A small proportion of Mycobacterium tuberculosis populations, irrespective of their experimental/host/environmental conditions, successfully survives under stress conditions for long periods by forming natural or stress/...A small proportion of Mycobacterium tuberculosis populations, irrespective of their experimental/host/environmental conditions, successfully survives under stress conditions for long periods by forming natural or stress/antibiotics-induced, phenotypically different but genotypically identical subpopulation(s). This review highlights and describes the complex, dynamic, and diverse spectrum of phenotypic heterogeneity of these subpopulations and the characteristics of their varied response and adaptations to stress/antibiotics for survival. These surviving subpopulations of M. tuberculosis were found in the tuberculosis patients before, during, and after treatment, in the people carrying latent bacilli, in infected animal models, infected macrophages ex vivo, and in in vitro cultures maintained under extremely diverse stress/antibiotics conditions. The phenotypes included slow-growing bacilli, viable but non-culturable bacilli, differentially culturable bacilli, differentially detectable bacilli, directly solid-media culturable and non-culturable bacilli, and bacilli of diverse morphology, size, shape, and stainability. There are naturally evolved subpopulations as well, which also survive and dynamically evolve under stress/antibiotics in vitro, in the host, and environment. The formation of subpopulations with such astounding phenotypic diversity, their stress/antibiotics response, and survival indicate stress/antibiotics-response-specific triggering of diverse biochemical pathways and mechanisms, reflecting the incredibly phenomenal metabolic plasticity of tubercle bacillus. These surviving subpopulations can undergo adaptive evolution under host/antibiotic stress and contribute to poor treatment response and outcome. The molecular and structural study of the features of the diverse phenotypes and the mechanisms of their formation and survival may help to design methods for their detection in patient samples and to formulate strategies for successful and favourable treatment outcome.
BACKGROUND: Individuals infected by Mycobacterium tuberculosis (Mtb) develop tuberculosis (TB) which is a chronic infectious disease with the main transmission route being the respiratory tract. Currently, 24% of TB pati...BACKGROUND: Individuals infected by Mycobacterium tuberculosis (Mtb) develop tuberculosis (TB) which is a chronic infectious disease with the main transmission route being the respiratory tract. Currently, 24% of TB patients are still not detected in time, which shows the shortcomings of current diagnostic methodology. METHODS: We developed a novel Whole Slide Imaging (WSI) platform for TB detection, integrating a proprietary Curved Surface Focus Algorithm (CSFA) for high-speed, full-slide digitization under oil immersion, and a two-stage deep learning AI pipeline (YOLOv5 for sensitive candidate detection and ResNet-18 for specific classification) for automated acid-fast bacilli (AFB) identification. We prospectively and retrospectively evaluated its diagnostic performance against conventional smear microscopy, culture, and Xpert MTB/RIF in 1097 patients. RESULTS: The results indicate that in the 1097 study population, WSI-TB showed an overall sensitivity of 42.43% and a specificity of 100.00%. Its sensitivity was higher than that of traditional acid-fast staining smear (18.80%) and culture method (30.36%). Compared with other methodologies, the sensitivity was significantly improved. In the sputum smear microscopy group with 600 visual fields, the positive rate of WSI-TB compared with manual microscopy was 42.43% versus 18.8%; in the sputum culture group, it was 43.46% vs 30.36%; in the Xpert group, it was 62.95% versus 44.26% CONCLUSIONS: The WSI-TB technology significantly improves the sensitivity of tuberculosis sputum smear testing while maintaining 100% specificity, providing a new approach to enhance TB detection rates.
