Abstract
To establish an integrated in vitro evaluation platform for identifying effective antibiotic combinations against rifampicin-resistant Mycobacterium tuberculosis (RR-TB), by simultaneously assessing synergistic drug interactions, intracellular bactericidal activity, macrophage apoptosis, and cytokine responses. Using this platform, we investigated the combinatory potential of linezolid (LZD) with five second-line antitubercular agents. The minimum inhibitory concentrations (MICs) of LZD and five second-line drugs were determined using the Alamar Blue microplate assay. Drug–drug interactions between LZD and cycloserine (CS), clofazimine (CFZ), bedaquiline (BDQ), moxifloxacin (MFX), or levofloxacin (LFX) were evaluated using checkerboard microdilution analysis. Drug pairs demonstrating in vitro synergy were further examined in a macrophage infection model. Intracellular bacterial burden was quantified by colony-forming unit (CFU) enumeration. Macrophage apoptosis was assessed using flow cytometry, and cytokine production (IL-12/23 p40, TNF-α, IL-6, and IL-10) was analyzed to characterize immune modulation. LZD demonstrated synergistic interactions with CS and CFZ, whereas no synergy was observed with BDQ, MFX, or LFX. The synergistic combinations LZD + CS and LZD + CFZ significantly reduced intracellular CFU counts, enhanced macrophage apoptosis, and altered cytokine responses, characterized by increased TNF-α and decreased IL-10 levels in infected macrophages. This study presents a comprehensive and mechanistically informative in vitro methodology for evaluating antibiotic combinations against RR-TB. The platform effectively integrates drug synergy testing with assessments of intracellular killing, apoptosis induction, and immune modulation, offering a promising approach for preclinical screening of novel anti-TB therapeutic strategies.
Data availability
The data that support the findings of this study are not publicly available due to privacy reasons but are available from the corresponding author upon request.
References
World Health Organization. Drug-resistant TB. In: 1.3 Drug-resistant TB - Global Tuberculosis Report 2024. Geneva: WHO. at (2024). https://www.who.int/teams/global-programme-on-tuberculosis-and-lung-health/tb-reports/global-tuberculosis-report-2024/tb-disease-burden/1-3-drug-resistant-tb
Kanabalan, R. D. et al. Human tuberculosis and Mycobacterium tuberculosis complex: A review on genetic diversity, pathogenesis and omics approaches in host biomarkers discovery. Microbiol. Res. 246, 126674 (2021).
Dechow, S. J. & Abramovitch, R. B. Targeting Mycobacterium tuberculosis pH-driven adaptation. Microbiology (Reading). 170, 001458 (2024).
Mohammadnabi, N. et al. Mycobacterium tuberculosis: The mechanism of pathogenicity, immune responses, and diagnostic challenges. J. Clin. Lab. Anal. 38, e25122 (2024).
Conradie, F. et al. Bedaquiline-pretomanid-linezolid regimens for drug-resistant tuberculosis. N Engl. J. Med. 387, 810–823 (2022).
Elbarbry, F. & Moshirian, N. Linezolid-associated serotonin toxicity: A systematic review. Eur. J. Clin. Pharmacol. 79, 875–883 (2023).
Howell, P. et al. Treatment of rifampicin-resistant tuberculosis disease and infection in children: Key Updates, challenges and opportunities. Pathogens 11, 381 (2022).
Nyang’wa, B. T. et al. Short oral regimens for pulmonary rifampicin-resistant tuberculosis (TB-PRACTECAL): an open-label, randomised, controlled, phase 2B-3, multi-arm, multicentre, non-inferiority trial. Lancet Respir Med. 12, 117–128 (2024).
Ding, P., Li, X., Jia, Z. & Lu, Z. Multidrug-resistant tuberculosis (MDR-TB) disease burden in China: A systematic review and spatio-temporal analysis. BMC Infect. Dis. 17, 57 (2017).
