Emergence of mutH V76G among longitudinal carbapenem resistant Klebsiella pneumoniae causing long-term colonization and recurrent infection disrupts DNA mismatch repair and results in a hypermutator phenotype.

Although hypermutation due to Mut protein mutations that disrupt DNA mismatch repair has been characterized in some bacteria, its mechanisms and consequences in Klebsiella pneumoniae remain poorly defined. We analyzed 11 longitudinal KPC-3 carbapenemase-producing, ST258 K. pneumoniae isolates collected over ~4 years from an immunocompromised patient with chronic colonization and recurrent infections. After ~3.3 years, isolates developed ceftazidime-avibactam (CZA)-resistance with restored carbapenem susceptibility, coinciding with emergence of a V76G substitution in a highly-conserved motif in the core of MutH endonuclease. Compared with earlier isolates, mutH V76G-carrying isolates showed greater within-host genomic diversification (69-179 vs. 2-12 SNP differences) and acquired bla KPC-3L169P, encoding an KPC Ω-loop substitution that mediates CZA resistance and re-establishes carbapenem susceptibility. mutH V76G isolates exhibited stepwise increases in meropenem-vaborbactam (MVB) and cefiderocol minimum inhibitory concentrations, plausibly linked to substitutions in KPC, OmpK36 porin, CirA iron transporter and/or EnvZ kinase. Clinical mutH V76G isolates and CRISPR-engineered mutH V76G mutants were hypermutators based on rifampin mutational frequencies. Using isogenic mutant and parent strains, we confirmed that mutH V76G accelerated evolution of CZA and MVB resistance in vitro and in vivo, promoted transfer and uptake of resistance plasmids, and improved bacterial fitness during mouse infections. Resistance evolution in mice recapitulated clinical trajectories, with bla KPC-3 and ompK36 mutations emerging under CZA and MVB exposure, respectively. Phenotypes of mutH V76G and mutH-null strains were comparable, indicating that the V76G substitution largely abrogates MutH function. Our findings reveal MutH-mediated hypermutation as an adaptive mechanism in K. pneumoniae, enabling rapid antibiotic resistance development and plasmid acquisition without fitness cost.