: The nucleotide MALDI-TOF–MS assay yielded highly consistent results compared to DST and WGS, suggesting that it is a promising tool for the rapid detection of sensitivity to RIF, INH, EMB, and MXF. [ABSTRACT FROM AUTHOR]

Study design: Systematic review. Introduction: Total hip arthroplasty (THA) is a well-acknowledged surgical intervention to restore a painless and mobile joint in patients with osteoarticular tubercular arthritis of the hip joint. However, there is still substantial uncertainty about the ideal management, clinical and functional outcomes following THA undertaken in patients with acute Mycobacterium tuberculosis (TB) hip infections. Aim of the study: To undertake a systematic review and evaluate existing literature on patients undergoing THA for acute mycobacterium tuberculosis arthritis of the hip. Methods: A systematic review of electronic databases of PubMed, EMBASE, Scopus, Web of Science and Cochrane Library was performed on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The search focused on "arthroplasty in cases with tuberculosis of hip joint" since inception of databases until July 2023. Data on patient demographics, clinical characteristics, treatment administered, surgical interventions and outcome, as reported in the included studies, were recorded. Median (range) and mean (standard deviation) were used to summarise the data for continuous variables (as reported in the original studies); and frequency/percentage was employed for categorical variables. Available data on Harris hip scores and complications were statistically pooled using random-effects meta-analysis or fixed-effect meta-analysis, as appropriate Results: Among a total of 1695 articles, 15 papers were selected for qualitative summarisation and 12 reporting relevant data were included for proportional meta-analysis. A total of 303 patients (mean age: 34 to 52 years; mean follow-up: 2.5 to 10.5 years) were included in our systematic review. In a majority of included studies, postero-lateral approach and non-cemented prosthesis were employed. Fourteen studies described a single-staged procedure in the absence of sinus, abscess and tubercular infection syndrome (TIS). All surgeries were performed under cover of prolonged course of multi-drug anti-tubercular regimen. The mean Harris hip score (HHS) at final follow-up was 91.36 [95% confidence interval (CI): 89.56–93.16; I2:90.44%; p<0.001]. There were 30 complications amongst 174 (9.9%) patients (95% CI: 0.06–0.13; p=0.14; I2=0%). Conclusion: THA is a safe and effective surgical intervention in patients with active and advanced TB arthritis of hip. It is recommended that the surgery be performed under cover of multi-drug anti-tubercular regimen. In patients with active sinus tracts, abscesses and TIS, surgery may be accomplished in a multi-staged manner. The clinical (range of motion, deformity correction, walking ability and pain scores), radiological (evidence of radiological reactivation and implant incorporation) and function outcome (as assessed by HHS) significantly improve after THA in these patients. [ABSTRACT FROM AUTHOR]

We examined literature on Mycobacterium tuberculosis (Mtb) subsequent to its genome release, spanning years 1999–2020. We employed scientometric mapping, entity mining, visualization techniques, and PubMed and PubTator databases. Most popular keywords, most active research groups, and growth in quantity of publications were determined. By gathering annotations from the PubTator, we determined direction of research in the areas of drug hypersensitivity, drug resistance (AMR), and drug-related side effects. Additionally, we examined the patterns in research on Mtb metabolism and various forms of tuberculosis, including skin, brain, pulmonary, extrapulmonary, and latent tuberculosis. We discovered that 2011 had the highest annual growth rate of publications, at 19.94%. The USA leads the world in publications with 18,038, followed by China with 14,441, and India with 12,158 publications. Studies on isoniazid and rifampicin resistance showed an enormous increase. Non-tuberculous mycobacteria also been the subject of more research in effort to better understand Mtb physiology and as model organisms. Researchers also looked at co-infections like leprosy, hepatitis, plasmodium, HIV, and other opportunistic infections. Host perspectives like immune response, hypoxia, and reactive oxygen species, as well as comorbidities like arthritis, cancer, diabetes, and kidney disease etc. were also looked at. Symptomatic aspects like fever, coughing, and weight loss were also investigated. Vitamin D has gained popularity as a supplement during illness recovery, however, the interest of researchers declined off late. We delineated dominant researchers, journals, institutions, and leading nations globally, which is crucial for aligning ongoing and evolving landscape of TB research efforts. Recognising the dominant patterns offers important information about the areas of focus for current research, allowing biomedical scientists, clinicians, and organizations to strategically coordinate their efforts with the changing priorities in the field of tuberculosis research. [ABSTRACT FROM AUTHOR]

