Search Results (1,130)

Carotenoid pigments are widely distributed in nature and play a crucial role in protecting organisms from photodynamic damage. However, the characterization of carotenoid production in clinically relevant mycobacteria has been limited due to the low sensitivity of conventional detection methods. We present a descriptive analysis of carotenoid production in seven mycobacterial isolates from the scotochromogenic, photochromogenic, and non-chromogenic groups. To achieve this, we used a combination of High-performance liquid chromatography with diode-array detection (HPLC-DAD) and Ultra-high performance liquid chromatography–mass spectrometry (UHPLC-MS) to detect carotenoids pigments. Mycobacterium tuberculosis (MTB) and Mycobacterium bovis (MB) (non-chromogenic mycobacteria) produced β-carotene when cultured in the absence of light, at levels comparable to those of photochromogenic mycobacteria such as M. marinum (MM) and M. kansasii (MK). The highest levels of carotenoids were found in scotochromogenic species M. avium (MAV) and M. gordonae (MGOR). Conversely, M. abscessus (MABS), a non-chromogenic species in which no β-carotene was detected, served as a negative control for matrix effects. As expected, the use of highly sensitive analytical techniques such as HPLC-DAD and UHPLC-MS significantly enhanced the detection of β-carotene compared to visual pigment assessment. These methods allowed the detection of basal β-carotene levels even in mycobacteria classified as non-chromogenic. The proposed analytical approach provides a robust research tool to understand the effects of different stimulus that may alter the cell physiology in terms of pigment production. Full article

Tuberculosis (TB) remains a significant worldwide health challenge due to the limitations of conventional treatments and the rising incidence of drug-resistant Mycobacterium tuberculosis strains. This review consolidates the advancements in nanotechnology-based therapeutics, inhalable formulations, CRISPR–Cas tools, host-directed therapies (HDTs), and nanoparticle-based vaccine development aimed at enhancing TB management. Novel nanocarriers such as liposomes, solid-lipid nanoparticles (SLNs), dendrimers, and polymeric nanoparticles (NPs) offer enhanced bioavailability of drugs, sustained release, as well as targeted delivery to infected macrophages, thereby reducing systemic toxicity and dosing frequency. Inhalable nanomedicines provide localized delivery to the pulmonary site, enhancing the concentration of the drug at the primary site of infection. CRISPR–Cas technology is emerging as a transformative approach to disabling drug-resistant genes and enhancing diagnostic precision. HDTs, including agents like vitamin D and metformin, show potential in modulating host immune responses and enhancing pathogen clearance. Nanoparticle-based vaccines, including mRNA and antigen-conjugated platforms, aim to overcome the limitations of the BCG vaccine by enhancing antigen presentation and eliciting stronger, longer-lasting immunity. Collectively, these modalities mark a shift toward more personalized, effective, and less toxic TB therapies. However, challenges such as regulatory approval, safety, scalability, and accessibility remain. This review highlights the integrated potential of nanomedicine, gene editing, and immunomodulation to transform TB care and combat drug resistance, paving the way for more robust and durable treatment strategies. Full article

Rv1899c, a previously identified HDAC1–ZBTB25-interacting protein of Mycobacterium tuberculosis, plays a crucial role in bacterial adaptation and immune modulation. Recombinant M. smegmatis-expressing Rv1899c (MS_ Rv1899c) showed enhanced survival under acidic and oxidative stress compared to vector controls, along with improved early intracellular growth in THP1-derived macrophages. This was accompanied by reduced reactive oxygen species (ROS), diminished cytokines associated with inflammation and downregulation of autophagy proteins ATG5, Beclin, and LC3, which ultimately skewed the immune response, suppressing the pro-inflammatory M1 macrophage population. Targeting Rv1899c with 3-aminobenzamide (3-AB) impaired intracellular bacterial survival and restored IL-12B expression, while its combination with the HDAC inhibitor C1994 significantly enhanced bacterial clearance. Structural modelling confirmed the high stereochemical quality of the Rv1899c macrodomain, and computational studies identified 3-AB as the strongest ligand (−5.75 kcal/mol), stabilized through hydrogen bonding and hydrophobic interactions with key residues. Molecular dynamics simulations conducted for 200 ns demonstrated stable protein–ligand interactions with consistent parameters, while MM/GBSA analysis indicated favourable binding energy (ΔG_bind = −6.6 kcal/mol), largely influenced by van der Waals and electrostatic forces. Together, these findings highlight Rv1899c as a mediator of stress resistance and immune evasion and propose it as a potential therapeutic target against M. tuberculosis. Full article

