Search Results (355)

Background/Objectives: Bacterial biofilms formed by Escherichia coli pose a significant challenge in veterinary medicine due to their intrinsic resistance to antibiotics. Antimicrobial peptides (AMPs) represent a promising alternative. AMPs exert their bactericidal activity by binding to negatively charged phospholipids in bacterial membranes via electrostatic interactions, leading to membrane disruption and rapid cell lysis. Methods: In vitro assays including MIC determination, biofilm eradication testing (crystal violet, colony counts, and CLSM), swimming motility, and EPS quantification were performed. CRISPR/Cas9 was used to construct and complement a kduD mutant. A transposon mutagenesis library was screened for biofilm-defective mutants. In an in vivo murine excisional wound infection model treated with the mouse cathelicidin-related antimicrobial peptide (CRAMP-34), wound closure and bacterial burden were monitored. Gene expression changes were analyzed via RT-qPCR. Results: CRAMP-34 effectively eradicated pre-formed biofilms of a clinically relevant, porcine-origin E. coli strain and promoted wound healing in the murine infection model. We conducted a genome-wide transposon mutagenesis screen, which identified kduD as a critical gene for robust biofilm formation. Functional characterization revealed that kduD deletion drastically impairs flagellar motility and alters exopolysaccharide production, leading to defective biofilm architecture without affecting growth. Notably, the anti-biofilm activity of CRAMP-34 phenocopied aspects of the kduD deletion, including motility inhibition and transcriptional repression of a common set of biofilm-related genes. Conclusions: This research highlights CRAMP-34 as a potent anti-biofilm agent and unveils kduD as a previously unrecognized regulator of E. coli biofilm development, which is also targeted by CRAMP-34. Full article

Background/Objectives: Discriminating bacterial from mammalian membranes remains a central challenge in antibiotic design. Bacterial membranes are enriched in phosphatidylethanolamine (PE), a lipid normally absent from the outer leaflet of mammalian cells, providing a signature for selective molecular engagement. We report a compact covalent ligand, 6-dimethylamino-4-ketohexanoic acid (DMAX), which targets PE via Schiff base formation, leveraging its tertiary amine to facilitate the reaction and strengthen ionic binding with the phosphate group. Methods: The reactivity of DMAX and PE was evaluated by computational simulations, and their interaction was examined by spectroscopic analyses (NMR and FT-IR) and an artificial membrane assay. The targeting ability of DMAX for live bacteria was determined by microscopy study, and its applicability to therapeutic system was tested in vitro under washed conditions that mimic rapid in vivo clearance. Results: Spectrometric analyses revealed the selective covalent interaction of DMAX and PE, consistent with the simulated results. Fluorescently labeled DMAX selectively binds PE-enriched model membranes and efficiently recognizes Gram-negative bacteria while sparing mammalian cells. Conjugation of DMAX to Gemifloxacin (Gem) significantly enhanced antibiotic efficacy by 10-fold compared with free Gem, even after rapid drug clearance, while maintaining safety in mammalian cells. Conclusions: These results identify DMAX as an efficient and versatile PE-targeting platform, enabling selective membrane anchoring to advance precision antibiotic strategies. Full article

One significant trend in the research of plant treatment methods is that regarding the use of natural-based methods in plant protection. In this study, antimicrobial activity and changes in MdPR10 gene expression were tested for a total of five plant pathogens in a model of apple fruits, where strawberry and ivy EOs were used. The vapor-phase chemical composition of both EOs was profiled using HS-GC-MS. qRT-PCR was applied for a bacterial response analysis, together with disk diffusion assays, and minimum inhibitory concentrations were determined. To elucidate the molecular basis of the antibacterial potential of essential oils (EOs), docking analyses were performed. For Xanthomonas arboricola and Pectobacterium carotovorum, the presence of EOs resulted in the downregulation of MdPR10. Strawberry EO was more effective against weakly virulent strains of bacteria; ivy EO had greater inhibitory effects. HS-GC-MS detected 13 volatiles in strawberry EO—dominated by ethyl butyrate, ethyl 2-methylbutanoate, ethyl hexanoate, and ethyl 3-methylbutanoate—and 16 in ivy EO, characterized by monoterpenes and monoterpenoids with 1,8-cineole as the principal component. P-cymene showed the most potent binding activity against D-alanine–D-alanine ligase. Ivy EO has the potential to be effective as a natural preservative alternative mainly in postharvest technology. Full article