Background: The tracheo-innominate fistula is a rare and potentially life-threatening entity that occurs in approximately less than 1% of patients after a tracheostomy. It occurs when the anterior wall of the trachea erodes and comes into contact with the posterior wall of the innominate artery or brachiocephalic trunk due to excessive pressure from the hyperinflation of the cuff over the mucosa, creating a fistulous tract. Clinically, it manifests as massive tracheal bleeding that puts the patient's life at imminent risk. Case presentation: We present the case of a 60-year-old Latin American male patient with a history of SARS CoV-2 pneumonia approximately 4 months earlier, who required prolonged orotracheal intubation and tracheostomy due to subglottic stenosis, which required tracheal dilations. The patient was admitted to the emergency department due to hemoptysis associated with hemodynamic instability and later on presented with massive tracheal bleeding. The chest-CT angiography evidenced a tracheo-innominate fistula that required surgical management. A concomitant Mycobacterium Tuberculosis infection was also diagnosed during his hospitalization. Conclusions: There are currently many gaps in our knowledge about the tracheo-innominate fistula, mainly in terms of its incidence following the SARS-CoV-2 pandemic, as well as the role that concomitant infections and their treatments, such as tuberculosis, play in the development of these events. During the pandemic, the cases of intubated patients and patients with tracheostomies increased, giving way to new and unexpected complications, we have yet to study in depth. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) remains one of the most afflictive bacterial infections globally. In high burden TB countries, surveillance, diagnosis and treatment of drug resistant TB (RR and X/MDRTB) display a crucial public health challenge. Therefore, we need new TB vaccines; diagnostic and therapeutic strategies to briskly prevent disease promotion; reduce drug-resistant TB and protect everyone from disease. The study identified various potent membrane and cell wall M. tuberculosis glycolipoproteins that are relevant for diagnostics, drug and vaccine discovery. A M. tuberculosis Proskauer and Beck broth culture was extracted for total proteins by ammonium sulfate method. After ConA-Affinity Chromatography reputed glycoproteins were collected followed by 2DE gel electrophoresis and LC Mass spectrometry. A total of 293 glycoproteins were identified using GlycoPP and IEDB database. Probable conserved trans-membrane protein (Rv0954), LpqN (Rv0583), PPE68 (Rv3873), Phosphate-binding protein (Rv0932c), PPE61 (Rv3532) and LprA (Rv1270c), had the highest glycosylation percentage value with 13.86%, 11.84%, 11.68%, 11.1%, 10.59% and10.2%, respectively. Our study discloses several dominant glycoproteins that play roles in M. tuberculosis survival, and immunogenicity. These include glycoproteins involved in antigenicity, transport and biosynthesis of M. tuberculosis cell envelope, pathogen-host interaction and drug efflux pumps, which are considered as a feasible drug targets or TB new vaccine candidates. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) remains one of the most afflictive bacterial infections globally. In high burden TB countries, surveillance, diagnosis and treatment of drug resistant TB (RR and X/MDRTB) display a crucial public health challenge. Therefore, we need new TB vaccines; diagnostic and therapeutic strategies to briskly prevent disease promotion; reduce drug-resistant TB and protect everyone from disease. The study identified various potent membrane and cell wall M. tuberculosis glycolipoproteins that are relevant for diagnostics, drug and vaccine discovery. A M. tuberculosis Proskauer and Beck broth culture was extracted for total proteins by ammonium sulfate method. After ConA-Affinity Chromatography reputed glycoproteins were collected followed by 2DE gel electrophoresis and LC Mass spectrometry. A total of 293 glycoproteins were identified using GlycoPP and IEDB database. Probable conserved trans-membrane protein (Rv0954), LpqN (Rv0583), PPE68 (Rv3873), Phosphate-binding protein (Rv0932c), PPE61 (Rv3532) and LprA (Rv1270c), had the highest glycosylation percentage value with 13.86%, 11.84%, 11.68%, 11.1%, 10.59% and10.2%, respectively. Our study discloses several dominant glycoproteins that play roles in M. tuberculosis survival, and immunogenicity. These include glycoproteins involved in antigenicity, transport and biosynthesis of M. tuberculosis cell envelope, pathogen-host interaction and drug efflux pumps, which are considered as a feasible drug targets or TB new vaccine candidates. [ABSTRACT FROM AUTHOR]