Drug-resistant Mycobacterium tuberculosis (Mtb) remains a major global health challenge, prompting the need for new therapeutics targeting essential bacterial proteins. The caseinolytic protein C1 (ClpC1) is a promising drug target, and accurate measurement of its ATPase activity is critical for understanding drug mechanisms. We optimized a sensitive luminescence-based ATPase assay and evaluated ClpC1 constructs with various tag positions and truncations. N-terminal tagging significantly impaired enzymatic activity, whereas C-terminal tagging had no effect; truncated domains showed reduced activity compared to native full-length (FL) ClpC1. Using the native FL-ClpC1, we assessed ecumicin (ECU) and five analogs via ATPase activity and surface plasmon resonance (SPR), using rufomycin (RUF) and cyclomarin A (CYMA) as controls. RUF and CYMA bound tightly (K_D = 0.006–0.023 µM) and inhibited Mtb growth (MIC₉₀ = 0.02–0.094 µM) but modestly stimulated ATPase activity (≤2-fold). In contrast, ECU and its analogs strongly enhanced ATPase activity (4–9-fold) despite slightly weaker binding (K_D = 0.042–0.80 µM) and growth inhibition (MIC₉₀ = 0.19 µM). The partial correlation among AC₅₀, K_D, and MIC values highlights the complementary value of enzymatic, biophysical, and cellular assays. Our assay platform enables mechanistic characterization of ClpC1-targeting compounds and supports rational antitubercular drug development. Full article

Tuberculosis remains a serious global public health challenge and requires the development of rapid, sensitive, and specific diagnostic tools for effective treatment and disease control. Bioimaging reporters are promising diagnostic tools that exploit the unique biochemical properties of Mycobacterium tuberculosis for real-time detection of viable cells from clinical samples. Moreover, these methods offer significant advantages over the conventional methods currently used in practice, including reduced assay time, increased specificity, and the ability to discriminate viable cells from dead cells. In this review, we highlight reporters of a different nature that the enable direct detection of Mycobacterium tuberculosis, eliminating complex sample preparation. Such reporters could serve as powerful tools in fluorescence microscopy, provide alternative strategies for automated culture-based diagnostic systems, and offer new approaches for developing point-of-care methods and diagnostic devices suitable for clinical practice. Full article

Mismatch repair (MMR) system alterations can trigger transient hypermutation, promoting adaptive mutations under stress, such as antibiotic exposure. While most organisms use MutS and MutL protein families for MMR, many archaea and actinobacteria, including the major human pathogen Mycobacterium tuberculosis, lack these components and instead rely on NucS, a structurally distinct enzyme driving a non-canonical MMR pathway. Given the role of MMR in mutation control, understanding how nucS expression is regulated could be essential for uncovering the molecular basis of antibiotic resistance development in mycobacteria. In this study, we characterized the nucS promoter and transcription start site in Mycobacterium smegmatis. We found that nucS expression declines during the stationary phase in both M. smegmatis and M. tuberculosis, paralleling replication activity and canonical MMR downregulation. Our data suggest that the alternative sigma factor σ^B may negatively regulate nucS expression during this phase. Additionally, we identified candidate compounds that may modulate nucS expression, underscoring its responsiveness to environmental cues. These findings enhance our understanding of mycobacterial stress responses and lay the groundwork for exploring antibiotic resistance mechanisms. Strikingly, our work reveals a case of double convergent evolution: both canonical (MutS/MutL) and non-canonical (NucS) pathways have independently evolved not only the same DNA repair function, but also similar regulatory frameworks for genome integrity preservation under stress conditions. Full article

For drug-susceptible TB, the WHO-endorsed first-line regimen (isoniazid, rifampicin, ethambutol, pyrazinamide) remains the global reference. Therapy must always be tailored to drug susceptibility, especially in MDR- and XDR-TB. HIV-associated mycobacterial infections—including Mycobacterium tuberculosis (TB), disseminated Mycobacterium avium complex (MAC), and Mycobacterium leprae (M. leprae)—remain leading causes of morbidity and mortality in people living with HIV (PLWH). TB continues to account for the highest burden of AIDS-related deaths worldwide, while MAC and leprosy complicate care in advanced immunosuppression. This review synthesizes current evidence on epidemiology, clinical features, and management challenges of HIV–mycobacterial co-infections. We discuss drug-susceptible and drug-resistant TB therapies, drug–drug interactions with antiretroviral therapy (ART), and the clinical impact of immune reconstitution inflammatory syndrome (IRIS). Beyond established regimens, we highlight host-directed strategies such as metformin, glutathione augmentation, mTOR modulation, and vitamin D; immunotherapies including interferon-γ, GM-CSF, and IL-7; and therapeutic vaccines (M72/AS01E, MTBVAC, VPM1002) as promising adjuncts. Distinct from guideline-focused overviews, this review emphasizes non-tuberculous mycobacterial disease (NTM, including MAC) and leprosy in PLWH and synthesizes host-directed and adjunctive strategies with their translational prospects, including ART compatibility and IRIS. By integrating TB, NTM, and leprosy across the HIV care continuum, we highlight opportunities not treated in detail elsewhere—particularly HDT-enabled approaches and implementation considerations in PLWH. Full article