Antibiotic-resistant infections remain a major challenge in cystic fibrosis (CF), where chronic Pseudomonas aeruginosa colonization drives lung infection. The overexpression of adhesion-related proteins and extracellular matrix components, including fibronectin (Fn), facilitates bacterial colonization. Recent evidence identifies the RNA-binding protein Human Antigen R (HuR) as a key regulator of this process, as it stabilizes Vav3 mRNA, promoting Fn deposition and the formation of bacterial docking platforms. Here, we report the synthesis, optimization, and functional evaluation of the HuR-targeted small-molecule (2S,3S)-BOPC1. Functional assays in CF human airway epithelial cells demonstrated that (2S,3S)-BOPC1 significantly reduced P. aeruginosa adhesion in a dose-dependent manner without detectable cytotoxic effects. These findings provide the first evidence that targeting HuR can disrupt the HuR–Vav3–Fn axis, reducing bacterial attachment. This host-directed approach represents a promising strategy to prevent chronic infections in CF without promoting antibiotic resistance. Full article

Background: Bacterial keratitis, a major cause of corneal blindness, is frequently associated with biofilm-forming pathogens such as Klebsiella pneumoniae. Cyclic-di-GMP (c-di-GMP) controls biofilm development, which increases antibiotic resistance and makes treatment more difficult, highlighting the need for innovative therapeutic approaches. Methods: This study investigated cinnamaldehyde as a potential ocular therapeutic using combined computational and experimental approaches. Molecular docking and in vitro assays (XTT, resazurin reduction, crystal violet staining, qRT-PCR, and fluorescence microscopy) were used to evaluate the anti-biofilm and immunomodulatory activities of cinnamaldehyde (CA) against Klebsiella pneumoniae. Results: CA inhibited biofilm formation in a dose-dependent manner (≈89% at 1000 µM; >50% at 250 µM), reduced bacterial attachment to contact lenses, and downregulated key biofilm genes (mrkA, mrkC, ybtS, bolA). Docking analysis revealed strong binding affinity to the mrkH regulator (−5.46 kcal/mol. CA maintained more than 80% corneal cell viability by increasing IL-10, suppressing inflammatory mediators (IL-1β, IL-6, and TNF-α), and improving bacterial clearance. Conclusions: This study combines computational docking, biofilm quantification, immune cell assays, and functional gene expression analyses to reveal the ability of cinnamaldehyde not only to suppress biofilm formation but also to enhance macrophage-mediated clearance and modulate corneal immune responses, a multi-target approach not previously described in the context of bacterial keratitis. Such effects highlight its potential as a novel ocular drug candidate for protecting corneal integrity in infectious keratitis. Full article

A novel series of Schiff bases (3a–3k), incorporating tranexamic acid (TXA) and phenol-derived aldehydes via imine linkers, was synthesized and structurally characterized. The antimicrobial activity of the compounds was evaluated against a range of clinically and environmentally relevant bacterial and fungal strains. Among them, derivatives 3i and 3k, bearing bromine and chlorine substituents on the phenol ring, exhibited the most potent antimicrobial effects, particularly against Penicillium italicum and Proteus mirabilis (MIC as low as 0.014 mg/mL). To elucidate the underlying mechanism of action, in silico molecular docking studies were conducted, revealing strong binding affinities of 3i and 3k toward fungal sterol 14α-demethylase (CYP51B), with predicted binding energies surpassing those of the reference antifungal ketoconazole. Additionally, UV-Vis and fluorescence spectroscopy assays demonstrated good stability of compound 3k in PBS and its effective binding to human serum albumin (HSA), respectively. ADMET and ProTox-II predictions further supported the drug-likeness, low toxicity (Class 4), and favorable pharmacokinetic profile of compound 3k. Collectively, these findings highlight TXA–phenol imine derivatives as promising scaffolds for the development of next-generation antimicrobial agents, particularly targeting resistant fungal pathogens. Full article

