Essential oils (EO) from Piper marginatum Jacq. exhibit biological activities dependent on chemical compositions, which vary mainly due to environmental factors. This study investigated the seasonal and regional variation in the chemical composition and antimicrobial activity of EO from P. marginatum leaves collected in three Brazilian regions across four seasons. Sixty compounds were identified, primarily terpenoids and phenylpropanoids. Multivariate analyses revealed seasonal variations and distinguished the Northeast region EO, which contained the isomers pipermargine (3.38%-14.56%) and 1,3,5-trimethoxy-2-[(Z)-prop-1-enyl]benzene (13.47%-14.87%)-reported for the first time in EO. The Central-West winter EO exhibited the broadest antimicrobial spectrum, with strong activity against Candida albicans (MIC 0.5 mg mL[-1]) and Bacillus subtilis (MIC 0.5 mg mL[-1]), and a bacteriostatic effect against Staphylococcus aureus. All EO inhibited Escherichia coli, with the Northeast winter EO showing the strongest activity (MIC 0.25 mg mL[-1]). This work highlights the importance of studying the chemical and biological variability of P. marginatum EO for potential applications.

The escalating environmental burden of conventional plastic food packaging has intensified the search for sustainable, biodegradable alternatives. However, single-component polysaccharide edible films often exhibit suboptimal mechanical and barrier properties, which limits their practical applicability. In this study, pullulan (PUL), konjac glucomannan (KGM) and low-acyl gellan gum (LGG) were blended as film-forming substrates with glycerol as a plasticiser. Single-factor experiments combined with Box-Behnken response surface methodology (RSM) were employed to optimise the formulation of ternary edible composite films. The effects of PUL, KGM and LGG concentrations on mechanical properties and water vapour permeability (WVP) were systematically investigated. The optimised film was comprehensively characterised for microstructure, physical barrier performance, antioxidant activity, soil disintegration, bacteriostatic capacity and plum preservation efficiency. The optimal formulation comprised 1.43 g/100 mL PUL, 0.24 g/100 mL KGM and 0.48 g/100 mL LGG. Under these conditions, the composite film exhibited a transparent and smooth appearance, with a continuous and dense internal microstructure. The comprehensive performance score (K) reached 0.805 ± 0.013, with a WVP of 2.75 ± 0.17 g·μm/(m[2]·h·kPa), light transmittance of 89.93% ± 1.26%, tensile strength (TS) of 3.05 ± 0.14 N/cm[2], elongation at break (EB) of 64.57% ± 0.58% and DPPH radical scavenging activity of 34.12% ± 0.96%. Storage trials on two plum varieties demonstrated that the composite coating effectively mitigated fruit weight loss, retarded peel browning and pulp softening, and maintained superior sensory quality throughout storage. Overall, the PUL/KGM/LGG ternary edible composite film exhibited favourable mechanical strength, promising water vapour barrier properties and moderate antioxidant activity, suggesting its potential as a useful reference for the further development and industrial adoption of biodegradable edible films in fruit and vegetable preservation.

Background/Objectives: The emergence of methicillin-resistant Staphylococcus aureus (MRSA) highlights the need for new antibacterial agents. Here, we report the identification and evaluation of a novel Gram-positive selective antibacterial: NDM-552. Methods: Antibacterial activity was determined by broth microdilution against a panel of Gram-positive and Gram-negative pathogens. Mechanistic studies included BOCILLIN-FL competition assays, microscale thermophoresis (MST), and antibiotic interaction with oxacillin and moenomycin. Additionally, time-kill analyses, frequency-of-resistance measurements, cytotoxicity assays, plasma stability studies, and a murine wound infection model were performed to characterize NDM-552. Results: NDM-552 exhibits activity against Gram-positive pathogens including S. aureus, Enterococcus faecium, and Staphylococcus epidermidis, with minimum inhibitory concentrations (MICs) of 1-2 µg/mL. NDM-552 displays no activity against Gram-negative bacteria. NDM-552 displays bacteriostatic activity against MRSA and additive interactions with oxacillin and moenomycin. The frequency of resistance in MRSA is low (2.39 × 10[-9]). BOCILLIN-FL competition assays and MST binding analysis identify the essential penicillin-binding protein 2 (PBP2) as the potential target of NDM-552. NDM-552 demonstrates acceptable cytotoxicity, favorable plasma stability, and reduces bacterial burden in a murine wound infection model. Conclusions: NDM-552 is a potent Gram-positive selective antibacterial agent that potentially targets PBP2-associated cell wall biosynthesis and exhibits in vivo efficacy against MRSA. Findings establish NDM-552 as a promising scaffold for the development of new antibacterial agents.

