Multidrug-resistantKlebsiella pneumoniae has emerged as a formidable healthcare challenge, driving the urgent need for alternative therapeutic strategies such as phage therapy. In this study, we isolated and comprehensively characterized a novel phage, designated vB_Kp_IME328 (IME328), which exhibits highly specific bactericidal activity against K62-type K. pneumoniae. Morphological analyses revealed that IME328 forms plaques surrounded by distinct translucent halos and possesses a long tail, clustering it within the Webervirus genus. Through whole-genome sequencing and bioinformatic analysis, we identified ORF3 as the gene encoding a putative tail-fiber depolymerase. This gene was successfully cloned and expressed, yielding the recombinant protein Dp3. Functional assays confirmed that Dp3 possesses robust depolymerizing and antibacterial activities. In vitro, Dp3 acted synergistically with serum to effectively eradicate the target bacteria. Furthermore, live/dead cell staining and cellular assays demonstrated that Dp3 significantly enhances the phagocytic and intracellular bactericidal capacities of macrophages. In vivo evaluations using a lethal murine bacteremia model (inoculated with 6 × 10[7] CFU) showed that Dp3 administration conferred 100% protection and survival, whereas all untreated infected mice succumbed to the infection. Together, our findings highlight the targeted anti-virulence properties of the depolymerase Dp3. By enhancing host-mediated immune clearance, Dp3 offers a promising therapeutic strategy against antibiotic-resistant K62-type K. pneumoniae infections.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is of clinical concern due to its resilience against antibiotics. Among potential alternatives for treatment, postbiotics from lactic acid bacteria (LAB) incite interest due to having strong antimicrobial activities whilst being generally recognised as safe. However, insights into their components and interactions remain limited. Weissella cibaria NM1 produces the proteinaceous postbiotic NM1 that inhibits K. pneumoniae ATCC13382. This study aims to compare the anti-K. pneumoniae activities of crude postbiotic NM1 and its partially-purified protein fractions via minimum inhibitory concentration (MIC) and time-kill assays. The purification of postbiotic NM1 revealed two cationic and six anionic fractions with yields of 0.03-1.08% and purification folds of 0.02-1.47 times. Crude postbiotic NM1 had the lowest MIC at 15.63 mg/mL, as compared to the MICs of its partially purified NM1 fractions at 125-250 mg/mL. The partially purified fractions were bacteriostatic, achieving reductions of 0.07-1.30 log10 CFU/mL within 1-8 h, but the crude postbiotic NM1 was bactericidal, achieving complete inhibition by 8 h of incubation. The purification of postbiotic NM1 highlights its richly diverse antimicrobial makeup that provide the robust activity retained in its crude form, showing potential for practical applications even before extensive purification.

The rising threat of carbapenem-resistant Enterobacteriaceae demands novel strategies to restore antibiotic efficacy. Here we show that compound amino acid injection (18AA), a clinically approved parenteral nutrition formulation, markedly potentiates ceftazidime-avibactam (CZA) against multidrug-resistant and carbapenem-resistant Escherichia coli. Checkerboard and Bliss independence analyses demonstrated strong synergy. Time-kill kinetics confirmed dose-dependent bactericidal enhancement across susceptible, multidrug-resistant, and carbapenem-resistant strains. In a panel of 113 clinical isolates, the combination significantly improved population-based killing efficacy (KE50 and KE90) compared to CZA alone. Moreover, 18AA prolonged the post-antibiotic effect, reduced mutation frequency, and delayed resistance development during serial passage over 15 cycles. Untargeted metabolomics revealed that 18AA induces profound metabolic reprogramming, with robust activation of the pyruvate cycle as the most prominent signature common to all tested strains; key biomarkers pyruvate, ketoglutarate, and succinate were identified. Mechanistically, 18AA boosted the pyruvate cycle, increased NADH production, and elevated proton motive force (PMF), thereby promoting intracellular CZA accumulation, which was abrogated by the PMF uncoupler CCCP. Collectively, our findings establish 18AA as a promising metabolism-based adjuvant that rejuvenates CZA activity via the pyruvate cycle and PMF-dependent uptake, offering a readily translatable strategy against hard-to-treat E. coli infections.

Antimicrobial resistance is an urgent global public health threat, killing at least 1.27 million people worldwide and associated with nearly 5 million deaths. Antibiotic resistance has been recently recognized by the WHO as a global health emergency. In this scenario, the research and development of novel derivatives specifically active against resistant pathogens became an urgent need. Pyrazole-based compounds have recently attracted significant interest for their potential antimicrobial activity, particularly against resistant Gram-positive strains such as methicillin-resistant Staphylococcus aureus (MRSA). Based on this evidence, we carried out an in-depth investigation of this chemical class by the synthesis of unreported compounds differently decorated on the pyrazole scaffold. The newly prepared derivatives were tested on a large panel of Gram-negative and Gram-positive strains. Interestingly, pyrazolyl ureas (PUs) 1c and 1h showed excellent minimum inhibitory concentration (MIC) values (8-16 μg/mL) against different Staphylococcus strains and were therefore further evaluated by minimum bactericidal concentration (MBC) determination and time-kill assays and for their ability to inhibit biofilm formation on several MRSA strains. Pyrazole 1c was shown to exert such inhibition at MIC and sub-MIC concentrations (1/2 MIC and 1/4 MIC), highlighting the pharmaceutical potential of this class of compounds against resistant MRSA. Finally, selected PUs did not affect GM-6114 cell viability, thus indicating the lack of cytotoxicity on healthy fibroblasts, and were predicted to possess favorable physicochemical, pharmacokinetic, and drug-likeness profiles.