UNLABELLED: Tubercular uveitis is an extrapulmonary form of TB characterized by ocular inflammation that clinically mimics other inflammatory eye diseases, including non-tubercular uveitis. Currently, no gold standard te...UNLABELLED: Tubercular uveitis is an extrapulmonary form of TB characterized by ocular inflammation that clinically mimics other inflammatory eye diseases, including non-tubercular uveitis. Currently, no gold standard test is available for the diagnosis of tubercular uveitis, and it is primarily based on clinical presentation. OBJECTIVE: To characterise the transcriptional landscape of vitreous fluid of tubercular uveitis and non-tubercular uveitis patients for biomarker identification and gaining insights into disease pathology. DESIGN: Vitreous fluid samples from uveitis patients were processed for isolation of cells and RNA sequencing to compare the transcriptomic profiles of TB and non-TB uveitis entities. Differentially expressed genes were identified using the criteria of false discovery rate (FDR) < 0.25, fold-change (FC) ≥ 2 or ≤ -2, and p-value <0.05 and the top dysregulated genes were selected for biomarker validation. Additionally, gene set enrichment analysis (GSEA) was performed to explore underlying disease mechanisms. RESULTS: RNA sequencing revealed distinct vitreous fluid gene expression patterns in TB uveitis and non-TB uveitis. Notably, MIR581 and MIR4762, encoding miR-581 and miR-4762, respectively, were among the most upregulated genes. Validation via qRT-PCR confirmed the upregulation of mature miR-4762-5p, supporting its potential as a diagnostic biomarker. Pathway enrichment analysis based on transcriptional profiling revealed significant downregulation of immune-related pathways in tubercular uveitis. CONCLUSION: Unique transcripts are associated with TB uveitis, identifying miR-4762 as a potential diagnostic biomarker. Further, downregulation of transcripts associated with immune-related pathways suggests that Mycobacterium tuberculosis (Mtb) may utilize complex molecular strategies to evade host immune responses and establish infection in the ocular environment.
Linezolid is an essential drug for treating multidrug-resistant and extensively drug-resistant tuberculosis, yet its long-term use is frequently limited by peripheral neuropathy, which in severe cases may be irreversible...Linezolid is an essential drug for treating multidrug-resistant and extensively drug-resistant tuberculosis, yet its long-term use is frequently limited by peripheral neuropathy, which in severe cases may be irreversible. To investigate biological factors underlying susceptibility, we analyzed transcriptomic profiles of peripheral blood mononuclear cells from 51 MDR/XDR-TB patients before and after six months of linezolid treatment, stratifying individuals into asymptomatic, mild, and severe neuropathy groups. Patients who later developed severe neuropathy already exhibited distinct transcriptional signatures at baseline, including evidence of immune dysregulation and impaired antioxidant defense, with downregulation of genes involved in immune receptor activity and the pentose phosphate pathway. Following treatment, these patients showed limited transcriptional responses compared with other groups, most notably a marked downregulation of IL10, a central anti-inflammatory cytokine, suggesting maladaptive systemic regulation. Using a machine learning-based feature selection approach, we derived an 11-gene predictive model that achieved strong performance with a cross-validated AUC of 0.93. Model interpretation using SHAP values highlighted HP and HSPA1B as the most influential predictors. Overall, our findings suggest that severe linezolid-induced peripheral neuropathy is predisposed by baseline immune and metabolic alterations and that patients with this complication fail to mount appropriate systemic responses during treatment.
Anemia is a common complication of tuberculosis (TB), closely associated with impaired host immune function and poor prognosis; however, its impact on the dynamic alterations of erythroid progenitor cells (EPCs) remains...Anemia is a common complication of tuberculosis (TB), closely associated with impaired host immune function and poor prognosis; however, its impact on the dynamic alterations of erythroid progenitor cells (EPCs) remains largely unexplored. In this study, we integrated and analyzed single-cell transcriptomic data from peripheral blood mononuclear cells (PBMC) of healthy controls, latent tuberculosis (LTB), TB, and disseminated tuberculosis (DTB) cohorts, as well as 18F-FDG-labeled TB lung tissues and adjacent uninvolved regions. At single-cell resolution, we identified ten EPCs clusters within PBMC, among which clusters 2-6 represented DTB-specific subpopulations (EPC_DTB). These EPC_DTB exhibited metabolic reprogramming (including glycolysis, arginine, and lipid metabolism) accompanied by upregulation of IFITM3/JUN and ribosomal genes, consistent with enhanced differentiation. EPC_DTB and EPC_TB interacted with macrophages, dendritic cells, and monocytes through the ANNEXIN and GALECTIN signaling pathways, while these immune cells reciprocally regulated EPCs subsets via the MIF signaling axis. In lung tissues, EPCs (12 clusters) displayed disease-specific characteristics, with clusters 0, 3, and 6 uniquely present in TB lesions. The EPC_High subpopulation within inflammatory foci showed significant upregulation of GZMA, IL32, CCL5, and CHI3L1, whereas EPC_Low communicated with immune cells, epithelial cells, and stromal cells through the MDK-NCL signaling pathway. Moreover, we established an LTB predictive model based on EPC_LTB signature genes in PBMC (GRIK3, S100B, ZDHHC19, and LRRN3). Our study uncovers the heterogeneity of EPCs across different TB stages and tissue compartments, delineates their bidirectional crosstalk with immune cells, and establishes a link among anemia, immune regulation, and EPCs biology, thereby proposing potential diagnostic biomarkers and therapeutic targets.
