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Here, we built a library of species representative of the genus\n                    <jats:italic>Mycobacterium<\/jats:italic>\n                    and determined their antibiotic resistance profiles, allowing for the first time systematic multispecies comparisons. Analyzing antibiotic resistance in the context of other closely related yet diverse organisms revealed species with truly exceptional traits as well as general principles underpinning antibiotic resistance. Among these, we reveal that intrabacterial accumulation of antibiotics does not correlate with their potency at the species level. Our data also reveals that rifamycin resistance in mycobacteria is dominantly caused by antibiotic modification, contrary to what has been observed in\n                    <jats:italic>Mycobacterium tuberculosis<\/jats:italic>\n                    . Our data provides a solid starting point for the exploration of novel determinants of antibiotic resistance. We illustrate the utility of this species-level approach to discovery of novel traits by characterizing a previously unrecognized rifamycin-inactivating enzyme group that is present in a wide range of bacterial genera.\n                  <\/jats:p>","DOI":"10.7554\/elife.101940","type":"journal-article","created":{"date-parts":[[2024,11,11]],"date-time":"2024-11-11T12:25:19Z","timestamp":1731327919000},"update-policy":"https:\/\/doi.org\/10.7554\/elife.101940","source":"Crossref","is-referenced-by-count":1,"title":["A macroevolution-inspired approach to reveal novel antibiotic resistance mechanisms"],"prefix":"10.7554","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8607-6887","authenticated-orcid":true,"given":"Fernanda T","family":"Subtil","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/04tnbqb63","id-type":"ROR","asserted-by":"publisher"}],"name":"Mycobacterial Metabolism and Antibiotic Research Laboratory, The Francis Crick 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