ID: 42072119
Title: Harmine Targets Peroxiredoxin 6 to Enhance Macrophage Immunity Against Pseudomonas plecoglossicida in Ayu (Plecoglossus altivelis).
Abstract: Pseudomonas plecoglossicida causes bacterial hemorrhagic ascites in ayu (Plecoglossus altivelis), a lethal disease characterized by abdominal distension with hemorrhagic ascites, multifocal organ hemorrhages, and histopathologically evident hepatocellular necrosis and inflammatory infiltration. The lack of effective treatments exacerbates mass mortalities, posing a significant threat to aquaculture. Given the severe pathogenesis of P. plecoglossicida infection-which involves bacterial colonization, tissue necrosis, and host immune dysregulation-effective therapeutic strategies are urgently needed. Through a screen of traditional Chinese medicine monomers, we identified harmine, an indole alkaloid derived from Peganum harmala seeds, as a potent agent against this pathogen. In vivo, harmine exhibited direct bactericidal activity by disrupting membrane integrity, as evidenced by increasing membrane permeability, and inhibiting biofilm formation. In an ayu infection model, harmine significantly increased host survival, reduced tissue bacterial load, and enhanced innate immunity by augmenting monocyte/macrophage phagocytosis and bactericidal capacity while suppressing pro-inflammatory cytokine release and apoptosis. Mechanistically, the Drug Affinity Responsive Target Stability assay was used to identify the molecular target of harmine, followed by functional validation through PRDX6-knockdown experiments. Harmine exhibited direct bactericidal activity by disrupting membrane integrity and inhibiting biofilm formation. In the ayu infection model, harmine significantly increased host survival, reduced tissue bacteria1 load, and enhanced innate immunity by augmenting monocyte/macrophage system and bactericidal capacity while suppressing pro-inflammatory cytokine release and apoptosis, the latter likely through modulation of PRDX6-mediated oxidative stress and downstream caspase signaling. Mechanistically, DARTS revealed that harmine binds to peroxiredoxin 6 (PRDX6), a multifunctional enzyme possessing peroxidase, phospholipase A2, and lysophosphatidylcholine acyltransferase activities. This binding liberates TNF receptor-associated factor 6 (TRAF6), facilitating its mitochondrial translocation and association with the ECSIT signaling integrator complex, thereby amplifying mitochondrial reactive oxygen species (mROS) production and potentiating macrophage-mediated bacterial killing. These findings establish harmine as a promising therapeutic candidate for controlling P. plecoglossicida infections and underscore the value of host-directed immunomodulation derived from natural products in aquaculture medicine.

ID: 42160310
Title: The Pseudomonas aeruginosa ribonuclease Ribocin cleaves eukaryotic ribosomes at helix 69 to inhibit host translation.
Abstract: Pseudomonas aeruginosa employs host translation inhibition as a virulence-enhancing strategy. We previously showed that the bacterium induces cleavage of Caenorhabditis elegans large ribosomal RNA at helix 69 (H69), part of a central intersubunit bridge and the ribosomal decoding center. In this study, we demonstrate that a previously uncharacterized ribonuclease, Ribocin, is necessary and sufficient for H69 cleavage. Recombinant Ribocin cuts H69 in worm and mammalian ribosomes, indicating that H69 cleavage by P. aeruginosa is phylogenetically conserved. In worms, mammalian cells, and rabbit reticulocyte lysates, H69 cleavage results in translation inhibition. Furthermore, Ribocin contributes to bacterial virulence toward C. elegans, triggers a major host response to translation inhibition, and operates in parallel with Exotoxin A-mediated translation inhibition. These findings unveil the first known nuclease that cleaves eukaryotic ribosomes at H69 and expand the understanding of host translation-inhibition by establishing targeted rRNA cleavage as a mechanism of host attack.

ID: 42294707
Title: From screening to treatment: in vitro and in vivo efficacy of phage-meropenem combination against highly virulent Carbapenem-resistant Acinetobacter baumannii.
Abstract: Carbapenem-resistant Acinetobacter baumannii (CRAB) is a hospital-acquired pathogen of significant clinical importance. Given the slow progress in antibiotic development, phages have emerged as a novel therapeutic strategy against CRAB infections due to their synergistic effects with antibiotics. This study aims to explore phage-antibiotic combination therapy targeting CRAB, thereby providing a theoretical foundation for its clinical translation. Through virulence assessments in Galleria mellonella larvae and mice, CRAB110 was identified as the most virulent strain among 199 clinical CRAB isolates. Transcription analysis revealed that its enhanced pathogenicity may be associated with dysregulated pro-inflammatory responses. Using CRAB110 as the host bacterium, the newly isolated phage, P2, was selected for further study due to its high lytic efficiency and broad host range. The CRAB110 mutant strain CRAB110-2N, treated with P2, exhibited increased drug susceptibility and attenuated virulence compared to the wild-type strain. Both in vitro and in vivo experiments demonstrated that the combination of P2 with antibiotics achieved superior bacterial clearance compared to phage therapy alone. Furthermore, whole-genome analysis of CRAB110-2N revealed a mutation in its ugd. The constructed CRAB110Δugd showed tolerance to P2; the quorum-sensing gene abaI was associated with phage susceptibility, with the CRAB110ΔabaI strain displaying insensitivity to P2. These findings provide experimental evidence supporting the clinical application of phage-antibiotic combination therapy. This study focused on phage-antibiotic combination therapy targeting carbapenem-resistant Acinetobacter baumannii (CRAB), a priority pathogen designated by the World Health Organization (WHO). A highly virulent strain, CRAB110, was screened from 199 clinical isolates, and a novel, highly lytic phage, P2, was isolated from sewage samples. CRAB110-2N, a mutant strain induced by P2 exposure, displayed increased antibiotic susceptibility and attenuated virulence. Key findings showed that the combination of P2 and meropenem achieved a 99% bacterial clearance rate in vitro and exerted significant synergistic effects in vivo. Genomic analysis identified mutations in the ugd gene, and knockout experiments confirmed its correlation with phage susceptibility. Additionally, the quorum-sensing gene abaI was found to be associated with phage susceptibility in CRAB. Collectively, these results provide critical experimental evidence for the clinical translation of this phage-antibiotic combination therapy.

ID: 42305438
Title: Phosphatase PTP-4 downregulates ATLN-1-mediated host defense against Pseudomonas aeruginosa infection in Caenorhabditis elegans.
Abstract: The p38 MAPK pathway governs the core innate immune response in Caenorhabditis elegans (C. elegans), yet the negative regulatory networks preventing immune overactivation-and their potential as therapeutic targets-remain elusive. We aimed to identify novel phosphatase checkpoints in host defense, elucidate their molecular mechanisms, and validate their druggability using small-molecule interventions. We performed a genome-wide RNAi screen of 166 phosphatase genes in C. elegans infected with Pseudomonas aeruginosa (PA14) to identify key regulators. The molecular mechanism was dissected using yeast two-hybrid screening, Co-IP, and mutagenesis assays. Furthermore, a small-molecule screen was performed to identify compounds capable of interfering with PTP-4 function, followed by Surface Plasmon Resonance (SPR), molecular docking, and structural biology approaches to characterize compound-PTP-4 interactions. PTP-4 was identified as a critical negative regulator of immunity. Genetic deficiency of ptp-4 significantly enhanced host resistance via hyperactivation of the p38 MAPK (PMK-1) pathway. Mechanistically, PTP-4 physically interacts with ATLN-1, an ER-shaping GTPase, through its phosphatase domain, thereby suppressing immune activation. Crucially, baicalin was identified through compound screening as an inhibitor of P. aeruginosa infection. Structural and biochemical analyses revealed a dual mechanism of action: baicalin directly binds to the PTP-4/ATLN-1 interface with high affinity (KD = 28.69 μM) to allosterically disrupt their complex formation, while concurrently downregulating PTP-4 protein expression. This "dual-hit" inhibition mimics the genetic loss of ptp-4, effectively unleashing the innate immune response. Our study uncovers the PTP-4/ATLN-1 axis in the control of P. aeruginosa infection. Importantly, we provide the first proof-of-concept that this axis is a druggable target. By demonstrating that baicalin boosts host immunity through the specific dismantling of the PTP-4/ATLN-1 complex, we highlight a novel therapeutic strategy for combating bacterial infections.

ID: 42306840
Title: Structural insights and therapeutic targets in Acinetobacter baumannii capsule biosynthesis.
Abstract: Gram-negative bacteria produce nonulosonic acids, a family of nine-carbon sugars that mimic host sialic acids and may contribute to bacterial virulence. The nonulosonic acid C8-epimeric 5,7-di-N-acetyllegionaminic acid (8-epi-Leg5,7Ac2) is a component of the K-locus 49 (KL49) capsule of hypervirulent Acinetobacter baumannii, and its epimerization is proposed to be mediated by ElaA, ElaB, and ElaC. We identified a KL49 strain that induces lethal sepsis in mice and found deletion of elaA, elaB, or elaC abolished lethality and markedly reduced capsule production. Crystal structures of ElaB, ElaA, and ElaC revealed conserved N-acetyl recognition underlying CMP-Leg5,7Ac2 synthesis and regioselective C8 epimerization. Together, these genetic, structural, and in vivo findings identify ElaABC as potential antivirulence targets in emerging hypervirulent KL49 A. baumannii strains.

ID: 42318016
Title: Microbial and host innate immune factors affecting the persistence of Staphylococcus aureus in cystic fibrosis airways.
Abstract: Chronic bacterial respiratory infections by Staphylococcus aureus are a hallmark of cystic fibrosis (CF) lung disease, affecting up to 80% of all people with CF by their mid-teens. S. aureus is able to survive and persist in the CF lung despite robust neutrophilic inflammation. As neutrophils are the immune system's front line of defense against bacteria, the persistent nature of S. aureus infections in CF indicates both a defect in the ability of neutrophils to kill S. aureus and an enhanced ability of the bacteria to survive. S. aureus persistence in the CF lung is driven by both microbial and host, genetic and microenvironmental, factors. There doesn't seem to be one unifying feature that makes S. aureus more virulent in the CF lung as several factors have been proposed to aide its survival. These include increased resistance to antibiotics, the ability to form small colony variants, biofilm formation, co-infection with Pseudomonas aeruginosa, and several virulence factors such as the accessory gene regulatory system, leukocidins, and staphylococcal protein A. A variety of host factors also affect the ability of neutrophils to kill S. aureus in the CF lung. Defects in the function of the cystic fibrosis transmembrane conductance regulator, the genetic cause of CF, affect phagolysosomal killing and lead to increased formation of neutrophil extracellular traps, which are less effective at killing S. aureus. Additionally, data suggest that factors within the CF lung microenvironment also affect neutrophilic killing of S. aureus. However, more research is needed to clearly identify what these environmental factors may be. This review article summarizes the current knowledge on the clinical relevance of S. aureus lung infections in CF, on microbial and host mechanisms promoting S. aureus survival in the CF lung, and on details of neutrophil-S. aureus interactions in CF. By understanding how S. aureus is able to survive in the CF lung and why neutrophils are unable to kill this bacterium, it could be possible to identify potential therapeutic targets to alleviate the consequences of S. aureus respiratory infection in CF.

