Eco-evolutionary responses to plasmid-dependent phage constrain the spread of multidrug resistance plasmids

Phage therapy offers a promising alternative to antibiotics for treating multidrug-resistant infections. Plasmid-dependent phages (PDPs) are particularly attractive as therapeutics because they can both kill targeted pathogens and prevent the further spread of antibiotic resistance genes encoded by plasmids. However, the evolutionary trajectories of multidrug-resistance (MDR) plasmids under the selective pressure of PDPs remain poorly understood, particularly in eco-evolutionary contexts that remain permissive to plasmid conjugation. We experimentally evolved populations of Escherichia coli carrying the MDR plasmid RP4 in the presence of the plasmid-dependent phage PRD1 under conditions where the benefits of conjugation were either strong or weak. When opportunities for conjugation were rare, PRD1 only transiently suppressed the conjugative plasmid population due to the rapid evolution of phage-resistant plasmids lacking conjugative ability. Increasing ecological opportunities for conjugation enhanced plasmid suppression and delayed the evolution of phage-resistant plasmids. PRD1 resistance was associated with plasmid loss and reduced conjugative ability, although this trade-off was complex because resistance mutations had heterogeneous effects on pilus production and conjugation. Mutations and IS-mediated inactivation in conjugation genes generated a spectrum of resistance phenotypes, from partial resistance (trbB, trbL) to complete resistance (virB4/trbE). Bioinformatic analysis of publicly available IncP plasmids revealed frequent truncations of the VirB4/TrbE protein, suggesting that plasmid-dependent phages may represent an important selective pressure shaping plasmid evolution in natural populations. Our results demonstrate an evolutionary trade-off between conjugative ability and phage resistance that cannot be easily circumvented by plasmids. Targeting multidrug resistance plasmids with PDPs is likely to drive loss of conjugation, limiting the transfer of antibiotic resistance genes in microbial communities.