Mastitis severely hinders dairy farming, with Streptococcus agalactiae (S. agalactiae) serving as a major causative pathogen. Prolonged antibiotic administration promotes the emergence of multidrug-resistant strains, thereby compromising mastitis control. Consequently, novel natural antibacterial agents are urgently needed. In this study, bacteriostatic assays identified the MIC of rosmarinic acid (RA) against S. agalactiae as 8 mg/mL. At the MIC, RA reduced the adhesion capacity of S. agalactiae by 16.6% and inhibited biofilm formation by 25.09%. RA also disrupted bacterial membrane architecture and markedly downregulated the expression of virulence factors. A mouse model of mastitis treated with varying doses of RA (25, 50, and 100 mg/kg) indicated that RA attenuated pathological damage to the mammary duct architecture and ameliorated inflammatory responses in S. agalactiae-infected mice. ELISA revealed that RA significantly reduced pro-inflammatory mediators, including IL-1α, IL-1β, IL-6, and TNF-α. RT-qPCR results indicated that mRNA expression levels of TLR2, NF-κB, AKT, and PI3K were substantially reduced following RA treatment, whereas apoptosis-related genes p53, RUNX1, and Bim exhibited distinct expression patterns. Western blotting analysis further indicated that RA modulated the phosphorylation of Bim, RUNX1, and p53 in mastitis tissues, suppressed the TLR2/NF-κB inflammatory signaling pathway, and regulated the PI3K/AKT cascade. By targeting multiple pathways, RA suppressed S. agalactiae proliferation and attenuated mammary gland inflammation, highlighting its potential as a green therapeutic agent for sustainable animal husbandry.

Globally, antimicrobial resistance (AMR) poses a serious threat to human health and is often associated with poor treatment outcomes. There is an urgent need for effective therapeutic options to address this escalating issue. This study deals with the design and synthesis of new analogs based on the hybridization of the 2-methylpyrimidin-4-amine scaffold with the 5-(substituted-benzylidene)-2,4-thiazolidinedione ring through a methyl linker 9a-n and 10. The antimicrobial evaluation against a diverse panel of Gram-positive, Gram-negative, and fungal strains investigated the promising activity of compounds 9d, 9j, and 9l. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), as well as cytotoxic assessments of the latter derivatives, suggested the bacteriostatic effect against P. aeruginosa of compound 9j, while compounds 9d and 9l demonstrated bactericidal activity against both P. aeruginosa and E. coli, all showing a favorable safety profile. The anti-virulence activity of the hits 9d and 9l was also assessed using various biofilm assays. They effectively suppressed microbial biofilm formation in P. aeruginosa and E. coli strains, with suppression values ranging from 45 to 58%. Furthermore, time-kill kinetics of compound 9l demonstrated bactericidal action in a time-dependent manner in E.coli species. The DNA gyrase B assay of the hits 9d, 9j, and 9l investigated their promising suppression impact with varying IC50 values ranging from 1.967 to 6.403 μg/mL. Molecular docking and molecular dynamic simulation were also carried out for 9j and 9l. Overall, our findings emphasize the pyrimidine-2,4-thiazolidinedione scaffold as a viable antimicrobial option against infections caused by P. aeruginosa and E. coli.