ID: 42340134
Title: Acinetobacter baumannii in the Age of Antimicrobial Resistance: Clinical Impact, Therapeutic Approaches, and Vaccine Development.
Abstract: Acinetobacter baumannii is a globally prevalent Gram-negative pathogen responsible for a substantial proportion of healthcare-associated infections, particularly in intensive care units. Its ability to persist in hospital environments, form biofilms on medical devices, and evade host immune responses has contributed to the rapid emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains. This narrative review was conducted through a comprehensive literature search of PubMed, Scopus, Web of Science, and Google Scholar, focusing on studies published primarily over the past two decades. The review summarizes global epidemiology, vulnerable populations, mechanisms of antimicrobial resistance, clinical and economic impacts, and current and emerging therapeutic strategies. The advances in alternative treatments, including antimicrobial peptides, bacteriophage therapy, and vaccine development, are also discussed. In addition to summarizing existing knowledge, this review critically examines the key limitations underlying current therapeutic and vaccine strategies, including rapid resistance evolution, immune evasion, and antigenic variability. It further identifies major translational gaps and proposes future directions integrating systems biology and multitarget approaches. Although preclinical studies demonstrate the promising efficacy of several vaccine candidates, clinical trials in humans remain necessary to confirm their safety and effectiveness.

ID: 42341576
Title: Genomic insights and antifungal potential of Pseudomonas species isolated from maple sap, including the novel species Pseudomonas acericola sp. nov. and Pseudomonas edsoni sp. nov.
Abstract: Fungal contamination poses a significant challenge in maple sap collection systems and postproduction processes, which affects the quality and shelf life of maple syrup. As an alternative to chemical treatments, microorganisms offer promising biocontrol potential. This study investigates nine Pseudomonas strains isolated from maple sap for their antifungal activity and genomic features. Whole-genome sequencing followed by comparative genomic analysis identified five distinct Pseudomonas species, including two previously uncharacterized taxa for which we propose the names Pseudomonas acericola sp. nov. and Pseudomonas edsoni sp. nov., in accordance with the nomenclatural guidelines of the SeqCode. Strain distributions from metagenome recruitment suggest they originate from sapwood, and previous metataxonomic data show that the amplicon sequence variant matching P. edsoni predominated maple sap samples. Genome mining using antiSMASH and BAGEL4 identified gene clusters associated with the synthesis of antifungal compounds, such as hydrogen cyanide, siderophores, cyclic lipopeptides, and ribosomally synthesized peptides. Antifungal assays demonstrated inhibitory activity against food spoilage fungi, with P. edsoni strains being active against Kluyveromyces lactis. The absence of activity in the cell-free supernatant and the presence of Type VI secretion systems in the genomes point toward contact-dependent mechanisms. Collectively, these findings reveal previously unrecognized taxonomic diversity and ecological specialization in maple sap-associated Pseudomonas, providing a basis for the rational development of Pseudomonas-based antifungal strategies in maple syrup production and quality control.

ID: 42341627
Title: The synthesis of 6'-N-acyl neamine-based amphiphiles: Synergistic activity against Pseudomonas aeruginosa via enhanced membrane permeability.
Abstract: Amphiphilic aminoglycosides offer a promising approach to combat antibiotic resistant Gram-negative bacteria. Here, a series of neamine-based amphiphiles bearing alkyl chains of varying lengths (C4-C16) was synthesized via a concise route through selective modification at the 6'-amino position of neamine. Antibacterial evaluation revealed a chain length-dependent activity, with the C14- and C15-alkyl neamine amphiphiles exhibiting the most potent antibacterial activity against ESKAPE pathogens. The C15 derivative displayed superior activity compared to neamine and synergized with multiple clinical antibiotics against P. aeruginosa. Mechanistic studies indicated that this synergy results from enhanced outer membrane permeability, and cytotoxicity assays confirmed low toxicity at therapeutically relevant concentrations.

ID: 42350929
Title: Essential Oils and Their Multifunctional Activities Against Pseudomonas aeruginosa: Current Evidence and Perspectives.
Abstract: Pseudomonas aeruginosa is an opportunistic pathogen responsible for acute and chronic infections and is characterized by a remarkable ability to develop antibiotic resistance. This has prompted increasing interest in alternative or complementary antimicrobial strategies, including the use of essential oils (EOs), which are complex mixtures of plant-derived volatile compounds with documented biological activities. This review aims to summarize and discuss the current evidence on the antimicrobial, antibiofilm, and anti-virulence activities of EOs against P. aeruginosa, with particular emphasis on their mechanisms of action, synergistic interactions with antibiotics, and limitation to the possible clinical applicability. A large number of studies, mainly conducted in vitro, indicate that EOs and their major constituents can impair P. aeruginosa viability and pathogenicity through multiple mechanisms. In addition, several EOs or purified terpene components exhibit synergistic effects when combined with conventional antibiotics, enhancing antimicrobial efficacy. However, in vivo evidence remains limited, largely restricted to topical infection models, while clinical studies in humans are currently lacking. Significant challenges related to chemical variability, toxicity, safety, standardization, and regulatory classification also emerge from the literature. Although EOs show considerable promise as antimicrobial and anti-virulence agents against P. aeruginosa, their current role should be viewed primarily as adjunctive or alternative strategies rather than as standalone systemic therapies. Future progress will depend on the development of standardized and safer formulations, advanced delivery systems, and well-designed in vivo and clinical studies.

ID: 42353674
Title: The ESKAPE Challenge: Understanding Resistance and Exploring Alternative Treatments.
Abstract: Antimicrobial resistance (AMR) constitutes a critical and escalating global public health challenge, severely limiting the potential of existing antimicrobial drugs and escalating infection-associated morbidity and mortality rates. This analysis focuses on the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species), which are prioritized by the World Health Organization (WHO) and represent a significant cause of nosocomial infections due to their extensive drug resistance. We provide an in-depth review of the global prevalence and specific antibiotic-resistant mechanisms of these pathogens. Due to the decline in the traditional antibiotic development pipeline, accelerated development of alternative therapeutic strategies is essential. The review comprehensively discusses innovative non-traditional therapies currently being explored to bypass traditional antibiotic limitations, such as phage therapy, antimicrobial peptides (AMPs), anti-virulence therapies, fecal microbiota transplantation (FMT), and targeted CRISPR-based approaches. Addressing the ESKAPE challenge requires a concerted, multi-sectoral strategy guided by the One Health principle, focusing on enhancing public awareness, improving surveillance and research, optimizing judicious antibiotic use, and cultivating sustainable investment in novel interventions.

ID: 42363205
Title: Epidemiological trends of multidrug-resistant organisms in a tertiary-care pediatric hospital, 2017-2023.
Abstract: Antimicrobial resistance (AMR) is a growing global threat, particularly for children under 5 years. This study is aimed to investigate temporal trends and describe the epidemiological characteristics of MDR bacteria isolated in hospitalized children from 2017 to 2023 in an academic children's hospital in Italy. We included all bacterial isolates obtained from blood or cerebrospinal fluid (CSF) cultures collected from pediatric patients evaluated in both inpatient settings and the Emergency Department between 1 January 2017 and 31 December 2023. The study included the main ESKAPE pathogens (including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa) and other relevant MDROs such as Escherichia coli. Data were extracted from the hospital microbiology data warehouse, and resistance proportions were calculated as MDR isolates over total isolates for each pathogen. Associations between year, patient characteristics, ward type, and MDRO infections were evaluated using univariable and multivariable logistic regression; trends over time were evaluated using the Cochrane-Armitage test for categorical variables. Out of 51,886 blood or CSF cultures, 1,528 (2.9%) were positive for microorganisms of interest, and 211 (13.8%) were MDROs. The highest resistance proportions were observed in Carbapenems-Resistant Pseudomonas Aeruginosa/Pseudomonas Aeruginosa (CRPA/PA, 52.8%), followed by Methicilln-Resistant Staphylococcus Aureus/Staphylococcus Aureus (MRSA/SA, 36.7%), Carbapenem-Resistant Klebsiella Pneumoniae/Klebsiella Pneumoniae (CRKP/KP, 12.6%), and Vancomycin-Resistant Enterococcus (VRE)/Enterococcus (VRE/E, 11.5%). Significant reductions were recorded for MRSA/SA (43.5% in 2017 to 25.7% in 2023; p = 0.001) and CRPA/PA (85.0% to 47.1%; p < 0.001), while CRKP/KP and VRE/E showed no significant temporal trends. Neonatology and PICU showed higher odds for MRSA isolation (p < 0.001) compared to medical wards. Onco-hematology displayed an adjusted OR of 3.0 (95% CI: 1.2-7.7) for VRE. Year was independently associated with CRPA, with adjusted ORs of 0.03 and 0.14 in 2020 and 2021 (p < 0.01) compared to 2017. Understanding local AMR patterns remains essential for guiding empirical therapy across heterogeneous hospital settings.

ID: 42363638
Title: Inhalable bacteriophage endolysins: a novel therapeutic strategy for drug-resistant bacterial pulmonary infections - a comprehensive review.
Abstract: Pulmonary infections caused by multidrug-resistant (MDR) bacteria pose a severe global health threat with high mortality rates, especially in hospital-acquired pneumonia. The stagnation of new antibiotic development underscores the urgent need for alternative therapeutics. This review summarizes recent advances in the application of bacteriophage endolysins against major MDR respiratory pathogens, including Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae. We focus on their mechanisms of action, synergistic effects with antibiotics, and efficacy against biofilms. Endolysins demonstrate potent and species-specific lytic activity against a broad spectrum of MDR bacteria. A key advantage is their low propensity for inducing resistance. Critically, when administered via optimized inhalation delivery systems, endolysins can achieve high local concentrations in the lungs - though this depends on factors such as the aerosol device, formulation properties and patient-related variables - effectively eradicating pathogens in animal models of pneumonia with minimal systemic toxicity. This direct pulmonary delivery approach bypasses many challenges associated with systemic administration and enhances therapeutic outcomes. Endolysins represent a promising paradigm shift in combating drug-resistant bacterial pulmonary infections. Their rapid lytic activity, synergy with conventional antibiotics, and suitability for inhalable formulation position them as a potent adjunct or alternative therapy. While challenges in stabilization and large-scale production remain, advancing inhalation delivery systems for endolysins holds immense potential to holds immense potential to revolutionize the treatment of recalcitrant respiratory infections.

ID: 42382725
Title: Single-dose dual-indication nanoparticle vaccine co-displaying Pseudomonas aeruginosa antigen and tetanus toxin fragment confers durable protection against mixed exposure.
Abstract: Trauma, disaster, and conflict-related injuries can create clinically important co-exposure to Pseudomonas aeruginosa (PA) and tetanus toxin, where disrupted barriers and contaminated wounds enable simultaneous bacterial infection and toxin entry. Until now no licensed PA vaccine exists and multi-dose schedules are difficult to complete in emergency settings, thus a single-dose, dual-indication vaccine that provides rapid and durable protection is urgently needed. Here, we engineered and characterized a lumazine synthase (LS) nanoparticle vaccine (rePO-TTFC@LS) that co-displays a PA antigen (rePO; PcrV-OprI fusion) and tetanus toxin fragment C (TTFC). In BALB/c mice, rePO-TTFC@LS enhanced antigen uptake and dendritic-cell activation relative to soluble antigens. After a single intramuscular immunization, rePO-TTFC@LS elicited rapid, high anti-rePO and anti-TTFC IgG responses together with increased IFN-γ/IL-4/IL-17A-secreting splenocytes. Vaccination reduced lung bacterial burden and pulmonary IL-1β/IL-6 and improved survival and clinical outcomes in an acute PA pneumonia model, which provided complete protection against lethal tetanus toxin challenge, and decreased PA colonization with improved survival in a femoral fracture-implant co-infection model. Elevated antibody titers and dual protection persisted to 28 weeks after one dose, and biosafety assessments showed no meaningful hemolysis or cytotoxicity and no abnormalities in hematology/serum biochemistry or multiorgan histopathology. Collectively, these results support rePO-TTFC@LS as a single-dose, durable dual-indication nanoparticle vaccine and highlight a modular strategy for combination vaccines suited to mixed-exposure emergency scenarios.

