Acidic <scp>pH</scp> Restricts Non‐Tuberculous Mycobacteria Replication

Non-tuberculous mycobacteria (NTM) are becoming increasingly prevalent in clinical settings with certain environmental species becoming opportunistic in both immunocompetent and immunodeficient hosts. However, the factors that drive this environmental-to-opportunistic switch remain poorly understood. NTM are environmental organisms that commonly thrive in acidic niches. Conversely, in host cells, acidic pH acts as a defence mechanism. Here, we investigated how acidic pH impacts NTM replication both in vitro and in cellulo. A set of 12 NTM, representing both environmental and opportunistic bacteria, was assessed for growth across a pH range of 4.5-8.0 in vitro, revealing that acid tolerance was species specific. Fluorescent NTM reporters were generated and used to track real-time live cell intracellular replication within human macrophages where acid tolerant species in vitro replicated with a higher efficiency in cellulo than acid sensitive NTM. Further neutralisation of the phagosomal pH using bafilomycin A1 (BafA1) led to enhanced intracellular replication and survival of NTM. This study demonstrated that specific environmental adaptations may determine the opportunistic potential of NTM whilst acidic pH within the host serves as a key host defence mechanism to restrict NTM growth.