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This lineage is subdivided into four extant main subclades A, B, C1 and C2 that exhibit distinct features in terms of invasiveness, antibiotic resistance and transmissibility. However, the evolutionary pathway from the generally susceptible ST131-B to the drug-resistant ST131-C clades remains poorly mapped. To fill this knowledge gap, we analysed in detail human clinical isolates obtained in Vietnam, designated as belonging to the generally neglected minor clade ST131-B0. We sequenced them using both short- and long-read technology and combined these data with a recently published high-resolution genomic collection to provide further insight into the evolutionary process and its timeline. Extensive genomic analyses established ST131-B0 as an intermediary progenitor in the evolutionary path leading from ST131-B to the ST131-C clades, associated with multiple type I pili switches, as well as the loss and gain of specific chromosomal genes representing diverse core functions such as metabolism, transcription, DNA binding and type II toxin\u2013antitoxin systems. Furthermore, all Vietnamese isolates of ST131-B0 unprecedentedly harboured\n                    <jats:italic>bla<\/jats:italic>\n                    <jats:sub>CTX-M<\/jats:sub>\n                    genes encoding extended-spectrum \u03b2-lactamases, a trait dominant in ST131-C clades and not previously observed in ST131-B0. Our study supports the hypothesis that the ST131-C clades have gradually evolved from ST131-B by reducing the host range with better adaptation to colonizing humans under selective conditions.\n                  <\/jats:p>","DOI":"10.1099\/mgen.0.001795","type":"journal-article","created":{"date-parts":[[2026,9,23]],"date-time":"2026-09-23T16:16:15Z","timestamp":1790180175000},"source":"Crossref","is-referenced-by-count":0,"title":["Mapping the evolutionary path towards multi-drug resistance in the pandemic Escherichia coli ST131 lineage"],"prefix":"10.1099","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3160-2042","authenticated-orcid":false,"given":"Anna K.","family":"P\u00f6ntinen","sequence":"first","affiliation":[{"name":"Department of Biostatistics, Faculty of Medicine, University of Oslo, Oslo, Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3656-533X","authenticated-orcid":false,"given":"Nguyen 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