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However, the factors that drive this environmental\u2010to\u2010opportunistic 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\u00a0vitro and\n                    <jats:italic>in cellulo<\/jats:italic>\n                    . A set of 12 NTM, representing both environmental and opportunistic bacteria, was assessed for growth across a pH range of 4.5\u20138.0 in\u00a0vitro, revealing that acid tolerance was species specific. Fluorescent NTM reporters were generated and used to track real\u2010time live cell intracellular replication within human macrophages where acid tolerant species in\u00a0vitro 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.\n                  <\/jats:p>","DOI":"10.1111\/mmi.70060","type":"journal-article","created":{"date-parts":[[2026,3,9]],"date-time":"2026-03-09T06:51:11Z","timestamp":1773039071000},"page":"375-388","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Acidic\n                    <scp>pH<\/scp>\n                    Restricts Non\u2010Tuberculous Mycobacteria Replication"],"prefix":"10.1111","volume":"125","author":[{"given":"Parise K.","family":"Lockwood","sequence":"first","affiliation":[{"name":"Host\u2010Pathogen Interactions in Tuberculosis Laboratory The Francis Crick Institute  London UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Luiz Pedro Sorio","family":"de Carvalho","sequence":"additional","affiliation":[{"name":"Department of Chemistry The Herbert Wertheim University of Florida Scripps Institute for Biomedical Innovation and Technology  Jupiter Florida USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rachel P. 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