{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,2]],"date-time":"2026-09-02T17:17:06Z","timestamp":1788369426215,"version":"build-2803163510"},"reference-count":46,"publisher":"American Society for Microbiology","issue":"9","license":[{"start":{"date-parts":[[2026,9,2]],"date-time":"2026-09-02T00:00:00Z","timestamp":1788307200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,9,2]],"date-time":"2026-09-02T00:00:00Z","timestamp":1788307200000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R21A1166847"],"award-info":[{"award-number":["R21A1166847"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["UM1AI104681"],"award-info":[{"award-number":["UM1AI104681"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Antimicrob Agents Chemother"],"published-print":{"date-parts":[[2026,9,2]]},"abstract":"ABSTRACT<\/jats:title>\n \n \n \n Hypermutation-driven evolution is a major contributor to antibiotic resistance in some bacterial pathogens, but its role in\n Klebsiella pneumoniae<\/jats:italic>\n remains poorly defined. We analyzed 11 KPC-producing ST258\n K. pneumoniae<\/jats:italic>\n isolates collected serially over ~4 years from a host with persistent colonization and recurrent infections. After >3 years, isolates acquired ceftazidime\u2013avibactam (CZA) resistance with restored carbapenem susceptibility, coinciding with emergence of a V76G substitution in MutH, a conserved endonuclease in the DNA mismatch repair pathway. Isolates carrying\n \n mutH\n V76G<\/jats:sup>\n <\/jats:italic>\n demonstrated sharp increases in within-host genetic diversification (69\u2013179 versus 2\u201312 SNPs), and accumulated mutations across multiple resistance-associated loci, including\n bla<\/jats:italic>\n KPC-3<\/jats:sub>\n ,\n ompK36<\/jats:italic>\n ,\n cirA<\/jats:italic>\n , and\n envZ<\/jats:italic>\n . Both clinical\n \n mutH\n V76G<\/jats:sup>\n <\/jats:italic>\n isolates and CRISPR-Cas9-engineered mutants (\n mutH<\/jats:italic>\n V76G<\/jats:sup>\n and\n mutH<\/jats:italic>\n null mutant) exhibited hypermutator phenotypes and showed accelerated acquisition of resistance or reduced susceptibility to CZA, meropenem\u2013vaborbactam (MVB), and cefiderocol. Using matched isogenic engineered strains, we confirmed that\n \n mutH\n V76G<\/jats:sup>\n <\/jats:italic>\n heightens the pace of resistance evolution\n in vitro<\/jats:italic>\n , enhances plasmid uptake and transfer, and increases bacterial fitness during mouse infections. Resistance pathways that emerged\n in vivo<\/jats:italic>\n paralleled those observed clinically, including\n bla<\/jats:italic>\n KPC-3<\/jats:sub>\n variants under CZA pressure and\n ompK36<\/jats:italic>\n mutations under MVB exposure. The similarity of\n \n mutH\n V76G<\/jats:sup>\n <\/jats:italic>\n and\n mutH<\/jats:italic>\n -null phenotypes indicates that the V76G substitution largely abolishes MutH function. These findings identify MutH-mediated hypermutation as an adaptive strategy in\n K. pneumoniae<\/jats:italic>\n that accelerates resistance to multiple last-line antibiotics and promotes horizontal gene transfer without apparent fitness cost.\n <\/jats:p>\n \n IMPORTANCE<\/jats:title>\n \n Antibiotic-resistant\n Klebsiella pneumoniae<\/jats:italic>\n is a major global health threat, and resistance to new \u201clast-line\u201d antibiotics is rising. In this study, we examined a rare collection of carbapenem-resistant\n K. pneumoniae<\/jats:italic>\n isolates obtained over 4 years from a single host, giving us an opportunity to observe how the bacterium evolved over time. During this period, the bacteria acquired a single nucleotide change in a DNA damage repair gene\n mutH<\/jats:italic>\n , which caused them to accumulate mutations far more rapidly than with wild-type\n mutH<\/jats:italic>\n . Through extensive genomic and experimental work, we found that this single change produced a hypermutator strain capable of quickly developing resistance to several key antibiotics, including ceftazidime\u2013avibactam and meropenem\u2013vaborbactam, and also reduced susceptibility to cefiderocol. Using laboratory-engineered strains, animal infection models, and detailed genetic analyses, we confirmed that the\n mutH<\/jats:italic>\n mutation accelerates resistance development and increases the bacterium\u2019s ability to acquire resistance plasmids. Importantly, the same types of mutations that appeared under laboratory conditions also emerged during infection. Our findings show how hypermutation can compromise even the newest antibiotics and highlight the need for surveillance of DNA repair defects in antibiotic-resistant\n K. pneumoniae<\/jats:italic>\n .\n <\/jats:p>\n <\/jats:sec>\n <\/jats:sec>","DOI":"10.1128\/aac.00189-26","type":"journal-article","created":{"date-parts":[[2026,8,5]],"date-time":"2026-08-05T13:36:16Z","timestamp":1785936976000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Emergence of\n mutH<\/i>\n V76G<\/sup>\n in carbapenem-resistant\n Klebsiella pneumoniae<\/i>\n disrupts DNA mismatch repair and results in a hypermutator phenotype"],"prefix":"10.1128","volume":"70","author":[{"ORCID":"https:\/\/orcid.org\/0009-0000-5055-0543","authenticated-orcid":false,"given":"Shaoji","family":"Cheng","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01an3r305","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Medicine, University of Pittsburgh","place":["Pittsburgh, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1750-4005","authenticated-orcid":false,"given":"Cornelius J.","family":"Clancy","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01an3r305","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Medicine, University of Pittsburgh","place":["Pittsburgh, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Giuseppe","family":"Fleres","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01an3r305","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Medicine, University of Pittsburgh","place":["Pittsburgh, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-7412-0199","authenticated-orcid":true,"given":"Hassan","family":"Badrane","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01an3r305","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Medicine, University of Pittsburgh","place":["Pittsburgh, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8327-4135","authenticated-orcid":true,"given":"Matthew J.","family":"Culyba","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01an3r305","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Medicine, University of Pittsburgh","place":["Pittsburgh, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anthony","family":"Newbrough","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01an3r305","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Medicine, University of Pittsburgh","place":["Pittsburgh, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5845-2235","authenticated-orcid":true,"given":"Liang","family":"Chen","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01y64my43","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Pharmacy and Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo","place":["Buffalo, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2065-3239","authenticated-orcid":true,"given":"M. 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