Shiga toxin-producing Escherichia coli (STEC) pose a significant threat to public health and effective methods of detection are needed. The use of naturally occurring bacteriophages (phages) to detect E. coli has been well documented. However, detecting multiple serotypes at the same time often required multiple phages specific to individual serotypes. To limit the burden of complex cocktails, this study aimed to engineer phages with an expanded host range that allows each phage to contribute to detection across multiple STEC serogroups. Kutterviruses, in the Ackermannviridae family, contain four tailspike proteins (TSPs), each of which confers tropism to a different bacterial strain. The modular nature of TSPs allows for mixing receptor-binding domains from diverse phage types. The host range of the Kuttervirus CBA120 was modified by replacing its native tailspike proteins (TSPs) with chimeric versions incorporating receptor-binding domains from related and unrelated phages. A structure-guided approach was utilized to overcome minimal sequence similarity between donor and recipient phages and achieve novel functional TSP chimeras. Two engineered phage variants were created that collectively detect five STEC serogroups: O26, O45, O103, O111, and O157. Spotting and luciferase assays confirmed that the replacement TSPs were functional and the phages had acquired new host ranges. This study demonstrates the feasibility of engineering Ackermannviridae phages with customized host ranges for detecting multiple STEC strains. This approach has potential applications in developing improved phage-based bacterial detection, therapy, and biocontrol. Full article

Peptidoglycan recognition proteins (PGRPs) are conserved pattern recognition receptors (PRRs) that play key roles in insect innate immunity by binding bacterial peptidoglycan (PGN) and activating downstream signaling pathways. The Dendrolimus kikuchii, a major defoliator of coniferous forests in southern China, has incompletely characterized immune defenses. This study systematically identified the PGRP gene family in D. kikuchii based on genome-wide data, identifying 10 PGRP genes with typical PGRP/Amidase_2 conserved domains, including 6 PGRP-S proteins and 4 PGRP-L proteins. Additionally, to further investigate the evolutionary relationships of these PGRP genes, a maximum likelihood (ML) phylogenetic tree was constructed using PGRP amino acid sequences from 6 different insect species, along with the 10 PGRP amino acid sequences from D. kikuchii. Phylogenetic analysis revealed that the DkikPGRP genes of D. kikuchii are distributed across distinct evolutionary branches and share high homology with PGRP genes from other insects, suggesting a close evolutionary relationship between the PGRP genes of D. kikuchii and those of other insect species. Transcriptome profiling revealed that DkikPGRP-S1, -S2, -S3, -S4, and -S5 were upregulated in the midgut, fat body, and hemolymph after Bt infection, showing tissue- and time-specific immune responses. Functional assays using siRNA knockdown demonstrated distinct roles of DkikPGRP-S4 and DkikPGRP-S5: DkikPGRP-S5 mainly promoted antimicrobial peptide (AMP) expression, including attacin, lebocin, lysozyme, and cecropin, whereas DkikPGRP-S4 showed a complex regulatory pattern, enhancing lebocin and lysozyme but suppressing attacin without affecting gloverin or cecropin. Silencing either gene significantly increased larval mortality upon Bt challenge. These results highlight the specialized immune regulatory functions of PGRPs in D. kikuchii, provide new insights into host–pathogen interactions, and suggest potential molecular targets for sustainable pest management strategies. Full article

Gold nanoparticles (AuNPs) synthesized via picosecond pulsed laser ablation were investigated as enhancers of methylene blue (MB)-mediated photodynamic therapy (PDT) against Escherichia coli. AuNPs produced at 532 and 1064 nm with frequencies of 20–50 kHz showed frequency- and size-dependent effects, with 50 kHz yielding the highest particle concentrations and smaller particles enhancing reactive oxygen species (ROS) generation. UV-Vis and fluorescence spectroscopy confirmed nanoparticle formation and plasmonic properties consistent with TEM measurements. Photobleaching assays demonstrated that AuNPs significantly increased MB singlet oxygen generation, while the efflux pump inhibitor INF-55 further amplified bacterial killing without altering net ROS yield. In vitro assays revealed that INF-55 combined with MB/AuNPs achieved ~59% higher bacterial deactivation compared to MB/AuNPs alone. Molecular docking confirmed stronger binding of INF-55 to the AcrB efflux pump (−9.1 kcal/mol) than MB, supporting its role as a competitive inhibitor that promotes intracellular MB retention. These findings establish a dual-action PDT strategy in which AuNPs enhance ROS production and INF-55 augments antibacterial efficacy via efflux pump inhibition. Together, this platform provides a proof of concept for future translation to biofilm- and tissue-based infection models, and potentially to localized clinical applications such as prosthetic joint, catheter-associated, or chronic wound infections where conventional sterilization or systemic antibiotics are insufficient. Full article