Khan, M. A. et al. MDR-TB in Pakistan: Challenges, efforts, and recommendations. Ann. Med. Surg. (Lond). 79, 104009 (2022).
Howard, N. C. & Khader, S. A. Immunometabolism during Mycobacterium tuberculosis infection. Trends Microbiol. 28, 832–850 (2020).
Salzer, H. J. F. et al. Personalized medicine for chronic respiratory infectious diseases: Tuberculosis, nontuberculous mycobacterial pulmonary diseases, and chronic pulmonary aspergillosis. Respiration 92, 199–214 (2016).
Lemus, D. et al. Antituberculosis drug resistance in pulmonary isolates of Mycobacterium tuberculosis, Cuba 2012–2014. MEDICC Rev. 19, 10–15 (2017).
Simões, M. F., Ottoni, C. A. & Antunes, A. Mycogenic metal nanoparticles for the treatment of mycobacterioses. Antibiotics. 9, 569 (2020).
Liu, C. X., Zhao, X., Wang, L. & Yang, Z. C. Quinoline derivatives as potential anti-tubercular agents: Synthesis, molecular docking and mechanism of action. Microb. Pathog. 165, 105507 (2022).
Micheletti, V. C. D., Kritski, A. L. & Braga, J. U. Clinical features and treatment outcomes of patients with drug-resistant and drug-sensitive tuberculosis: A historical cohort study in Porto Alegre, Brazil. PLoS One. 11, e0160109 (2016).
Getahun, M., Blumberg, H. M., Ameni, G., Beyene, D. & Kempker, R. R. Minimum inhibitory concentrations of rifampin and isoniazid among multidrug and isoniazid resistant Mycobacterium tuberculosis in Ethiopia. PLoS One. 17, e0274426 (2022).
Sharma, A. et al. Estimating the future burden of multidrug-resistant and extensively drug-resistant tuberculosis in India, the Philippines, Russia, and South Africa: A mathematical modelling study. Lancet Infect. Dis. 17, 707–715 (2017).
Singh, P. K. & Jain, A. Limited scope of shorter drug regimen for MDR TB caused by high resistance to fluoroquinolone. Emerg. Infect. Dis. 25, 1760–1762 (2019).
Gao, J. et al. Stepwise selection of mutation conferring fluroquinolone resistance: Multisite MDR-TB cohort study. Eur. J. Clin. Microbiol. Infect. Dis. 40, 1767–1771 (2021).
Wasserman, S., Meintjes, G. & Maartens, G. Linezolid in the treatment of drug-resistant tuberculosis: The challenge of its narrow therapeutic index. Expert Rev. Anti Infect. Ther. 14, 901–915 (2016).
Lee, J. K. et al. Substitution of ethambutol with linezolid during the intensive phase of treatment of pulmonary tuberculosis: A prospective, multicentre, randomised, open-label, phase 2 trial. Lancet Infect. Dis. 19, 46–55 (2019).
Bagheri-Yarmand, R. et al. Combinations of tyrosine kinase inhibitor and ERAD inhibitor promote oxidative stress–induced apoptosis through ATF4 and KLF9 in medullary thyroid cancer. Mol. Cancer Res. 17, 751–760 (2019).
Cox, D. J. et al. Inhibiting histone deacetylases in human macrophages promotes glycolysis, IL-1β, and T helper cell responses to Mycobacterium tuberculosis. Front. Immunol. 11, 1609 (2020).
Singh, P. et al. Computational modeling and bioinformatic analyses of functional mutations in drug target genes in Mycobacterium tuberculosis. Comput. Struct. Biotechnol. J. 19, 2423–2446 (2021).
Bakhtiyariniya, P., Khosravi, A. D., Hashemzadeh, M. & Savari, M. Detection and characterization of mutations in genes related to isoniazid resistance in Mycobacterium tuberculosis clinical isolates from Iran. Mol. Biol. Rep. 49, 6135–6143 (2022).
Tan, Z. M. et al. Novel approaches for the treatment of pulmonary tuberculosis. Pharmaceutics 12, 1196 (2020).
