Impaired Membrane Energetics Underlies Collateral Sensitivity to Amphenicols During Meropenem Resistance Evolution in...

Collateral sensitivity, an evolutionary trade-off accompanying antibiotic resistance, offers a potential strategy to counteract multidrug resistance. Here, we characterized collateral sensitivity profiles in plasmid-bearing and plasmid-free Escherichia coli (E. coli) lineages evolved under diverse antibiotic selection pressures. Across six independent meropenem (MEM)-evolved lineages derived from two distinct genetic backgrounds, a conserved collateral sensitivity to amphenicols consistently emerged during adaptive laboratory evolution (ALE). Whole genome sequencing identified recurrent mutations in envZ, mrdA and yghB, with yghB as an important genetic determinant associated with amphenicol sensitivity. Functional characterization of yghB disruption revealed that loss of YghB function affected membrane integrity and proton motive force (PMF) homeostasis, accompanied by altered efflux-associated activity, increased intracellular accumulation of amphenicols and altered Mg²⁺ homeostasis. In the ΔyghB background, these alterations were further associated with disrupted ribosome homeostasis and reduced translational capacity. Restoration of Mg²⁺ partially recovered ribosome-associated processes and amphenicol resistance in the ΔyghB mutant, suggesting that Mg²⁺ limitation contributes to yghB-associated amphenicol sensitivity. Furthermore, amphenicol treatment improved infection outcomes against MEM-resistant mutants in both Galleria mellonella and murine septicemia models. Collectively, MEM resistance evolution in E. coli generates robust collateral sensitivity to amphenicols, in which recurrent yghB mutations represent an important genetic basis. Functional analysis of yghB disruption further suggests that altered membrane energetics, efflux-associated processes and Mg²⁺-dependent ribosome homeostasis may contribute to this collateral sensitivity, revealing a potential therapeutic opportunity based on evolutionary trade-offs.