Clofazimine, moxifloxacin and terizidone/cycloserine play an important role in the treatment of drug-resistant tuberculosis (DR-TB). Personalized therapy guided by model-informed precision dosing (MIPD) can be a powerful...Clofazimine, moxifloxacin and terizidone/cycloserine play an important role in the treatment of drug-resistant tuberculosis (DR-TB). Personalized therapy guided by model-informed precision dosing (MIPD) can be a powerful tool to improve treatment outcomes, minimize adverse effects and combat the emergence of resistance. To set up an MIPD workflow, a population pharmacokinetic model (popPK model) is required. In this study, an external evaluation of popPK models of the three aforementioned drugs was carried out, using pharmacokinetic data from a cohort of patients with DR-TB, in order to identify the model with the best predictive performance. The best performing models (Abdelwahab et al. for clofazimine, Chirehwa et al. for moxifloxacin and Mulubwa and Mugabo for terizidone/cycloserine) were selected to calculate the area under the concentration-time curve (AUC, total exposure). An interoccasion variability (IOV, variability across dosing occasions) of AUC was quantified (13.4%CV (clofazimine), 16.1%CV (moxifloxacin), 14.5%CV (cycloserine)) indicating that using samples from one dosing occasion for AUC calculations may be sufficient to guide potential dose adjustment. Various single sampling schemes to estimate AUC were evaluated, but a unified timepoint for all drugs could not be determined. Known pharmacodynamic targets (AUC/MIC, or T>MIC) were attained in almost all patients and dosing occasions.
BACKGROUND/OBJECTIVES: Tuberculous pleuritis (TP), a common manifestation of Mycobacterium tuberculosis infection, poses challenges in differentiating microbiologically positive (PEMP-MT) from negative (PEMN-MT) pleural...BACKGROUND/OBJECTIVES: Tuberculous pleuritis (TP), a common manifestation of Mycobacterium tuberculosis infection, poses challenges in differentiating microbiologically positive (PEMP-MT) from negative (PEMN-MT) pleural effusions due to the limited sensitivity of traditional diagnostic methods. METHODS: Proteomics analysis using iTRAQ, non-targeted metabolomics, parallel reaction monitoring (PRM), and machine learning were employed to diagnose PEMN-MT or PEMP-MT. A validation cohort of 63 PEMN-MT and 28 PEMP-MT patients underwent ELISA experiments. Receiver operating characteristic (ROC) curves evaluated the predictive value of LDH and LV218 individually and in combination. RESULTS: Differentially expressed proteins (DEPs) and metabolites (DEMs) were identified using bioinformatics tools and pathway enrichment analyses. A machine learning model utilizing six biomarkers (LV218, F13A, RET4, LV321, TBA1C, and LDH) demonstrated excellent diagnostic performance with an AUROC of 0.987 and an AUPR of 0.974, distinguishing PEMP-MT from PEMN-MT. ROC curve analysis showed that both LDH and LV218, alone and in combination, provided strong predictive value for distinguishing the two groups. CONCLUSION: LDH and LV218 are promising biomarkers for differentiating microbiologically positive and negative pleural effusions in tuberculous pleuritis. These biomarkers, particularly when combined, could improve diagnostic accuracy and clinical management.
Tuberculosis (TB) remains the world's deadliest infectious disease, with treatment increasingly complicated by the emergence of multidrug-resistant strains (MDR-TB). This study conducted structure-based drug screening ta...Tuberculosis (TB) remains the world's deadliest infectious disease, with treatment increasingly complicated by the emergence of multidrug-resistant strains (MDR-TB). This study conducted structure-based drug screening targeting Mycobacterium tuberculosis protein kinase B (MtPknB), a serine/threonine kinase essential for M. tuberculosis survival and proliferation, to identify novel anti-TB drug candidates. From the ChemBridge library, a hierarchical screening pipeline integrating docking and molecular dynamics simulations identified candidate compounds. Among these, a quinoline-pyridine hybrid chemical demonstrated antibacterial activity against Mycobacterium smegmatis (IC = 31.8 μM) without toxicity to Escherichia coli or mammalian cells. MM-PBSA and ab initio fragment molecular orbital (FMO) analyses revealed LEU17, VAL25, and MET155 as key stabilizing residues in the MtPknB active site. ProLIF interaction fingerprinting confirmed stable hydrophobic and van der Waals interactions formed by the quinoline-pyridine hybrid chemical. SwissADME and ProTox-3.0 predictions indicated favorable drug-like properties for the quinoline-pyridine hybrid chemical, despite potential toxicity risks. Structure-activity relationship analysis of the quinoline-pyridine hybrid chemical analogs demonstrated that subtle variations in hydrophobic interactions and substituent positioning significantly influence antibacterial potency. These findings position these chemicals as promising lead compounds for MtPknB-targeted anti-TB drug development.
