TL;DR
A systematic ESKAPE virome resource is established, phages’ dual roles in targeting resistant pathogens and curbing resistance spread are demonstrated, and mechanistic support for phage therapy clinical application is provided.
BACKGROUND The global rise of multidrug resistant (MDR) ESKAPE pathogens represents a serious threat to antimicrobial therapy. While phage therapy has re-emerged as a promising alternative, its effectiveness may be compromised by bacterial defense systems, particularly those encoded on plasmids. Comprehensive surveillance of the distribution, diversity, and mobilome context of plasmid-encoded defense systems in ESKAPE pathogens remains key to the design of effective phage therapies. RESULTS We analyzed 7,330 dereplicated plasmids from ESKAPE pathogens to characterize the prevalence, diversity, and co-occurrence of plasmid-encoded antiphage defense systems. Conjugative plasmids, especially from Enterobacter spp. and K. pneumoniae, harbored the highest prevalence and diversity of defense systems. Defense-positive plasmids showed larger sizes, higher GC content, and frequent co-occurrence of resistance genes, especially from β-lactam, aminoglycoside, and sulfonamide classes, along with transposable elements such as IS6, IS3, and Tn3. Random forest and correlation analyses confirmed TEs and ARGs as dominant predictors of defense system occurrence. Network analysis revealed structured and partially conserved interactions among defense genes, TEs, and ARGs. RM and CBASS systems were frequently linked to beta-lactam and aminoglycoside resistance genes, as well as TEs such as IS6 and IS3. Recurrent associations such as RM-IS6, RM-IS1380, CBASS-IS3 and RM-OXA suggest shared horizontal transfer mechanisms. CONCLUSIONS Plasmid-encoded antiphage defense systems in ESKAPE pathogens are widespread, structured, and linked to ARGs and mobile genetic elements. These findings highlight the contribution of plasmids to the dissemination of phage-resistance traits, underscore the importance of the mobilome in shaping phage-resistance landscapes in multidrug-resistant pathogens, and support the incorporation of plasmid defense profiling into phage therapy design.
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.
ABSTRACT Vibrio sinaloensis has been reported in association with disease events in aquaculture, yet its genomic features and non-antibiotic suppression strategies remain largely unexplored. Here, we report genomic characterization of a multidrug-resistant V. sinaloensis strain, ZZ006, isolated from diseased shrimp and evaluate phage-mediated suppression of this strain in vitro. The ZZ006 genome harbors multiple putative virulence-associated secretion systems and antimicrobial resistance genes, suggesting chromosomal carriage of traits potentially relevant to host association and antimicrobial tolerance. Using this isolate and its phage-resistant derivatives as hosts, we isolated and characterized three novel bacteriophages (VS1, VS2, VS3) with complementary infection strategies. Their combined application efficiently suppressed ZZ006 growth in vitro and reduced the emergence of phage-resistant populations compared with individual phages. Whole-genome resequencing of cocktail-resistant isolates revealed recurrent mutations in pilus- and pseudopilin-associated genes, consistent with receptor-level phage resistance. This study provides genomic characterization of an aquaculture-associated V. sinaloensis isolate and establishes an in vitro framework for phage cocktail suppression and resistance-evolution analysis, supporting future evaluation of phage-based biocontrol in aquaculture-relevant settings. IMPORTANCE Vibrio sinaloensis has been detected in diseased aquaculture animals, and multidrug-resistant isolates may complicate disease management. By integrating genomic analysis with experimental phage therapy, this work characterizes a multidrug-resistant V. sinaloensis isolate from diseased shrimp and evaluates in vitro phage-mediated suppression of this isolate and its phage-resistance evolution. These findings offer actionable solutions for mitigating antibiotic resistance and enhancing sustainability in aquaculture systems. Vibrio sinaloensis has been detected in diseased aquaculture animals, and multidrug-resistant isolates may complicate disease management. By integrating genomic analysis with experimental phage therapy, this work characterizes a multidrug-resistant V. sinaloensis isolate from diseased shrimp and evaluates in vitro phage-mediated suppression of this isolate and its phage-resistance evolution. These findings offer actionable solutions for mitigating antibiotic resistance and enhancing sustainability in aquaculture systems.
The global rise of antibiotic resistance (AR) is driven by clinical misuse and complex ecological dynamics within the environmental resistome. In addition to bacteria and free DNA, bacteriophages play a critical role in disseminating antibiotic resistance genes (ARGs) via transduction. Anthropogenically impacted matrices—such as wastewater, sludge, and agricultural effluents—act as major hotspots where dense microbial communities and selective pressures promote ARG persistence. However, monitoring these environments remains challenging due to ARG diversity and the lack of standardized surveillance frameworks. The discovery of crAssphage, a highly abundant and human-specific gut virus, offers a promising solution. Because the best indicator for a phage is another phage, crAss-like phages are ideal fecal indicators due to their high persistence and environmental stability. Notably, the isolation of new crAssBcn phages has enabled the functional characterization of their infectivity, confirming their robustness against environmental stressors and disinfection. Furthermore, crAss-like phages show exceptional human specificity and strongly correlate with ARG abundance in fecally contaminated matrices, often outperforming traditional bacterial indicators. Comparative analyses across wastewater, sludge, and food matrices demonstrate that crAssphage mirrors ARG trends, remaining detectable even when conventional bacteria decline. These findings support integrating crAss-like phages as functional indicators in environmental surveillance frameworks. This approach simplifies monitoring human-derived ARGs and provides insight into phage-mediated mobilization. While geographic variability must be considered, their performance as fecal and ARG-associated markers underscores their value for One Health-oriented strategies to track antibiotic resistance dissemination in aquatic systems.
The increasing clinical evidence shows that synthetic biology can effectively overcome the inherent defects of natural bacteriophages, confirming the safety and initial efficacy of bacteriophage therapy.
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