Mutational basis of ceftazidime-borrelidin A collateral sensitivity in <i>Escherichia coli</i>

The rapid emergence of antimicrobial resistance in bacterial pathogens threatens the efficacy of nearly all available antibiotics. One evolution-informed strategy to limit the emergence of resistance is the exploitation of collateral sensitivity, whereby resistance to one compound results in increased sensitivity to another. Here, we investigate the collateral sensitivity relationship between the cephalosporin antibiotic ceftazidime and the natural product borrelidin A in Escherichia coli. Previously, we found that borrelidin A prevented the evolution of clinical ceftazidime resistance during laboratory selection. In this study, we further characterized the genomic and phenotypic consequences of evolution under collateral sensitivity in these evolved populations. Co-dosing with 128µM borrelidin A significantly reduced the evolution of ceftazidime resistance, while preserving fitness in the absence of drug. Overall co-dosed strains had reduced resistance and cross-resistance to all tested antibiotics. Whole-genome sequencing revealed that co-dosing suppressed the accumulation of single nucleotide polymorphism and insertion/deletion mutations in known resistance-associated genes yet resulted in an increased number of mobile-element insertion mutations. In one case we find a possible novel borrelidin A resistance mutation. Our results suggest that ceftazidime-borrelidin A co-dosing limits both resistance and cross-resistance through selection against costly resistance mutations. While borrelidin A itself is cytotoxic, our findings highlight the promise of targeting bacteria-specific vulnerabilities to curb the emergence of multidrug resistance. These findings contribute to the growing body of evidence supporting collateral sensitivity-informed approaches as practical strategies to mitigate antimicrobial resistance in bacterial populations.