Mycobacteriophages in the Treatment of Mycobacterial Infections: From Compassionate Use to Targeted Therapy

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This study highlights the potential application of mycobacteriophages as a targeted therapeutic strategy against drug-resistant mycobacterial infections, including multidrug-resistant (MDR)/extensively drug-resistant (XDR) tuberculosis and nontuberculous mycobacterial (NTM) diseases. By leveraging the natural specificity and bactericidal activity of phages, this approach offers a viable alternative or adjunct to conventional antibiotics, particularly in cases where resistance or patient comorbidities limit treatment efficacy. The findings support the further exploration of phage-based methods as a complementary tool in managing mycobacterial diseases.

Abstract

This review addresses the urgent need for alternative strategies to combat drug-resistant mycobacterial infections, including multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis, as well as non-tuberculous mycobacterial (NTM) diseases. Traditional antibiotics are increasingly limited by resistance, toxicity, and poor efficacy, particularly in immunocompromised patients. A comprehensive literature search was conducted using PubMed, Scopus, and Google Scholar, covering publications primarily from 2000 to 2025. Only articles published in English were included to ensure consistency in data interpretation. Search terms included “mycobacteriophages,” “phage therapy,” “drug-resistant mycobacteria, “diagnostic phages,” and “phage engineering.” The review examines the therapeutic and diagnostic potential of mycobacteriophages—viruses that specifically infect mycobacteria—focusing on their molecular biology, engineering advances, delivery systems, and clinical applications. Evidence suggests that mycobacteriophages offer high specificity, potent bactericidal activity, and adaptability, positioning them as promising candidates for targeted therapy. Although significant obstacles remain—including immune interactions, limited host range, and regulatory challenges—rapid progress in synthetic biology and delivery platforms continues to expand their clinical potential. As research advances and clinical frameworks evolve, mycobacteriophages are poised to become a valuable asset in the fight against drug-resistant mycobacterial diseases, offering new precision-based solutions where conventional therapies fail.

1. Introduction

Tuberculosis (TB) remains one of the top 10 causes of death worldwide and the leading cause from a single infectious agent, ranking above HIV/AIDS. According to the World Health Organization (WHO) Global Tuberculosis Report 2024, an estimated 10.8 million people fell ill with TB in 2023, with approximately 1.6 million deaths globally [1]. The burden of TB is disproportionately borne by low- and middle-income countries, with countries such as India, Indonesia, China, the Philippines, Pakistan, and Nigeria accounting for two-thirds of the global cases [1]. The emergence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) has exacerbated this public health crisis. MDR-TB, resistant to at least isoniazid and rifampicin, and XDR-TB, which further resists fluoroquinolones and injectable second-line drugs, present significant challenges to effective treatment, leading to increased mortality, longer treatment durations, and substantially higher costs [2].

The decline in the efficacy of anti-TB medications stems from several critical factors. Indiscriminate and inappropriate use of antibiotics in both community and clinical settings accelerates the development of resistance. Additionally, many patients fail to complete the full course of treatment due to drug toxicity, prolonged regimens, socioeconomic barriers, and limited access to healthcare, which promotes the selection of resistant mycobacterial strains [3]. Toxic side effects of second-line drugs, including ototoxicity, nephrotoxicity, and hepatotoxicity, further undermine adherence and treatment success [4,5]. The ongoing evolution of resistance mechanisms in Mycobacterium tuberculosis (M. tuberculosis) necessitates the urgent development of alternative or adjunctive therapies [6]. Beyond TB, non-tuberculous mycobacterial (NTM) infections, collectively referred to as mycobacterioses, are increasingly recognized as significant clinical challenges, particularly in immunocompromised populations but also among immunocompetent individuals [7,8]. NTM infections are caused by diverse environmental mycobacteria distinct from M. tuberculosis and M. leprae, with an intrinsic resistance to many antibiotics and prolonged, often toxic treatment regimens [8,9]. The rising incidence of drug-resistant NTM infections further emphasizes the need for novel and effective treatment approaches [8].

Against this backdrop, bacteriophage therapy (phage therapy) has regained interest as a promising “old-new” strategy to combat drug-resistant mycobacterial diseases [10]. Phages are viruses that specifically infect and lyse bacterial cells and were originally explored as therapeutic agents in the early 20th century before antibiotics became widespread [11]. Recent advances in molecular biology, genomics, and genetic engineering have revitalized this field, offering the potential for precise and effective bacterial targeting with minimal disruption of the host microbiota [12,13]. Recent advances in molecular biology and genetic engineering—such as BRED (bacteriophage recombineering of electroporated DNA), which enables precise genetic modifications in phage genomes, and CRISPR (clustered regularly interspaced short palindromic repeats)—Cas (CRISPR associated protein) systems used for sequence-specific genome editing, have greatly expanded the toolbox available for the creation of genetically modified bacteriophages. Notably, mycobacteriophages have shown efficacy against drug-resistant Mycobacterium strains, making phage therapy a promising adjunct or alternative to conventional treatments for MDR-TB, XDR-TB, and resistant mycobacterioses [12,14]. In an era of escalating antibiotic resistance and limited therapeutic options, phagotherapy thus represents a promising and innovative strategy that warrants further rigorous scientific investigation and clinical development.

This article aims to comprehensively evaluate the current landscape of mycobacteriophage research with a focus on their potential therapeutic and diagnostic applications. Specifically, we seek to: (1) assess recent advances in phage engineering and delivery methods; (2) identify critical challenges such as phage resistance, regulatory hurdles, and limitations in clinical translation; and (3) highlight key knowledge gaps and propose future research directions necessary to realize the clinical potential of mycobacteriophages. By addressing these objectives, this review contributes to a deeper understanding of the evolving role of phage therapy in combating the global threat posed by drug-resistant mycobacterial diseases.

2. Study Methodology

This review was conducted following a systematic approach aimed at gathering and evaluating current information on the use of mycobacteriophages in the treatment and diagnosis of mycobacterial diseases. Literature searches were performed in major scientific databases, including PubMed, Scopus, and Google Scholar. The timeframe considered spanned publications primarily from 2000 up to early 2025 to capture the most recent advances in the field. Searches used combinations of keywords such as “mycobacteriophages”, “phage therapy”, “drug-resistant mycobacteria”, “diagnostic phages”, and “phage engineering”. Only articles published in English were included to ensure consistency in data interpretation. The review encompassed original experimental studies, scientific reviews, clinical reports, and expert opinions. Publications with limited access to full texts and those not directly related to mycobacteriophages were excluded. All selected articles were critically appraised for methodological quality and relevance to the objectives of this review. This methodology ensures that the review provides a comprehensive and up-to-date overview of the state of knowledge regarding mycobacteriophage applications, while also identifying existing research gaps and future directions.