Background: Tuberculosis (TB) is a leading cause of death among people living with HIV (PLHIV). While body mass index (BMI) affects TB risk, its association with latent tuberculosis infection (LTBI) in PLHIV is unclear. High-transmission settings, such as prisons, may further increase LTBI risk, yet this relationship has not been studied across both prison and community populations of PLHIV. Methods: We conducted a dual cross-sectional study of PLHIV in Jiangsu Province, China, recruiting participants from a prison hospital in 2021 and community healthcare facilities from July to November 2023. BMI was calculated from measured height and weight. LTBI was identified by a positive ESAT6-CFP10 (EC) skin test or the QuantiFERON-TB Gold In-Tube (QFT-GIT) assay. Logistic regression and generalized additive models (GAMs) assessed the association between BMI and LTBI, adjusting for demographic, clinical, and behavioral confounders. Results: A total of 1799 PLHIV were included in the analysis, of whom 343 (19.07%) were recruited from prison settings and 1456 (80.93%) from community-based screening. The overall prevalence of LTBI was 13.79% (n = 248). Obesity (BMI ≥ 28 kg/m²) was linked to a significantly lower risk of LTBI (adjusted OR = 0.47, 95% CI: 0.23–0.95, p = 0.036), particularly when identified by EC testing (adjusted OR = 0.13, 95% CI: 0.03–0.54, p = 0.005). The BMI–LTBI association followed a nonlinear “U-shaped” pattern, with the lowest prevalence in individuals who were obese. Among those with CD4+ T cell counts < 500 cells/μL, the inverse association between obesity and LTBI was even more marked (adjusted OR = 0.20, 95% CI: 0.05–0.83, p = 0.027). Conclusion: In summary, obesity is significantly associated with a lower risk of LTBI among PLHIV, with an approximate 54% risk reduction. This inverse relationship was most pronounced when using the EC skin test. Full article

Bovine tuberculosis (bTB) is mainly caused by Mycobacterium bovis, which affects cattle, leading to significant economic losses. In Chile, the vaccination with the M. bovis Bacillus Calmette-Guérin (BCG) strain has been implemented in dairy herds with high prevalence of bTB. This study evaluated non-specific protection associated with BCG on the detection of pathogen-associated genes (nsp5, stx1, stx2, invA, IS1081) and mortality related to diarrhea and pneumonia in calves. A total of 186 calves from a commercial dairy farm were enrolled and grouped as vaccinated (n = 96) and non-vaccinated (n = 90). The BCG Russia strain (2–5 × 10⁵ UFC) was inoculated subcutaneously within the first 30 days after birth. Animals were monitored through fecal sampling at 3 and 6 months of age for molecular detection of gene sequences. A logistic regression analysis showed differences in detection rates of the stx1 sequence at 3 months, with a higher risk for the non-vaccinated individuals (OR 2.91, CI 1.42–5.94, p = 0.03) and for those born in the cold season (OR 9.55, CI 2.02–45.11, p = 0.004). A Kaplan–Meier survival analysis showed a significant difference in deaths in vaccinated calves compared with non-vaccinated animals (p = 0.018), suggesting that BCG confers non-specific protection during the first 3 months after birth, in field conditions. Full article