Direct sputum whole genome sequencing (dsWGS) can revolutionize Mycobacterium tuberculosis (Mtb) diagnosis by enabling rapid detection clinically relevant resistance mutations and strain diversity without the biohazard o...Direct sputum whole genome sequencing (dsWGS) can revolutionize Mycobacterium tuberculosis (Mtb) diagnosis by enabling rapid detection clinically relevant resistance mutations and strain diversity without the biohazard of culture. We searched PubMed, Web of Science, and Google Scholar, identifying 8 studies meeting inclusion criteria for testing protocols for dsWGS. Utilising meta-regression, we identified factors positively associated with dsWGS success, including higher Mtb bacillary load, mechanical disruption, enzymatic/chemical lysis and sequencing volume. Decontamination with sodium hydroxide (NaOH) was negatively associated with dsWGS success (OR = 0.00032, 95 % CI: 1.33 × 10ˆ-6-0.077; p = 0.004), likely due to its harsh effects on Mtb cells. Mechanical lysis (OR = 6120, 95 % CI: 7.23-5.18 × 10ˆ6; p = 0.011) and enzymatic/chemical lysis (OR = 131, 95 % CI: 1.68-1.03 × 10ˆ4; p = 0.028) were positively associated with sequencing success, as was heat inactivation (OR = 4.66, 95 % CI: 1.24-17.5; p = 0.023). Total sequencing volume was also strongly associated with dsWGS success (OR = 10.35, 95 % CI: 4.43-24.2; p = 6.53 × 10ˆ-8). In addition to these effects, we also observed high variability in pre-processing approaches, highlighting the need for standardized practices and identified pre-processing steps including decontamination and DNA extraction as priorities for further optimization. Considering the strong association between Mtb load and successful dsWGS, protocols for optimal sputum sample collection, handling, and storage could also further enhance the success rate of dsWGS.
Tuberculosis, primarily caused by Mycobacterium tuberculosis (Mtb), remains a leading global health issue. We investigate the interplay between Mtb infection and various programmed cell death (PCD) in active pulmonary tu...Tuberculosis, primarily caused by Mycobacterium tuberculosis (Mtb), remains a leading global health issue. We investigate the interplay between Mtb infection and various programmed cell death (PCD) in active pulmonary tuberculosis (ATB). Using GSE19491 and GSE107994 datasets, we identified 1306 overlapping differentially expressed genes (DEGs) in peripheral blood from ATB patients and healthy controls. Gene set variation analysis revealed that, except for cuproptosis, the PCD pathways: necroptosis, apoptosis, pyroptosis, and ferroptosis were significantly elevated in ATB patients. Weighted Gene Co-expression Network Analysis further identified 392 PCD-associated hub genes. KEGG and GO analyses highlighted key functional enrichments in immune responses, cellular stress, and PCD pathways. Moreover, we found a positive correlation between PCD types and specific immune cell populations. Additionally, by integrating DEGs of peripheral blood samples and lung granuloma tissues with PCD-associated hub genes, we identified 30 PCD-related genes in ATB patients. RT-qPCR results demonstrated significantly elevated GCLC, RBCK1, ZEB1, and EIF2AK2 levels, alongside lowered PLA2G4C and CAMK2G levels in patients' peripheral blood. These findings underscore the critical role of PCD pathways in modulating the immune response during Mtb infection. Future mechanistic studies are required to definitively establish the causal roles of these pathways in regulating cell death and bacterial control.