Evaluation of the biological activity of food wastes is essential to identify promising bioactive compound sources for food applications. This study comparatively analyzed bioactive compounds and biological activities of Amazonian agro-industrial fruit wastes: acerola pomace (AP), açaí seed (AS), mango peel (MP), and pupunha peel (PP), aiming at their valorization as natural antioxidants and antimicrobials. Extracts were obtained by dynamic maceration using ethanol and methanol, and thermal treatment using water and diluted sulfuric acid at mass/solvent ratios of 1:10 and 1:20 (w/v). Total phenolic content (TPC), total flavonoid content (TFC), antioxidant activity (AA), and major phenolic compound profile by HPLC-DAD were determined. Antibacterial activity was assessed by broth microdilution. Extracts showing antimicrobial potential were further evaluated for cytogenetic safety using the Allium cepa assay. Ethanolic and methanolic AS extracts (ASE and ASM) at 1:10 (w/v) showed the highest TPC (2,252.20 and 2240.00 mg GAE/100 g, respectively). ASM showed the highest TFC (1112.31 mg/100 g), while ASE showed the highest AA (25661.90 µmol TE/100 g). Catechin was the predominant phenolic compound in ASE 1:10 (29.86 ± 0.64 mg/100 g). Only ASE and ASM showed antibacterial activity, with bactericidal effects against Staphylococcus aureus and bacteriostatic activity against Proteus mirabilis and Pseudomonas aeruginosa. ASM exhibited genotoxic and mitodepressive effects, whereas ASE demonstrated the lowest cytogenetic alterations at the bactericidal concentration. ASE represents a promising renewable source of bioactive compounds for high-value products, supporting sustainable waste management. PRACTICAL APPLICATIONS: Amazonian fruit residue extracts, especially those obtained from açaí seeds, showed potential as sustainable sources of natural antioxidant and antibacterial compounds. The ethanolic açaí seed extract may be explored as a functional ingredient or natural additive in food products, contributing to cleaner-label formulations, improved oxidative stability, and extended shelf life. This study also highlights the importance of selecting extraction methods that combine bioactivity with biological safety for future food applications.

The timely identification of drug-resistant pathogens and rapid differentiation of antibacterial mechanisms are critical in combating the escalating crisis of antimicrobial resistance. However, traditional broth culturing methods are inherently time-consuming and lack single-cell sensitivity. In this study, we reported a highly responsive biosensing platform based on single-entity impact electrochemistry (SIE) to rapidly differentiate bacterial activity at the single bacterium level. This method utilized p-benzoquinone (BQ) and BQ-K3Fe(CN)6 as redox mediators that were continuously reduced by the bacterial respiratory chain. Upon the collision of an individual bacterium with the ultramicroelectrode, these mediators were re-oxidized, generating transient current spikes that serve as a direct indicator of cellular viability. By tracking these distinct electrochemical signals, we successfully discriminated the responses of Escherichia coli to three antibacterial agents. Specifically, the heavy metal stress induced by Pb2+ exposure resulted in a progressive decline in collision spike amplitude, with the total transferred charge decreasing from (7.51 ± 0.48) × 10-2 pC to (2.50 ± 0.14) × 10-2 pC per bacterium. The bacteriostatic effect of doxycycline yielded negligible immediate signal change due to its protein synthesis inhibition mechanism. In contrast, the membrane-disrupting effect of polymyxin B triggered a rapid cessation of collision signals alongside an increase in total current amplitude due to the leakage of intracellular redox contents. Based on these unique electrochemical signatures, we successfully identified polymyxin B-resistant E. coli mutants and achieved rapid validation of alternative antibiotics against this drug-resistant strain. This work expanded the analytical capabilities of SIE, offering a highly efficient platform for bacterial phenotypic screening and drug resistance monitoring.

