Tomas Correa Zuluaga

Background: Mycobacterium tuberculosis (Mtb) is the acid-fast pathogen responsible for tuberculosis (TB), characterized by a mycolic acid-rich cell wall that makes it resistant to many standard antibiotics and allows it to persist within host macrophages.^1,2 The emergence of multidrug-resistant (MDR-TB) and extensively drug-resistant (XDR-TB) strains has made frontline regimens increasingly ineffective, worsened by the pathogen’s ability to transition into a latent state and evade immune clearance for decades.^1,4 Bacteriophages are viruses that naturally attack bacteria and offer a lytic mechanism of action entirely distinct from conventional antibiotics, making them a promising non-traditional strategy against refractory TB.^3,4 However, more research is needed to understand why phages such as D29 demonstrate high lytic efficiency on agar plates but reduced efficacy in liquid cultures and within human primary macrophages, likely due to Mtb’s inhibition of phagosome-lysosome fusion and manipulation of intracellular membrane trafficking.^1,3

Objective: This literature review evaluates the evidence for bacteriophage-based strategies against antibiotic-resistant Mtb, examining aerosolized delivery, engineered lytic cocktails, phage-derived enzymatic agents, bactericidal activity under pathophysiological stress, and efficacy in humanized granuloma models.

Search Methods: PubMed was used to search for articles published between 2019 and 2024 using the following MeSH terms and keywords: “bacteriophage therapy,” “Mycobacterium tuberculosis,” “phage cocktail,” “mycobacteriophage,” “antibiotic resistance,” “lysin,” and “granuloma.”

Results: Five primary studies were identified and reviewed. Carrigy et al. demonstrated that nebulized bacteriophage D29 can be delivered to the deep lung at approximately 1 PFU/alveolus, significantly reducing Mtb bacterial burden in the lungs and spleens of mice when administered prophylactically^.5 Guerrero-Bustamante et al. showed that genome engineering can convert temperate phages into purely lytic variants; a resulting five-phage cocktail effectively killed all tested clinical Mtb isolates, including MDR strains, without impacting first-line antibiotics such as isoniazid and rifampicin^.6 Hurst-Hess et al. investigated Lysin B (LysB) from bacteriophage D29, finding that this phage-encoded lipase hydrolyzes trehalose-6,6’-dimycolate (TDM), a key mycolic acid component of the outer membrane; pulmonary delivery of aerosolized recombinant D29-LysB produced a significant reduction in pulmonary bacterial burden in mice^.7 Jeyasankar et al. assessed phages D29, TM4, and DS6A against virulent Mtb under conditions simulating the TB lesion microenvironment (acidic pH, hypoxia, and biofilm formation) demonstrating that these phages maintained high bactericidal activity even under these pathophysiological stressors.⁸ Yang et al. utilized NSG-SGM3 humanized mice capable of forming caseous necrotic granulomas and found that DS6A therapy significantly reduced Mtb burden across pulmonary and extrapulmonary tissues, with direct lysis of bacilli sequestered within host macrophages confirmed, while D29 showed limited efficacy in this chronic infection model.¹

Conclusion: Collectively, these studies establish a preclinical framework supporting bacteriophage therapy as an effective approach against drug-resistant Mtb.^1,5,6 Aerosolized delivery, engineered lytic cocktails, and phage-derived enzymes each provide distinct mechanisms to overcome the mycolic acid cell wall and intracellular refuge that cause conventional antibiotics to be ineffective, particularly in children. the heterogeneous lesion compartments where frontline agents like isoniazid and rifampicin fall below bactericidal concentrations.^1,4,7 The demonstrated bactericidal activity under pathophysiological stress and penetration of human-like granulomas by DS6A further support the clinical translatability of targeted phage selection.^1,8 Research going forward should focus on vivo cocktail optimization, long-term safety and immunogenicity evaluation, and dose-escalation studies to establish the optimal delivery required for human clinical trials against MDR and XDR tuberculosis.^3,4

Works Cited

1. 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
2. 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
3. Hatfull GF. Mycobacteriophages. Microbiol Spectr. 2014;2(4):VMBF-0001-2014. doi:10.1128/microbiolspec.VMBF-0001-2014
4. Strathdee SA, Hatfull GF, Mutalik VK, Schooley RT. Phage therapy: from biologic mechanisms to future directions. Cell. 2023;186(1):17-31. doi:10.1016/j.cell.2022.11.017
5. Carrigy NB, Larsen SE, Reese V, et al. Prophylaxis of Mycobacterium tuberculosis H37Rv infection in a preclinical mouse model via inhalation of nebulized bacteriophage D29. Antimicrob Agents Chemother. 2019;63(12):e00871-19. doi:10.1128/AAC.00871-19
6. Guerrero-Bustamante CA, Dedrick RM, Garlena RA, Russell DA, Hatfull GF. Toward a phage cocktail for tuberculosis: susceptibility and tuberculocidal action of mycobacteriophages against diverse Mycobacterium tuberculosis strains. mBio. 2021;12(3):e00973-21. doi:10.1128/mBio.00973-21
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. Jeyasankar S, Kalapala YC, Sharma PR, Agarwal R. Antibacterial efficacy of mycobacteriophages against virulent Mycobacterium tuberculosis. BMC Microbiol. 2024;24(1):320. doi:10.1186/s12866-024-03474-3
9. Strydom N, Gupta SV, Fox WS, et al. Tuberculosis drugs’ distribution and emergence of resistance in patient’s lung lesions: A mechanistic model and tool for regimen and dose optimization. PLoS Med. 2019;16(4):e1002773. doi:10.1371/journal.pmed.1002773