Mycobacterium tuberculosis (M. tuberculosis) has developed some strategies to evade host immune responses through ubiquitination, thereby facilitating persistent mycobacterial infection. The Rv3717 protein has been identified as a peptidoglycan (PG) amidase that contributes to mycobacterial survival, but its exact mechanism is still unclear. The findings of this study indicate that Rv3717 inhibits mycobacterial clearance within pulmonary epithelial cells. To elucidate the molecular mechanisms by which Rv3717 facilitates persistent infection, we identified intracellular candidates interacting with Rv3717 using co-immunoprecipitation (Co-IP) combined with liquid chromatography–mass spectrometry (LC-MS/MS). The unique proteins are categorized into three functional networks: mRNA splicing, the immune system process, and the translation process through Protein–Protein Interaction (PPI) analysis. The candidate interacting proteins of Rv3717 are involved in interleukin-2 enhancer-binding factor 2 (ILF2) and TAF15, as well as the polyubiquitin chain (UBC) and E3 ubiquitin ligase TRIM21. Our results suggest that intracellular Rv3717 is likely to influence biological processes through the potential interacting proteins. Our findings confirmed that Rv3717 interacted with interleukin enhancer-binding factor 2 (ILF2) through Co-IP and immunofluorescence assays. Furthermore, Rv3717 was verified to bind with ubiquitin and be degraded through the proteasome system. More importantly, the ubiquitination of Rv3717 accelerated the proteasomal degradation of ILF2 and downregulated the expression of IL-2. This study is the first to propose that the ubiquitination of the mycobacterial membrane vesicle-associated protein Rv3717 facilitates the proteasomal degradation of ILF2, resulting in the downregulation of IL-2 expression. Overall, the role of intracellular Rv3717 in promoting mycobacterial survival is associated with its ubiquitination and the proteasomal degradation of ILF2. Full article

Bacterial lysates have emerged as promising immunomodulatory agents that can enhance innate immune responses. Given the crucial role of macrophages in recognizing and controlling intracellular pathogens such as Mycobacterium tuberculosis, this study aimed to evaluate the immunological effects of selected bacterial lysates on human monocyte-derived macrophages (MDMs). We examined the ability of commercial bacterial lysates, Pulmonarom, Ismigen, Uro-Vaxom, and a lysate of M. tuberculosis H37 Ra (LMtb) to stimulate the production of key pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-8. In addition, we investigated whether these lysates could modulate the expression of bactericidal/permeability-increasing protein (BPI), a critical antimicrobial effector, and assessed their ability to reduce the intracellular burden of mycobacteria and induce autophagy. The results demonstrate diverse immunostimulatory profiles among the lysates, highlighting differences in both inflammatory and antimicrobial responses that may be relevant for host-directed therapeutic strategies against tuberculosis. Notably, beyond the in vitro antimycobacterial activity observed for BPI, this protein was also found to be elevated in both serum and bronchoalveolar lavage fluid from patients with active TB, reflecting local and systemic immune activation. Furthermore, the reduction in BPI levels after treatment suggests its potential utility for following the dynamics of infection. Full article

The Mycobacterium tuberculosis complex (MTBC) represents one of the most significant bacterial pathogen groups affecting both animals and humans worldwide. This review provides a comprehensive analysis of MTBC species distribution across different animal hosts and evaluates current laboratory diagnostic methodologies for pathogen detection and identification. The complex comprises seven primary species: Mycobacterium bovis, M. caprae, M. tuberculosis, M. microti, M. canettii, M. africanum, and M. pinnipedii, each exhibiting distinct host preferences, geographical distributions, and pathogenic characteristics. Despite sharing >99% genetic homology, these species demonstrate variable biochemical properties, morphological features, and pathogenicity profiles across mammalian species. Current diagnostic approaches encompass both traditional culture-based methods and advanced molecular techniques, including whole genome sequencing. This review emphasises the critical importance of rapid, accurate detection methods for effective tuberculosis surveillance and control programmes in veterinary and public health contexts. Full article

Background/Objectives: The diagnosis of Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacterial (NTM) infections is accomplished by three main diagnostics methods: smear microscopy, culture, and molecular testing. Diagnostic algorithms used by laboratories can significantly impact clinical and infection control management. Current Canadian Tuberculosis Standards recommend the use of nucleic acid amplification testing (NAAT) for smear-positive patients and smear-negative patients upon request. An alternative algorithm is to utilize NAAT in the Panel approach on all samples, pulmonary and extrapulmonary, to potentially reduce time to diagnosis and treatment. This alternative approach was implemented in November 2019 at the Newfoundland and Labrador Public Health and Microbiology Laboratory (NL PHML) using a laboratory-developed multiplex real-time PCR (LDT m-qPCR) assay targeting Mycobacterium spp. (Myco spp.) and MTBC, performed in parallel with smear and culture. Methods: To investigate the impact of this alternate testing approach, we conducted an observational retrospective analysis of laboratory diagnostic and treatment data, recognizing that temporal changes in epidemiology, clinical practice, and laboratory workflow may also have influenced outcomes. To complete this, study data from three years before and four years after implementation were gathered. Results: The sensitivity/specificity of the smear, m-LDT qPCR-MTBC, m-LDT qPCR-Myco spp., and culture assays in this study were 18.1%/100%, 96.7%/99.8%, 47.6%/99.0%, and 96.8%/100%, respectively. The gold standard utilized for these calculations was clinical diagnosis for active MTBC disease and culture for NTM infections, recognizing that the use of clinical diagnosis may introduce subjectivity. The Panel approach reduced the time to diagnosis of tuberculosis MTBC by 29 days (p < 0.0001) for NL PHML, and when modelled for a laboratory with rapid culture identification, diagnosis was reduced by 14 days (p = 0.003). Among non-empirically treated tuberculosis patients, the time to treatment was decreased by 25.5 days (p < 0.001). For NTM infections, rapid diagnostics only affected one patient’s treatment. This finding agrees with clinical management guidelines, which do not routinely utilize rapid diagnostics for the diagnosis of disease or treatment decisions. The cost implications of additional NAAT testing were calculated to be an increase of CAD 23.62 per sample. Conclusions: Our findings support the adoption of a molecular assay for MTBC as an initial diagnostic tool to decrease time to diagnosis and time to treatment, depending on local epidemiology and irrespective of smear status. Utilizing a molecular assay for genus level identification of NTM had minimal impact on clinical management suggesting its limited diagnostic utility in a broad population setting. Full article