Antibiotic resistance has renewed interest in bacteriophages as antimicrobials, yet the complexity of phage-host interactions often leads to inconsistent infection outcomes, underscoring the need to unravel the molecular basis of phenotypic diversity among closely related phages. Here, we compared two closely related T7-like phages, polyvalent phages C5 and N30, both belonging to Teseptimavirus, which share 89.67% genomic identity and an identical host range. While C5 exhibited a higher adsorption rate, plaque size, and efficiency of plating, N30 showed stronger bacteriostatic activity and larger burst sizes, highlighting the importance of a comprehensive evaluation of phage infection traits for therapeutic or biocontrol applications. Comparative genomics revealed high overall similarity between phages C5 and N30 but notable divergence in a hypothetical protein and a histidine-asparagine-histidine (HNH) endonuclease. Broader analysis across Teseptimavirus further uncovered two distinct genomic positions of the HNH endonuclease: either adjacent to virion assembly proteins in the genome middle or next to the terminase large subunit at the genome end, suggesting functional specialization in capsid assembly and DNA packaging. Tertiary structure prediction and comparison of the HNH endonucleases revealed moderate structural similarity despite moderate sequence divergence. Furthermore, a single nucleotide substitution in the HNH gene of N30 markedly increased its efficiency of plating in one tested host strain, demonstrating that terminase-associated HNH endonuclease plays a pivotal role in plaque formation. Together, these findings provide the first evidence of dual HNH endonuclease positioning within Teseptimavirus and identify the HNH endonuclease as a key genetic determinant of plaque formation, offering new insights into the molecular basis of phage infection variability and host adaptation.IMPORTANCEClosely related bacteriophages often display strikingly different infection phenotypes, yet the genetic basis of this variation remains poorly understood. Here, we uncover a previously unrecognized feature within the genus Teseptimavirus: the HNH endonuclease can occupy two distinct genomic positions, either associated with virion assembly or DNA packaging modules, suggesting functional specialization. We further demonstrate that this enzyme is closely linked to infection phenotypes, as even a single nucleotide substitution in the HNH gene significantly alters the efficiency of plating. These findings identify the HNH endonuclease as a key determinant of phage infectivity and reveal that its genomic context may shape functional outcomes. This work provides new insight into how subtle genetic differences drive phenotypic divergence among closely related phages and highlights the HNH endonuclease as a promising target for rational phage optimization in therapeutic and biocontrol applications.

Photocatalysis is an efficient approach for antibiotic remediation, but rapid carrier recombination remains a key challenge. Here, we construct an oxygen-vacancy rich Cu/DUT-67 Schottky junction photocatalyst for antibiotics degradation. Surface oxygen vacancies provide abundant adsorption sites, while the interfacial built-in electric field drives directional electron migration, suppressing carrier recombination. The optimized Cu/DUT-67-2 achieves tetracycline and oxytetracycline removal efficiencies of 84.05% within 150 min and 83.87% within 120 min, respectively, substantially outperforming pristine Dresden University of Technology-67 (DUT-67). Mechanistic studies show that h+ and ·O2- synergistically dominate the degradation process. Cu/DUT-67-2 also exhibits excellent stability, and gel microspheres containing the catalyst enable continuous antibiotic removal under illumination. Molecular reactivity and high-performance liquid chromatography-mass spectrometry (HPLC-MS) analyses elucidate the reactive sites, degradation intermediates, and degradation pathways of the antibiotics. Toxicological evaluation and bean sprout germination tests confirm effective degradation with significantly reduced ecological toxicity. Meanwhile, the bacteriostatic rates of Cu/DUT-67-2 against E. coli and S. aureus reach 98.82% and 98.68%, respectively. This work integrates defect regulation with interface engineering for synergistic adsorption and catalysis, offering a feasible strategy for antibiotic pollution remediation.

This study employed linear unbranched β-1,3-D-glucan from Poria cocos (PCPA) as the raw material and synthesized a series of quaternized polysaccharides (QPCPA) with tunable degrees of substitution via a stepwise pH-controlled nucleophilic substitution method using glycidyltrimethylammonium chloride (GTMAC) as the quaternary ammonium reagent. Using Staphylococcus aureus and Escherichia coli as test strains, the antibacterial activity and underlying mechanisms were investigated. The results demonstrated that increasing the GTMAC feed ratio significantly enhanced the degree of substitution and surface positive potential of QPCPA, accompanied by improved water solubility but a slight reduction in thermal stability. QPCPA exhibited concentration-dependent bacteriostatic/bactericidal activity, with QPCPA-10 (DS = 0.962) possessing the highest degree of substitution displaying optimal antibacterial performance. Mechanistic studies confirmed that positively charged QPCPA electrostatically adsorbs to the negatively charged bacterial membrane, triggering membrane depolarization, intracellular Reactive Oxygen Species accumulation, membrane disruption, and massive leakage of nucleic acids and proteins, ultimately leading to bacterial death. This research establishes a facile modification strategy for constructing cationic antimicrobial polysaccharides from natural Poria cocos polysaccharides, demonstrating promising application potential in food preservation, antimicrobial biomaterials, and antibacterial coatings.

