Myrah N Savul

Background: The bacterial infection Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb), which remains the leading infectious cause of death from a single pathogen worldwide.¹ Mtb is transmitted through inhalation and internalized by alveolar macrophages, persisting in a drug-tolerant state within granulomas, organized immune structures that attempt to contain but rarely eliminate infection.¹ Treatment of drug-susceptible TB requires at least six months of multidrug therapy and achieves cure in roughly 85% of cases.² For multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB, success rates fall to approximately 57%, and strains resistant to every available antibiotic have been documented in at least four countries.² These outcomes illustrate a fundamental limitation of current pharmacologic approaches and have driven interest in alternative treatment strategies. Mycobacteriophages are viruses that specifically infect and lyse mycobacteria, providing a mechanism of bacterial killing that is entirely independent of antibiotic mechanisms of action.^2,3 Their therapeutic potential depends on whether they can survive and remain active within the biological environments encountered during human infection.³

Objective: This literature review investigated existing evidence for mycobacteriophage therapy against Mtb, including systemic and pulmonary delivery methods, strategies to expand phage host range, approaches to avoid phage resistance, efficacy under granuloma-mimicking conditions, and performance in humanized immune models.

Search Methods: The online PubMed database was used to search for papers published between 2019 to 2025 using the following words or phrases: “mycobacteriophage,” “bacteriophage tuberculosis,” “phage therapy Mycobacterium tuberculosis,” “MDR-TB treatment,” and “humanized mouse model tuberculosis.”

Results: The literature review examined five papers, each addressing a different barrier to the clinical translation of phage therapy. Carrigy et al. delivered aerosolized phage D29 prophylactically via a nose-only nebulizer system and found a significant reduction in pulmonary Mtb burden at 24 hours (p < 0.05). Splenic bacterial counts were unchanged at three weeks (p > 0.1), suggesting early lymphatic dissemination of Mtb occurs before pulmonary phage activity can intervene and that inhaled prophylaxis alone is unable to prevent systemic spread.⁴ Guerrero-Bustamante et al. screened 220 phages against 17 Mtb lineages and found that most displayed lineage-restricted activity. A five phage cocktail demonstrated broader strain coverage and reduced frequency of resistant outgrowth compared to monotherapy, though efficacy against M. africanum strains was lower, suggesting regional customization may be necessary.⁵ Jeyasankar et al. assessed phage activity under low oxygen and an acidic pH, both conditions modeling the granuloma interior. A three-phage cocktail of D29, TM4, and DS6A produced a 4-log CFU reduction within seven days (p < 0.05) and sustained bacterial suppression past 100 days, compared to approximately 65 days with single-phage treatment.⁶ The cocktail also demonstrated lytic activity against a rifampicin and isoniazid-resistant strain (M. tuberculosis mc28251), supporting phage relevance in the MDR-TB challenge.⁶ Hurst-Hess et al. investigated Lysin B (LysB), a phage-encoded esterase that hydrolyzes trehalose-6,6’-dimycolate (TDM), a glycolipid essential for mycobacterial outer membrane integrity and virulence, operating independent of phage infection. LysB killed over 99.9% of Mtb cells within 30 minutes in vitro. Aerosolized delivery in SCID mice produced a 20-fold reduction in pulmonary Mycobacterium abscessus bacterial burden compared to controls.⁷ The result in SCID mice support its potential in HIV-TB coinfected patients. Yang et al. tested phages D29 and DS6A in a humanized mouse model reconstituted with human hematopoietic stem cells. D29 demonstrated lytic activity on 7H10 agar but failed to kill Mtb in 7H9 liquid culture or in vivo. DS6A achieved complete eradication of intracellular Mtb within primary human macrophages by day 10 and after given intravenously, reduced bacterial burden across the lungs, liver, and spleen (p < 0.01), with total splenic eradication in 66.7% of treated animals. The increased bacterial clearance in the spleen relative to the lungs was attributed to phage distribution to filter organs following IV administration. Anti-phage IgM antibodies were detected via ELISA but did not appear to neutralize therapeutic activity during the 20 day period.⁸

Works Cited: The studies reviewed support mycobacteriophage therapy as a biologically feasible strategy for drug-resistant TB. Phage cocktails outperformed monotherapy in resisting bacterial regrowth and phage-derived enzymes like LysB may circumvent host-range limitations. Phage DS6A demonstrated activity within a humanized immune environment. IV delivery was most effective for systemic disease while aerosolized delivery mainly addressed pulmonary infection, suggesting a need for combined delivery in order to achieve broad organ clearance. Limitations include the absence of mature granuloma models, short treatment durations, and unanswered questions about whether long-term adaptive IgG immune responses will limit phage activity with repeated dosing. Future work should address phage penetration into caseous granuloma centers, longer-term immunologic outcomes, and regional customization of phage cocktails to account for global Mtb diversity.

Work Cited

1. Warner DF, Barczak AK, Gutierrez MG, Mizrahi V. Mycobacterium tuberculosis biology, pathogenicity and interaction with the host. Nat Rev Microbiol. 2025;23(12):788-804. doi:10.1038/s41579-025-01201-x
2. Allué-Guardia A, Saranathan R, Chan J, Torrelles JB. Mycobacteriophages as potential therapeutic agents against drug-resistant tuberculosis. Int J Mol Sci. 2021;22(2):735. doi:10.3390/ijms22020735
3. Bonacorsi A, Ferretti C, Di Luca M, Rindi L. Mycobacteriophages and their applications. Antibiotics (Basel). 2024;13(10):926. doi:10.3390/antibiotics13100926
4. Carrigy NB, Larsen SE, Reese V, Pecor T, Harrison M, Kuehl PJ, Hatfull GF, Sauvageau D, Baldwin SL, Finlay WH, Coler RN, Vehring R. 2019. Prophylaxis of Mycobacterium tuberculosis H37Rv infection in a preclinical mouse model via inhalation of nebulized bacteriophage D29. Antimicrob Agents Chemother 63:e00871-19. https://doi.org/10.1128/AAC.00871-19.
5. Guerrero-Bustamante CA, Dedrick RM, Garlena RA, Russell DA, Hatfull GF. 2021. Toward a phage cocktail for tuberculosis: susceptibility and tuberculocidal action of mycobacteriophages against diverse Mycobacterium tuberculosis strains. mBio 12: e00973-21. https://doi.org/10.1128/mBio .00973-21.
6. Jeyasankar S, Kalapala YC, Sharma PR, Agarwal R. Antibacterial efficacy of mycobacteriophages against virulent Mycobacterium tuberculosis. BMC Microbiol. 2024;24(1):320. Published 2024 Sep 4. doi:10.1186/s12866-024-03474-3
7. Hurst-Hess K, Walz A, Yang Y, et al. Intrapulmonary treatment with mycobacteriophage LysB rapidly reduces Mycobacterium abscessus burden. Antimicrob Agents Chemother. 2023;67(6):e0016223. doi:10.1128/aac.00162-23
8. Yang F, Labani-Motlagh A, Bohorquez JA, et al. Bacteriophage therapy for the treatment of Mycobacterium tuberculosis infections in humanized mice. Commun Biol. 2024;7(1):294. doi:10.1038/s42003-024-06006-x