Although homeless segment of the society could be the hotspots for tuberculosis (TB) transmission, there is little data on TB in homeless individuals in Ethiopia. The objective of this study was to investigate the molecular epidemiology and drug sensitivity of Mycobacterium tuberculosis (M. tuberculosis) isolated from homeless individuals in Addis Ababa, Ethiopia. The study was conducted on 59 M. tuberculosis isolates, which were recovered by the clinical screening of 5600 homeless individuals and bacteriological examination of 641 individuals with symptoms of pulmonary tuberculosis (PTB). Region of difference-9 (RD9) based polymerase-chain reaction (PCR), Spoligotyping and 24-loci Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat (MIRU-VNTR) typing were used for genotyping of the isolates. In addition, drug sensitivity test was performed on the isolates using BD Bactec Mycobacterial Growth Inhibition Tube (MGIT) 960. Fifty-eight of the 59 isolates were positive by spoligotyping and spoligotyping International type (SIT) 53, SIT 37, and SIT 149 were the dominant spoligotypes; each consisting of 19%, 15.5%, and10.3% of the isolates, respectively. The majority of the isolates (89.7%) were members of the Euro-American (EA) major lineage. MIRU-VNTR identified Ethiopia_3, Delhi/CAS, Ethiopia_2, TUR, X-type, Ethiopia_H37Rv-like strain, Haarlem and Latin-American Mediterranean (LAM) sub lineages. The proportion of clustering was 77.6% (45/58) in spoligotyping while it was 39.7% (23/58) in 24-loci MIRU-VNTR typing. Furthermore, the proportion of clustering was significantly lowered to 10.3% (6/58) when a combination of spoligotyping and 24-loci MIRU-VNTRplus was used. The recent transmission index (RTI) recorded by spoligotyping, 24-loci MIRU-VNTR typing, and a combination of the two genotyping methods were 58.6%, 27.6% and 5.2%, respectively. Young age and living in groups were significantly associated with strain clustering (P < 0.05). The drug sensitivity test (DST) result showed 8.9% (4/58) of the isolates were resistant to one or more first line ant-TB drugs; but multidrug resistant isolate was not detected. Clustering and RTI could suggest the transmission of TB in the homeless individuals, which could suggest a similar pattern of transmission between homeless individuals and the general population. Hence, the TB control program should consider homeless individuals during the implementation of TB control program. [ABSTRACT FROM AUTHOR]

The S genotype (first described in Sicily, hence the name) is a part of the Euro-American lineage of Mycobacterium tuberculosis. Here, we report a method for its detection based on PCR-RFLP of the Rv0557 gene. The method was optimized on strains with known whole genome sequences and further applied to screening the DNA collections from Russia, Bulgaria, China, Japan, and Vietnam. PCR-RFLP analysis confirmed the S genotype in strains with the spoligotypes SIT34 (ancestral to S) and SIT1253 (Yakutia strains). Strains from Bulgaria with abridged spoligoprofiles SIT4 and SIT125 were also assigned to the S genotype. The developed method provides for rapid detection of the M. tuberculosis S genotype and reliably resolves phylogenetic ambiguity of the disputed spoligotypes. [ABSTRACT FROM AUTHOR]