Mycobacteriophages are viruses that selectively infect mycobacteria. Due to their specificity and lytic activity, they are considered a promising tool for controlling mycobacterial infections, especially amid rising antibiotic resistance. Their significant genetic diversity and unique biological properties have generated sustained global research interest, supported by successes in treating complex infections caused by non-tuberculous mycobacteria. However, data on phage populations and applied research results in the Russian Federation remain fragmented, necessitating systematization to assess the genuine potential. This review is based on literature data and the authors’ personal results, summarizing current knowledge on mycobacteriophage biology and analyzing the prospects for their therapeutic and diagnostic application. It examines data on mycobacteriophage taxonomic and genomic diversity in a global context, describes the first characterized Russian isolates, including phages Vic9 and Yasnaya_Polyana, and details a domestic collection of 15 phages. The collection aligns with global trends and includes unique findings, such as the phage Arbat with an anomalously large genome and extremely low similarity to known phages. In the therapy context, the review explores strategies for creating effective therapeutic agents, including the design of phage cocktails and genetic modification to switch them to a lytic state. It presents a critical analysis of international clinical experience using phages in parallel with mycobacterioses and highlights fundamental barriers to phage therapy for tuberculosis. Special attention is paid to domestic delivery systems, particularly liposomal formulations of phage D29, which have demonstrated high efficacy against M. tuberculosis, including multidrug-resistant strains, and a favorable safety profile in preclinical studies. In the diagnostics section, the evolution of phage-based methods is traced — from biological amplification tests to reporter systems — and the Russian-developed test system “TB-Phage-LCh” for phenotypic tuberculosis drug susceptibility testing is presented. Despite regulatory complexities and the need to study immunogenicity, this work demonstrates that Russian Federation possesses a solid scientific foundation for developing this field. Further research focused on in-depth characterization of domestic isolates, optimization of therapeutic platforms, and conducting controlled clinical trials could lead to creating effective phage-based drugs and diagnostic tools capable of holding an important place in the arsenal against drug-resistant mycobacterial infections.

The increasing prevalence of drug-resistant Mycobacteriumtuberculosis (Mtb) and nontuberculous mycobacteria (NTM) underscores the need for novel strategies. Mycobacteriophages are attracting growing interest for therapeutic and diagnostic applications. Here, we describe four new subcluster A3 phages (Cat, Goose1D, Goose1X, and Shep) and investigate their genomic diversity and lytic activity. The phages share >98% nucleotide identity with known A3 members but carry distinct deletions within the lysogeny module: Cat and Goose1X have different repressor-loss variants, and Goose1D lacks the integrase gene, whereas Shep retains an intact module. All phages lysed M. tuberculosis H37Rv, with efficiencies of plating (EOPs) of 10⁻²–10⁻¹, but showed limited activity against NTM. Cat was the most effective against drug-resistant Mtb isolates (EOPs of 10⁻¹–1), remained stable across a broad pH and temperature range, but had a low burst size (8 PFU (plaque-forming units/cell). In lysogeny assays, 6% of Cat-exposed M. smegmatis colonies exhibited a lysogen-like phenotype, but no phage DNA was detected, suggesting spontaneous resistance. Sequencing of one strain revealed an H535Q substitution in the pyruvate carboxylase MSMEG_2412. These findings highlight lysogeny module plasticity in closely related phages and position Cat, with its naturally occurring repressor deletion and potent lytic activity, as a promising candidate against drug-resistant tuberculosis.