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 pathogens is a serious threat to global health, demanding novel treatment strategies. Antibiotics based on programmable antisense oligomers (asobiotics) offer an attractive solution, as their specificity can be quickly updated to target resistant bacteria. To understand the genetic architecture of resistance to asobiotics, we used laboratory evolution assays to identify mutations that decrease susceptibility to antisense peptide nucleic acids (PNAs) in four major gram-negative pathogens: Escherichia coli, Klebsiella pneumoniae, Salmonella enterica, and Pseudomonas aeruginosa. Reduced susceptibility depended on the cell-penetrating peptide (CPP) conjugated to the PNA, with reduced uptake emerging as a common adaptation only against the (KFF)3-K CPP. Accordingly, sbmA was consistently mutated across all species treated with (KFF)3-K-conjugated PNAs. We further identified mutations related to translation, peptide transport, and the cell envelope, generating new hypotheses on the cellular response to CPP-PNA conjugates. By contrast, for (RXR)4XB-acpP we observed only a modest resistance increase, driven by mutations in the PNA binding site that could be readily bypassed by changing the PNA sequence. These findings show that CPP identity strongly determines robustness against resistance evolution, and that laboratory evolution can illuminate the mechanisms of action of asobiotics.