3. Mycobacteriophages—General Characteristics and Overview

Bacteriophages (phages) are viruses that infect and lyse bacterial cells. They are considered the most abundant and diverse biological entities on the planet, with an estimated 10³¹ viral particles and over 10⁸ distinct genotypes [15,16,17]. Phages inhabit virtually all environments containing bacteria, including soil, water, wastewater, and clinical specimens [18,19]. The existence of bacteriophages was first observed in the late 19th and early 20th centuries by researchers such as E. Hankin, F. Twort, and F. d’Hérelle, the latter of whom formally introduced the concept of “bacteriophages” and pioneered early therapeutic use [20,21,22,23]. While phage therapy saw some application in the 1930s and 1940s, its popularity waned with the rise in antibiotics. However, due to growing antibiotic resistance, interest in phages has resurged, particularly because of their species- or even strain-specific lytic activity and minimal disruption to host microbiota [24,25,26,27]. Bacteriophage classification is governed by the International Committee on Taxonomy of Viruses (ICTV), which groups phages based on genome type (DNA or RNA), strandedness (single or double), capsid morphology, and presence or absence of a lipid envelope [28,29,30]. Detailed information on the general classification of bacteriophages is provided in

Table 1. General classification of bacteriophages.

Family	Nucleic Acid Type	Representative Phage	Characteristic Features	Typical Host	Application	Year/Author of Classification [Reference]
Myoviridae	dsDNA, linear	TM4	Complex structure, contractile tail	Mycobacterium sp.	a genetic tool and diagnostic reporter for Mycobacterium tuberculosis and M. smegmatis	1944/Delbrück M. [31]
Siphoviridae	dsDNA, linear	L5	Complex structure, long flexible tail	Mycobacterium sp.	widely used in mycobacterial genetics as a tool for transduction and gene delivery	1951/Lederberg E. [32]
Podoviridae	dsDNA, linear	Φ29	Complex structure, short tail	Bacillus sp.	widely used in molecular biology for its high-fidelity DNA polymerase and DNA replication studies	1965/Reilly B. [33]
Tectiviridae	dsDNA, linear	PRD1	Isometric structure, lipoprotein vesicle	Escherichia coli	a model to study virus assembly, DNA packaging, and evolution of membrane-containing viruses	1984/Bamford D. [34]
Corticoviridae	dsDNA, circular	PM2	Isometric structure	Alteromonas sp.	a model to study membrane-containing viruses and virus evolution in aquatic environments	1968/Espejo R. Canelo E. [35]
Plasmaviridae	dsDNA, circular	L2	No capsid, lipoprotein envelope	Acholeplasma sp.	a model to study phage–host interactions in wall-less bacteria (Mollicutes) and horizontal gene transfer	1971/Gourlay R. [36]
Cystoviridae	dsRNA, linear, and segmented	Φ6	Isometric structure, lipoprotein envelope	Pseudomonas sp.	a model for studying segmented double-stranded RNA viruses and virus evolution	1973/Vidaver A. [37]
Inoviridae	ssDNA, circular	fd	Helical structure	Escherichia coli	used in phage display technology and studies of virus assembly and secretion	1959/Hoffmann-Bering H. [38]
Leviviridae	ssRNA, linear	MS2	Isometric structure	Escherichia coli	a model for RNA virus replication, translational regulation, and phage display systems	1961/Clark A. [39]

Abbreviations: dsDNA—double-stranded DNA; dsRNA—double-stranded RNA; ssDNA—single-stranded DNA; and ssRNA—single-stranded RNA. The classification follows the guidelines established by the International Committee on Taxonomy of Viruses (ICTV), with key references including [31,32,33,34,35,36,37,38,39].

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Mycobacteriophages were first isolated in 1946 from environmental samples containing Mycobacterium smegmatis, a fast-growing, nonpathogenic species often used as a model organism [40]. In 1954, phages infecting Mycobacterium tuberculosis were identified, highlighting the potential of these viruses in tuberculosis research and treatment [41]. Mycobacteriophages have since been recovered from a wide range of ecological sources, including soil, freshwater, marine environments, and clinical materials [17,41]. Structurally, mycobacteriophages typically consist of an icosahedral head and a tail apparatus that facilitates host recognition and DNA injection (Figure 1). The head, formed by dozens of capsomere subunits, contains the viral genome, while the tail—ranging in length from several nanometers to over 800 nm—often includes specialized fibers or baseplates essential for the adsorption to the bacterial cell wall [42,43,44]. Most mycobacteriophages belong to the order Caudovirales and possess linear double-stranded DNA genomes [45,46,47,48]. These phages exhibit either long, flexible non-contractile tails (family Siphoviridae) or contractile tails (family Myoviridae) [45,46,47,48]. Due to their specificity, diversity, and ease of genetic manipulation, mycobacteriophages are not only important in fundamental microbiological research but are also being explored as diagnostic tools and therapeutic agents, particularly in cases of drug-resistant Mycobacterium infections.

Most mycobacteriophages belong to the Siphoviridae family, within which there is a strong variation in the length of the non-contractile tail and the symmetry of the phage head. The genomes of mycobacteriophages from this family range from 22 to 140 kbp. Mycobacteriophages from the Myoviridae family have shorter, contractile tails, which allow them to penetrate the bacterial membrane and inject their genetic material into the host cell. The genomes of these phages range from 30 to 500 kbp [43,53].

The first complete mycobacteriophage genome was sequenced in 1993. It belonged to phage L5, which is closely related to phage L1, a virus that infects both fast- and slow-growing species of mycobacteria. The next fully sequenced phage was mycobacteriophage D29, which shared sequence similarity with phage L5, particularly in the genes coding for structural elements of the virion [54]. The third sequenced genome was that of phage TM4, isolated from M. avium [43]. However, it was soon found that this phage could infect not only M. avium but also M. smegmatis and M. tuberculosis [55]. Comparative analysis of the TM4 genome showed significant differences from the genomes of mycobacteriophages L5 and D29. By 2010, a total of 60 complete mycobacteriophage genomes had been deposited in GenBank, originating from various geographical regions and environments. However, it is worth noting that more than half of these were isolated from the western Pennsylvania region of the USA. The limited number of phage isolations from other countries does not appear to be due to their absence in the environment, but rather the costly and time-consuming process of their isolation [43].