Bovine tuberculosis (bTB) is an important cattle disease with major public health and economic impacts. Mycobacterium bovis, its causative agent, is thought to persist in non-replicative forms within the host, similar to...Bovine tuberculosis (bTB) is an important cattle disease with major public health and economic impacts. Mycobacterium bovis, its causative agent, is thought to persist in non-replicative forms within the host, similar to Mycobacterium tuberculosis, leading to chronic or latent infection. In this study, we used the Wayne model-an in vitro system that gradually depletes oxygen-to mimic the hypoxic conditions M. bovis may encounter during latency. Growth analysis showed that part of the bacterial culture remained viable but non-replicative under hypoxia, while another fraction likely lysed, as indicated by declining optical density during late hypoxia and reduced colony-forming units. Secreted proteome analysis identified 36 proteins detected exclusively in culture supernatants, with Cut3, SapM, and Cdh accumulating more under hypoxia (p < 0.05, FDR = 0.25). In the cellular proteome, 288 proteins showed differential accumulation (p < 0.05, FDR = 0.25), with 172 more abundant under hypoxia. Under oxygen depletion, M. bovis increased proteins related to nitrogen and lipid metabolism, purine biosynthesis, carbon metabolism, anaplerotic pathways, and several DosR regulon proteins. Aerated cultures showed higher levels of proteins involved in transcription, translation, DNA replication, and virulence. Protein secretion decreased under hypoxia. Overall, M. bovis remodels its proteome to persist in a viable, non-replicative state.
INTRODUCTION: One of the biggest challenges associated with tuberculosis is the emerging drug resistance which emphasizes the need of alternative therapeutic approaches. In recent years, multitargeting approach for TB th...INTRODUCTION: One of the biggest challenges associated with tuberculosis is the emerging drug resistance which emphasizes the need of alternative therapeutic approaches. In recent years, multitargeting approach for TB therapy has garnered considerable attention. The Antigen85 complex of proteins represent key enzymes which are involved in Mtb cell wall biosynthesis. This study aims at investigating the antimycobacterial effects of Centella asiatica and its two compounds - asiatic acid and quercetin against these three proteins and validate them as multi targets for drug targeting. METHODS: Three-fold validation approach included in-silico studies, in-vitro inhibitory studies against MtbH37Ra strain and inhibition studies against recombinant enzymes. RESULTS: Displaying good bioavailability, asiatic acid and quercetin exhibited favorable binding affinities. Disruption of the integrity of the cell membrane of MtbH37Ra was observed through electron microscopy and flow cytometry. Both compounds and extract showed inhibitory activity against the enzymes and microorganism and were found to act synergistically in conjunction with isoniazid. Asiatic acid demonstrated bactericidal activity. The reduced reaction rate of the recombinant enzymes implied their function and activity were hampered, which was confirmed through a fluorometric assay. CONCLUSION: These results highlight the potential of asiatic acid and quercetin as multitarget inhibitors of the Mtb Antigen85 complex.
Tuberculosis (TB), caused by Mycobacterium tuberculosis, exhibits pronounced sex differences in incidence and disease progression, with adult males disproportionately affected. Increasing evidence indicates that sex ster...Tuberculosis (TB), caused by Mycobacterium tuberculosis, exhibits pronounced sex differences in incidence and disease progression, with adult males disproportionately affected. Increasing evidence indicates that sex steroid hormones estrogen, progesterone, and testosterone modulate immune responses critical for MTB control. This narrative review synthesizes findings from both human and animal studies using PubMed, ScienceDirect, and Google Scholar. Effective host defense against MTB relies on pro-inflammatory cytokines, including interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6, whereas anti-inflammatory cytokines such as IL-4, IL-5, and IL-10 are associated with reduced bacterial control and disease progression. Sex steroid hormones regulate both the magnitude and balance of these immune responses in a dose, stag-, and context-dependent manner. Estrogen enhances Th1-mediated immunity at physiological concentrations but may favor Th2-biased responses at supraphysiologic levels, such as during pregnancy. Progesterone contributes to immune homeostasis at basal concentrations but suppresses dendritic cell function and Th1 immunity at elevated levels. Testosterone consistently attenuates Th1 immunity and enhances anti-inflammatory pathways. Human epidemiologic and clinical studies support these trends, showing adult males are more susceptible to active TB, while women experience increased risk during pregnancy. However, circulating hormone data in TB patients are inconsistent, highlighting the need for longitudinal, hormone-aware studies. Overall, sex hormone mediated immune modulation influences TB susceptibility and pathogenesis, and future research should adopt sex and hormone-dose-aware designs to optimize host-directed therapies.
