Diggle, S. | bioRxiv Science

bioRxiv2026

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 StatementPan-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 pathogens 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.

microbiology↗

bioRxiv2026

Biophysical Constraints Dictate the Stability of Social Traits in Pseudomonas aeruginosa Aggregates

The maintenance of cooperative behaviors within microbial populations remains an evolutionary puzzle, particularly in spatially structured environments like those found in chronic infection. In Pseudomonas aeruginosa, quorum sensing (QS) directs the production of costly public goods that are vulnerable to exploitation by non-producing cheaters. However, the biophysical mechanisms that stabilize this cooperation in complex environments remain poorly understood. Here, we elucidate how the biophysical properties of the bacterial cell surface govern the micron-scale architecture of bacterial aggregates to promote or constrain the stability of cooperation. Using a polymer-structured growth environment, we demonstrate that cell surface hydrophobicity acts as a primary determinant of spatial organization, where hydrophilic wild-type cells assemble into "stacked" aggregates, whereas hydrophobic O-specific antigen-deficient cells form dense "clumps". In mixed populations, distinct surface properties exhibit phase-separation-like immiscibility, segregating the cells at the micron scale. We show that this segregation directly suppresses cheater fitness by physically sequestering cooperative clusters. Specifically, hydrophobic QS-deficient cells were effectively excluded by hydrophilic QS cooperators, limiting their access to public goods. Furthermore, invasion experiments revealed that hydrophilic cells are inherently fitter, capable of unidirectionally invading populations with a hydrophobic cell surface and independent of social dynamics. Conversely, the need to gain access to nutrient resources can fine-tune these barriers, enabling hydrophilic QS-deficient cells to cluster proximally to hydrophobic cooperators. These findings establish that bacterial cell surface traits impact strongly on microbial social interactions and offer new insights into the maintenance and loss of cooperative traits in chronic infections such as those found in chronic lung infections. Significance StatementCooperative behaviors are essential for the survival of pathogenic bacteria like Pseudomonas aeruginosa, yet they are continually threatened by "cheater" cells that exploit shared public good resources. We demonstrate that the physical properties of the cell surface, specifically hydrophobicity, act as a key architect of microbial social structure. In polymer-rich environments, differences in surface hydrophobicity drive a distinct "oil-and-water" segregation between cell populations. This micron-scale phase separation physically sequesters cooperative cells, insulating them from exploitation by cheaters. Consequently, spatial structure serves as a biophysical stabilizer of cooperation, limiting the metabolic advantages typically held by cheaters. Our findings demonstrate that microbial social evolution is governed not only by genetic strategies, but by fundamental biophysical constraints that dictate the spatial limits of exploitation.

microbiology↗