Antimicrobial resistance (AMR) poses a global health threat, which is becoming more challenging due to the involvement of bacterial virulence mechanisms such as quorum sensing (QS) and biofilm formation. These systems regulate pathogenic traits and shield bacteria from conventional therapies. Phytocompounds offer promising antivirulence strategies by disrupting QS and biofilms without exerting selective pressure. In this study, emodin, a natural anthraquinone, was evaluated for its anti-QS and antibiofilm efficacy. Emodin inhibited violacein production by 63.86% in C. violaceum 12472. In P. aeruginosa PAO1, it suppressed pyocyanin (68.04%), pyoverdin (48.79%), exoprotease (58.55%), elastase (43.13%), alginate (74.12%), and rhamnolipids (56.37%). In S. marcescens MTCC 97, emodin reduced prodigiosin (55.94%), exoprotease (48.80%), motility (83.27%), and cell surface hydrophilicity (41.20%). Biofilm formation was inhibited by over 50% in all three bacteria, highlighting emodin’s potential as a broad-spectrum antibiofilm agent. Molecular docking analyses indicated that emodin exhibited affinity towards QS regulatory proteins CviR, LasR, and SmaR, implying a possible competitive interaction at their ligand-binding sites. Subsequent molecular dynamics simulations confirmed these observations by demonstrating structural stability in emodin-bound proteins. The collective insights from in vitro assays and computational studies underscore the potential of emodin in interfering with QS-mediated virulence expression and biofilm development. Such findings support the exploration of non-antibiotic QS inhibitors as therapeutic alternatives for managing bacterial infections and reducing dependence on traditional antimicrobial agents. Full article

This paper proposes a method for E. coli detection in a microplate format using low-molecular-weight compounds that specifically interact with the lipopolysaccharides (LPSs) of E. coli cell walls. These compounds can amplify analytical signals by binding to multiple repeating cell surface structures, while the selectivity for E. coli is ensured by preliminary cultivation on selective media, such as Endo or MacConkey agar. 3-Aminophenylboronic acid (APBA) was selected as the binding reagent for detecting E. coli LPSs. Conjugates of streptavidin (STP) and bovine serum albumin (BSA) with APBA and conjugates of biotin and soybean trypsin inhibitor (STI) and BSA were synthesized. The conditions for the sequential formation of “sandwich” type complexes (BSA-APBA conjugate/E. coli/STP-APBA/STI–biotin/STP–peroxidase) and their colorimetric detection using chromogenic peroxidase substrate were determined. The detection limit was 3 × 10² cells/mL, and the range of quantitative determination covered five orders of magnitude—from 10³ to 10⁸ cells/mL. The developed assay was successfully tested using inactivated cells of pathogenic E. coli strains, confirming its potential for application. The assay was demonstrated to have universality, with the ability to detect E. coli, other bacterial pathogens, and LPS alone. This method could be adopted for the quantitative determination of different specific bacterial species using different selective media. Full article

Introduction: Bacterial infections, especially those caused by multidrug-resistant strains, remain a major health concern. This study investigates 4-chlorobenzyl p-coumarate, assessing its antibacterial mechanism, pharmacokinetic profile, and potential to modulate antimicrobial resistance. Methods: In silico studies were conducted, including molecular docking, molecular dynamics simulations, and pharmacokinetic predictions, alongside in vitro assays assessing efflux pump inhibition, antibiotic modulation, and bacterial DNA analysis. Results: The compound showed higher binding affinity and complex stability with the enzyme phosphatidylglycerol phosphate synthase, while also exhibiting reduced residue fluctuations and better flexibility with the NAD⁺-dependent DNA ligase. Molecular interactions with the efflux proteins MepA and NorA were also observed. Pharmacokinetic predictions indicated a favorable profile, including suitability for oral administration. Experimentally, the compound inhibited the MepA and NorA efflux pumps, modulated the activity of the antibiotics ciprofloxacin and norfloxacin, and reduced DNA concentration in treated cells. Conclusions: The findings suggest that the compound acts through dual mechanisms, with a prediction of activity by disrupting phosphatidylglycerol synthesis and DNA replication while inhibiting and modulating MepA and NorA efflux pumps. Full article