The hypoxia-tolerant group (T group) large yellow croaker (Larimichthys crocea) exhibits high survival rates when infected with Aeromonas hydrophila. To elucidate the underlying mechanism, this study explored the potential new role of proline hydroxylase domain-containing protein 1 (PHD1) in immunity, and examined the differences in PHD1/IKKβ/NF-κB signaling pathway between the T group and normal group following A. hydrophila challenge. Our results found that LcPhd1 responds to LPS stimulation in head kidney macrophages of L. crocea and affects the phosphorylation of IKKβ and NF-κB nuclear translocation in the classical immune signaling pathway. Co-IP and site-directed mutagenesis further demonstrated that this effect may occur through the modulation of the proline residue in the LXXLAP sequence of LcIKKβ. Additionally, PHD1 inhibited macrophage polarization into the M1 type, including reducing the production of bactericidal substances, such as inflammatory factors, ROS, as well as diminishing the macrophage functions, such as migration, invasion, and phagocytosis. Following A. hydrophila infection, the PHD1 expression in the head kidney of T gorup L. crocea decreased, while IKKβ phosphorylation and p65 nuclear translocation increased. In conclusion, these results indicated that PHD1 may regulate macrophage polarization through the IKKβ/NF-κB signal cascade and contribute to the resistance of T gorup L. crocea to A. hydrophila infection.

Antimicrobial resistance poses a major global health challenge, highlighting the need for alternative antimicrobial strategies. In this study, silver-containing colloidal aggregates were synthesized using an extract from Artocarpus heterophyllus leaves and evaluated for their antimicrobial activity and effects on microbial survival under adaptive conditions. Silver-containing colloidal aggregates were characterized for size, structure, and stability, followed by antimicrobial assessment using Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal/Fungicidal Concentration (MBC/MFC) assays. The synthesized silver-containing colloidal aggregates exhibited an average size of 444.00 ± 32.84 nm, a polydispersity index of 0.81 ± 0.16, and a zeta potential of -24.28 ± 10.66 mV. Functional group analysis indicated that phytochemicals mediated the reduction of Ag[+] ions. Antimicrobial activity was observed against Escherichia coli, Staphylococcus aureus, and Candida albicans, with MIC values of 80, 640, and 320 mg l-1, respectively, and MBC/MFC values of 1280 mg l-1 (E. coli and C. albicans) and 5120 mg l-1 (S. aureus). Adaptive laboratory evolution (ALE) experiments under gradually increasing silver-containing colloidal particle concentrations showed a progressive decline in microbial growth, with no detectable survival at higher exposure levels. These findings suggest that silver-containing colloidal aggregates exhibit antimicrobial activity and may limit short-term microbial adaptation under the tested conditions.

The multidrug resistance (MDR) problem in Streptococcus suis (S. suis) is becoming increasingly severe, necessitating the development of novel antibacterial agents and strategies. In this study, seven elaiophylin derivatives were isolated from Streptomyces sp. WS-30248, and the antibacterial activity and mechanism of action of the principal compound, elaiophylin, against S. suis were systematically evaluated for the first time. Through comprehensive approaches including in vitro efficacy assays, biofilm inhibition and eradication tests, bacterial membrane integrity analysis, reactive oxygen species (ROS) level detection, and a mouse infection model, elaiophylin was found to exhibit significant antibacterial activity against multiple clinically MDR S. suis strains. Its minimum inhibitory concentration (MIC) was as low as 0.5 μg/mL, and complete bactericidal activity was achieved within 24 h at 4× MIC. The compound effectively inhibited and eradicated bacterial biofilms, directly killing embedded cells within the biofilm matrix. Mechanistic studies revealed that elaiophylin functions through multiple synergistic pathways, including disruption of bacterial membrane integrity and induction of massive ROS accumulation, thereby interfering with the proton motive force (PMF), depleting intracellular ATP, and blocking energy metabolism. In the mouse infection model, the elaiophylin-treated group showed a significantly increased survival rate of 60% and effectively reduced bacterial loads in tissues. This study demonstrates that elaiophylin is a highly promising natural candidate drug with multi-target synergistic effects, offering a new strategy to combat MDR S. suis and biofilm-associated infections.