Background/Objectives: Tuberculosis (TB) remains one of the most pressing global health challenges, ranking among the leading infectious causes of mortality worldwide. Medicinal plants possess antimycobacterial potential, warranting the isolation and characterization of their bioactive compounds to address bacterial infections. The study aimed to determine five selected traditional medicinal plants’ in vitro antioxidant and antibacterial activities and the isolation of active phytoconstituents. Methods: Powdered leaf material was extracted using n-hexane, dichloromethane, acetone, methanol, and water. The quantity of phytochemicals and antioxidants was determined using colorimetric assay, The antimycobacterial activity and combination effects were determined using microbroth dilution assay. Cell viability was determined using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] MTT reduction assay. Bioassay-guided fractionation was used to isolate bioactive compounds. Results: Polar solvents had high extraction yields, and all extracts had varying phytoconstituents. Active extracts were selected for fractionation and isolation of pure compounds using gradient elution column chromatography. Rhoicissus tridentata water extracts had the highest total phenolic (335.20 ± 8.26 mg GAE/g) and tannin (103.48 ± 7.36 mg GAE/g) content, while Rosmarinus officinalis (45.90 ± 11.04 mg QE/g) methanol extract had the highest total flavonoid. Ximenia caffra had promising antioxidant activity. R. officinalis had prominent antimycobacterial. Rhoicissus tridentata had the highest percentage cell viability. Two compounds were isolated, and they were active against Mycobacterium smegmatis with minimum inhibitory concentration values ranging from 0.125 to 0.25 mg/mL. Conclusions: The selected medicinal plants contain phytochemicals with antioxidant and antimycobacterial activities, supporting their pharmacokinetic studies and evaluation against Mycobacterium tuberculosis H37Rv. Full article

Cell division is critical for the survival, growth, pathogenesis, and antibiotic susceptibility of Mycobacterium tuberculosis (Mtb). However, the regulatory networks governing the transcription of genes involved in cell growth and division in Mtb remain poorly understood. This study aimed to investigate the impact of BlaI overexpression on cell division and growth in Mtb and elucidate the underlying mechanisms. Mycobacterium smegmatis mc²155 was used as the model organism. Recombinant strains overexpressing BlaI were constructed. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), ethidium bromide and Nile red uptake assays, minimum inhibitory concentration (MIC) determination, drug resistance analysis, quantitative real-time PCR (qRT-PCR) assays, and electrophoretic mobility shift assay (EMSA) were employed to assess changes in bacterial morphology, cell wall permeability, antibiotic susceptibility, gene transcription levels, and the interaction between BlaI and its target genes. Overexpression of BlaI disrupted bacterial division in M. smegmatis, leading to growth delay, cell elongation, and formation of multi-septa. It also altered the lipid permeability of the cell wall and enhanced the sensitivity of M. smegmatis to β-lactam antibiotics. BlaI overexpression affected the transcription of cell division-related genes, particularly downregulating ftsQ. Additionally, BlaI negatively regulated the transcription of Rv1303—a gene co-transcribed with ATP synthase-encoding genes—inhibiting ATP synthesis. This impaired the phosphorylation of division complex proteins, ultimately affecting cell division and cell wall synthesis. Overexpression of BlaI in Mtb interferes with bacterial division, slows growth, and alters gene expression. Our findings identify a novel role for BlaI in regulating mycobacterial cell division and β-lactam susceptibility, providing a foundation for future mechanistic studies in M. tuberculosis, with validation required to assess relevance to clinical tuberculosis—though validation in M. tuberculosis and preclinical models is required. Full article