Bacterial meningitis involves complex molecular networks, including microRNA-mediated regulation of inflammatory responses; however, the specific role of Ovis aries microRNA-125b (oar-miR-125b) in this process remains poorly understood. In this study, using a lamb model of Enterococcus faecalis-induced meningitis, we observed significant downregulation of oar-miR-125b, which inversely correlated with its newly identified target, Tumor Necrosis Factor Superfamily Member 4 (TNFSF4). Dual-luciferase reporter assays confirmed that oar-miR-125b directly binds to the 3′ Untranslated Region (3′UTR) of TNFSF4 but not to the 3′UTRs of Kelch Like Family Member 31 (KLHL31) or NF-κB Inhibitor Interacting Ras Like 2 (NKIRAS2). Mechanistically, decreased oar-miR-125b expression relieves its repression of TNFSF4, leading to NF-κB pathway activation and blood–brain barrier disruption. Collectively, our results demonstrate that oar-miR-125b serves as a key anti-inflammatory regulator in bacterial meningitis by targeting TNFSF4 and constraining NF-κB signaling, highlighting its potential as a therapeutic target for attenuating neuroinflammation in meningitis. Full article

The excessive use of conventional antibiotics in aquaculture has created significant challenges, making it essential to explore and develop effective alternatives. Antimicrobial peptides (AMPs) have gained attention as potential therapeutic agents owing to their wide-ranging antibacterial effects and their ability to address pathogens resistant to conventional drugs. PpRcys1 is an antimicrobial peptide that mainly targets bacterial cell membranes, exhibiting a minimum inhibitory concentration of 8–32 μM. Its antibacterial activity should be further optimized. Before such optimization, however, it is crucial to identify the key amino acid residues that determine its functional activity. In this study, molecular dynamics simulations indicated that arginine 40 (ARG40), lysine 55 (LYS55), lysine 90 (LYS90), and lysine 93 (LYS93) play critical roles in the interaction between PpRcys1 and bacterial membranes. To investigate this further, these residues were mutated to serine, producing the mutant peptide PpRcys1_RMRK. Compared with PpRcys1, the mutant peptide PpRcys1_RMRK showed a significant reduction in antibacterial activity. Results from molecular dynamics simulations, Western blot, and ELISA demonstrated a marked decrease in its ability to bind to bacterial cell membranes. Membrane permeation assays, cell membrane depolarization experiments, and scanning electron microscopy revealed that PpRcys1 could not compromise the integrity of the bacterial membrane after losing ARG40, LYS55, LYS90 and LYS93. These findings highlight the critical roles of ARG40, LYS55, LYS90, and LYS93 in sustaining the antibacterial activity of PpRcys1. This study provides important initial insights into the structure–activity relationship of PpRcys1 and establishes a theoretical foundation for its future optimization. Full article

The production of β-lactamases is the main mechanism underlying carbapenem resistance. This study combined in silico and in vitro approaches to identify potential polyphenols as carbapenemase inhibitors. Molecular docking, molecular dynamics, and ADMET prediction were performed to assess the binding affinity, stability, and safety of quercetin, kaempferol, caffeic acid, and 3,4-dihydroxybenzoic acid against KPC-2, NDM-1, and OXA-48 carbapenemases. In vitro antibacterial assays and checkerboard analyses were conducted against Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa to assess antibacterial and synergistic effects. Then, the inhibition of the β-lactam hydrolytic activity was confirmed. In silico results showed that quercetin, kaempferol, and caffeic acid exhibited strong binding affinity and consistent stability towards the targets. Therefore, quercetin and kaempferol showed the strongest affinities (−8.0 kcal/mol) and stable interactions with key catalytic residues. ADMET profiles indicated good pharmacokinetic behavior and low acute toxicity. In vitro assays revealed that the polyphenols exhibited MIC values ranging from 12.5 to 25 mg/L and MBC values of 25–50 mg/L. Combined with cefotaxime, they enhanced bacterial susceptibility and inhibited β-lactam hydrolysis, with quercetin achieving complete inhibition at 200 mg/L. These findings highlight the potential of the four polyphenols as natural β-lactamase inhibitors. Further enzyme kinetics and in vivo studies are needed to confirm their therapeutic relevance. Full article