Despite the isolation of approximately 2400 different mycobacteriophage types from numerous clinical and environmental mycobacterial species, by 2012, the genomes of 221 mycobacteriophages had been fully sequenced. These phages exhibited significant nucleotide sequence variability [56,57]. It is estimated that the genome length of mycobacteriophages ranges from approximately 42 kbp to 150 kbp, with the average length being about 70 kbp. The average GC content of these genomes is 63.7%, with exceptions for phages such as Wildcat (59%) and Rosebush (69%). Mycobacteriophages also vary in the number of tRNA genes they carry. It has been found that about two-thirds of them do not contain these genes, while the remaining one-third (mainly from the Myoviridae family) may encode 30 or more tRNA genes [44]. A comparative analysis of the sequenced genomes of mycobacteriophages revealed a high level of diversity, even greater than that observed in phages infecting Pseudomonas or Staphylococcus species [44]. This diversity may arise from the large number of mycobacterial species susceptible to phage infection, or as suggested, it could be due to differences in laboratory procedures used during phage isolation [44]. As of the latest update from PhagesDB, mycobacteriophages are grouped into more than 30 clusters, labeled from A to AE, based on nucleotide sequence similarity and gene content [58]. Each cluster contains phages with related genomic architecture, and several large clusters—such as cluster A—are further divided into subclusters (e.g., A1 to A9). In addition to these clusters, over 100 singletons have been identified. These are phages whose genomes do not share a sufficient similarity with any other known phages to be assigned to an existing cluster. This classification system continues to evolve as new genomes are sequenced, and it is expected that further diversity, including entirely new clusters and subclusters, will emerge [56]. Therefore, current cluster designations should be regarded as provisional and subject to regular updates.

Mycobacteriophage typing has revealed that some phages have a broad host range (e.g., D29), while others infect only a single species of bacteria (e.g., Barnyard, DS6A). This is because phages are capable of recognizing specific receptors on the surface of the bacterial cells they infect [42,50]. Mycobacteriophages can follow two distinct infection pathways: lytic or lysogenic, with most of them operating in a mild manner (e.g., L5, phages from cluster A), while others lead to the death of the infected cell (e.g., D29). There are also mycobacteriophages, such as Bxz1 and its related phages, whose infection pathway is still unclear [42].

To improve the clinical relevance of mycobacteriophages—particularly in the context of treating M. tuberculosis—genetic engineering is increasingly applied to enhance phage safety, efficacy, and precision. A significant proportion of naturally occurring mycobacteriophages are temperate, meaning they can integrate into the host bacterial genome via site-specific recombination [59,60]. Such lysogenic phages pose inherent risks for therapeutic applications due to their potential to mediate horizontal gene transfer, promote bacterial persistence, or elicit unintended genomic modifications in the target host. To address these concerns, temperate phages can be genetically engineered to adopt an obligately lytic lifestyle. This is typically achieved through targeted deletions or disruptions of key regulatory genes, such as integrase and repressor. The integrase enzyme mediates the site-specific recombination between the phage genome and the host chromosome, a critical step for lysogeny. Its removal prevents genomic integration, thus blocking the establishment of lysogeny. Similarly, disruption of the repressor gene eliminates the ability to maintain the lysogenic state, ensuring that the phage proceeds exclusively through the lytic cycle and consistently induces host cell lysis [61,62]. Further advances in synthetic biology have allowed for the customization of phage genomes to include functional payloads such as CRISPR-Cas systems, antimicrobial peptides, or reporter genes [63,64]. These engineered additions enable phages to not only enhance the intracellular killing of M. tuberculosis—which predominantly resides within macrophages—but to also serve as innovative diagnostic tools through fluorescence or reporter-based detection [63,64,65]. Delivery of these engineered phages into infected tissues remains a critical bottleneck, but nanoparticle encapsulation or liposomal formulations have shown promise in facilitating phage entry into macrophages, thereby overcoming one of the major biological barriers in TB therapy [65].

A cornerstone of modern mycobacteriophage research is the SEA-PHAGES program (Science Education Alliance–Phage Hunters Advancing Genomics and Evolutionary Science). This initiative has led to the discovery, isolation, and genome sequencing of thousands of mycobacteriophages by undergraduate researchers worldwide [66]. The sequenced phages—many of which are archived in public databases such as PhagesDB.org—serve as a critical resource for therapeutic development [58]. Not only does this collection enable the identification of candidate phages with lytic activity against mycobacterial pathogens, but it also provides valuable insights into phage evolution, comparative genomics, host-range specificity, and resistance mechanisms [67,68]. The SEA-PHAGES dataset, by virtue of its scale and diversity, forms the taxonomic and functional foundation of the mycobacteriophage field. It offers a unique platform for understanding genomic mosaicism, exploring gene content relationships among phage clusters, and designing recombinant phages tailored to specific clinical applications. In particular, phage selection and genetic modification strategies are increasingly informed by comparative analyses within this database, allowing for the rational design of phage cocktails and engineered variants suitable for therapeutic deployment.

Despite significant scientific progress, several challenges still hinder the widespread adoption of mycobacteriophage therapy for tuberculosis. Foremost among these is the difficulty of effectively delivering phages to intracellular M. tuberculosis residing within human tissues, particularly within granulomatous lesions. Furthermore, phages are susceptible to immune system clearance, which may reduce their bioavailability after systemic administration [69]. Regulatory hurdles, including the standardization of production, quality control, and clinical trial design, also remain substantial barriers to clinical translation. Nevertheless, the successful use of engineered phages in compassionate treatment cases—for example, in patients with disseminated M. abscessus infections—demonstrates that personalized phage therapy is feasible and potentially translatable to M. tuberculosis, especially in multidrug-resistant or refractory cases [61,70].

Having characterized the structural and biological diversity of mycobacteriophages, the discussion now turns to their translational potential. The following sections examine their emerging roles in diagnostics and therapy, the technological advances driving phage-based applications, and the key challenges and future perspectives shaping the field.

4. The Use of Mycobacteriophages in Diagnostics and Therapy

The escalating problem of antimicrobial resistance arises from a complex interplay between the intrinsic adaptability of microorganisms and anthropogenic factors. Bacteria have evolved a variety of mechanisms to withstand antimicrobial agents, including efflux pumps, enzymatic degradation, target modification, biofilm formation, and metabolic dormancy. These innate strategies are further amplified by environmental pressures, most notably the widespread and often indiscriminate use of antibiotics in human medicine, veterinary practice, agriculture, and aquaculture. Selective pressure from subtherapeutic or inappropriate antibiotic use, such as incomplete courses of treatment or the prophylactic use in livestock, accelerates the emergence and dissemination of resistant strains. Horizontal gene transfer facilitates the rapid spread of resistance determinants within and across bacterial species, compounding the problem. The resulting proliferation of multidrug-resistant and extensively drug-resistant organisms, including M. tuberculosis, poses a critical challenge to global health.

Concurrently, a stagnation in the development of new antibiotic classes has led to growing dependence on older, less effective treatments [27]. This imbalance between resistance evolution and therapeutic innovation underscores the urgent need for alternative antimicrobial strategies.

Among emerging alternatives, phage therapy has gained renewed attention as a targeted and potentially effective approach to combat drug-resistant infections, including those caused by M. tuberculosis. Unlike antibiotics, bacteriophages offer high specificity for their bacterial hosts and can evolve alongside them, potentially limiting the long-term impact of resistance. Nevertheless, phage therapy is not without limitations. Issues such as a narrow host range, potential for immune neutralization, regulatory hurdles, and the need for individualized matching between phages and pathogens present significant challenges to widespread clinical implementation. Phage therapy involves the administration of bacteriophages through various routes, depending on the infection site. These include intravenous, oral, topical (e.g., wound dressings), intratissue, rectal, or parenteral delivery methods [71]. To provide a clearer perspective on its clinical potential, the key advantages and disadvantages of phage therapy are summarized in Figure 2.

