Lethal permeabilization of host bacteria to small-molecule compounds during phage penetration.

Tailed phages (Caudoviricetes) inject their double-stranded linear genomic DNA across the bacterial envelope to initiate infection. During this DNA entry process, ionic fluxes and membrane depolarization may occur, potentially leading to increased vulnerability to antibiotic molecules. Here, we report that phage DNA translocation combined with certain small molecules triggers rapid and phage-specific cell permeabilization leading to bacterial growth arrest. We use live-cell fluorescence microscopy to visualize the accumulation of otherwise excluded compounds such as anthracyclines and propidium iodide. We show that the influx and accumulation of these compounds depend on both the presence of an inner-membrane receptor and persistent membrane depolarization. We further demonstrate that phage-induced permeabilization enhances the efficacy of certain antibiotics, enabling synergistic elimination of antibiotic-resistant bacteria. Our results provide a mechanistic foundation for combining phages with small-molecule therapeutics and offer insights into how phage entry relies on and alters host physiology.