ID: 42383755
Title: The gac system integrates physical and chemical cues to promote plant root attachment.
Abstract: Plants host complex communities of microbes that are attached to root surfaces. While many studies have sampled mutant populations after prolonged incubation on roots to identify bacterial genes that enable long-term colonization, the molecular mechanisms governing the early stages of root attachment remain less understood. Here, we developed an in vitro root culture system that enables controlled and scalable investigation of bacterial attachment to root tissue. We used this platform to perform a genome-wide screen for root attachment determinants in the plant-associated bacterium Pseudomonas protegens Pf-5. Our results reveal that the gacSA two-component system functions as a sensory integration hub for coordinating early root attachment. Mutations that disrupt gacS or gacA cause severe root attachment defects despite having no effect on abiotic surface attachment in standard biofilm assays. Mutation of flagellar assembly genes enhances root attachment by mimicking surface contact and activating the gac system. In parallel, chemical cues released by roots stimulate surface attachment in a gac-dependent manner. By integrating these signals, the gac system activates cyclic-di-GMP-mediated attachment programs that drive the transition from planktonic to sessile behavior required for root association. We build on this model to show that manipulating flagellar surface-sensing sustains the attachment of Pf-5 in the presence of a synthetic bacterial community, suggesting a strategy to improve the attachment of beneficial microbes on crops. These findings establish a mechanistic framework linking surface sensing, global regulation, and root attachment in a beneficial rhizobacterium.IMPORTANCEPlant roots are covered with diverse microbes that strongly influence plant health. Growing in association with roots has many benefits, but how bacteria attach to root tissue remains poorly understood. We developed a system to study how the plant-associated bacterium Pseudomonas protegens Pf-5 attaches to roots. We found that physical contact with the root surface and chemical cues released by roots both enhance attachment to roots. A sensory system called gacSA is responsible for integrating physical and chemical cues to activate a root attachment program. Variant bacteria that prematurely activate the gac system attach more effectively even in the presence of other bacteria on roots, suggesting that the root attachment pathway we characterized could serve as a strategy to use beneficial bacteria in agriculture.

ID: 42390031
Title: Biocontrol Efficacy of Endophytic Bacteria from Lawsonia inermis L. Against Phytopathogens Fusarium oxysporum and Rhizoctonia solani.
Abstract: Bioactive compounds from endophytic bacteria contribute significantly to drug discovery and plant defense. Medicinal plants, such as Lawsonia inermis L., serve as a rich source of microbial endophytes. To isolate and characterise endophytic bacteria from L. inermis leaves and evaluate their bioactive metabolites for antimicrobial and biocontrol potential. Endophytic bacteria were isolated from surface-sterilised leaves of L. inermis. Morphological and molecular identification, including 16S rRNA gene sequencing and phylogenetic analysis, identified the isolates as Bacillus subtilis, Pseudomonas mosselii, and Sphingomonas aquatilis. Metabolite profiling was performed using Fourier transform infrared spectroscopy (FTIR) and gas chromatography-mass spectrometry (GC-MS). Antifungal activity was tested against Fusarium oxysporum f. sp. lycopersici and Rhizoctonia solani using dual-culture assays. The endophytes demonstrated significant antagonistic activity, inhibiting the growth of fungal mycelia. FTIR spectra indicated the presence of carboxylic, ester, ketone, and alkane functional groups. GC-MS analysis of active fractions revealed several known antimicrobial and pharmaceutical compounds, supporting the observed bioactivity. Endophytic bacteria from L. inermis produce bioactive secondary metabolites with potent antifungal activity. These findings suggest their potential as sources of novel biocontrol agents and therapeutic compounds, contributing to the medicinal properties of the host plant. Résumé Contexte:Les composés bioactifs produits par les bactéries endophytes contribuent de manière significative à la découverte de nouveaux médicaments et aux mécanismes de défense des plantes. Les plantes médicinales, telles que Lawsonia inermis L., constituent une source importante d’endophytes microbiens. Cette étude avait pour objectif d’isoler et de caractériser les bactéries endophytes présentes dans les feuilles de L. inermis et d’évaluer le potentiel antimicrobien et de biocontrôle de leurs métabolites bioactifs.Méthodes:Les bactéries endophytes ont été isolées à partir de feuilles de L. inermis préalablement stérilisées en surface. L’identification morphologique et moléculaire, incluant le séquençage du gène 16S rRNA et l’analyse phylogénétique, a permis d’identifier les isolats comme étant Bacillus subtilis, Pseudomonas mosselii et Sphingomonas aquatilis. Le profilage des métabolites a été réalisé à l’aide de la spectroscopie infrarouge à transformée de Fourier (FTIR) et de la chromatographie en phase gazeuse couplée à la spectrométrie de masse (GC-MS). L’activité antifongique a été évaluée contre Fusarium oxysporum f. sp. lycopersici et Rhizoctonia solani par des essais de culture duale.Résultats:Les endophytes ont démontré une activité antagoniste significative en inhibant la croissance mycélienne des champignons. Les spectres FTIR ont révélé la présence de groupes fonctionnels carboxyliques, esters, cétones et alcanes. L’analyse GC-MS des fractions actives a mis en évidence plusieurs composés connus pour leurs propriétés antimicrobiennes et pharmaceutiques, confirmant ainsi l’activité biologique observée.Conclusion:les bactéries endophytes de L. inermis produisent des métabolites secondaires bioactifs dotés d’une forte activité antifongique. Ces résultats suggèrent leur potentiel en tant que nouvelles sources d’agents de biocontrôle et de composés thérapeutiques, contribuant aux propriétés médicinales de la plante hôte.

ID: 42390567
Title: Disrupting virulence: in silico discovery of MexL inhibitors to block pyocyanin biosynthesis in Pseudomonas aeruginosa.
Abstract: Pseudomonas aeruginosa is an opportunistic pathogen known for its intrinsic antibiotic resistance and ability to form biofilms, primarily regulated by the redox-active compound, pyocyanin. The transcriptional activator (Multidrug efflux regulator L) MexL plays a crucial role in the biosynthesis of pyocyanin by upregulating key genes such as phz1, phz2, and phzM. Targeting MexL may serve as a novel anti-virulence strategy by suppressing pyocyanin production and attenuating pathogenicity of P. aeruginosa. This study aimed to identify potential inhibitors of MexL using in silico approaches to disrupt its regulatory function and reduce pyocyanin synthesis. The findings suggest that the selected compounds could serve as effective inhibitors of MexL, offering a promising strategy to suppress pyocyanin biosynthesis and reduce P. aeruginosa virulence. For this purpose, molecular docking was conducted between the binding pocket of the MexL protein and compounds selected based on pharmacokinetic properties from the FooDB, NPASS, and LOTUS databases. Furthermore, molecular dynamics simulations were performed on the selected MexL-ligand complexes to evaluate the stability of the protein-ligand complex within a simulated solvated environment under defined conditions. The virtual screening by molecular docking identified four top-scoring ligands FDB000380, LTS0012415, and NPC253096 and ZINC14780782 with binding energies of -8.6, -7.5, -7.1, and -8.8 kcal/mol respectively, showing strong affinities toward the MexL binding pocket. These ligands demonstrated key molecular interactions with residues critical for regulatory function of MexL protein. Molecular dynamics simulations (300 ns) of the three lead MexL-ligand complexes (FDB000380, LTS0012415, and NPC253096) revealed exceptional structural stability, with low backbone RMSD values (0.240-0.309 nm), minimal residue fluctuations, stable radius of gyration, and consistent solvent-accessible surface area profiles. Persistent protein-ligand hydrogen bond networks (occupancy 72.1-100%) confirmed stable polar interactions throughout the trajectories. MM-GBSA and MM-PBSA binding free energy calculations demonstrated superior affinities over the endogenous ligand pyocyanin (ΔG =  - 15.30 to - 21.75 kcal/mol versus - 12.57 kcal/mol). Principal component analysis further revealed restricted conformational dynamics and compound-specific free energy landscapes, collectively supporting these three compounds as thermodynamically stable lead inhibitors of MexL-mediated pyocyanin biosynthesis in Pseudomonas aeruginosa.

ID: 42396178
Title: Low concentrations of tetrasodium EDTA cause significant killing of biofilm-associated Pseudomonas aeruginosa in high-validity models of chronic wound and cystic fibrosis lung infections - but not in a model of endotracheal tube colonization.
Abstract: Pseudomonas aeruginosa is a pathogen notorious for its antimicrobial resistance and is currently classified as a high-priority pathogen for which new drugs are needed. Tetrasodium EDTA (tEDTA) is one of the new antimicrobial compounds that have been shown to have good antibacterial and antibiofilm efficacy against P. aeruginosa. Due to the diversity and highly drug-tolerant nature of P. aeruginosa biofilms in different infection environments, it is important to carry out pre-clinical testing of new antibiofilm agents against this pathogen in media and models that accurately mimic diverse infection environments. In this study, we used different high-validity media and biofilm models that mimic chronic wounds, endotracheal tubes and cystic fibrosis lung infections to assess the efficacy of tEDTA against P. aeruginosa biofilms. We report that different infection environments influence the susceptibility of both planktonic and biofilm forms of P. aeruginosa to tEDTA. The highest tolerance to tEDTA was observed in the media and biofilm model that mimics the endotracheal tube environment. In conclusion, we show that although different infection environments influence the efficacy of tEDTA against P. aeruginosa biofilms, it has good potential for use as an alternative antimicrobial in treating P. aeruginosa-associated biofilm infections.

ID: 42397450
Title: Molecular characterization and genotypic diversity of extensively drug-resistant Acinetobacter baumannii from endotracheal aspirates in a pediatric intensive care unit in Sulaimani, Iraq.
Abstract: Extensively drug-resistant (XDR) Acinetobacter baumannii poses a significant threat in pediatric intensive care units (PICUs), particularly in regions with limited surveillance data. This study characterized the bacterial profile of endotracheal aspirate samples from a PICU in Sulaimani, confirmed A. baumannii identification using blaOXA-51 PCR, assessed antimicrobial resistance patterns, and determined genotypic diversity using ERIC-PCR. From August 2023 to March 2025, 100 consecutive Gram-negative isolates were recovered from mechanically ventilated pediatric patients. VITEK2 was used for identification and susceptibility testing. XDR Acinetobacter isolates underwent molecular confirmation via blaOXA-51 PCR, and confirmed isolates were genotyped using ERIC-PCR with UPGMA analysis. Among the 100 total isolates, A. baumannii accounted for 21 isolates alongside Pseudomonas aeruginosa (n = 33) and Klebsiella pneumoniae (n = 20). Given its clinical significance, A. baumannii was selected for further molecular characterization. Overall, 26 isolates exhibited XDR phenotypes, 12 of which were A. baumannii by VITEK2; 10 were confirmed by blaOXA-51 PCR (83.3%). All confirmed isolates were resistant to β-lactams, carbapenems, aminoglycosides, and fluoroquinolones, while remaining susceptible to colistin. ERIC-PCR revealed one dominant clone (Cluster A, 50%), two minor clusters (20% each), and one singleton (10%), indicating moderate genetic diversity with evidence of sustained clonal circulation. This study provides the first molecular epidemiological characterization of XDR A. baumannii in an Iraqi PICU, revealing clonal persistence of a successful epidemic strain and critical gaps in phenotypic identification. These findings emphasize the need for routine molecular surveillance, targeted infection control, and antimicrobial stewardship in resource-limited settings.

