Mapping the genomic landscape of antibiotic susceptiblity in Escherichia coli

Antibiotic treatment failure is frequently driven by bacterial insusceptibility, yet understanding of the full repertoire of genes contributing to this in Gram-negative bacteria remains incomplete. We used TraDIS-Xpress, a high-density transposon mutagenesis approach, to systematically define the genetic and physiological determinants of Escherichia coli K-12 susceptibility to 13 antibiotics spanning all major drug classes and multiple concentrations. Beyond known resistance determinants, our data reveal that the E. coli core genome plays a central role in modulating susceptibility. Disruption of metabolic, energy-generating, growth-associated, and amino acid biosynthetic pathways commonly enhanced survival under antibiotic stress, indicating that reduced metabolic activity and growth modulation broadly promote survival during exposure to bactericidal drugs. In contrast, pathways involved in DNA replication, peptidoglycan and lipopolysaccharide synthesis, aminoacyl-tRNA biosynthesis, riboflavin metabolism, and terpenoid backbone biosynthesis consistently aided survival. Experimental validation using defined mutants confirmed that disruption of selected metabolic and respiratory pathways confers survival benefits across multiple antibiotic classes. Together, this work provides a genome-scale framework for understanding antibiotic susceptibility in E. coli , identifies conserved physiological pathways underpinning survival, and offers new opportunities for strategies aimed at reversing insusceptibility and improving antibiotic efficacy.