R-pyocin-mediated selection restores aminoglycoside sensitivity in a pan-drug resistant Pseudomonas aeruginosa isolate
- 1Georgia Institute of Technology
Abstract
The global escalation of antibiotic resistance is a critical threat necessitating 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 as conventional biocides but as precise selective forces to drive an evolutionary trap. We subjected the laboratory strains PAO1 and PAK and the clinical pan-drug-resistant (PDR) wound isolate MRSN 6220 to R-pyocin selective pressure. Under sustained R-pyocin pressure, every pyomelanogenic variant we recovered evaded R-pyocin killing through large-scale chromosomal deletions spanning 250-388 kbp. 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. These deletions also eliminate critical virulence factors and biofilm clusters, including the hcnABC, exoY, phzABCDEFG, and cup operons. In a Galleria mellonella model, the resulting brown mutants were non-lethal, and in infected murine wound tissue treated ex vivo with gentamicin they exhibited a significant 3-log reduction in bacterial load, whereas the pan-drug-resistant ancestor did not. This work establishes a framework for utilizing R-pyocins as evolutionary steering agents that couple the acquisition of R-pyocin resistance to the reversion of multidrug resistance into an attenuated, biofilm-deficient, and clinically manageable state.
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BibTeX
@misc{estrada2026rpyocinmediated,
title = {{R-pyocin-mediated selection restores aminoglycoside sensitivity in a pan-drug resistant Pseudomonas aeruginosa isolate}},
author = {I. Estrada and D. Campbell and G. S. Welch and P. Smith and J. B. Goldberg and K. P. Rumbaugh and S. P. Diggle},
howpublished = {bioRxiv},
year = {2026},
month = aug,
doi = {10.64898/2026.08.04.742823},
url = {https://www.biorxiv.org/content/10.64898/2026.08.04.742823v3},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - R-pyocin-mediated selection restores aminoglycoside sensitivity in a pan-drug resistant Pseudomonas aeruginosa isolate
AU - I. Estrada
AU - D. Campbell
AU - G. S. Welch
AU - P. Smith
AU - J. B. Goldberg
AU - K. P. Rumbaugh
AU - S. P. Diggle
PY - 2026
DA - 2026/08/04
PB - bioRxiv
DO - 10.64898/2026.08.04.742823
UR - https://www.biorxiv.org/content/10.64898/2026.08.04.742823v3
AB - The global escalation of antibiotic resistance is a critical threat necessitating 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 as conventional biocides but as precise selective forces to drive an evolutionary trap. We subjected the laboratory strains PAO1 and PAK and the clinical pan-drug-resistant (PDR) wound isolate MRSN 6220 to R-pyocin selective pressure. Under sustained R-pyocin pressure, every pyomelanogenic variant we recovered evaded R-pyocin killing through large-scale chromosomal deletions spanning 250-388 kbp. 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. These deletions also eliminate critical virulence factors and biofilm clusters, including the hcnABC, exoY, phzABCDEFG, and cup operons. In a Galleria mellonella model, the resulting brown mutants were non-lethal, and in infected murine wound tissue treated ex vivo with gentamicin they exhibited a significant 3-log reduction in bacterial load, whereas the pan-drug-resistant ancestor did not. This work establishes a framework for utilizing R-pyocins as evolutionary steering agents that couple the acquisition of R-pyocin resistance to the reversion of multidrug resistance into an attenuated, biofilm-deficient, and clinically manageable state.
ER -