Phase‐Separated Nano‐Antibiotics Enhanced Survival in Multidrug‐Resistant Escherichia coli Sepsis by Precise Periplasmic EcDsbA Targeting

Abstract Disulfide bond (Dsb) proteins, especially DsbA, represent a promising but as‐yet‐unrealized target in combating multidrug‐resistant (MDR) bacteria because their precise subcellular targeting through multibarrier remains a significant challenge. Here, a novel heterogenization‐phase‐separated nano‐antibiotics ( NCefoTs ) is proposed, through the co‐assembly of enzyme‐inhibiting lipopeptides (ELp component), membrane‐recognizing and disrupting lipopeptides (MLp component), and cefoperazone. The self‐sorting components of MLp “concentrated island‐liked clusters” on the surface of NCefoTs promote the efficient penetration of NCefoTs through the outer membrane. Triggered by the DsbA, the precisely spatiotemporal engineered NCefoTs transform to nanofibers in situ and further significantly enhance the inhibition of DsbA. The hydrolytic activity of β‐lactamase and the motility function of flagella are thereby impeded, confirming the efficacy of NCefoTs in restoring susceptibility to antibiotics and inhibiting infection dissemination. By these synergistic effects of NCefoTs , the minimum inhibitory concentration of antibiotics decreases from over 300 µM to 1.56 µM for clinically isolated E. coli MDR. The survival rate of sepsis‐inflicted mice is significantly enhanced from 0% to 92% upon encapsulation of cefoperazone in NCefoTs , which rapidly eliminates invading pathogens and mitigates inflammation. The universally applicable delivery system, based on an “on demands” strategy, presents a promising prospect for undruggable antibiotic targets in the periplasm to combat MDR bacteria.