ID: 42398007
Title: Marked variations in the effectiveness of commercial disinfectants against clinically relevant antibiotic-resistant bacteria and fungi.
Abstract: Effective disinfection of noncritical hospital surfaces may be essential for limiting the transmission of antibiotic-resistant bacteria (ARB), which seem to persist despite clear disinfection guidelines and raise concern about the potency of real-world disinfectants and about the consistency of disinfection practices. This study compared the bactericidal efficacy of eight commercially available disinfectants - alcohols, hydrogen peroxide, quaternary ammonium compounds (QACs), and chlorhexidine (CHX) - against multidrug-resistant clinical isolates of Klebsiella pneumoniae, Acinetobacter baumannii, Staphylococcus aureus, Enterococcus faecium, carbapenem-resistant Pseudomonas aeruginosa, and model organism Candida albicans as the most frequent causes of hospital-acquired infections in Croatia. Several formulations, particularly those based on QACs and CHX, retained full bactericidal activity even when diluted 10-32 times after only 1 min of contact, while alcohols were ineffective when diluted more than twice. However, all the formulations were effective when applied at full concentration. These findings highlight marked differences in the intrinsic bactericidal strength of commercial products, especially if not applied properly, which may explain variable outcomes observed in routine hospital disinfection practices. These issues may be overcome by rotating different disinfectant classes during routine surface decontamination and by optimising disinfectant strength to improve ARB and fungal control in clinical settings. Učinkovita dezinfekcija nekritičnih bolničkih površina mogla bi biti ključan korak u smanjenju brojnosti bakterija otpornih na antibiotike (ARB) u zdravstvenim institucijama. Unatoč jasnim smjernicama za dezinfekciju, ove su bakterije i dalje prisutne u bolničkom okolišu, što izaziva zabrinutost glede učinkovitosti i dosljednosti dezinfekcijske prakse. Ovdje predstavljena studija usporedila je baktericidnu učinkovitost osam komercijalno dostupnih dezinficijenasa iz skupine alkohola, vodikova peroksida, kvaternih amonijevih spojeva (QAC) i klorheksidina (CHX), prema višestruko otpornim bolničkim izolatima bakterija Klebsiella pneumoniae, Acinetobacter baumannii, Staphylococcus aureus, Enterococcus faecium i Pseudomonas aeruginosa te prema gljivici Candida albicans kao najčešćih uzročnika bolničkih infekcija u Hrvatskoj. Spojevi na bazi QAC-ova i CHX-a, zadržali su punu baktericidnu aktivnost čak i kada su bili razrijeđeni 10 – 32 puta te nakon samo jedne minute kontakta. Alkoholi su, na primjer, izgubili učinkovitost kada su bili razrijeđeni više od dva puta. No, treba naglasiti da su sve formulacije bile potpuno učinkovite kada su se primjenjivale u punoj, preporučenoj koncentraciji. Rezultati upućuju na značajne razlike u baktericidnoj snazi komercijalnih proizvoda, posebice ako se ne primjenjuju pravilno, što može objasniti prisutnost bakterija i nakon čišćenja. Predlaže se stoga rutinska izmjena različitih klasa dezinficijensâ tijekom tretiranja nekritičnih površina radi što učinkovitijeg smanjenja prijenosa bakterija otpornih na antibiotike i gljivice u bolnicama.

ID: 42400505
Title: Prevalence and genotypic distribution of virulence factor genes and antibiotic susceptibility profiles in clinical Pseudomonas aeruginosa isolates from a public hospital in Nanyang, China.
Abstract: Pseudomonas aeruginosa remains a critical global health concern, particularly in low- and middle-income countries, owing to its high carriage of diverse virulence factor genes (VFGs) and multidrug resistance. Here, we analyzed the prevalence and genotype distribution of VFGs in P. aeruginosa isolated from Nanyang Central Hospital (NYCH), China, along with their antibiotic susceptibility profiles. The study involved 79 multidrug-resistant (MDR) and 96 non-MDR (nMDR) strains and the following 11 key VFGs (nan1, pilA, aprA, phzS, plcH, toxA, lasB, exoS, exoU, exoT, exoY). Antibiogram profiling showed near-universal resistance to at least one antibiotic class. MDR strains exhibited 23 distinct antibiotic resistance patterns, compared with 13 patterns in nMDR strains. A moderate negative correlation was observed between β-lactams/β-lactamase inhibitors and fluoroquinolone resistance. Moreover, statistical analysis revealed that the phzS gene displayed the most significant prevalence difference between MDR and nMDR subgroups (P < 0.001). Finally, 28 distinct genotypes were identified in nMDR isolates, and 26 genotypes appeared in MDR strains. There was a statistically significant correlation found between the presence of genotype XX (r = 0.481, P < 0.001) and genotype XXVIII (r = -0.320, P < 0.001) in two subgroups. Collectively, our findings provide data support for understanding the pathogenic mechanism of local P. aeruginosa, and are of great significance for guiding clinical anti-infection treatment and infection control.IMPORTANCEPseudomonas aeruginosa is an opportunistic pathogen notorious for causing difficult-to-treat infections, particularly among hospitalized patients in resource-limited healthcare settings. This study analyzed clinical isolates from a major hospital in China to investigate the prevalence and genotypic distribution of virulence factor genes (VFGs) and antibiotic susceptibility profiles of P. aeruginosa. Our results showed that multidrug-resistant (MDR) strains displayed more complex and diverse resistance profiles than non-MDR (nMDR) strains. Both MDR and nMDR isolates exhibited high genotypic diversity. Notably, a statistically significant association was observed between genotype XX and genotype XXVIII across the two subgroups. These findings elucidate the molecular basis underlying the variable treatment difficulty of P. aeruginosa infections and offer valuable insights for guiding empirical antibiotic selection. Characterizing local bacterial features, including antibiotic susceptibility profiles and the distribution of clinically relevant virulence factors, may improve understanding of regional resistance trends and support antimicrobial stewardship and infection control efforts.

ID: 42401050
Title: Dual role of HCN produced by the endophyte Pseudomonas urmiensis A32 in biocontrol and the defence response of pepper.
Abstract: Plant-associated Pseudomonas species are widely recognised for their ecological versatility and potential as sustainable biocontrol and plant growth-promoting agents. Among their diverse metabolites, hydrogen cyanide (HCN) is traditionally considered a potent antimicrobial compound, yet its broader role in plant-microbe interactions remains underexplored. This study investigated Pseudomonas urmiensis A32, an endophytic bacterium previously shown to enhance drought tolerance and disease resistance in pepper (Capsicum annuum). Whole-genome analysis revealed genes involved in nutrient acquisition, stress protection, and competitive exclusion, including secondary metabolite clusters for carotenoid and HCN biosynthesis. Wild-type A32 showed increased tolerance to oxidative stress, highlighting a redox-active role for HCN. Seed inoculation with A32 reduced disease symptoms and pathogen load in pepper plants challenged with Xanthomonas euvesicatoria, indicating that HCN contributes not only to direct pathogen antagonism but also to modulation of host immune responses. Biochemical analyses showed decreased activity of antioxidant enzymes and increased phenylalanine ammonia-lyase activity. Gene expression analyses revealed upregulation of PR1 and the antimicrobial peptide gene DEF1, indicating enhanced activation of defence genes. These findings demonstrate that HCN acts as a multifunctional metabolite that fine-tunes the balance between reactive oxygen species detoxification and signalling, and integrates microbial biocontrol with host immune priming. In summary, this work highlights the ecological adaptability of P. urmiensis A32 and its potential as an effective bioinoculant for sustainable crop protection.

ID: 42401059
Title: The overlooked risk of horizontal transfer of plasmid-borne antibiotic resistance genes induced by organophosphate esters in aquaculture environments.
Abstract: In recent years, the emergence of new environmental pollutants has drawn increasing attention to the plasmid-mediated conjugation transfer of antibiotic resistance genes (ARGs) induced by these contaminants. The widespread application of organophosphate esters (OPEs) has led to their frequent detection in aquaculture water, posing potential risks to the aquatic ecosystems and human health. In this study, we revealed that exposure to five types of OPEs (0.1-1000 µg/L) promoted the dissemination of RP4 plasmid from Escherichia coli to Pseudomonas alcaligenes, a multidrug-resistant bacterium isolated from actual aquaculture wastewater. Through the application of fluorescence detection, scanning electron microscopy, RT-qPCR, and RNA-seq techniques, we clarified at the cellular and molecular levels that OPEs promoted plasmid conjugation transfer by inducing reactive oxygen species (ROS) accumulation, activating the stress response (SOS), enhancing cell membrane permeability, improving intracellular energy supply, regulating the expression of conjugation-related genes, and activating the transfer apparatus encoded by RP4. Specifically, TBEP was selected as a representative OPE to systematically explore its impact on plasmid dissemination. Notably, a frequently overlooked aspect was that exposure to TBEP also enhanced the genetic stability and expression persistence of ARGs. Our findings emphasized that OPEs released from aquaculture facilities might act as driving factors to promote intergenus horizontal transfer of ARGs in actual aquaculture environments-a potential risk that has not yet been fully recognized.

ID: 42402019
Title: Photosynthesis-driven interactions in the phycosphere enhance bacterial extracellular superoxide production.
Abstract: Extracellular superoxide generated by heterotrophic bacteria influences aquatic redox transformations, yet its regulation within the phycosphere remains poorly understood. Here, we show that the cyanobacterium Microcystis aeruginosa enhances extracellular superoxide production by Pseudomonas sp. QJX-1 under illumination. Cocultivation increased extracellular superoxide production approximately threefold over bacterial monoculture, with maximum enhancement during carbon starvation. Membrane separation localized detectable superoxide production to the bacterial chamber, indicating that diffusible algal products stimulated bacterial superoxide production. Mechanistically, illuminated cocultures showed 20.4% higher photocurrent than algal monocultures and accumulated the highest extracellular NADP(H) levels, indicating intensified photosynthesis-associated extracellular redox activity. Reduced nicotinamide cofactors stimulated diphenyleneiodonium-sensitive QJX-1-associated superoxide production, consistent with an oxidoreductase-associated route for one-electron reduction of molecular oxygen. In parallel, coculture accumulated catecholate siderophores, consistent with intensified microscale Fe competition; this siderophore-enhanced response required metabolically active bacterial cells and was attenuated by respiratory-chain inhibition. Representative extracellular redox-active and algal photoactive components further enhanced the bacterial response, with cytochrome c and chlorophyll a stimulating QJX-1-associated superoxide production under defined conditions. Functionally, during the carbon-starvation interval coinciding with peak superoxide production, the apparent sulfamethoxazole removal rate constant in coculture was 2.6 times that observed for QJX-1 alone. Moreover, quenching extracellular reactive oxygen species impaired metabolic activity under diverse chemical stressors and reduced carbon, nitrogen, and phosphorus substrate utilization. Collectively, these findings identify photosynthesis-dependent phycosphere interactions as regulators of bacterial superoxide production, linking biogenic reactive oxygen species to contaminant transformation, chemical-stress resilience, and elemental nutrient turnover.

