Metabolic mutations reduce antibiotic susceptibility of E. coli by pathway-specific bottlenecks

Metabolic variation across pathogenic bacterial strains can impact their susceptibility to antibiotics and promote evolution of antimicrobial resistance (AMR). However, little is known about how metabolic mutations influence metabolism and which pathways contribute to antibiotic susceptibility. Here, we measured antibiotic susceptibility of 15,120 Escherichia coli mutants, each with a single amino acid change in one of 346 essential proteins. Across all mutants, we observed modest increases of the minimal inhibitory concentration (2- to 10-fold) without any cases of major resistance. Most mutants that showed reduced susceptibility to either of the two tested antibiotics carried mutations in metabolic genes. The effect of metabolic mutations on antibiotic susceptibility was antibiotic- and pathway-specific: mutations that reduced susceptibility against the β-lactam antibiotic carbenicillin converged on purine nucleotide biosynthesis, those against the aminoglycoside gentamicin converged on the respiratory chain. Additionally, metabolic mutations conferred tolerance to carbenicillin by reducing growth rates. These results, along with evidence that metabolic bottlenecks are common among clinical E. coli isolates, highlight the contribution of metabolic mutations for AMR.