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Bacteriophages (phages), which are viruses that infect and kill bacteria, are actively being explored as an alternative approach for treating mycobacterial diseases. Several compassionate-use cases of phage therapy for drug-resistant NTM infections exhibit favorable outcomes. To further the development of phage therapy, it is important to recognize and avoid conditions that negatively impact phage activity during phage production, storage, formulation, or treatment. Conversely, there is a need to inactivate free phages in certain preclinical phage therapy experiments. In this study, we investigated three mycobacteriophages BPs\u0394\n                      <jats:italic toggle=\"yes\">33<\/jats:italic>\n                      HTH-HRM10, Muddy, and ZoeJ\u0394\n                      <jats:italic toggle=\"yes\">45<\/jats:italic>\n                      from compassionate-use NTM treatment cases for their sensitivity to a variety of conditions that included temperature, acid pH, detergents, mucus, and phage inactivating buffers. Several conditions resulted in dramatic and rapid reductions in the level of active phage, while others had no effect. We also observed different sensitivities between the phages. The results provide valuable information to support further investigation and development of these phages as therapeutics.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>Bacteriophages (phages) offer a promising alternative therapy for treating drug-resistant mycobacterial infections. For the successful implementation of phage therapy, it is important to recognize conditions that inactivate the phages. Here, we studied three mycobacteriophages from recent compassionate-use phage therapy cases for their sensitivity to a range of conditions that may be encountered in production, storage, formulation, or treatment. The results demonstrate sensitivity to some conditions and tolerance to others, and they additionally reveal phage-specific differences in sensitivities, highlighting the need for direct evaluation of individual therapeutic phages during development.<\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.02655-25","type":"journal-article","created":{"date-parts":[[2026,2,11]],"date-time":"2026-02-11T14:00:10Z","timestamp":1770818410000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":1,"title":["Inactivating conditions of therapeutic mycobacteriophages"],"prefix":"10.1128","volume":"14","author":[{"given":"Andrew","family":"Wiggins","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03k1gpj17","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Microbiology, Immunology, and Pathology, Mycobacteria Research Laboratories, Colorado State 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