R-pyocin-mediated selection reverses pan-drug resistance in Pseudomonas aeruginosa
The global escalation of antibiotic resistance is a critical threat necessitating the development of innovative strategies to provide new therapeutic options and restore the efficacy of conventional drugs. Pseudomonas aeruginosa exemplifies this challenge by utilizing a robust genomic resistome to persist in clinical settings. Here, we demonstrate that R-pyocins (phage-like bactericidal particles) can be leveraged not merely as conventional biocides, but as precise selective forces to drive an evolutionary “checkmate” strategy. We subjected the laboratory strains PAO1 and PAK and the clinical pan-drug-resistant (PDR) wound isolate MRSN 6220 to R-pyocin selective pressure. To evade R-pyocins targeting the host lipopolysaccharide (LPS) core, resistance consistently emerges through large-scale chromosomal deletions spanning 250-388 kbp. Crucially, these deletions encompass a conserved region harboring the galU gene (essential for LPS synthesis), the hmgA gene (yielding a pyomelanogenic ‘brown’ phenotype), and the mexXYZ multidrug efflux operon. While the loss of galU confers broad cross-resistance to R-pyocins by likely truncating the LPS receptor, the concurrent excision of mexXY induces profound collateral sensitivity to aminoglycosides. Furthermore, these large deletions systematically eliminate critical virulence factors and biofilm clusters, including the hcnABC, exoY, phzABCDEFG, and cup operons. In Galleria mellonella and murine chronic wound models, the resulting brown mutants were rendered non-lethal and exhibited a significant 3-log reduction in bacterial load following gentamicin treatment. Ultimately, this work establishes a framework for utilizing R-pyocins as potent evolutionary steering agents to force the predictable reversion of multidrug resistance into an attenuated, biofilm-deficient, and clinically manageable state. Significance Statement Pan-drug-resistant (PDR) pathogens demand novel strategies that both kill and restore antibiotic efficacy. Here, we describe an evolutionary ‘checkmate’ for Pseudomonas aeruginosa, where selection for R-pyocin resistance drives large-scale (∼300 kb) chromosomal remodeling. Although these deletions confer R-pyocin immunity via loss of the galU gene, they simultaneously collapse the pathogen’s virulence and defense. Crucially, the excision of the mexXY efflux operon resensitizes PDR strains to conventional aminoglycosides, while the collateral loss of critical virulence and biofilm clusters abrogates pathogenesis. By coupling resistance acquisition to substantial fitness costs, our work establishes a framework for using R-pyocins to force predictable evolutionary trade-offs, driving the reversion of multidrug resistance to an attenuated, biofilm-deficient, and clinically manageable state.