In polymicrobial infections, phage cocktails—mixtures of different phages—are often used to enhance therapeutic efficacy [74]. These cocktails can be delivered in liquid or aerosol form, particularly in treating burn wound infections caused by pathogens like Pseudomonas aeruginosa or Staphylococcus aureus [79]. Recently, combination therapy involving antibiotics and phages has shown promise in reducing the likelihood of bacteria developing a resistance [80]. Phage therapy is also cost-effective and, to date, has shown a favorable safety profile in humans. In addition to therapeutic use, phage solutions are used for disinfection purposes, including the decontamination of surgical tools and hospital surfaces [81].

Despite their ability to lyse pathogenic bacteria, phages lost popularity in Western Europe following the advent of antibiotics. Early phage studies were often poorly understood and yielded inconsistent results, which led many researchers to abandon this line of investigation [78,82]. However, phage research continued in Eastern Europe and the former Soviet Union, where therapeutic use was further explored. For example, in the mid-20th century, an institute in Georgia tested phage susceptibility in bacteria such as Staphylococcus, Clostridium, Streptococcus, Proteus, and Pseudomonas [83,84]. In Poland, the Phage Therapy Center was established in 2005 at the Hirszfeld Institute of Immunology and Experimental Therapy in Wrocław. This was one of the first ethically approved phage therapy centers in Europe [85]. The center houses a large phage collection—more than 300 well-characterized strains—effective against at least 14 pathogenic bacterial species, including multidrug-resistant variants. The extensive phage bank enables personalized phage therapy, allowing the targeted treatment of infections involving Staphylococcus, Klebsiella, E. coli, Pseudomonas, and Salmonella [86]. The center’s work reinforces the value of phage therapy as a potential alternative or complement to antibiotics, especially in light of the growing global threat of antimicrobial resistance.

In the case of M. tuberculosis, the slow-growing nature of the pathogen, its thick and lipid-rich cell wall, and the ability to persist in latent forms within macrophages create additional barriers to effective antibiotic penetration and action. Infections caused by M. tuberculosis and NTMs represent some of the most severe and difficult-to-control diseases globally. TB affects over 10 million people annually, leading to approximately 1.6 million deaths worldwide [1]. In parallel, NTMs—including species such as M. avium, M. abscessus, M. ulcerans, M. xenopi, and M. chimaera—also pose a significant burden on public health systems [87]. The emergence of drug-resistant mycobacterial strains—including MDR and XDR variants—has complicated treatment outcomes. These strains often resist not only first-line drugs (e.g., isoniazid and rifampicin) but also second-line options. The prevalence of MDR M. tuberculosis is steadily increasing, particularly in low- and middle-income countries, where treatment regimens are often interrupted or delayed [88,89].

In this context, the need for rapid, accurate, and cost-effective diagnostics is urgent. Traditional culture-based diagnostic techniques, such as solid media culturing and Ziehl–Neelsen staining, remain the gold standard but are time-consuming (8 to 16 weeks) and often lack sensitivity [90,91,92]. Although molecular tools like GeneXpert offer high diagnostic performance, their high cost limits accessibility in resource-poor settings [93]. Immunodiagnostic tests are further complicated by mycobacterial immune evasion mechanisms, which can lead to false-negative or inconclusive results [94].

4.1. Phage-Based Diagnostic Strategies for Mycobacteria

In response to these diagnostic limitations, phage-based diagnostic approaches are gaining traction [14]. Since the first identification of mycobacteriophages in 1947, researchers have explored their use in detection, species identification, and drug susceptibility testing for TB [40,95]. These assays offer several advantages: rapid turnaround, minimal instrumentation, and low operating costs. Two primary phage-based diagnostic strategies are currently in use: Phage Reporter Assays (PRA) and Phage Amplification Assays (PAA) [96]. Both methods rely on viable mycobacterial cells to support phage replication or reporter gene expression.

The PRA uses genetically modified mycobacteriophages that carry reporter genes such as luciferase (Fflux or luc) or fluorescent proteins like GFP or ZsYellow [51,96]. When a modified phage infects a viable host cell, the reporter gene is expressed, resulting in bioluminescence (measurable by luminometer) or fluorescence (detectable by microscopy or flow cytometry), depending on the gene used (Figure 3). The presence of a light emission indicates metabolically active bacteria in the sample [51,96]. Although PRA is sensitive and fast, its specificity may be limited, as some phages—such as TM4—can infect multiple Mycobacterium species (M. tuberculosis, M. bovis, and M. avium-intracellulare) [96]. In culture-based differentiation of mycobacterial species, the chemical inhibitor p-nitro-α-acetylamino-β-hydroxyl-propiophenone (NAP) is employed to enhance specificity by selectively inhibiting the growth of M. tuberculosis complex strains, which facilitates their distinction from non-tuberculous mycobacteria that may otherwise interfere with diagnostic accuracy [51,97]. Parallel tests with and without NAP allow species differentiation based on the presence or absence of a luminescent signal. Other mycobacteriophages utilized in these assays include L5, Che12, D29, and the highly specific DS-6A [96,97]. In drug susceptibility testing, fluorescent phage-based tools offer a real-time assessment of bacterial response to antimicrobials, further enhancing the clinical value of phage diagnostics [98,99,100]. Banaiee et al. demonstrated that tests based on luciferase reporter phages (LRP) exhibit comparable sensitivity, specificity, and rapidity to the MGIT 960 and BACTEC 460 systems for the detection of antibiotic-resistant M. tuberculosis in Mexico [100]. Furthermore, the LRP assay is up to five times faster than the BACTEC 460 method. Following these findings, luciferase phage-based diagnostics could be implemented in reference laboratories for mycobacterial identification in developing countries, potentially enhancing access to reliable testing [100].

The second test, PAA, uses non-recombinant phages to indirectly detect the presence of mycobacteria through morphological changes (bald spots) generated on a bacterial lawn [51]. This test most commonly employs the D29 phage, capable of infecting M. tuberculosis, M. smegmatis, M. kansasii, M. bovis, and M. leprae [96]. To perform the test, host cells are incubated with a high-titer mycobacteriophage suspension. Infection leads to cell lysis and the release of progeny phages, indicated by bald spots (plaque-forming units, PFU) on an M. smegmatis lawn (Figure 4). More than 20 PFUs suggest the presence of mycobacteria. This test is available commercially as the FASTPlaque^TM TB test and the PhageTek MB assay kit [51,96]. The amplification method detects Mycobacterium in sputum samples. After initial processing and 24 h of broth incubation, a specific mycobacteriophage (Actiphage, D29) infects bacterial cells [101]. Iron ammonium sulfate is added to inactivate extracellular phages, ensuring only replicating intracellular phages remain. This step confirms the presence of viable mycobacteria, since phages replicate only in metabolically active bacteria. Progeny phages are detected by plating on an indicator strain (M. smegmatis) and observing lysis [102,103]. A meta-analysis of 13 studies showed that PAA offers high specificity (0.83 to 1.00), variable sensitivity, and a slightly higher specificity compared to standard culture [104]. Zhu et al. confirmed in a multicenter study that PhaB had a higher sensitivity than the Löwenstein–Jensen (LJ) culture and smear microscopy (p < 0.05), especially in smear-negative samples [105].