ID: 42402172
Title: Characterization and Therapeutic Potential of a Novel Lytic Phage-Derived Endolysin PA16cLys Against Uropathogenic Pseudomonas aeruginosa Biofilms.
Abstract: Pseudomonas aeruginosa (P. aeruginosa) is a leading cause of urinary tract infections (UTIs). The escalating misuse of antibiotics has led to the emergence of multidrug-resistant strains, necessitating novel therapeutic approaches. Bacteriophages and their encoded lytic enzymes offer unique advantages for antimicrobial therapy and show promising potential for future applications. This study focuses on the characterization of uropathogenic P. aeruginosa phages and their lytic enzymes, investigating their biological properties and potential therapeutic applications. We isolated a novel lytic bacteriophage, vB_PaeS_PA16c (PPA16c), which demonstrated favorable biological characteristics, including a 34% biofilm eradication capacity within 6 h. The phage-encoded endolysin PA16cLys exhibited superior antibacterial efficacy compared to the phage itself. Notably, it achieved significant biofilm reduction (~63%) after 1 h treatment without requiring outer membrane permeabilizers. This endolysin exhibited enhanced antibacterial efficacy in eradicating mature P. aeruginosa biofilms. Regarding host range, PA16cLys displayed broader lytic activity. Both the phage and endolysin demonstrated cross-order lytic activity against other pathogenic bacteria. Crucially, PA16cLys maintained potent inhibitory activity against phage-resistant mutants. These findings provide a theoretical foundation for utilizing phages and lytic enzymes against uropathogenic P. aeruginosa and establish a research basis for developing novel antimicrobial therapy.

ID: 42402899
Title: Mechanistic insights into the inhibition of biofilm formation and antibacterial action of endophytic Streptomyces strain KJ7TB2 isolated from the Indian Sundarbans Mangrove estuary.
Abstract: To explore the mechanism involved in the inhibition of biofilm formation and antibacterial action of endophytic Streptomyces sp. isolated from the Indian Sundarbans Mangrove Forest. In this study, Streptomyces sp. KJ7TB2 was isolated from the Indian Sundarbans. An ethyl acetate extract was subjected for GC-MS profiling. The mechanisms of antibacterial and antibiofilm action were also explored. In-silico molecular docking and dynamics simulation analyses were performed. The GC-MS analysis of the extract revealed 14 distinct bioactive secondary metabolites. The extract exhibited potent MIC values of 250 μg/mL and 125 μg/mL against Escherichia coli MTCC 1195 and Pseudomonas aeruginosa ATCC 27853. Antibiofilm investigation further revealed inhibition of biofilm formation in both pathogens especially in E. coli MTCC 1195, likely by disrupting initial surface adhesion and exopolysaccharide synthesis. Also, the extract inhibited swarming motility, suggesting interference with flagella-driven motility and quorum sensing. The brine shrimp lethality assay confirmed a favorable safety profile, recording limited mortality (36.84% at 2×MIC over 24 h). In-silico molecular docking and dynamics simulation analyses exhibited robust affinity for two key biofilm-associated proteins, exhibiting significant binding energy (ΔG). The study highlights Streptomyces sp. KJ7TB2 as a potential source for the discovery of safe, potent therapeutics. Exploring a Mangrove-derived microbe to fight against drug-resistant germs. Antibiotic resistance is a serious global health risk. Many antibiotics do not work as well as before. Because of this, bacterial infections are becoming harder to treat. Scientists are looking for new medicines from natural sources. In this study, we found a rare, helpful microbe from the mangrove forests of the Indian Sundarbans. These forests contain many useful microorganisms. A rare bacteria called Streptomyces sp. KJ7TB2 was collected and grown in the laboratory. After cultivation of the bacteria and collecting its extract, chemical studies found 14 useful compounds. The extract showed strong activity against harmful bacteria. It also stopped the bacteria from building protective walls around themselves. It blocks the signals of the bacteria use to talk with each other. Computational studies also supported these results. Safety tests showed very low toxicity of the extract. This means it may be safe for future studies. Overall, this study suggests that Streptomyces sp. KJ7TB2 could be a useful natural source for new treatments against drug-resistant bacterial infections.

ID: 42402928
Title: Assessment of the enhanced antibacterial activity of snail mucus, thymol, and ferulic acid in a rat model of burn wounds infected with Staphylococcus aureus and Pseudomonas aeruginosa.
Abstract: To develop and evaluate a novel topical formulation - comprising snail mucus, thymol, and ferulic acid (STF) - for treating wounds infected with multidrug-resistant bacteria, and to compare its therapeutic efficacy against silver sulfadiazine. The in vitro antimicrobial properties of STF were analyzed using checkerboard assays to determine the fractional inhibitory concentration (FIC) index. The cytotoxicity of STF was determined using the MTT viability assay using L929 fibroblasts. The in vivo efficacy of STF was assessed using a rat excisional wound model, and wound closure rates were measured at 14 days post-infection. STF showed an additive interaction in the in vitro model (FIC index: 0.717-0.936) and possessed low cytotoxicity (IC50: 705.4 µg/mL) compared to the individual compounds. In the in vivo model, the group treated with STF showed significantly faster wound healing and decreased inflammation compared to all other groups, including the silver sulfadiazine group. The combination of snail mucus in STF is a highly effective remedy for infected wounds, providing complementary benefits in vivo by coupling antimicrobial activity with pro-regenerative functions, and demonstrates superiority over silver sulfadiazine.

ID: 42404584
Title: Pseudomonas aeruginosa Virulence Bacteriophage Isolated From Inflammatory Mouse Feces Exhibits Bactericidal Activity in Infected Wounds of a Mouse Model.
Abstract: Recently, bacteriophages have risen as a potent therapy for superbug infections. The mammal gut demonstrates an interesting source of virulence bacteriophages. The gut with inflammation is phage-rich; therefore, we primarily aimed to prove the concept that an inflammatory gut is a possible source of effective phages and to evaluate the efficacy of the candidate phage against Pseudomonas aeruginosa in vitro and in a mouse model of infected wounds. The gut microbiome of cecal ligation and puncture (CLP) sepsis mice, an animal model of inflammation, showed a dominant presence of Podoviruses. CLP bacteriophages (CLP Φ1-Φ4), of which the CLP Φ4 possessed the broadest bactericidal activity (viable bacterial cell reduction in time-kill study) against P. aeruginosa isolates. The CLP Φ4 specifically killed the Pseudomonas aeruginosa clinical (PACL) strain with two huge burst events. Although the CLP Φ4 had no effect on ex vivo mouse bone marrow-derived macrophage (BMDM) cytokine gene expression and cytokine production, the CLP Φ4 attenuated the severity of the P. aeruginosa-infected wound mouse model after treatment. P. aeruginosa PACL exhibited significantly pathogenic characteristics in a mouse model, including excessive bacterial loads (in wounds and internal organs, indicating the systemic infection due to localized infected wound with P. aeruginosa), increased IL-6 cytokine (in serum), upregulated IL-6 expression (in wounds), and immune cell infiltration (in wounds), indicating severe inflammation. In the CLP Φ4 treatment alone, the wound tissues upregulated IL-10 expression and recruited inflammatory cells. Interestingly, the three-day CLP Φ4 treatment was adequate to eradicate P. aeruginosa PACL in the wounds and other internal organs. After treatment, the mouse serum cytokine showed a remarkably decreased IL-6. Likewise, IL-6 downregulation and IL-10 upregulation were demonstrated in the treated wounds, suggesting an anti-inflammatory shift. These results demonstrated the effectiveness (bacterial wound and internal organ clearance and cytokine modulation) of the CLP Φ4 in the P. aeruginosa-infected wound and systemic infection. Finally, the CLP Φ4 isolation verified a proof of concept that the irritated gut acts as a source of bacteriophages. The gut virome was a promising and interesting source of antimicrobial and immunomodulating bacteriophage.

ID: 42405072
Title: Citrus sinensis zest-mediated silver nanoparticles for rapid dye removal and antimicrobial properties.
Abstract: Growing interest in sustainable and renewable nanotechnology has accelerated the development of green synthesis strategies that are both environmentally friendly and economically viable. In this study, we report a simple, low-cost, and eco-conscious approach for synthesizing silver nanoparticles (AgNPs) using an aqueous zest extract of Citrus sinensis (Cs), which naturally serves as both the reducing and stabilizing agent. The phytochemicals present in the extract-including ascorbic acid, phenolic acids, flavonoids, terpenoids, and polysaccharides-effectively reduce Ag+ ions to stable AgNPs. The formation of AgNPs was confirmed through UV-Vis spectroscopy, revealing a characteristic surface plasmon resonance peak at 420 nm. FESEM imaging demonstrated predominantly spherical nanoparticles with uniform crystallite morphology and rough surface features, while TEM analysis showed an average particle size of approximately 15 ± 5.3 nm. XRD patterns confirmed the polycrystalline nature of the particles with diverse d-spacings, and EDX analysis indicated high purity (93.91% Ag) of the synthesized material. Compared to previously reported green synthesized nanoparticle systems, the biosynthesized AgNPs displayed excellent catalytic activity, achieving dye removal efficiencies of 98.93% (within 18 min) for methyl orange (MO) and 92.15% (within 30 min) for methylene blue (MB) under mild conditions, highlighting the advantage of waste-derived plant extract-mediated synthesis. Moreover, they exhibited strong antibacterial activity, showing notable inhibition against Gram-negative bacteria (E. coli,Pseudomonas spp.,K. pneumoniae, andSalmonella spp.) compared with Gram-positive strains (S. aureus andB. cereus), as assessed by inhibition zone measurements.

ID: 42405460
Title: Sustainable Synthesis of Bio-Based Furanic and Aromatic Amines Using an Optimized Whole-Cell Transaminase-Decarboxylase Cascade in E. coli RARE.
Abstract: The development of efficient biocatalytic routes for synthesizing primary amines is often hindered by the unfavorable thermodynamic equilibrium of enzymatic transamination. In this study, we present a highly effective bienzymatic cascade coupling the (S)-selective ω-transaminase from Pseudomonas psychrotolerans (PpS-TA) with pyruvate decarboxylase from Zymomonas mobilis (ZmPDC). This integrated system uses ZmPDC to irreversibly remove the pyruvate coproduct, thereby shifting the equilibrium toward the desired amine. Optimization of the purified enzyme system resulted in a twofold increase in conversion compared to the transaminase alone. The cascade exhibited broad substrate scope across various substituted benzaldehydes and simultaneously enabled the formation of valuable (R)-acyloin byproducts through the carboligation activity of ZmPDC. To enhance industrial applicability, the cascade was transferred to a whole-cell system using an engineered Escherichia coli strain (RARE), which minimizes undesired endogenous aldehyde reduction. The coexpressed whole-cell biocatalyst exhibited superior productivities at substrate loadings up to 50 mM without the need for organic cosolvents. Finally, the utility of this approach was demonstrated through the chemo-enzymatic synthesis of the bio-based platform molecule 2,5-bis(aminomethyl)furan from d-fructose, achieving an exceptional 90% conversion on a 500-mL preparative scale. This integrated strategy provides a sustainable and scalable framework for producing high-value aromatic and furanic amines.

ID: 42405543
Title: Metagenomic Analysis of Thoracic Empyema Etiology Through Next-Generation Sequencing Enhances Conventional Culture Techniques.
Abstract: This study aimed to analyze the microbiome of thoracic empyema using metagenomic methods and compare the results with conventional culture methods to increase diagnostic accuracy and enhance antibiotic therapy. This study involved 30 patients with thoracic empyema from hospitals in Riau Province, Indonesia. Pleural fluid samples were collected for culture analysis and identification using the Vitek 2 compact system and metagenomic analysis. Patient clinical data were also collected. Culture methods showed a 40.0% positive rate, with Gram-negative bacteria (Klebsiella pneumoniae and Pseudomonas aeruginosa) predominating. Metagenomics showed a 56.7% positive rate, identifying a more diverse microbiome, including fungi (29.4% abundance), other Gram-negative bacteria (26.5%), and anaerobic bacteria (22.5%). Comparison of the two methods showed 36.7% complete agreement and 23.3% partial agreement, with 40% disagreement, with a Kappa coefficient of 0.416 and P-value of 0.016 (P<0.050). Metagenomic NGS offers significant advantages in detecting the microbiome of thoracic empyema, particularly fungi and anaerobic bacteria, which are often missed by conventional culture methods. This has the potential to improve diagnostic accuracy and optimize antibiotic therapy. Further research with larger sample sizes is needed.

