Coupling metabolic enhancement to plasmid spread enables programmable antimicrobial control

The rise of multidrug-resistant pathogens underscores the need for precise antimicrobial strategies beyond conventional antibiotics. Conjugation-based approaches offer a powerful yet underexploited means of delivering targeted genetic interventions within microbial communities. Here, we combine selective killing modules with optimized conjugative vectors to target antibiotic-resistant pathogens and clinically relevant resistance plasmids. First, we engineered toxin-intein modules, programmable cassettes that restrict toxic activity to specific regulatory contexts. We developed modules targeting Shigella spp., Salmonella enterica, and bacteria carrying the resistance plasmid pOXA-48, demonstrating tunable and selective activity at both species and strain levels. To identify the optimal delivery platform, we compared mobilizable and conjugative plasmids, finding that conjugative systems consistently outperform mobilizable ones by approximately one order of magnitude. We then streamlined the broad-host-range plasmid RP4 and enhanced its functionality by incorporating the metabolic fos locus, a type IV pilus operon or both features combined. Integration of the anti-pOXA-48 module into these vectors enabled effective suppression of resistance plasmid spread in complex microbial communities. Together, these results advance the use of conjugative plasmids as programmable antimicrobial platforms and identify delivery efficiency and ecological competitiveness as key determinants of the success of such interventions.