4.2. In Vitro Studies

Bacteriophages demonstrate a strong potential for therapeutic use against Mycobacterium due to their specificity. Kalapala et al. showed that phages could infect and lyse antibiotic-resistant bacteria and work synergistically with antibiotics. A cocktail of D29, TM4, and DS6A phages inhibited the growth of slow-growing M. tuberculosis for several weeks [106]. Phage cocktails designed to target M. tuberculosis typically consist of obligately lytic mycobacteriophages capable of infecting and lysing the bacterium through a classical lytic cycle. This involves adsorption, genome injection, replication, and eventual lysis of the host cell, releasing progeny virions. The approach is particularly relevant for M. tuberculosis, which is known for its slow growth and ability to persist in a dormant state. While the lytic cycle remains the primary mechanism of action, some phages may also exert indirect effects, such as metabolic disruption or interference with biofilm integrity, which can sensitize otherwise recalcitrant cells. The effectiveness of such cocktails depends not only on phage–host compatibility, but also on the physiological state of the bacterial population—actively replicating cells are generally more susceptible to productive infection than dormant or nutrient-limited ones [107,108].

Certain phage proteins may enhance antibiotic effectiveness. Li et al. demonstrated that gp39 from phage SWU1 increases M. smegmatis cell wall permeability, facilitating antibiotic penetration and enhancing treatment efficacy [109]. However, antibiotics like kanamycin, hygromycin, or streptomycin inhibit phage infection, while spectinomycin does not [110].

4.3. Animal Studies

Early efforts to treat M. tuberculosis and M. bovis BCG infections in guinea pigs using mycobacteriophages in the 1960s failed due to septic shock, likely from rapid bacterial lysis [111]. In 1991, the DS6A phage showed therapeutic efficacy in guinea pigs [112]. Broxmeyer et al. (2002) used the TM4 phage via M. smegmatis vectors to treat M. tuberculosis and M. avium infections [41]. In 2006, the D29 phage directly administered to infected animals without intermediaries showed lytic activity against M. tuberculosis [112]. Aerosolized phage delivery is being explored for respiratory infections, including those in birds and potentially in cystic fibrosis patients [112].

4.4. Clinical Studies in Humans

The first successful human phage therapy for a disseminated antibiotic-resistant infection was reported by Dedrick et al. in 2019 [61]. A 15-year-old cystic fibrosis patient developed a multidrug-resistant M. abscessus subsp. massiliense infection post-lung transplant. After failing antibiotic treatment, she received intravenous phage therapy with three phages (Muddy, ZoeJ, and BP), two of which were genetically modified for lytic activity. Administered twice daily for six months, the therapy was well-tolerated with no severe side effects. Clinical improvement was observed, including wound healing, improved organ function, and weight gain. Follow-up imaging showed reduced inflammation, and no M. abscessus was isolated from samples during or after treatment. Notably, no resistance to the phages developed [61].

4.5. Phage–Antibiotic Combination Therapy

Due to declining antibiotic efficacy, phage–antibiotic combination therapy (PAS—phage–antibiotic synergy) has gained attention [113]. This strategy can lower antibiotic dosages and slow resistance development [109]. Kalapala et al. observed reduced M. smegmatis growth when rifampicin was combined with phages, outperforming antibiotics alone [106]. Cristinziano et al. showed synergy between phages and beta-lactams (azibactam + meropenem) against M. abscessus GD276B [114]. However, aminoglycosides like kanamycin can inhibit phage infection, making their combined use inadvisable [110]. Some studies suggest a limited synergy. Jeyasankar et al. found that phage plus rifampicin or isoniazid yielded no improvement or worse outcomes than phage monotherapy, indicating a lack of synergy [115]. Despite these caveats, more than 10,000 mycobacteriophages have been identified, offering a vast therapeutic potential [116]. While resistance can develop, resistant strains often exhibit reduced virulence, slower growth, and increased antibiotic sensitivity [117].

5. New Technologies to Support the Development of Phage Therapy

According to the Actinobacteriophage database, known also as PhagesDB, there are 14,433 mycobacteriophages with 2618 genomes fully sequenced. For the vast majority of them (14,392 phages), M. smegmatis is the target microorganism, whereas strictly M. tuberculosis is the only host for one mycobacteriophage named DS6A included in the Singleton cluster [58]. A few mycobacteriophages with a broad host range have been described as effective against both tuberculous and non-tuberculous mycobacteria, such as phages G1, J1, and D1 infecting M. tuberculosis, M. avium, M. fortuitum, and M. kansasii or phages Bxz2, D29, and L5 active towards M. tuberculosis, M. avium, M. ulcerans, M. fortuitum, and M. chelonae [118]. Thus, it is evident that when thinking about phage therapy against tuberculosis, there is a need to use modern molecular methods and technologies for phage engineering to develop the phages efficiently infecting TB strains. Nature has prompted the idea since comparative phage genome analysis reveals their extensive mosaicism. Although the mycobacteriophages are currently grouped into 34 clusters (A–Z, AA–AE) and seven singletons based on genome similarity (phages sharing at least 35% of their genes are grouped within the same cluster), many genes are shared between clusters. This mosaicism probably arises from exchanging genetic modules between phages presented in the same host cell via horizontal gene transfer. There is speculation that this mechanism may be an evolutionary strategy of phages for better adaptation to the host organism and to avoid the defense systems [118,119,120]. Mohammed et al. experimentally documented how mosaicism might have developed by the isolation of recombinant mycobacteriophages formed between a Butters prophage (cluster N) and an infecting Island3 genome (subcluster I1) [120]. Unfortunately, the same strategy is used by bacteria to adapt to changing environmental conditions and to ensure survival in the presence of biocidal agents, including phages. It is estimated that up to 10% of the M. tuberculosis genome may have been acquired by an evolutionarily recent horizontal gene transfer. What is more, phage-mediated gene transfer seems to play a significant role in the rearrangement of mycobacterial genomes, and mycobacteriophages have become the genetic tools to engineer mycobacteria [119,121].

