Mulkern, A. J. | Search bioRxiv

bioRxiv2026

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. SignificanceThis 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.

microbiology↗

bioRxiv2024

A systematic identification of resistance determinants to antisense antibiotics suggests adaptation strategies dependent on the delivery peptide

The rise of antimicrobial resistance (AMR) among human pathogenic microbes is a serious threat to global health, calling for the development of novel treatment strategies. Antibiotics based on programmable antisense oligomers (asobiotics) offer an attractive solution to the "arms-race", as their specificity can be quickly updated and tailored to target resistant bacteria. In order to understand the genetic architecture of resistance to asobiotics, we employed laboratory evolution assays to identify mutations that decrease susceptibility to antisense peptide nucleic acid (PNA) against four major gram-negative pathogens: Escherichia coli, Klebsiella pneumoniae, Salmonella enterica, and Pseudomonas aeruginosa. We observed that the reduction in susceptibility upon asobiotics treatment was dependent on the specific cell penetrating peptide (CPP) being conjugated to the PNAs, suggesting that reduced uptake is a common adaptation strategy only against the (KFF)3-K CPP. We in fact observed that sbmA was frequently mutated in all tested species when treated with (KFF)3-K conjugated PNAs. We further identified mutations related to translation, peptide transport and cell envelope, which provide new hypotheses on cellular response to CPP-PNAs conjugates. Furthermore, for (RXR)4XB-acpP we observed a modest increase in resistance only when mutations in the PNA binding site were induced, which could easily be bypassed by changing the PNA sequence. These findings indicate that the specific identity of the CPP used plays a key role in determining its robustness against the evolution of resistance, and that laboratory evolution can illuminate the remaining gaps in our knowledge on the mechanisms of action of asobiotics.

microbiology↗