ID: 42406057
Title: Aloin of Aloe vera disrupts pseudomonas aeruginosa biofilm formation: exploring nutritional therapeutics.
Abstract: Pseudomonas aeruginosa is responsible for several infections due to its remarkable antibiotic resistance, facilitated by its ability to form biofilms. In this study, Aloin, an anthraquinone present in the plant Aloe vera, was tested for antibiofilm activity against P. aeruginosa PAO1. Aloin demonstrated efficient biofilm-inhibiting capacity at a sub-MIC dose of 4.8 mM, along with/without GEN at 0.002 µg/µL (sub-MIC), in an additive manner, determined by the FICI test. Furthermore, it reduced biofilm-associated carbohydrates by 66.9%, proteins by 66.22%, and extracellular DNA by 69.57%. In silico analysis revealed that Aloin interacts with key P. aeruginosa proteins, such as RhlI (Glide Score: -6.035 kcal/mol; Glide e-model score: -60.414 kcal/mol), LasI (Glide Score: -4.407 kcal/mol; Glide e-model score: -35.286 kcal/mol), LasR (chain A) (Glide Score: -3.764 kcal/mol; Glide e-model score: -36.012 kcal/mol), LasR (chain B) (Glide Score: -5.015 kcal/mol; Glide e-model score: -46.672 kcal/mol), PqsR (Glide Score: -4.701 kcal/mol; Glide e-model score: -25.491 kcal/mol), PelA (Glide Score: -6.097 kcal/mol; Glide e-model score: -53.723 kcal/mol), and PelB (Glide Score: -5.043 kcal/mol; Glide e-model score: -53.128 kcal/mol) by forming strong hydrogen bonds. In silico simulation studies also substantiated the Aloin-protein binding results. The production of virulence factors, such as exoprotease and pyocyanin, was reduced by 71% and 74%, respectively. Furthermore, CLSM and SEM analysis showed a decrease in biofilm thickness from 498 μm to 72 μm and a reduced denser structure in the presence of Aloin. These results suggest that Aloin from Aloe vera has the potential to be an effective biofilm inhibitor without toxicity, with broad applicability.

ID: 42406312
Title: Genomic landscape of 340 virulent Acinetobacter bacteriophages reveals anti-CRISPR-enriched candidates for therapeutic prioritization.
Abstract: Carbapenem-resistant Acinetobacter baumannii (CRAB) represents a critical global health threat for which existing antibiotics are increasingly inadequate. This study aimed to establish a comprehensive genomic framework for the rational prioritization of virulent Acinetobacter bacteriophages as therapeutic candidates. We performed large-scale comparative genomic analysis of 340 virulent Acinetobacter bacteriophages, integrating phylogenetic reconstruction, pangenome analysis, CRISPR spacer-based host interaction mapping, Anti-CRISPR protein identification, and systematic antimicrobial resistance (AMR) gene screening. Genome sizes spanned a nearly 20-fold range, with a significant negative correlation between genome size and GC content (R² = 0.139, ρ = -0.630). Phylogenetic analysis revealed extensive divergence across multiple lineages with no dominant clade. Pangenome analysis identified 20,982 unique protein families, of which 76.2% were cloud genes, confirming a highly open genome architecture. CRISPR spacer matching yielded 1,480 high-confidence matches across 100 phage genomes, providing molecular evidence of broad historical infectivity. Anti-CRISPR profiling identified Acinetobacter phage XC1 as an exceptional therapeutic candidate harboring 55 predicted Anti-CRISPR proteins with canonical regulatory locus architecture. AMR screening identified 21 distinct AMR gene homologs (Loose RGI hits, 22.5 to 47.1% amino acid identity) distributed heterogeneously across the dataset, confirming abundant therapeutically clean candidates while flagging a subset warranting further scrutiny before therapeutic exclusion. These findings provide a multi-criteria genomic framework for rational phage candidate prioritization against multidrug-resistant Acinetobacter infections, with direct implications for evidence-based phage therapy development.

ID: 42407157
Title: CRISPR-based diagnostics for ESKAPE drug-resistant bacteria: From proof-of-concept to point-of-care.
Abstract: The ESKAPE pathogens, including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species, represent a major challenge in the antimicrobial resistance (AMR) crisis. These pathogens are progressively acquiring pan-resistance and spreading beyond traditional healthcare settings, yet conventional diagnostic methods remain ill-equipped for point-of-care (POC) deployment due to slow processing times and limited adaptability. CRISPR/Cas systems feature programmable target specificity and intrinsic signal amplification, enabling rapid and accurate nucleic acid detection. Despite these advantages, the translation of CRISPR-based assays for ESKAPE pathogens from proof-of-concept to practical POC tools faces major challenges. Current research is fragmented, and key trade-offs between sensitivity, multiplexing performance and operational simplicity have not been fully addressed. This review offers a critical assessment of the field, moving beyond a simple summary of existing studies to analyze how various CRISPR systems (Cas9, Cas12, Cas13, and Cas14) and amplification strategies address the demands of POC testing. We identify key barriers to clinical application, particularly sample preparation, multiplex detection, reagent stability, and discuss emerging solutions such as microfluidic integration, lyophilized reagents development, and artificial intelligence-driven data interpretation. By focusing on the central question of how to transition from benchtop research to bedside application, this review provides a strategic framework for advancing next-generation CRISPR diagnostics capable of rapid, precise, and real-time detection of drug-resistant ESKAPE pathogens in the fight against AMR.

ID: 42409485
Title: Analysis of the antibacterial mechanism and antivirulence efficacies of citral against Shewanella putrefaciens and Pseudomonas fluorescens in mono- and co-culture.
Abstract: Shewanella putrefaciens and Pseudomonas fluorescens, as significant specific spoilage organisms in aquatic products, can regulate cell density and a variety of physiological processes through quorum sensing (QS). This research examined the antibacterial mechanism and antivirulence efficacies of citral (CT), as a quorum sensing inhibitor (QSI), against S. putrefaciens and P. fluorescens in mono- and co-culture. Furthermore, the application of CT to large yellow croaker preservation was investigated. Results revealed that CT exerted its antibacterial activities in a dose-dependent manner. CT increased intracellular oxidative stress levels and malondialdehyde (MDA) content, decreased ATP content and peroxidase activity, and promoted the damage of cell membrane, especially in S. putrefaciens group. These two bacteria had a synergistic effect in causing spoilage through biofilm formation and motility. The antivirulence efficacies of CT against the co-culture group were weaker than those of in the mono-culture groups. CT was also found to suppress the expression of key virulence regulators in S. putrefaciens and P. fluorescens virulence, including Autoinducers-2 (AI-2) production in the QS system. Furthermore, CT had the potential capability in the preservation of large yellow croaker. Consequently, these results proved CT could combat bacteria and had antivirulence efficacies against S. putrefaciens and P. fluorescens in mono- and co-culture.

ID: 42409517
Title: Synergistic inhibition mechanism of slightly acidic electrolyzed water combined with ε-polylysine hydrochloride against Pseudomonas fluorescens.
Abstract: This study explored the synergistic inhibition mechanism on combining slightly acidic electrolyzed water (SAEW) and ε-polylysine hydrochloride (ε-PLH) against Pseudomonas fluorescens, a key spoilage bacterium in aquatic products. A significant synergistic antibacterial effect was achieved with the fractional inhibitory concentration index being 0.5 when the two agents were combined at their respective 1/4 of minimum inhibitory concentration. Mechanistic studies revealed that the combined treatment severely disrupted bacterial membrane integrity and potential, inhibited biofilm formation, and caused extensive cell lysis in P. fluorescens PF08. Fourier transform infrared spectroscopy further indicated that the combined treatment caused the most severe damage to cell wall peptidoglycan, proteins, and polysaccharides. Non-targeted metabolomics disclosed that SAEW primarily disturbed histidine and lysine metabolisms, while ε-PLH interfered with arginine and glutathione metabolisms, and their combination triggered broader metabolic reprogramming in the strain, demonstrating a multi-target antibacterial mechanism. This study provides a theoretical basis for the application of the SAEW-ε-PLH composite green antibacterial agent in the preservation of aquatic raw materials and their prefabricated meal products which is perishable due to contamination by P. fluorescens.

ID: 42410210
Title: Non-canonical gene amplifications facilitate adaptive evolution in bacteria.
Abstract: Gene amplification, a common route to bacterial adaptation, often occurs through recombination between two copies of an insertion sequence (IS) element flanking a genomic region. Alternative non-canonical structures have also been proposed, in which a duplication is formed by a single IS element whose two ends join two distant chromosomal loci. However, the prevalence of such non-canonical structures and their role in bacterial adaptive evolution remain unclear. Here we developed AmpliFinder, a computational tool that uses short-read sequencing data to systematically identify pairs of IS-chromosome junctions that correspond to the two ends of the same IS element yet map to distant genomic loci flanking amplified regions. Applying AmpliFinder to 10,347 laboratory-evolved Escherichia coli and Acinetobacter baumannii isolates, we identified 113 distinct de novo IS-associated amplifications and found that non-canonical amplifications are the most abundant mode of amplification. We validated the inferred architectures using ultra-long-read sequencing and propose a model for non-canonical amplification formation supported by the observation of nested intermediate structures. Quantifying enrichment for antibiotic-resistance genes in amplicons, we find that non-canonical amplifications more effectively and narrowly amplify genes under selection. These results highlight the role of non-canonical IS-based amplifications in the adaptive evolution of bacteria.

ID: 42411263
Title: Dual Sub‑MIC Copper-Gentamicin Stress Drives Strain‑Specific, Non‑Additive Phenotypic Shifts in Pseudomonas aeruginosa.
Abstract: Exposure to redox‑active metals and sub‑inhibitory antibiotics represents a significant selective pressure shaping bacterial physiology and antimicrobial susceptibility. Here, we investigated how four genetically and phenotypically distinct Pseudomonas aeruginosa strains respond to sub‑MIC copper (Cu) and gentamicin (GE) stress, individually and in combination (Cu + GE). Across all strains, Cu acted as the dominant envelope‑active stressor, increasing biofilm biomass and extracellular DNA (eDNA) release, suppressing twitching and swarming motility, reducing quorum‑sensing‑linked protease activity, and enhancing pyomelanin production. GE alone produced limited physiological changes but modulated Cu‑driven outputs during co‑exposure, including attenuation of Cu‑induced eDNA release and strain‑specific shifts in motility and pigmentation. Early transcriptional profiling revealed consistent Cu‑dependent repression of lasI and mvfR, induction of the metal‑responsive regulator czcR, and downregulation of oprD under Cu or Cu + GE, corresponding to reduced imipenem inhibition zones when Cu was present during susceptibility testing. Combined Cu + GE exposure produced non‑additive, emergent effects that diverged from single‑stressor responses and varied across strains. These findings demonstrate that sub‑inhibitory Cu creates an envelope‑centered regulatory landscape into which gentamicin‑derived signals are integrated, generating heterogeneous and context‑dependent phenotypes. This work underscores the importance of metal-antibiotic interactions in shaping bacterial adaptation and highlights limitations of single‑stressor models for predicting antimicrobial behavior in combination.