The complete sequencing of mycobacteriophages’ genomes, understanding the function of individual genes and developing tools that enable genetic modification, have opened the way to phage engineering. One possibility is to delete fragments of the genome of wild-type phages to change their properties. For example, removing part of the repressor gene can change the replication mode of the phages from temperate, with the capacity to integrate into bacterial genomes, to lytic, which is necessary for therapeutic phages. Dedrick et al. [122], using the BRED (Bacteriophage Recombineering of Electroporated DNA) methodology, described by Marinelli et al. [123] constructed the ZoeJΔ45 mutant with the deletion of repressor gene 45. Due to that, the ZoeJΔ45, infecting a broad spectrum of mycobacterial hosts (M. smegmatis, M. tuberculosis, M. avium, M. bovis BCG, and M. interjectum) as parental phage ZoeJ, could not develop the lysogenic cycle [122]. Similar engineered mycobacteriophages BPsΔ33HTH_HRM10 and D29_HRMGD40 combined with antibiotics were used in vivo to treat a M. abscessus multidrug-resistant lung infection in a patient with cystic fibrosis (CF) [124]. BPsΔ33HTH_HRM10 is a host range mutant (HRM) of an engineered lytic derivative of phage BPs developed by removing part of the repressor gene, rendering it lytic [61,124]. D29_HRMGD40 is an HRM of the D29 lytic mycobacteriophage isolated on an M. abscessus strain GD40, which, unlike the D29 parent, infects several M. abscessus clinical isolates [122,124]. CF affects the function of several organs, including the lungs, that predispose patients to develop chronic, recurrent, multispecies, biofilm-associated bacterial pulmonary infections, most commonly caused by drug-resistant P. aeruginosa, S. aureus, and Bulholderiacepacia complex strains. Chronic inflammation, pulmonary tissue damage, and progressive respiratory insufficiency heavily limit the quality of life and finally cause the premature death of CF patients [124,125,126,127]. A summary of the case report of phage therapy in a 26-year-old man with CF described by Nick et al. [124] is presented in

Table 2. Recent in vivo models of phage therapy against Mycobacterium spp. infections.

Model	Target	Infection	Phage	Therapy Mode	Effects	Reference
Human (CF patient, genotype H199Y/2184insA)	M. abscessus	M. abscessus lung disease, and chronic lung infection with multidrug-resistant (MDR) P. aeruginosa and methicillin-resistant S. aureus (MRSA)	BPsΔ33HTH_HRM10 (a host range mutant of engineered lytic phage BPs with the deleted part of the repressor gene); D29_HRMGD40 (a host range mutant of lytic phage D29)	Phage cocktail administered intravenously (109 to 108 pfu/mLin PBS) twice daily alongside continued antibiotic therapy	Clinical improvements: Decreased lung nodules by day 81, sputum cultures converting to negative by day 118, and the patient underwent successful lung transplantation on day 379	[124]
Mouse (humanized NSG-SGM3 mice expressing human genes for IL-3, GM-CSF, and KITLG)	M. tuberculosis	M. tuberculosis lung infection	Unmodified DS6A	Phage solution administered intravenously (1 × 1011 pfu/dose for a total of 10 doses)	Improvement in the mice’s condition: Increased body weight, improved pulmonary function, and reduced inflammatory markers and M. tuberculosis load in organs (lungs, spleen) with complete eradication in 6 of 9 mice	[13]
Mouse (C57BL/6 mice)	M. tuberculosis	Uninfected mouse to determine the impact of repeated mucosal or systemic delivery on anti-M. tuberculosis response	FionnbharthΔ45Δ47 (engineered lytic phage Fionnbharth with deleted integrase gene 45 and the repressor gene 47)	Phage solution administered weekly for 6 weeks via inhalation or intravenous injection	Inhalation: Phages were delivered across all lungs, well tolerated, and did not induce robust neutralizing humoral immunity Intravenous injection: Growing magnitude of neutralizing IgG and IgA response	[128]

Abbreviations: CF—cystic fibrosis; GM-CSF—granulocyte-macrophage colony stimulating factor; HRM—host range mutant; IL-3—interleukin 3; KITLG (KIT ligand, SCF)—the ligand of the tyrosine-kinase receptor encoded by the KIT locus; and NSG mouse—NOD scid gamma mouse with immunodeficiency.

.

However, there is limited progress in applying phage therapy against M. tuberculosis infections, and the few in vivo studies described so far have involved only animal models (Table 2). First of all, anti-M. tuberculosis activity tested in vitro is not common among mycobacteriophages and appears to be restricted mainly to cluster K, G, and sub-clusters A2 and A3, including the studied TM4, L5, and D29 mycobacteriophages [118,119]. Fortunately, the number of mycobacteriophages being discovered constantly increases, and they frequently expand or switch the host range. Howell et al., analyzing the genomes and predicting the host ranges of P mycobacteriophages, found that subcluster P1 phages are mostly limited to Mycobacterium spp., infecting mainly M. abscessus, M. marinum, and M. smegmatis, but also M. fortuitum, M. gilvum, and M. intracellulare. Meanwhile, subclusters P2–P6 may infect additional bacterial genera, including Gordonia, Rhizobium, Corynebacterium, Clostridioides, and Clostridium. Most cluster P phages have a conserved integration-dependent immunity system, involving integrase (Int), repressor (Rep), and Cro proteins, which controls the switch between lytic and lysogenic cycles and probably also determines host range. So, genetic switch complexity may influence host range evolution [129]. Although the temperate nature of most P phages limits their use in bacteriophage therapy, these observations bring hope for the development of genetic engineering to alter mycobacteriophage specificity and enhance their effectiveness. Dunne et al. used a temperate L. monocytogenes phage PSA, with a very narrow host range restricted to serovar 4b Listeria, as a model for host range re-programming based on receptor binding protein (RBP) modifications [130]. They identified Gp15 as the PSA RBP, showed its crystal structure, and, combining synthetic biology with structure-guided approaches, prepared PSA mutants containing chimeric RBPs that exhibited reprogrammed host specificity and changed plating efficiency [130].

Mycobacteriophages are a promising alternative to antibiotics and chemotherapeutics to treat the infections caused by Mycobacterium spp. However, their use in treating TB still seems to be a long way off. Of course, some unfavorable phenomena impacting treatment efficacy, such as bacterial resistance to phages or different susceptibility patterns resulting from bacterial morphotypes, should also be considered. Bacterial resistance to phages usually arises through mutations affecting the bacterial surface or internal components that interfere with the phage adsorption or lifecycle. However, mutants that totally disrupt phage adsorption have yet to be isolated among Mycobacterium strains [121]. Meanwhile, different susceptibility happens and may be a consequence of the mycobacterial morphotype. Dedrick et al. have demonstrated that smooth strains of M. abscessus are usually less susceptible to phage infection because of high glycopeptidolipid (GPL) content on the cell surface, whereas rough strains are more phage sensitive [131]. Therefore, we will probably never find appropriate phages for all M. tuberculosis strains. Still, in light of the crisis of antibiotic therapy, we must try to expand the therapeutic repertoire to combat bacterial infections, in which the application of phage cocktails or a combination of phages and drugs targeting the bacterial cell surface may prove to be very valuable.