In the present study, a series of new pyridine-embedded 1,3,4-oxadiazole derivatives (OXn series) bearing terminal long-chain alkoxy groups like decyloxy, dodecyloxy, tetra decyloxy, and hexadecyloxy groups have been sys...In the present study, a series of new pyridine-embedded 1,3,4-oxadiazole derivatives (OXn series) bearing terminal long-chain alkoxy groups like decyloxy, dodecyloxy, tetra decyloxy, and hexadecyloxy groups have been systematically synthesized. Further, the presence of these long-chain alkoxy groups in the OXn series would help to improve the overall molecular lipophilicity and ability to penetrate the lipid-rich mycobacterial cell membrane. Molecular docking has been performed against the mycobacterial InhA enzyme to gain an insight into the possible interactions with the protein, which could pave the way for our endeavor to identify potent antitubercular candidates. Also, these compounds were evaluated for their in vitro antitubercular activities. Among the screened compounds of OXn series, the compound (OX-14) have exhibited potent antitubercular activity against Mycobacterium tuberculosis H37Rv strain with MIC value 32.0 μg/mL and IC value of 10.4 μg/mL. We believe that further optimization of this molecule may lead to potent antitubercular agents.
The World Health Organization (WHO) has highlighted the need for new diagnostic tests for pulmonary tuberculosis (TB) that use easily obtainable samples, such as blood, to provide rapid and affordable results suitable fo...The World Health Organization (WHO) has highlighted the need for new diagnostic tests for pulmonary tuberculosis (TB) that use easily obtainable samples, such as blood, to provide rapid and affordable results suitable for primary healthcare settings. To address this, we evaluated the diagnostic potential of 34 markers quantified by Luminex in supernatants of whole-blood cultures, either stimulated or not with an apolar lipid extract from Mycobacterium tuberculosis (Mtb). The study included 20 patients with pulmonary TB and 20 symptomatic respiratory (SR) non-TB individuals. In unstimulated cultures, eight biomarkers (IL-18, IL-1RA, IL-1β, IL-8, IP-10, MIP-1β, SDF-1α, and TNF-α) differentiated TB patients from SR - non-TB patients, with areas under the ROC curve (AUC) ranging from 0.71 to 0.82. Combinatorial analyses with four-marker panels, namely, IL-18 + IL-1β, IL-1RA + IL-18, IL-18 + IL-1β + IL-1RA and IL-18 + IL-1β + IL-1RA + MIP-1β, revealed AUCs of 0.84-0.90, specificities above 90 % and sensitivities between 70 % and 75 %. The addition of the lipid extract to the whole-blood culture did not improve the discriminatory power of the panels. Validation of the IL-1RA + IL-18 combination by ELISA in an independent group (21 TB patients and 33 SR patients) yielded an AUC of 0.76, a sensitivity of 62 %, a specificity of 88 %, and an accuracy of 78 %. The collective elevation of these cytokines suggests an interplay between pro- and anti-inflammatory pathways in the host response. Although the selected biomarker panels showed moderate diagnostic performance in the ELISA test, other combinations may be useful in helping to predict TB progression or monitor treatment outcomes.
Tuberculosis (TB), caused by members of the Mycobacterium tuberculosis complex (MTBC), remains a significant global health concern. While zoonotic transmission of M. bovis from cattle to humans is well documented, revers...Tuberculosis (TB), caused by members of the Mycobacterium tuberculosis complex (MTBC), remains a significant global health concern. While zoonotic transmission of M. bovis from cattle to humans is well documented, reverse zoonotic transmission of M. tuberculosis from humans to cattle has received far less attention. This review provides the first comprehensive synthesis of M. tuberculosis infections in cattle, drawing on evidence from farms, households, and slaughterhouses where human-animal contact is most intense. Available data indicate that such spillover events are uncommon compared with M. bovis infection, and that infectious humans-via aerosols or sputum-contaminated feed or environments-represent the primary, and likely exclusive source of infection for cattle, as no sustained cattle-to-cattle transmission has been reported. Experimental M. tuberculosis infection in cattle consistently demonstrates an attenuated phenotype, with mild pathology and low bacterial loads. However, the identification of multidrug-resistant and pre-extensively drug-resistant M. tuberculosis strains in cattle raises a potential future concern regarding cross-species transmission, despite the fact that transmission back to humans has not been observed yet. Enhancing routine molecular diagnostics is vital for precise MTBC differentiation and a better grasp of cross-species transmission dynamics. A unified One Health strategy, which combines human, animal, and environmental monitoring, is essential to track and address this emerging threat.