{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,4,12]],"date-time":"2026-04-12T11:47:25Z","timestamp":1775994445995,"version":"3.50.1"},"posted":{"date-parts":[[2025,4,11]]},"group-title":"Microbiology","reference-count":63,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,4,11]],"date-time":"2025-04-11T00:00:00Z","timestamp":1744329600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/publicdomain\/zero\/1.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,4,11]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  Gene amplifications are thought to be common in bacterial populations, providing a rapid reversible mode of adaptation to diverse stresses, including the acquisition of antibiotic resistance. We previously showed that the opportunistic pathogen\n                  <jats:italic>Staphylococcus aureus<\/jats:italic>\n                  evolves resistance to the dual-targeting fluoroquinolone delafloxacin (DLX) that inhibits both the DNA gyrase and DNA topoisomerase IV via gene amplifications of an efflux pump encoding gene\n                  <jats:italic>sdrM<\/jats:italic>\n                  . However, the pathways that control the formation or selection of gene amplifications, and consequently adaptive trajectories, remain understudied, especially in gram-positive bacteria like\n                  <jats:italic>S. aureus<\/jats:italic>\n                  . Here, we show that specific DNA repair and chromosomal separation pathways alter the frequency of formation and selection of gene amplifications in\n                  <jats:italic>S. aureus<\/jats:italic>\n                  . Through a screen of 36 mutants deficient in various DNA processes, we found that while\n                  <jats:italic>sdrM<\/jats:italic>\n                  amplification was still the almost universal path to DLX resistance, other mutations that increased\n                  <jats:italic>sdrM<\/jats:italic>\n                  expression reduced the selection frequency of\n                  <jats:italic>sdrM<\/jats:italic>\n                  amplifications, demonstrating the critical role of\n                  <jats:italic>sdrM<\/jats:italic>\n                  in DLX resistance. We found that similar to other bacteria, the formation and loss of\n                  <jats:italic>sdrM<\/jats:italic>\n                  amplifications required a functional RecA recombinase, but multiple other mutants in pathways required for amplifications in other species still exhibited frequent\n                  <jats:italic>sdrM<\/jats:italic>\n                  amplifications, suggesting that\n                  <jats:italic>S. aureus<\/jats:italic>\n                  may have alternate routes of amplification formation. Finally, mutants in the tyrosine recombinase XerC that is involved in chromosomal separation were deficient for\n                  <jats:italic>sdrM<\/jats:italic>\n                  amplifications, indicating that XerC is a novel modulator of amplification formation, maintenance, or selection. Thus, our work sheds light on genetic factors that alter gene amplification-mediated evolutionary trajectories to antibiotic resistance in\n                  <jats:italic>S. aureus<\/jats:italic>\n                  and can potentially unlock mechanisms by which such evolution of resistance can be inhibited.\n                <\/jats:p>","DOI":"10.1101\/2025.04.10.647645","type":"posted-content","created":{"date-parts":[[2025,4,12]],"date-time":"2025-04-12T01:30:41Z","timestamp":1744421441000},"source":"Crossref","is-referenced-by-count":0,"title":["Genetic determinants of gene amplifications alter frequency and evolutionary trajectory of antibiotic resistance in\n                  <i>Staphylococcus aureus<\/i>"],"prefix":"10.64898","author":[{"given":"Kalinga Pavan T.","family":"Silva","sequence":"first","affiliation":[{"name":"Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1231-670X","authenticated-orcid":false,"given":"Anupama","family":"Khare","sequence":"additional","affiliation":[{"name":"Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2026041204035095000_2025.04.10.647645v1.1","doi-asserted-by":"publisher","DOI":"10.15252\/embr.202051034"},{"key":"2026041204035095000_2025.04.10.647645v1.2","doi-asserted-by":"publisher","DOI":"10.1038\/s41579-021-00649-x"},{"key":"2026041204035095000_2025.04.10.647645v1.3","doi-asserted-by":"publisher","DOI":"10.1016\/S0140-6736(21)02724-0\/ATTACHMENT\/4504FC2B-08AE-46D6-BD75-80811DF8B5E6\/MMC1.PDF"},{"key":"2026041204035095000_2025.04.10.647645v1.4","doi-asserted-by":"crossref","first-page":"1199","DOI":"10.1016\/S0140-6736(24)01867-1","article-title":"Global burden of bacterial antimicrobial resistance 1990\u20132021: a systematic analysis with forecasts to 2050","volume":"404","year":"2024","journal-title":"The Lancet"},{"key":"2026041204035095000_2025.04.10.647645v1.5","doi-asserted-by":"publisher","DOI":"10.1038\/nrmicro2200"},{"key":"2026041204035095000_2025.04.10.647645v1.6","doi-asserted-by":"publisher","DOI":"10.1128\/microbiolspec.gpp1123-0057-2018"},{"key":"2026041204035095000_2025.04.10.647645v1.7","doi-asserted-by":"publisher","DOI":"10.1038\/nrmicro2174"},{"key":"2026041204035095000_2025.04.10.647645v1.8","doi-asserted-by":"crossref","unstructured":"Silva, K. 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