Engineered mycobacteriophages can also be used for TB diagnostics, e.g., to determine the presence of viable tubercle bacilli, drug susceptibility, or treatment efficacy. In 2009, Piuri et al. described the construction of fluoromycobacteriophages based on TM4, able to infect a broad range of mycobacterial hosts, including fast-growing and slow-growing strains such as M. tuberculosis [132]. Fluorophage derivatives of TM4 were developed by gfp or ZsYellow gene fusion with the constitutive M. bovis BCG Hsp60 promoter in plasmid derivatives of pYUB854, transferred then into the phAE87 shuttle phasmid to generate phAE87::hsp60-EGFP and phAE87::hsp60-ZsYellow, respectively. Both engineered fluoromycobacteriophages could infect M. smegmatis mc2155 and mark these bacteria fluorescently, allowing the detection by fluorescence microscopy or flow cytometry in as few as 100 bacterial cells. The effect was specific for mycobacteria, and M. smegmatis was only labeled in a mixed culture containing M. smegmatis and E. coli. However, since the TM4 phage infects a broad range of mycobacterial hosts, the constructed fluoromycobacteriophages detect both M. smegmatis and M. tuberculosis strains, which was described as a limitation for TB diagnostics [132]. Therefore, an addition of p-nitrobenzoic acid (PNB) was next proposed during the diagnostic assay with fluoromycobacteriophages for discrimination between the M. tuberculosis complex and atypical or non-tuberculous mycobacteria strains [133]. Rondón et al. used a second-generation fluoromycobacteriophage, mCherrybombφ, a derivative of TM4 with the optimized expression of an mCherrybombgene in mycobacteria, with an improved fluorescent signal and shorter time to detection of M. tuberculosis using fluorimetry (fluorescence microscopy; flow cytometry) [133]. The results obtained were very promising. Compared to the reference culture method for detecting M. tuberculosis in sputum samples, the sensitivity and specificity of mCherrybombφ after recovering the sample for 96 h reached 91.98 and 98.96%, respectively. Additionally, M. tuberculosis cultures preincubated with PNB (500 μg/mL) before overnight infection with mCherrybombφ had quenched fluorescence. In contrast, phage infection and expression of mCherrybomb were not affected in the non-tuberculous mycobacteria strains tested [133]. Das et al. constructed TM4 expressing the highly sensitive BRET-nanoluciferase-based reporter, GeNL (TM4::GeNL), obtaining LRP for drug screening. It was demonstrated that the M. leprae luminescence output decreased dose dependence when exposed to rifampicin, dapsone, and Q203 for 24 and 48 h, followed by TM4::GeNL infection [134]. M. leprae transformed by the ColE1-integration proficient plasmid expressing GeNL was also detected in mice using an in vivo imaging system [134].

To provide a clear overview of the evolution of mycobacteriophage research, Figure 5 presents a chronological timeline highlighting major discoveries and advancements.

6. Problems, Challenges, and the Future of Phage Therapy

Despite the growing interest in mycobacteriophages as a promising alternative to combat antibiotic-resistant mycobacterial infections, several significant challenges continue to hinder their full clinical implementation. While preliminary results are encouraging, biological, technological, and regulatory barriers must be addressed to realize their therapeutic potential.

6.1. Biological Challenges and Possible Solutions

The successful implementation of mycobacteriophage therapy is limited by several biological obstacles that compromise its reliability and scalability. One of the most critical challenges is the high host specificity of mycobacteriophages, which typically infect only narrowly defined strains of Mycobacterium. This necessitates rapid and precise pathogen identification, which in turn requires access to advanced diagnostics and well-maintained phage libraries—resources often unavailable in urgent or resource-limited settings. The time and cost involved in matching phages to pathogens can thus delay or preclude timely treatment.

Adding to this complexity, Mycobacterium species exhibit an impressive capacity to develop a resistance to phages. Mechanisms include modifications or loss of surface receptors, restriction–modification systems, DNA exonucleases, abortive infection systems, and the CRISPR-Cas adaptive immune system [118,135,136,137,138,139,140,141]. These defenses hinder phage adsorption, degrade phage genomes, or interrupt the infection cycle altogether—paralleling the dynamic resistance seen with antibiotics and necessitating the continuous adaptation of phage formulations.

The first step in the phage infection cycle is the adsorption of the phage to specific surface receptors on the bacterial cell envelope. In Mycobacterium, these receptors may include proteins, glycopeptidolipids, mycolic acids, or complex glycolipids such as trehalose dimycolate and trehalose polyphleates (TPPs). Mutations or deletions in genes encoding these surface molecules can effectively block phage attachment, rendering the bacterium resistant [137]. Furthermore, mycobacteria possess innate immune systems capable of degrading foreign genetic material, including phage DNA. Restriction–modification (R-M) systems recognize specific DNA motifs and cleave non-methylated phage DNA while preserving self-DNA. Although not all mycobacteria harbor highly active R-M systems, their presence can significantly reduce phage replication efficiency [135]. Additionally, some mycobacterial strains possess adaptive immune elements such as CRISPR-Cas systems, which provide sequence-specific immunity by targeting and cleaving previously encountered phage genomes. However, functional CRISPR-Cas systems are rare in Mycobacterium spp., and their role in phage defense remains unclear. Abortive infection (Abi) systems offer population-level protection by inducing cell death in infected bacteria before phage replication is completed [142]. This form of “altruistic suicide” limits phage propagation within bacterial communities. While classical Abi systems have not been fully characterized in mycobacteria, similar mechanisms may exist and often involve toxin–antitoxin (TA) modules. TA systems are widespread in Mycobacterium spp. and are known to contribute to stress adaptation and bacterial persistence through the induction of dormant or non-replicating states. This dormancy may reduce susceptibility to phage infection by limiting the host’s metabolic activity required for phage replication [143].

A particularly formidable obstacle is the intracellular localization of M. tuberculosis, which survives and replicates within macrophages by evading phagosome maturation and neutralizing host defenses [144]. This intracellular niche protects bacteria from extracellular phages and reduces the likelihood of phage-mediated clearance [70]. Moreover, the presence of granulomas and biofilms, as well as the slow growth rate of M. tuberculosis, further limits phage access and efficacy [57].

The genetic and phenotypic heterogeneity of mycobacterial clinical isolates further complicates the application of phage therapy. Differences in cell wall composition, growth rate, or defense systems between strains may lead to variable phage susceptibility, necessitating highly personalized phage preparations and extensive in vitro testing prior to treatment [131].

The host immune response poses another barrier to therapeutic success. Depending on the patient’s immunological status, phage type, dose, and route of administration, immune reactions may neutralize phages or provoke inflammatory or allergic responses. In some cases, increased anti-phage antibody titers have been associated with treatment failure and relapse, requiring a return to conventional antibiotics [145].

