Plasmid-plasmid interactions reshape extracellular vesicle cargo and antimicrobial resistance gene transfer potential

Plasmids are major drivers of bacterial horizontal gene transfer, including transfer of antimicrobial resistance. Plasmid dissemination extends beyond conjugation to include extracellular vesicle (EV)-mediated transport. Although bacterial EVs can carry plasmid DNA, mechanisms governing which plasmids are incorporated into EVs remain poorly understood. Here, we tested whether principles of conjugative plasmid transfer, including effects of plasmid mobility type and plasmid-plasmid interactions, extend to EV-mediated export. Using model conjugative (pKJK5) and mobilizable (RSF1010) resistance plasmids in two independent hosts, we quantified plasmid incorporation into EVs under single- and dual-plasmid conditions. Despite its lower intracellular copy number, the conjugative pKJK5 was preferentially incorporated into EVs, exceeding RSF1010 by 10- to 23-fold under single-plasmid carriage. Under co-residence, this pattern reversed: RSF1010 enrichment increased by 13- to 39-fold, whereas pKJK5 incorporation decreased by 2- to 7-fold, strongly deviating from stochastic expectations based on intracellular abundance, with effects conserved across hosts. This indicates that mobilizable plasmids exploit conjugative transfer machinery to access membrane-associated sites of preferential EV formation, thereby increasing their likelihood of EV incorporation. This demonstrates that EV cargo composition is not stochastic but is actively reshaped by plasmid-specific properties and plasmid-plasmid interactions, revealing a previously unrecognized mechanism influencing gene transfer in microbial communities.