Mycobacterial infections caused by tuberculous and non-tuberculous strains pose significant treatment challenges, especially among immunocompromised patients. Conventional antibiotic therapies often fail due to bacterial resistance, highlighting the need for alternative therapeutic strategies. Mycobacteriophages are emerging as promising candidates for the treatment of mycobacteria. This review comprehensively explores phage isolation, characterization, and clinical applications. Despite the need for more extensive in vitro and in vivo studies, existing evidence shows their efficacy against both sensitive and antibiotic-resistant mycobacterial strains, even under disease-mimicking conditions, particularly when used in cocktails to minimize resistance development. Mycobacteriophages can be engineered and evolved to overcome limitations associated with lysogeny and narrow host range. Furthermore, they exhibit activity in ex vivo and in vivo infection models, successfully targeting mycobacteria residing within macrophages. Delivery methods such as bacterial and liposomal vectors facilitate their entry into human cells. Considering the potential for phage-treatment-induced bacterial resistance, as described in this review, the combination of mycobacteriophages with antibiotics shows efficacy in countering mycobacterial growth, both in the laboratory setting and in animal models. Interestingly, phage-encoded products can potentiate the activity of relevant antibiotics. Finally, the application of phages in different compassionate cases is reported. The positive outcomes indicate that phage therapy represents a promising solution for the treatment of antibiotic-resistant mycobacteria.

Background/Objectives: The ability of bacteria to form biofilms makes them more tolerant to traditional antibiotics. Given the lack of new antibiotic development, drug repurposing offers a strategy for discovering new treatments. Auranofin (AF), a gold-based compound indicated for the treatment of rheumatoid arthritis, shows promising antibacterial activity. This study investigates the antimicrobial and antibiofilm activity of AF and its two derivatives in which the thiosugar ligand is replaced by acetylcysteine (AF-AcCys) or naproxen (AF-Napx), against Staphylococcus aureus and Staphylococcus epidermidis. Methods: AF was conjugated by transmetalation with either naproxen or acetylcysteine. Assessments of their stability in DMSO/H₂O and lipophilicity expressed as the LogP were performed. The antimicrobial activity of AF and its analogues were investigated by broth microdilution assay to determine the minimum inhibitory concentration (MIC) and versus biofilm to obtain the minimum bactericidal biofilm concentration (MBBC) and minimum biofilm eradication concentration (MBEC). Results: AF derivatives were found to be stable in a DMSO/H₂O mixture for 48 h. AF-Napx showed a LogP = 1.25 ± 0.22, close to AF, while AF-AcCys had a LogP = −0.95. MIC values of S. aureus and S. epidermidis were ranging from 2 µM to 0.25 µM, and ≤0.12 µM, respectively. Both AF and AF-Napx maintained efficacy against biofilm-embedded S. aureus and S. epidermidis at non-cytotoxic concentrations, with AF-Napx demonstrating lower MBBC values for S. epidermidis. Conclusions: AF, and especially its naproxen conjugate, holds potential as a therapeutic agent for treating biofilm-associated infections caused by S. aureus and S. epidermidis, particularly in device-related infections where both infection and inflammation are present.

Pseudomonas aeruginosa is a major opportunistic pathogen with high levels of antibiotic resistance. Phage therapy represents a promising alternative for the treatment of difficult infections both alone and in combination with antibiotics. Here, we isolated and characterized three novel lytic myoviruses, Cisa, Nello, and Moonstruck. Genomic analysis revealed that Cisa and Nello belong to the Pbunavirus genus, while Moonstruck is a novel Pakpunavirus species. All lacked lysogeny, virulence, or resistance-associated genes, supporting their therapeutic suitability. Phage Nello and Moonstruck were active against P. aeruginosa Pa3GrPv, isolated from a patient with lung infection candidate for phage therapy. Moonstruck exhibited superior lytic activity with ciprofloxacin sub-MIC value (0.125 µg/mL), achieving bacterial suppression for 48 h. However, to improve the lytic efficacy of the phages on the clinical isolate, phage adaptation via serial passage was investigated. The killing efficacy of Nello was enhanced, whereas Moonstruck showed a less consistent improvement, suggesting phage-specific differences in evolutionary dynamics. Sequencing of the evolved phages revealed point mutations in tail-associated genes, potentially linked to a better phage–host interaction. These results support the use of phage–antibiotic combinations and directed evolution as strategies to enhance phage efficacy against drug-resistant infections. Overall, these findings support the therapeutic potential of the newly isolated phages in treating P. aeruginosa lung infections.