Emergence of mutH V76G in carbapenem-resistant Klebsiella pneumoniae disrupts DNA mismatch repair and results in a hypermutator phenotype

ABSTRACT Hypermutation-driven evolution is a major contributor to antibiotic resistance in some bacterial pathogens, but its role in Klebsiella pneumoniae remains poorly defined. We analyzed 11 KPC-producing ST258 K. pneumoniae isolates collected serially over ~4 years from a host with persistent colonization and recurrent infections. After >3 years, isolates acquired ceftazidime–avibactam (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 mutH V76G demonstrated sharp increases in within-host genetic diversification (69–179 versus 2–12 SNPs), and accumulated mutations across multiple resistance-associated loci, including bla KPC-3 , ompK36 , cirA , and envZ . Both clinical mutH V76G isolates and CRISPR-Cas9-engineered mutants ( mutH V76G and mutH null mutant) exhibited hypermutator phenotypes and showed accelerated acquisition of resistance or reduced susceptibility to CZA, meropenem–vaborbactam (MVB), and cefiderocol. Using matched isogenic engineered strains, we confirmed that mutH V76G heightens the pace of resistance evolution in vitro , enhances plasmid uptake and transfer, and increases bacterial fitness during mouse infections. Resistance pathways that emerged in vivo paralleled those observed clinically, including bla KPC-3 variants under CZA pressure and ompK36 mutations under MVB exposure. The similarity of mutH V76G and mutH -null phenotypes indicates that the V76G substitution largely abolishes MutH function. These findings identify MutH-mediated hypermutation as an adaptive strategy in K. pneumoniae that accelerates resistance to multiple last-line antibiotics and promotes horizontal gene transfer without apparent fitness cost. IMPORTANCE Antibiotic-resistant Klebsiella pneumoniae is a major global health threat, and resistance to new “last-line” antibiotics is rising. In this study, we examined a rare collection of carbapenem-resistant K. pneumoniae 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 mutH , which caused them to accumulate mutations far more rapidly than with wild-type mutH . 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–avibactam and meropenem–vaborbactam, and also reduced susceptibility to cefiderocol. Using laboratory-engineered strains, animal infection models, and detailed genetic analyses, we confirmed that the mutH mutation accelerates resistance development and increases the bacterium’s 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 K. pneumoniae .