Ramachandran, G. & Swaminathan, S. Safety and tolerability profile of second-line anti-tuberculosis medications. Drug Saf. 38, 253–269 (2015).
Jagielski, T. et al. A close-up on the epidemiology and transmission of multidrug-resistant tuberculosis in Poland. Eur. J. Clin. Microbiol. Infect. Dis. 34, 41–53 (2015).
Lee, S. M. et al. Resistance mechanisms of linezolid-nonsusceptible enterococci in Korea: Low rate of 23S rRNA mutations in Enterococcus faecium. J. Med. Microbiol. 66, 1730–1735 (2017).
Tang, M. et al. The properties of linezolid, rifampicin, and vancomycin, as well as the mechanism of action of pentamidine, determine their synergy against gram-negative bacteria. Int. J. Mol. Sci. 24, 13812 (2023).
Dodd, C. E., Pyle, C. J., Glowinski, R., Rajaram, M. V. S. & Schlesinger, L. S. CD36-mediated uptake of surfactant lipids by human macrophages promotes intracellular growth of Mycobacterium tuberculosis. J. Immunol. 197, 4727–4735(2016).
Beutler, M. et al. Rapid Tuberculosis diagnostics including molecular first- and second-line resistance testing based on a novel microfluidic DNA extraction cartridge. J. Mol. Diagn. 23, 643–650 (2021).
Liebenberg, D., Gordhan, B. G. & Kana, B. D. Drug resistant tuberculosis: Implications for transmission, diagnosis, and disease management. Front. Cell. Infect. Microbiol. 12, 943545 (2022).
Zhang, T. et al. The global, regional, and national burden of tuberculosis in 204 countries and territories, 1990–2019. J. Infect. Public Health. 16, 368–375 (2023).
Chen, Y. et al. Resistance to second-line antituberculosis drugs and delay in drug susceptibility testing among multidrug-resistant tuberculosis patients in Shanghai. Biomed. Res. Int. 2016, 2628913 (2016).
Coutinho, A. E. & Chapman, K. E. The anti-inflammatory and immunosuppressive effects of glucocorticoids, recent developments and mechanistic insights. Mol. Cell. Endocrinol. 335, 2–13 (2011).
Van’t Hoog, A. H. et al. The potential of a multiplex high-throughput molecular assay for early detection of first and second line tuberculosis drug resistance mutations to improve infection control and reduce costs: a decision analytical modeling study. BMC Infect. Dis. 15, 473 (2015).
Liang, Y. N. et al. MiR-124 contributes to glucocorticoid resistance in acute lymphoblastic leukemia by promoting proliferation, inhibiting apoptosis and targeting the glucocorticoid receptor. J. Steroid Biochem. Mol. Biol. 172, 62–68 (2017).
Matlow, A. G., Harrison, A., Monteath, A., Roach, P. & Balfe, J. W. Nosocomial transmission of tuberculosis (TB) associated with care of an infant with peritoneal TB. Infect. Control Hosp. Epidemiol. 21, 222–223 (2000).
Funding
This project was funded by Medical Science Research Project of Hebei [No.20260817].
Author information
Authors and Affiliations
Contributions
D.C.: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Writing—original draft, Writing—review and editing; N.L.:Investigation, Formal analysis, Writing—review and editing; X.X. R.: Conceptualization, Methodology, Investigation, Writing—review and editing.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Ethical approval
This study did not involve human participants or animals. The standard strain of Mycobacterium tuberculosis H37Rv (ATCC 27295) was obtained from the Key Laboratory of Pulmonary Diseases, Hebei Chest Hospital, and therefore did not require ethical approval.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
About this article
Cite this article
Cui, D., Li, N. & Ren, X. Integrated platform for linezolid combinations against rifampicin-resistant Mycobacterium tuberculosis: synergy, macrophage apoptosis, and immune modulation. Sci Rep (2026). https://doi.org/10.1038/s41598-026-44422-7
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41598-026-44422-7
