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

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.