Targeting nontuberculous mycobacteria with phages: optimization of in vitro host range assays

The clinical implementation of mycobacteriophages is hampered by the lack of standardized in vitro methods and inconsistent host range results, limiting reliable assessment of their therapeutic potential. Nontuberculous mycobacterial (NTM) infections are frequently difficult to treat, emphasizing the urgent need for alternative or adjunctive strategies to antibiotics. To facilitate reproducible translational research, a detailed and systematic understanding of in vitro assay conditions is essential. Here, we comprehensively evaluated commonly used liquid and solid media for their suitability in supporting growth of multiple NTM strains and subsequent phage application. We compared bacterial growth kinetics, aggregation tendencies, and the efficiency of phage-induced lysis, including plaque formation, across three liquid media and corresponding solid agars. Our results demonstrate that Middlebrook-based media and agar consistently support robust NTM growth, reduce bacterial clumping, and enable reproducible detection of phage activity, outperforming TSB-based alternatives despite higher cost and labor requirements. These findings provide a standardized framework for in vitro NTM cultivation and phage testing, which is critical for reliable evaluation of host range, infection dynamics, and the development of mycobacteriophage-based therapies.