A systematic identification of resistance determinants to antisense antibiotics suggests adaptation strategies dependent on the delivery peptide

Next-Generation Strategies to Encounter Antimicrobial Resistance (AMR): From Lariocidin to Gene Editing and Nanotechnology-Based Approaches

The escalation of antimicrobial resistance (AMR) represents a serious global threat to public health, with AMR-associated mortality estimated to increase by 70% by 2050. As pathogens evolve through enzymatic inactivation, target modification, efflux-mediated clearance, biofilm formation, and broader genetic adaptation, conventional therapies are increasingly compromised, while the antibiotic development pipeline remains critically constrained by high discovery and development costs, weak commercial incentives, and the escalating complexity of resistance mechanisms. This review comprehensively synthesizes advanced pharmacological and biotechnological innovations designed to circumvent these entrenched resistance mechanisms. We highlight the development of novel therapeutic classes, particularly lariocidin, which disrupts bacterial protein synthesis via a previously unexploited ribosomal-binding site. Moreover, we critically evaluate molecular interventions, emphasizing CRISPR/Cas-based gene silencing and genome editing as precise tools to neutralize specific resistance determinants, such as the mecA gene in methicillin-resistant Staphylococcus aureus (MRSA). Concurrently, we explore the integration of engineered nanoparticles to revitalize existing antimicrobials by overcoming biofilm barriers, improving drug solubility, and enabling targeted delivery. Collectively, mastering the evolving AMR landscape requires a multidimensional framework that seamlessly integrates these novel molecular targets with advanced rapid diagnostics and robust international governance.

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.

Adaptive Response of Escherichia coli to Pexiganan: Insights from Genomic Analysis

Background/Objectives: Antimicrobial peptides (AMPs) are considered alternatives to classical antibiotics due to limited resistance development in bacteria. However, bacteria can develop resistance to AMPs through evolutionary adaptation, including oligosaccharide modifications and multidrug efflux pumps. Further research is needed to elucidate the defense mechanisms employed against AMPs to address the emerging resistance problem. Pexiganan is a cationic peptide with effective broad-spectrum antimicrobial activity. The aim of this study is to elucidate the genomic and transcriptomic basis of E. coli’s evolutionary adaptation to pexiganan. Methods: The E. coli ATCC BAA-2523 strain became resistant to pexiganan via evolutionary adaptation methodologies. Whole-genome and transcriptome analyses of resistant and susceptible populations were conducted using Nanopore sequencing and Illumina RNA sequencing, respectively. Results: Resistance development became particularly evident after 15 µg/mL, and the bacteria demonstrated the ability to grow even at high doses (up to 1000 µg/mL). Increases in expression levels of classical ARGs such as emrB, acrF, OXA, sul2, and dfrA14 in the pexiganan-resistant strain indicate that bacteria have the potential for broad-spectrum resistance to other antibiotics alongside pexiganan. Missense mutations have been identified in the phosphatidylserine synthase and cardiolipin synthase genes, which are involved in membrane biosynthesis. Increased expression was observed in the membrane-bound lytic murein transglucosylase (mltF), the murein hydrolase activator (EnvC), and the Antigen 43 (Ag43) gene. Conclusions: Pexiganan may not only target the cell membrane but also trigger the bacterium’s overall transcriptional response and cross-resistance and MDR systems. Upregulation of multidrug efflux pumps, lytic murein transglucosylase, the murein hydrolase activator and the Antigen 43 gene might be associated with resistance. In addition, the missense mutation was detected in the membrane biosynthesis genes pssA and clsB. In vivo infection models, targeted functional genomics, and comprehensive phenotypic cross-resistance testing will be required to validate our results.

Phage and CRISPR based precision antimicrobials: a dual strategy against multidrug-resistant bacteria

This review critically examines bacteriophage-based antimicrobials, CRISPR-Cas therapeutic systems, and their emerging integration as CRISPR-armed phages, highlighting their comparative advantages, current limitations, and future potential as promising targeted antimicrobial approach platforms requiring further clinical validation.

Transposon mutagenesis uncovers the genetic landscape of streptomycin susceptibility and implicates SbmA in aminoglycoside uptake in Escherichia coli

Interactions of antiphage defense systems in the ESKAPE pathogen plasmids.

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.