Riboflavin biosynthesis is required for in vitro survival of the human pathogen Mycobacterium tuberculosis ( Mtb ). However, despite the lack of a known transporter, growth can be rescued by exogenous riboflavin. The riboflavin biosynthesis pathway is also predicted essential in vivo , but whether riboflavin levels available in the host can support survival has not been directly tested. Here we constructed a set of inducible CRISPR interference (CRISPRi) knockdown and targeted gene deletion strains for known riboflavin biosynthesis genes ( ribA2, ribG, ribH, ribC ) as tools to characterize riboflavin requirements, uptake, and metabolite changes and to assess in vivo essentiality. We found that riboflavin, but not flavin adenine dinucleotide or flavin mononucleotide, rescued auxotrophy for all strains tested. Further, riboflavin uptake did not show strong evidence of being dependent on active or facilitated transport, supporting the mechanism of passive diffusion. Targeted metabolite profiling after removal of riboflavin from growth medium confirmed reduced riboflavin levels. While other riboflavin intermediates were not detected, significant accumulation of aromatic amino acids (Phe, Tyr) was observed across all assayed strains, as well as alteration in a vitamin B9 metabolite. Selecting the ribC knockout as a representative strain, we found that riboflavin depletion had a bacteriostatic effect as late as 3 weeks after removal. Unexpectedly, Δ ribC lacked infectivity in an aerosol mouse infection, suggesting that the potential to scavenge riboflavin from the host is not sufficient to survive in vivo . Overall, our results show that altered metabolism upon loss of riboflavin biosynthesis leads to compromised Mtb infectivity.

Antimicrobial resistance involving opportunistic bacterial and fungal pathogens along with persistent biofilm-associated infections possess a critical global healthcare challenge. In this study, we have reported a cationic amphiphilic peptide-based hydrogelator with a disulfide backbone and tryptophan side-chain residues that exhibited potent antimicrobial efficacy against several multidrug resistant bacterial and fungal strains. This C-terminally lipidated peptide was self-assembled into a thixotropic hydrogel in Tris-HCl buffer at physiological pH of 7.46. Subsequently, the morphology of the hydrogelators was well characterized by atomic force microscopy, field-emission gun transmission electron microscopy, and field-emission scanning electron microscopy, and it was apparent that the gelators formed a nanofibrous network. Bioactivity evaluation demonstrated that the peptide gelator solution showed broad-spectrum antibacterial activity with rapid bactericidal action and sustained bacteriostatic behavior against a wide range of Gram-positive and Gram-negative bacterial strains. In addition, pronounced antifungal activity was observed across multiple pathogenic fungal species. Detailed mechanistic study revealed a multimodal antimicrobial action involving membrane permeation, trans-membrane depolarization, and reactive oxygen species generation, which ultimately led to microbial cell flocculation. Interestingly, this injectable hydrogel also displayed strong antibiofilm performance against bacterial strains like methicillin-resistant Staphylococcus aureus and Klebsiella pneumoniae and also against fungal pathogens such as Aspergillus niger and fluconazole-resistant Candida albicans. Both extracellular matrix degradation and subsequent planktonic microbial decay effectively inhibited biofilm growth and disrupted mature biofilm microcolonies. Notably, the peptide hydrogel showed nonsignificant cytotoxicity toward normal eukaryotic cells such as HEK-293 (human embryonic kidney) and thus can be promoted as efficient biocompatible soft-material. Collectively, this study focuses on antimicrobial and antibiofilm properties of an injectable lipopeptide hydrogel, highlighting its potential as a promising biomaterial for anti-infective surface-coating and infection-resistant biomedical applications.