DNA-damaging combination treatments impose genotype-specific constraints on hypermutator evolvability

Bacterial hypermutator strains drive rapid evolution of antibiotic resistance in chronic infections. Inspired by cancer therapy approaches that exploit synthetic lethality by targeting DNA repair deficiencies in hypermutator tumours, we tested whether pairing a conventional antibiotic with a secondary DNA-damaging agent could constrain hypermutator evolution in bacteria. Using high-throughput experimental evolution of Escherichia coli repair-deficient strains, we evolved populations under carbapenem selection in combination with ciprofloxacin or mitomycin C. Strains lacking oxidative damage repair, double-strand break repair, or transcription-coupled repair showed significantly reduced evolvability, particularly under constant antibiotic pressure and increasing genotoxic stress. However, mismatch repair (MMR) hypermutators, the predominant clinical genotype, did not show reduced evolvability under these combination treatments. This is consistent with pathway orthogonality: MMR does not repair the structural DNA lesions induced by ciprofloxacin or mitomycin C, and the elevated mutation supply of MMR-deficient strains may allow rapid adaptation despite background DNA damage. Our findings demonstrate that combination strategies can constrain the evolvability of specific repair-deficient genotypes in vitro , but success requires matching DNA damage type to specific repair vulnerabilities. This work establishes proof of principle for genotype-directed antimicrobial strategies that exploit DNA repair vulnerabilities to constrain hypermutator evolution. Significance This work demonstrates that combining DNA-damaging agents with antibiotics can constrain resistance evolution in specific hypermutator genotypes, but not others. These findings establish that evolution-informed antimicrobial strategies must be genotype-specific, opening new paths for precision approaches to delay or prevent the evolution of antibiotic resistance by exploiting DNA repair vulnerabilities.