{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,10,27]],"date-time":"2026-10-27T03:01:01Z","timestamp":1793070061978,"version":"4.5.0"},"posted":{"date-parts":[[2025,7,11]]},"group-title":"Evolutionary Biology","reference-count":24,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,7,11]],"date-time":"2025-07-11T00:00:00Z","timestamp":1752192000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000928","name":"Welch Foundation","doi-asserted-by":"crossref","award":["I-2082-20240404"],"award-info":[{"award-number":["I-2082-20240404"]}],"id":[{"id":"10.13039\/100000928","id-type":"DOI","asserted-by":"crossref"}]},{"name":"NIGMS Grant","award":["R01GM125748"],"award-info":[{"award-number":["R01GM125748"]}]},{"DOI":"10.13039\/100000057","name":"National Institute of General Medical Sciences","doi-asserted-by":"crossref","award":["R35GM133631"],"award-info":[{"award-number":["R35GM133631"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"crossref"}]},{"name":"NIH National Institute for Allergy and Infectious Diseases","award":["R01AI178692"],"award-info":[{"award-number":["R01AI178692"]}]},{"name":"National Science Foundation","award":["MCB-1943442"],"award-info":[{"award-number":["MCB-1943442"]}]},{"name":"NIH","award":["R01GM125748"],"award-info":[{"award-number":["R01GM125748"]}]},{"name":"CZI Theory Institute Without Walls"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,7,11]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>The rapid evolution of extended-spectrum \u03b2-lactamases (ESBLs) represents a global health threat, undermining the efficacy of \u03b2-lactams, the most extensively used antibiotic class. To elucidate the evolutionary dynamics underlying \u03b2-lactam resistance, we constructed a comprehensive combinatorial mutant library comprising all 55,296 possible TEM-1 \u03b2-lactamase variants integrating 18 clinically observed mutations across 13 key residues. Over eight million empirical fitness measurements were obtained under selection pressure with both a native antibiotic substrate (ampicillin) and a novel antibiotic (aztreonam), establishing the largest experimentally determined fitness landscape for antibiotic resistance to date. Through graph-theoretic and epistatic analyses, we discovered that selection with ampicillin resulted in weak epistasis, with mutants rarely surpassing the fitness of the wild-type enzyme. Conversely, aztreonam selection elicited extensive higher-order epistasis, generating a rugged fitness landscape characterized by increased phenotypic unpredictability. Interpretable machine-learning analyses identified context-dependent epistatic interactions necessary for achieving high-level aztreonam resistance. Further evolutionary statistical analyses, including direct coupling analysis and latent generative landscapes, showed that top-performing TEM-1 variants consistently adhered to conserved epistatic patterns found in naturally occurring \u03b2-lactamases. Our findings demonstrate that higher-order epistasis critically shapes fitness landscape ruggedness when enzymes adapt to novel substrates, whereas adaptations to native substrates exhibit predictably smoother landscapes. This integrated experimental and computational framework provides a foundation for predictive evolutionary pharmacology, enabling assessments of newly developed \u03b2-lactams or emerging \u03b2-lactamase variants for their potential contribution to ESBL evolution. Importantly, incorporating graph-theoretically informed evolutionary constraints can strategically disrupt evolutionary pathways, presenting a viable approach to mitigate the rise of antibiotic resistance.<\/jats:p>","DOI":"10.1101\/2025.07.08.663783","type":"posted-content","created":{"date-parts":[[2025,7,12]],"date-time":"2025-07-12T07:40:22Z","timestamp":1752306022000},"source":"Crossref","is-referenced-by-count":3,"title":["Higher-order epistasis drives evolutionary unpredictability toward novel antibiotic resistance"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3397-7294","authenticated-orcid":false,"given":"Ilona Kamila","family":"Gaszek","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1473-0819","authenticated-orcid":false,"given":"Muhammed Sadik","family":"Yildiz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Devin","family":"Meng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jose Alberto","family":"de la Paz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-3112-2632","authenticated-orcid":false,"given":"Sophia Maria","family":"Alvarez","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6208-1561","authenticated-orcid":false,"given":"Faruck","family":"Morcos","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8680-2685","authenticated-orcid":false,"given":"Milo","family":"Lin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4883-7681","authenticated-orcid":false,"given":"Erdal","family":"Toprak","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025071504501831000_2025.07.08.663783v1.1","unstructured":"Wright, S. (1932). The roles of mutation, inbreeding, crossbreeding and selection in evolution. D.F. Jones , ed. 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