ID: 42412003
Title: Host-directed antimicrobial peptide biomaterials for combating Gram-negative bacterial infections.
Abstract: Antimicrobial resistance (AMR) represents a defining crisis in modern infectious disease medicine. Gram-negative microbes such as carbapenem-resistant Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae are becoming more resistant than ever. Traditional methods of treatment using antibiotics are ineffective because of the presence of several envelope layers, active efflux pumps, and biofilm production. In contrast, bacteriolytic therapy can exacerbate host immunopathology owing to systemic endotoxin release. Host-defense peptides are thus being explored for their ability to kill bacteria, neutralize lipopolysaccharide (LPS), and modulate innate immune responses in a single treatment; however, clinical use remains hindered by their low stability, high binding to serum proteins, and narrow therapeutic index. In this review, we will discuss the use of biomaterial platforms in the design of peptide-based therapeutics to not only increase their efficacy by reducing peptide clearance but also optimize the immunological environment in which these peptides operate. We will discuss the role of various characteristics, such as surface chemistry, mechanical stiffness, and protein corona, in influencing macrophage polarization and downstream signaling pathways. In addition, we point out a significant limitation within this area of research: many preclinical studies continue to use minimum inhibitory concentration and hemolysis values, which fail to account for the host-directed effects that make these approaches significant. For future progress in this realm, new endpoints are required, along with more clinically relevant animal models and a true incorporation of peptide design into biomaterials engineering.

ID: 42412160
Title: Lactobacillus strains in the prevention and disruption of antimicrobial-resistance in pathogenic microbial biofilms: a review of mechanisms and clinical perspective.
Abstract: The growing prevalence of antibiotic resistance, coupled with the heightened drug tolerance exhibited by biofilm-associated infections, represents a major public health challenge and demands the development of innovative therapeutic interventions. Probiotics, particularly the genus Lactobacilli, offer an effective, multifaceted strategy that targets biofilm-associated pathogens through competitive exclusion, secretion of antimicrobial metabolites, disruption of quorum sensing, enzymatic degradation of extracellular polymeric substances, and host immunomodulation. Studies demonstrate robust efficacy of probiotics against clinically challenging pathogens, including methicillin-resistant Staphylococcus aureus, multidrug-resistant Pseudomonas aeruginosa, Clostridioides difficile, Helicobacter pylori, and pathogenic Escherichia coli. Postbiotics, particularly cell-free supernatants and purified bacteriocins, offer standardized, shelf-life probiotic products. Notably, synergistic combinations with conventional antibiotics, phytochemicals, or bacteriophages enhance antimicrobial activity while enabling dose-reduction strategies to mitigate the development of resistance. However, compelling preclinical evidence, clinical translation remains constrained by the imperative for strain-specific characterization, methodologically robust randomized controlled trials, and comprehensive safety validation. This study critically mentions current mechanisms underlying Lactobacillus-mediated anti-biofilm strategies and identifies priority research essential to accelerating clinical application.

ID: 42412311
Title: Using the Damage Response Framework as a Lens to Discuss Acinetobacter baumannii Host-Pathogen Interactions: A Narrative Review and a Call to Action.
Abstract: The Damage Response Framework (DRF) is a conceptual model that evaluates the outcome of host-pathogen interactions (HPI) by assessing the total damage sustained by the host. The DRF emphasizes that disease results from cumulative host injury arising from the interplay between microbial virulence and the host immune response, rather than from the pathogen alone. HPI has been described as a "tug of war" between a pathogen and a host; the DRF improves this analogy by grounding it in reality, as both excessive immune activation and insufficient pathogen clearance can lead to host damage and disease. Acinetobacter baumannii is a Gram-negative pathogen known for its extensive drug resistance and immunomodulatory capacities. The interaction among isolate-specific virulence traits, antimicrobial resistance mechanisms, and host immune status determines the pattern and extent of damage, illustrating central tenets of the DRF. Different A. baumannii isolates may therefore produce varied clinical outcomes across different host conditions, depending on the extent of damage they cause. This narrative review discusses A. baumannii infections from the perspective of the DRF, highlighting how applying the DRF can inform our current understanding of A. baumannii pathogenesis by assessing A. baumannii HPI. We also provide brief insights into how the DRF can inform future research and treatment strategies that integrate host and microbial factors to minimize overall damage and improve host outcomes. A. baumannii infections are difficult to treat and can result in outcomes ranging from asymptomatic colonization to fatal disease. This narrative review examines these differences through the Damage Response Framework (DRF) to improve understanding of A. baumannii infections. The DRF emphasizes that disease results not only from the microbe but also from the interaction between microbial factors and the body’s immune response. When the total damage from the microbe and the immune response exceeds the body's ability to repair, hosts become sick. Hosts with weak immune responses can become very ill because their immune systems cannot control the infection effectively. Conversely, patients with strong immune responses can also become severely ill when their immune systems damage vital tissues and organs in an effort to eliminate the bacteria. This narrative review draws on existing literature to show that different A. baumannii isolates can cause varying levels of illness, depending on the host’s response to the infection. The review also briefly describes the importance of considering antimicrobial therapy as a potential source of host damage and the utility of the DRF in optimizing therapeutic strategies.

ID: 42414298
Title: Role of starvation survival response mechanisms on ribosome integrity, antibiotic tolerances, and virulence of Pseudomonas aeruginosa biofilms.
Abstract: Bacterial biofilms contain physiologically diverse subpopulations of cells, including cells that are nutrient stressed or dormant. We determined how two dormancy pathways, ribosome hibernation and the stringent response, contribute to the survival and antibiotic tolerance of Pseudomonas aeruginosa biofilms. Analyses of whole biofilms and single cells showed that these pathways have differing effects on biofilm cell physiology. Ribosome hibernation, mediated by hibernation promoting factor (HPF), is essential for optimal survival and resuscitation of starved biofilm cells. In the absence of HPF, starved cells progressively lose ribosome integrity. However, loss of HPF does not increase the sensitivity of P. aeruginosa biofilm cells to ciprofloxacin or tobramycin. In contrast, the stringent response, mediated by RelA and SpoT, is not required for viability or ribosome integrity in starved biofilm cells, but does affect biofilm antibiotic tolerance. In a plant model of biofilm infection, disruption of either ribosome hibernation or the stringent response reduced bacterial virulence. The results show that ribosome hibernation preserves ribosomal integrity necessary for recovery from starvation and for pathogenesis, while the stringent response is required for growth arrest, antibiotic tolerance, and pathogenesis. These two ribosome-mediated pathways play distinct yet complementary roles in regulating dormancy and persistence of P. aeruginosa biofilms.

ID: 42414520
Title: Single pass inactivation of airborne multidrug resistant bacteria using electron beam irradiation with mechanistic evidence.
Abstract: Airborne and aerosol transmission of multidrug-resistant (MDR) bacteria pose a major challenge in healthcare environments. This study evaluated the performance of an electron-beam irradiation (EBI) system for inactivating airborne MDR bacteria under controlled laboratory conditions. To perform this assessment, clinical isolates of MDR Pseudomonas aeruginosa, Acinetobacter sp., Klebsiella sp., and methicillin-resistant Staphylococcus aureus (MRSA) were aerosolized to simulate realistic airborne transmission and subsequently introduced into the EBI system. Downstream bacterial aerosols were collected using liquid impingers and quantified by culture-based enumeration. CFU data were log₁₀-transformed prior to analysis. The EBI system reduced airborne bacterial loads by 4.01 log₁₀ for P. aeruginosa, 3.64 log₁₀ for Acinetobacter sp., 3.64 log₁₀ for Klebsiella sp., and 5.37 log₁₀ for MRSA, corresponding to disinfection efficiencies of 99.98-99.999%. Scanning electron microscopy (SEM) and elemental mechanistic study revealed morphological damage, including membrane rupture, surface collapse, and loss of structural integrity, suggesting that electron impact and electroporation may be major mechanisms of inactivation. These results demonstrate that EBI is a rapid and effective method for mitigating airborne MDR bacterial threats, with potential for deployment in healthcare, laboratory, and other high-risk indoor environments.

ID: 42414953
Title: Dual antimicrobial and anticancer functions of artemisinin-loaded PEG400-oa nanoparticles: synergistic activity with ciprofloxacin and mechanistic insights from molecular docking.
Abstract: Artemisinin, a bioactive compound from Artemisia annua with potent antioxidant, antimicrobial and anticancer properties, faces limited clinical utility due to poor solubility and low bioavailability. Given the rising prevalence of multidrug-resistant (MDR) Pseudomonas aeruginosa alternative therapeutic strategies are essential. This study synthesized and characterized artemisinin-loaded micelle/liposome nanoparticles (Art/PEG400-OA NPs) and evaluated their antibacterial, antibiofilm, and anticancer efficacy. Artemisinin was successfully encapsulated within PEG400-OA NPs and characterized via FT-IR, FE-SEM, TEM, TGA, and zeta potential measurements. The nanoparticles exhibited uniform morphology, particle sizes of 40.8 nm (TEM) and 149.8 nm (FE-SEM), high encapsulation efficiency (92.14%), and strong thermal and colloidal stability. Synergistic and partial synergistic interactions between Art/PEG400-OA NPs and ciprofloxacin were observed in MDR bacterial samples, significantly inhibiting biofilm formation. Quantitative RT-PCR analysis revealed the downregulation of efflux pump genes (mexA, mexB, mexX, mexY, oprM) and biofilm-associated genes (algD, pelA) following combination therapy. In AGS gastric cancer cells, Art/PEG400-OA NPs induced dose- and time-dependent cytotoxicity (IC₅₀: 67.75, 62.25 and 54.98 µg/mL at 24, 48 and 72 h), increased apoptosis (31.76% ± 4.2%), and reduced wound-healing migration (56.65% ± 4.26%). Expression analysis demonstrated upregulation of tumor-suppressive miR-34a, and downregulation of its targets (CTNNB1, TGFB1, MMP2 and MMP9). Conversely, miR-181a was downregulated, leading to the upregulation of apoptotic target genes (TP53, CASP8, CASP9). Molecular docking supported strong artemisinin binding to key bacterial and oncogenic proteins. Overall, Art/PEG400-OA nanoparticles represent a potent dual-action nanotherapeutic platform against antibiotic-resistant infections and gastric cancer.

ID: 42418271
Title: Beyond bacterial communication: host factors that modulate quorum sensing and virulence in human pathogens.
Abstract: Quorum sensing (QS) coordinates collective bacterial behavior, but during infection, its activity is also shaped by host biology. This review examines how host-derived factors modulate QS-linked virulence in enteric pathogens, Pseudomonas aeruginosa and Staphylococcus aureus. Across these systems, host control operates through three recurring modes: activation or signal mimicry, enzymatic signal degradation, and extracellular sequestration or receptor-level interference. Catecholamines and related metabolites can feed into bacterial sensory pathways and promote virulence signaling; PONs (paraoxonases) and other host enzymes can degrade autoinducers; and lipoproteins, immune mediators, and epithelial surveillance systems can intercept or reinterpret quorum signals. Together, these data support the view that the host is an active signal-processing environment that reshapes colonization, persistence, and tissue damage, and they point to host-inspired anti-virulence strategies that target signaling rather than viability.