Aminoglycosides, including gentamicin, are potent bactericidal antibiotics that are widely utilized for the treatment of severe gram-negative bacterial infections. These drugs demonstrate a concentration-dependent bactericidal effect, whereby higher concentrations result in more rapid bacterial elimination. Despite their efficacy, determining optimal dosing regimens is challenging due to significant inter-individual pharmacokinetic (PK) variability and adverse drug reactions, such as nephrotoxicity and ototoxicity. This study developed a simplified, user-friendly PK-predictive system for target concentration intervention of gentamicin. A semi-automatic platform was developed for the generation of patient-specific NONMEM PK datasets using Shiny and the estimation of individual PK parameters using maximum a posteriori Bayesian estimation. The system provides a dosing assessment based on Bayesian PK predictions, with results presented in detailed reports generated via LaTeX. The predictability of the PK model was confirmed via external validation using clinical PK data from 22 patients who underwent therapeutic drug monitoring consultations at the Asan Medical Center. In the external validation the mean prediction error was -0.19 mg/L (95% confidence interval, -0.88 to 0.41) and the root mean squared prediction error was 2.85 mg/L, and 86.4% of observed concentrations fell within the model-predicted 95% prediction interval on the individual prediction check plots. These results support the applicability of the model to this population, within the limits of precision reported, for dose individualisation. The platform architecture may also be adaptable to other drugs with a narrow therapeutic index.

Aminoglycoside antibiotics bind RNA with high affinity through networks of amine and hydroxyl contacts, yet whether this multivalent binding can drive macroscopic RNA phase transitions has never been tested. Here we show that aminoglycosides are a class of small-molecule RNA condensers, and we take neomycin B (neoB), an FDA-approved member of the family, as a representative drug through which to dissect the mechanism. NeoB induces concentration-dependent phase separation of poly(A), poly(U), and total E. coli RNA, and kanamycin, apramycin, and gentamicin condense RNA as well. Condensate size and density are tunable by pH and ionic strength, which independently modulate neoB protonation and screening of interdroplet repulsion. NeoB forms more stable condensates than spermine despite spermine's larger effective charge at physiological pH, whereas the amine-free polyol fucitol fails to condense RNA. Molecular dynamics simulations attribute neoB's greater efficiency to additional hydrogen bonds donated by its hydroxyl groups. The chemical complexity that aminoglycosides evolved for RNA recognition thus also drives a macroscopic RNA phase transition that may contribute to bactericidal activity and cellular toxicity.

The global rise of multidrug-resistant (MDR) pathogens has created an urgent need for alternative antimicrobial agents. In this study, we evaluated the antibacterial activity of LTX-315, a synthetic cationic 9-mer peptide originally developed as an oncolytic agent, against six ATCC reference strains and six clinical isolates, including methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae, MDR Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Burkholderia cepacia. LTX-315 exhibited broad-spectrum antibacterial activity, with MICs of 8-32 μM against reference strains and as low as 4 μM against several MDR clinical isolates, whereas chloramphenicol and kanamycin were ineffective. Double-disk diffusion assays indicated synergistic interactions with aminoglycosides, chloramphenicol, and vancomycin, and checkerboard assays confirmed synergy-to-additivity interactions with gentamicin and vancomycin against S. aureus (FICI 0.38-0.75), yielding fourfold to 16-fold reductions in antibiotic dose. These findings demonstrate that LTX-315 retains potent activity against clinically relevant MDR pathogens and may serve as both a standalone antimicrobial peptide and a potential antibiotic adjuvant.

Methicillin-resistant Staphylococcus aureus (MRSA) pneumonia is a life-threatening infectious disease, yet current antibiotic-centered therapies remain insufficient to achieve satisfactory therapeutic outcomes. Herein, a lysostaphin-bioengineered cascade nanoreactor, LO-PDA/Zn@CA, is developed for the integrated treatment of MRSA pneumonia. This system consists of cinnamaldehyde (CA)-loaded, Zn[2+]-chelated polydopamine nanoparticles coated with lysostaphin-engineered bacterial outer membrane vesicles. Upon reaching MRSA-infected lung lesions, CA disrupts the biofilm matrix and compromises bacterial membranes, thereby weakening extracellular protective barriers. Zn[2+] subsequently activates surface-displayed lysostaphin to specifically hydrolyze pentaglycine cross-bridges within the MRSA peptidoglycan cell wall, further breaching bacterial integrity and promoting intrabacterial penetration of CA and Zn[2+]. The released Zn[2+] induces lethal DNA damage-associated responses, collectively amplifying bactericidal efficacy through a stepwise antibacterial cascade. In parallel, the PDA/Zn nanocore scavenges excessive reactive oxygen species, suppresses glycolytic reprogramming, and downregulates H3K27ac-dependent transcription of pro-inflammatory genes, thereby attenuating macrophage M1 polarization and mitigating pulmonary inflammatory injury. In vitro and in vivo studies demonstrate that LO-PDA/Zn@CA effectively eliminates planktonic MRSA, disrupts established biofilms, and alleviates MRSA-induced pulmonary inflammation with favorable biosafety. By integrating pathogen-specific bacterial eradication with inflammation resolution in a cascade manner, this work provides a versatile nanotherapeutic strategy for the treatment of drug-resistant MRSA pneumonia.