Coupling metabolic enhancement to plasmid spread enables programmable antimicrobial control

The rise of multidrug-resistant pathogens underscores the need for precise antimicrobial strategies that extend beyond conventional antibiotics. Conjugation-based approaches offer a powerful yet underexploited means of delivering targeted genetic interventions directly within microbial communities. In this work, we combined selective killing modules with rationally optimized conjugative vectors to target antibiotic-resistant pathogens and clinically relevant antimicrobial resistance plasmids. First, we engineered and validated toxin-intein modules, programmable cassettes that restrict toxic activity to highly specific regulatory contexts. Specifically, we developed and validated modules targeting Shigella spp., Salmonella enterica, and bacteria carrying the resistance plasmid pOXA-48, demonstrating a tunable system capable of selective activity at both the species and strain levels. To identify the most effective delivery platform, we compared mobilizable and conjugative systems and found that, in vitro, conjugative plasmids consistently outperformed mobilizable ones by approximately one order of magnitude. To further optimize delivery, we streamlined the broad-host-range plasmid RP4 and enhanced its functionality by incorporating either the metabolic fos locus, which confers a fitness advantage to cells carrying the delivery vehicle; a type IV pilus operon that promotes mating-pair stabilization and enables efficient conjugation in liquid environments; or both features combined. Using these engineered RP4 derivatives, we integrated the toxin-intein module targeting pOXA-48 and evaluated its performance in complex microbial communities. In this setting, the RP4 variant carrying both the fos locus and the type IV pilus operon effectively blocked the spread of pOXA-48. Together, this work advances the use of conjugative plasmids as robust and programmable platforms to combat antibiotic resistance and enable microbiome engineering. Beyond introducing highly specific antimicrobial modules and a new generation of optimized conjugative vectors, our results identify ecological competitiveness and plasmid transfer dynamics as critical determinants of the success of such interventions.