ID: 42419223
Title: Viscum album subsp. austriacum-mediated biosynthesis of magnesium nanoparticles: In vitro anticancer, antibiofilm, and antioxidant activities with computational validation.
Abstract: Viscum album L. (European mistletoe) is a medicinally significant hemiparasitic plant with a rich bioactive phytochemical profile. Despite growing interest in its pharmacological properties, studies integrating nanoparticle biosynthesis, comprehensive biological evaluation, and computational analysis remain scarce for its austriacum subspecies. In this study, magnesium nanoparticles (VA-MgNPs) were green-synthesized using the ethanol extract of Viscum album subsp. austriacum as a bioreducing and capping agent, and their biological activities were comparatively assessed alongside the crude extract and Mg(NO3)2·6H2O precursor control. LC-HR/MS analysis identified quinic acid (10,027.49 μg/g) and chlorogenic acid (5294.12 μg/g) as the dominant phenolic constituents. The synthesized VA-MgNPs were characterized by FT-IR, UV-Vis, FE-SEM, TEM, and XRD analyses, confirming nanocrystalline formation with a mean crystallite size of approximately 66.47 nm. Antioxidant evaluation using DPPH, ABTS, and FRAP assays revealed that the crude extract retained superior radical scavenging capacity, which is attributed to the consumption of free phenolic hydroxyl groups during nanoparticle formation. In contrast, VA-MgNPs demonstrated markedly enhanced anticancer activity against breast (MCF-7: IC50 = 124 μg/mL; MDA-MB-231: IC50 = 96 μg/mL) and colorectal (HCT116: IC50 = 105 μg/mL; HT-29: IC50 = 135 μg/mL) cancer cell lines, with lower cytotoxicity toward non-tumorigenic cells, indicating selective antiproliferative action. Although antibacterial activity was limited across tested strains, VA-MgNPs exhibited significant antibiofilm activity, achieving up to 75% inhibition against Pseudomonas aeruginosa. Molecular docking simulations revealed that chlorogenic acid and its magnesium complex displayed strong binding affinities toward antioxidant- and antimicrobial-related protein targets, with binding energies reaching -8.5 kcal/mol, mechanistically supporting the observed biological activities. These findings demonstrate that VA-MgNPs are a promising nanotherapy platform, particularly for antibiofilm and anticancer applications.

ID: 42419610
Title: Nanomedicine strategies against multidrug-resistant Acinetobacter baumannii: Delivery, biofilm disruption, and translational considerations.
Abstract: Acinetobacter baumannii has become a high-priority global problem because it causes hospital-acquired infections and ventilator-associated pneumonia, and multidrug resistance, environmental survivability, and biofilm formation severely restrict therapeutic choices. Antimicrobial nanomedicine has become a major focus as a translational approach. This review critically explores nanotechnology-based interventions for A. baumannii. Multimodal antibacterial and antibiofilm activities have been demonstrated in inorganic nanoparticles, such as silver, gold, and copper-based systems, with an emphasis on their translational therapeutic potential and nano-bio interactions. Polymer- and lipid-based nanocarriers focus on optimising pharmacokinetics, controlled release, and targeted delivery, supporting improved bioavailability and site-specific exposure. Carbon nanomaterials offer alternative antibacterial mechanisms but raise concerns regarding biodegradability and safety. Overall, antimicrobial nanomedicine can be considered an exposure- and delivery-focused system with significant potential for treating persistent drug-resistant A. baumannii infections, highlighting the translational, toxicological, and regulatory challenges relevant to clinical adoption.

ID: 42420271
Title: Immune protection and persistence in Pseudomonas aeruginosa infections by pyruvate cross-feeding.
Abstract: Chronic infections by Pseudomonas aeruginosa in people with cystic fibrosis are characterized by persistent inflammation and oxidative stress, yet the mechanisms enabling bacterial persistence are not fully understood. Here, we identify a persistence mechanism mediated by pyruvate secretion resulting from mutations in the pyruvate dehydrogenase complex in clinical isolates of P. aeruginosa, with putative analogous mutations also identified in Staphylococcus aureus, Haemophilus influenzae and Stenotrophomonas maltophilia. These mutations lead to elevated extracellular pyruvate, which dampens host inflammatory responses and favors bacterial persistence. Pyruvate exerts multiple roles: scavenges reactive oxygen species such as H2O2, suppresses host immune activation both in airway epithelial cells and macrophages, and increases bacterial survival during phagocytosis. This metabolic crosstalk promotes bacterial persistence while reducing epithelial and macrophage inflammatory responses. Our findings reveal pyruvate as a bacterial immunometabolite that mimics host antioxidant defenses, reshaping the infection niche to favor long-term colonization. This work highlights the broader role of secreted metabolites in host-pathogen interactions and suggests new strategies targeting metabolic pathways to manage chronic infections.

ID: 42420327
Title: Pulmonary mRNA-LNP vaccines for rapid and durable protection against bacterial infection.
Abstract: Pulmonary bacterial infections remain a major clinical challenge. Although vaccination reduces infection rates and mortality, the vulnerable post-vaccination immunity gap can still result in infection and vaccine failure. In addition, effective vaccines are unavailable for many clinically important bacterial pathogens. Here, we report a pulmonary mRNA-lipid nanoparticle (mRNA-LNP) vaccine incorporating an ionizable lipid engineered for localized high-level expression, which elicits both rapid and durable protection against bacterial lung infections in female mice, effectively bridging this critical window of vulnerability. Intratracheal delivery of mRNA-LNP rapidly primes lung neutrophils and macrophages into a transcriptionally pre-activated state, enhancing their phagocytic activity and enabling rapid, antigen-independent bacterial clearance during the early post-vaccination period (approximately 1-7 days). Subsequently, vaccination induces potent antigen-specific adaptive responses, conferring sustained protection against both laboratory and clinical drug-resistant Pseudomonas aeruginosa strains. Single-cell transcriptomics and immune profiling reveal coordinated activation of innate and adaptive immune programs. This dual-phase immune response exemplifies a paradigm-shifting vaccine design that integrates innate and adaptive immunity to confer both immediate and long-term protection. Our findings establish a mechanistic basis for rapid antibacterial defense and highlight pulmonary mRNA-LNP vaccination as a promising strategy for combating respiratory infections.

ID: 42422065
Title: Machine learning-driven discovery of antimicrobial peptides against Pseudomonas aeruginosa.
Abstract: Antibiotic resistance has become a global health crisis, driving the urgent need for novel antibacterial strategies. Antimicrobial peptides (AMPs) have emerged as promising therapeutic candidates due to their broad-spectrum activity and low propensity for resistance development. However, conventional discovery approaches remain time-consuming and inefficient for large-scale screening. In this study, we developed an integrated prediction framework that combines deep learning with classical machine learning methods to enable rapid and accurate identification of AMPs. The model was applied to screen over 250,000 artificially generated peptide sequences. Candidate peptides were selected based on physicochemical descriptors and activity-associated features, followed by solid-phase synthesis and minimum inhibitory concentration assays against Pseudomonas aeruginosa. Mechanistic investigations were conducted using scanning electron microscopy to visualize membrane damage, molecular dynamics simulations to probe peptide-membrane interactions, and transcriptomic profiling to assess bacterial stress responses and metabolic pathway alterations. Ten promising antibacterial candidates were successfully identified and experimentally validated. All selected peptides exhibited measurable activity against P. aeruginosa, with several showing potent inhibitory effects. Microscopic and simulation analyses confirmed that the peptides exert their antibacterial effects primarily through membrane disruption. Transcriptomic data further revealed that these peptides interfere with key metabolic pathways and activate bacterial stress response systems. Our findings demonstrate that the integrated deep learning-machine learning pipeline offers an efficient and reliable approach for large-scale AMP discovery. The mechanistic insights gained from this study not only validate the predicted candidates but also provide a foundation for rational optimization of peptide-based therapeutics. This comprehensive strategy holds promise for accelerating the development of next-generation alternatives to conventional antibiotics.

ID: 42422733
Title: Convergent GenX biodegradation by genomically designed and functionally screened synthetic bacterial consortia.
Abstract: GenX (hexafluoropropylene oxide dimer acid) has been introduced as a replacement for legacy polyfluoroalkyl substances (PFASs) compounds; however, accumulating evidence indicates this C6 ether-PFAS may exhibit comparable toxicity to PFOA while persisting in contaminated water supplies. Biological remediation offers genuine mineralization potential but has been constrained by the exceptional stability of carbon-fluorine bonds. Here, we demonstrate that synthetic bacterial consortia assembled through rational genomic design (GENIA) or microfluidic selection (Community G) achieve rapid GenX biodegradation through metabolic complementarity among functionally specialized taxa. Both consortia removed >80% of GenX within 3 days (GENIA: 94.6%; Community G: 80.6%), with defluorination confirmed by fluoride release (82.6 and 69.6%, respectively). First-order kinetic modeling yielded rate constants of k = 0.87 day-1 (GENIA) and k = 0.64 day-1 (Community G), corresponding to half-lives under 1.1 days. Despite pronounced taxonomic divergence, GENIA maintained a stable composition, whereas Community G underwent dramatic restructuring, enriching for specialists of Salmonella, Pseudomonas, and Stenotrophomonas. Comparative genomic analysis revealed functional equivalence, with no significant differences in PFAS-degrading gene content (p = 0.844). Multi-guild community architecture emerged wherein primary degraders (40%-60% abundance) harboring haloacid dehalogenases performed initial C-F bond cleavage, detoxification specialists possessing glutathione S-transferases and fluoride exporters (>90% universal distribution) processed toxic intermediates, and metabolic support taxa enhanced resilience. Molecular docking simulations predicted favorable binding of GenX across phylogenetically diverse dehalogenases (ΔG = -7.0 to -8.1 kcal/mol), suggesting thermodynamic compatibility with enzymatic catalysis. These results are predictive and computational; docking cannot confirm enzymatic activity or substrate turnover. Experimental validation (enzyme kinetics, purified protein assays) is required. Critically, GENIA activity did not disrupt indigenous soil microbiomes. The data support that GENIA does not cause detectable diversity loss, a necessary but not sufficient condition for ecological compatibility. These findings establish that functional gene diversity, distributed across metabolically complementary taxa through either rational design or adaptive selection, enables efficient PFAS bioremediation, converting recalcitrant "forever chemicals" into biodegradable substrates through engineered microbial complexity.

ID: 42422903
Title: Spectroscopic discrimination of bacterial species of variable pathogenicity through explainable machine learning.
Abstract: Rapid and accurate identification of bacterial pathogens and their degree of pathogenicity is essential for guiding antimicrobial therapy. Current culture-based methods require a timeframe of at least 24-48 h for species-level identification alone, which often leads to substantial disease progression and increases the propensity of antimicrobial resistance (AMR). Surface-Enhanced Raman Spectroscopy (SERS) shows promise in mitigating these drawbacks by providing a fast, non-invasive, label-free method to capture the biochemical fingerprints of bacteria achievable under clinical settings. However, the molecular complexity of SERS spectra, which has been an impediment for conventional data analysis, demands robust computational frameworks for reliable species-level identification. Here, we employ a comprehensive SERS analysis scheme with supervised machine learning and explainable artificial intelligence (XAI) to discriminate five clinically relevant pathogens, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), and Enterococcus faecalis, and to probe their degree of pathogenicity from a biomarker perspective. Among the tested models - Support Vector Machine (SVM), k-Nearest Neighbour (kNN), Random Forest (RF), and a 1D Convolutional Neural Network (CNN) - CNN achieved near-perfect classification accuracy, capturing subtle and spectrally relevant variations often inaccessible to traditional algorithms. Notably, the 1D-CNN also achieved 100% discrimination between MRSA and methicillin-sensitive S. aureus - two strains of the same species differing only in resistance phenotype. To ensure transparent decision-making and eliminate the black-box nature of machine learning models, SHapley Additive exPlanations (SHAP) analysis was applied to both RF and CNN models, which facilitated the convergence of both frameworks on the same discriminatory Raman regions, revealing conserved biochemical determinants of species identity. Complementary MCR-ALS decomposition further resolved the spectra into interpretable biochemical components and provided the rubric for biochemical differentiation. Together, this study aims to demonstrate an end-to-end explainable workflow that couples SERS with interpretable AI, offering a rapid, transparent approach for pathogenic disease diagnosis.