Activity of a Three-Phage Combination against Mycobacterium Tuberculosis in Disease-Relevant Conditions

Background: 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, and stationary-phase conditions in vitro.<br><br>Methods: For time-kill experiments in normal, hypoxic and acidic (pH5·5) conditions, Mtb H37Rv was cultured to exponential phase. An inoculum of 106 colony forming units (CFU)/milliliter (mL) was used. Hypoxia was induced using gas-generating sachets in airtight containers. For stationary growth experiments, Mtb was cultured for 14-21 days and stock was used undiluted. Treatments included control medium, single phages (Fred313cpm-1Δ33, FionnbharthΔ45Δ47and Muddy HRM N0157-2), and phage combinations (multiplicity of infection of 10:1); rifampicin 0·1 µg/mL and isoniazid 0·1 µg/mL. Liquid cocultures were sampled over time. Supernatants were evaluated for phage titers on soft agar overlays containing M. smegmatis mc2155. The remaining cellular fraction was treated for phage inactivation, and ten-fold serial dilutions plated in triplicate to assess Mtb CFU on Middlebrook 7H11 medium.<br><br>Findings: In normal Mtb growth conditions, phage concentrations increased around day seven followed by a significant reduction in CFU, which was maintained over 31 days. Regrowth was observed with rifampicin and isoniazid which was prevented by phage addition. Individual phages were 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.<br><br>Interpretation: 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.