Comparative evaluation of culture media for cultivation of Mycobacterium smegmatis mc²155 in the assessment of antimycobacterial activity

The growing prevalence of drug-resistant mycobacteria, including non-tuberculous species, highlights the urgent need for novel therapeutic approaches, among which phage therapy represents a promising strategy. However, the efficacy of antimycobacterial agents is critically dependent on cultivation conditions, making the choice of an optimal culture medium and a reliable model strain (Mycobacterium smegmatis mc²155) essential for accurate activity assessment and the development of effective therapeutics. The aim of the study was to conduct a comparative evaluation of the efficacy of culture media for cultivation of the reference strain Mycobacterium smegmatis mc²155 and for maintaining antimycobacterial activity. Materials and methods. The reference strain Mycobacterium smegmatis mc²155 was used in this study. Inoculations were performed on liquid media: Middlebrook 7H9 supplemented with 10% oleic acid–albumin– dextrose–catalase enrichment and Tween 80, Shkolnikova medium, Sauton's medium, and a modified medium with reduced salt content enriched with glucose and yeast extract. A bacteriophage lysate obtained from a compost sample by enrichment culture on M. smegmatis mc²155 was used as a standardized indicator of lytic activity. The phage lysate titer was 3.7∙10⁷ plaque-forming units per milliliter. Antimycobacterial activity was assessed by adding 0.25 mL of bacterial culture of M. smegmatis mc²155 (final concentration 3.7∙10⁸ colony-forming units per milliliter) and 0.25 mL of phage lysate to tubes containing 5 mL of the corresponding medium. Results. In Middlebrook 7H9 and modified broth supplemented with phage lysate, a decrease in optical density was observed as early as 4 hours, progressing by 48 hours (0.009 ± 0.028). Both media demonstrated a heterogeneous response: in 9 out of 10 replicates, optical density decreased to 0.008–0.009, while in one replicate it remained high (0.080–0.107), indicating the survival of a cell subpopulation, potentially attributable to persister formation or transition to pseudolysogeny. By contrast, no significant decrease in optical density was recorded in Shkolnikova and Sauton's media (optical density remained at 0.168–0.237). Statistical analysis confirmed no significant differences between Middlebrook 7H9 and modified broth (p = 0.92), while both media significantly outperformed Shkolnikova and Sauton's media (p < 0.001). Conclusion. The modified broth is not inferior to the commercial Middlebrook 7H9 medium in supporting antimycobacterial activity (p > 0.05) and may serve as a cost-effective alternative for phage research. These findings underscore the need for standardization of culture media in antimycobacterial activity evaluation and open up opportunities for optimizing screening protocols.

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