Understanding the interplay between phage biology and bacterial defense mechanisms will be crucial for designing robust, durable therapeutic strategies. Further research into host–pathogen–phage dynamics, supported by genomic and proteomic tools, is essential to unlock the full potential of phage therapy against drug-resistant mycobacterial infections. To address these limitations, innovative delivery strategies are being explored. Liposomal encapsulation enhances phage stability, enables intracellular delivery, and protects against immune clearance [146]. Surface ligand-functionalized liposomes may improve the targeted uptake by infected macrophages [147]. Aerosolized phage formulations offer a non-invasive method to deliver phages directly to the lungs—the primary site of TB infection [148]. Other vehicles, such as hydrogels and polymer-based carriers, provide a localized, sustained phage release at infection sites [149]. The genetic engineering of phages also shows promise. For example, the development of trehalose polyphleate (TPP)-independent mutants allows the infection of Mycobacterium strains that have lost these essential glycolipids—a known resistance mechanism. Wetzel et al. demonstrated that point mutations in tail spike proteins of BPs and Muddy phages can restore infectivity against TPP-deficient strains, offering a strategy to overcome phage resistance in clinical contexts [137]. Phage cocktails, composed of multiple strains with diverse host ranges and mechanisms of action, help suppress resistance emergence and are particularly effective in treating chronic infections with heterogeneous bacterial populations [11,135,150]. Moreover, phage–antibiotic combination therapy has shown synergistic effects in preclinical studies, enhancing bacterial clearance, inhibiting biofilm formation, and reducing required antibiotic doses [106,118,151].

The integration of these advanced delivery platforms and engineered phages into personalized therapeutic regimens could significantly enhance the clinical potential of mycobacteriophage therapy. However, overcoming the biological complexity of the host–phage–pathogen triad remains a prerequisite for widespread and consistent clinical success.

6.2. Regulatory and Standardization Barriers

The clinical translation of mycobacteriophage therapy faces considerable regulatory barriers. A major obstacle is the absence of harmonized national and international guidelines, alongside the lack of publicly accessible global phage banks. These gaps hinder standardized development, approval, and distribution processes. In the European Union, mycobacteriophages are classified as biological medicinal products and are subject to rigorous standards for safety, quality, and efficacy—comparable to those governing vaccines. This classification necessitates lengthy and expensive clinical trials. Regulatory ambiguity further complicates matters, particularly in the case of locally administered preparations such as aerosols. It remains unclear whether such therapies should be classified as innovative medicinal products or as personalized interventions, which affects their regulatory pathways. As a result, phage therapy in many EU countries is restricted to experimental or “compassionate use” contexts—permitted only in narrowly defined cases where conventional treatments have failed. Similar regulatory limitations are observed in the United States and parts of Asia, including India and China [11,152,153]. Compounding these issues is the absence of standardized procedures for phage isolation, purification, and quality control. Given the high specificity of phages to particular bacterial strains, therapies often require personalized formulations. This level of customization challenges reproducibility and limits integration into current pharmaceutical regulatory models.

Unlike traditional antibiotics, phages are self-replicating biological entities capable of co-evolving with their bacterial hosts. This unique nature raises additional concerns regarding quality control and regulatory oversight [154]. Although agencies such as the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have begun to develop specific regulatory pathways, these remain limited and inconsistent across jurisdictions [155]. One of the most pressing regulatory issues is also the personalized character of phage therapy. As phage cocktails often need to be tailored to individual patients’ infections, traditional approval models based on fixed compositions are inadequate [156]. Moreover, therapeutic phage formulations may require frequent updates as bacterial resistance emerges, further complicating regulatory compliance.

Despite these challenges, interest in mycobacteriophage-based treatments is growing rapidly. The rise in drug-resistant mycobacterial infections has fueled a surge in preclinical research, early-phase clinical trials, and patent activity [157,158]. Establishing harmonized regulatory frameworks and production standards is essential for improving access, ensuring safety and efficacy, and facilitating broader clinical adoption.

6.3. Cost–Benefit Analysis and Geographic Prioritization

The implementation of mycobacteriophage therapy requires the careful consideration of cost–benefit ratios. While initial development, diagnostic, and production costs are high, especially due to personalized phage preparations, the potential to effectively treat MDR-TB and other difficult infections could significantly reduce long-term healthcare expenses by shortening the treatment duration, lowering hospital stays, and reducing the use of expensive and toxic antibiotics.

Currently, regions with the highest burden of drug-resistant tuberculosis, such as parts of Sub-Saharan Africa, Southeast Asia, and Eastern Europe, represent priority locations where this therapy could have the greatest impact. However, limited healthcare infrastructure and diagnostic capacities in many of these areas present additional challenges to deploying such advanced therapies, emphasizing the need for investment in local diagnostic and phage production capabilities.

7. Limitations of the Review

This review has provided a comprehensive overview of the current state of mycobacteriophage therapy, including biological challenges, technological advances, regulatory considerations, and clinical potentials. Nevertheless, several limitations must be acknowledged. Firstly, the available literature is limited in scope, with relatively few clinical studies and many preclinical reports predominantly using animal models or in vitro systems. This restricts the generalizability of conclusions and underscores the need for robust clinical trials. Secondly, heterogeneity in methodologies, phage preparations, and patient populations among studies complicates direct comparisons and meta-analyses. Furthermore, gaps exist in understanding the long-term safety, immunogenicity, and pharmacokinetics of mycobacteriophage therapy. Lastly, the rapid evolution of phage-resistance mechanisms and the variability of bacterial populations mean that the continuous research and adaptation of phage cocktails will be essential, a factor not yet fully addressed in the current literature.

8. Conclusions

In light of the escalating global threat posed by drug-resistant mycobacterial infections, mycobacteriophages have emerged as a promising and scientifically grounded therapeutic option. This review has highlighted their high specificity, potent bactericidal effects, and engineering flexibility, which collectively position them as strong candidates for the targeted intervention in MDR/XDR tuberculosis and NTM diseases. Advancements in synthetic biology, phage engineering, and delivery systems are steadily overcoming longstanding barriers, offering renewed hope for effective, personalized phage-based therapies. Nonetheless, several critical challenges remain—particularly those related to immune responses, limited host range, phage resistance, and the absence of standardized clinical and regulatory frameworks. Moving forward, multidisciplinary efforts should focus on expanding genetically diverse phage libraries, optimizing delivery methods for intracellular pathogens, elucidating host–phage immune dynamics, and establishing uniform clinical guidelines. In parallel, the diagnostic potential of phage-derived tools warrants further exploration to enable the rapid, point-of-care detection of mycobacterial infections. Ultimately, mycobacteriophages represent more than a theoretical solution—they are becoming a practical and potentially transformative element of next-generation infectious disease management. With continued scientific and clinical investment, phage therapy may soon take its place as a core component of precision medicine in the global battle against drug-resistant mycobacterial diseases.