Counterselection against β-lactamase-expressing bacteria via an activatable photosensitizer that accumulates in resistant pathogens.

The rapid global spread of antimicrobial resistance via β-lactamase (bla) demands targeted strategies that selectively eliminate resistant pathogens without exacerbating resistance. Herein, we report BIN-3I, a photosensitizer (PS) that enables bla-selective activation and-more importantly-covalent retention and accumulation within resistant bacteria, allowing potent photodynamic eradication of bla-expressing pathogens and exerting counter-selection pressure. BIN-3I adopts an enzyme-triggered "one-to-multi" design: Upon bla hydrolysis, it undergoes a hydrophilic-to-hydrophobic and low-to-high permeability transition, facilitating uptake and generating a reactive quinone methide intermediate that covalently binds intracellular proteins, leading to >2,000-fold accumulation within resistant bacteria, thiol depletion and light-triggered reactive oxygen species generation. In vitro, BIN-3I demonstrated bla-specific activation, selective enrichment in bla-expressing MRSA, and potent photodynamic killing (>99.999% reduction), outperforming the corresponding uncaged photosensitizer. Crucially, it selectively eradicates bla-producing MRSA within mixed populations, exerting counter-selection pressure against resistant strains. The PS also activates and accumulates in bla-expressing Gram-negative Enterobacter cloacae, albeit with reduced killing efficiency compared to Gram-positive bacteria. In vivo, it exhibited prolonged retention at infection sites and targeted imaging capability in murine myositis and abscess models. Notably, BIN-3I-mediated photodynamic therapy effectively cleared MRSA infections across multiple models-including thigh infection, wound, and biofilm-associated infections-achieving >5-log reduction in bacterial load and surpassing vancomycin in efficacy. This work presents a targeted antimicrobial platform that exploits bacterial resistance mechanisms to achieve species-specific eradication, offering a promising strategy to combat multidrug-resistant infections and alleviate the selection pressure that drives the enrichment of resistant strains.