Janssen, S.; Larsen, S. E.; Torres, M. P.; Beldjenna, M.; Guerrero Bustamante, C.; Florian, I.; Smytheman, T.; Guo, T.; van Wijk, R.; Hatfull, G. F.; Diacon, A. H.; Coler, R.; van Ingen, J.

Phage therapy offers promise to combat antimicrobial resistance, including drug-resistant tuberculosis (TB). Understanding phage activity against Mycobacterium tuberculosis (Mtb) adapted to physiologic microenvironments, such as hypoxia and acidity in granulomas, is essential since these conditions induce non-replicating states. We evaluated a phage combination against Mtb under hypoxic, acidic (pH 5.5), and stationary-phase conditions in vitro. In planktonic Mtb growth conditions, phage concentrations increased around day seven followed by a significant reduction in Mtb H37Rv load, which was maintained over 31 days. Phage addition prevented regrowth was observed with rifampicin and isoniazid alone. Individual phage stability was differentially affected by acidic media conditions, resulting in variability of antimycobacterial activity. In hypoxic conditions and stationary growth experiments, phage titers remained stable over time with no change in mycobacterial load compared to controls. Model-based predictions were able to adequately capture phage-mycobacterial interactions with and without rifampicin. The lack of antimycobacterial activity in assays with non-replicating mycobacteria suggest that phages need actively replicating mycobacteria to exert lytic activity. Stable phage concentrations in assays with non-replicating mycobacteria suggests low grade phage replication in these conditions. Established models can support future study design through simulations of different experimental scenarios.