Search Results (210)

Linezolid represents a critical last-resort treatment for severe multidrug-resistant (MDR) Gram-positive bacterial infections. Rising linezolid resistance in Enterococcus isolates threatens its efficacy; this study characterized the molecular features and transfer potential of plasmid-encoded linezolid resistance genes optrA and poxtA in a linezolid-resistant Enterococcus asini isolate from chickens. An E. asini strain was isolated during a surveillance program focusing on drug-resistant Gram-positive bacteria in poultry. PCR screened linezolid resistance genes, conjugation and plasmid stability assays evaluated gene transferability and stability, and whole-genome sequencing (WGS) was performed using both the Illumina and Nanopore platforms. We present the first detection of optrA and poxtA genes in E. asini recovered from chicken feces in China. Sequence analysis of the complete genome showed that poxtA and optrA were situated on two distinct plasmids. The poxtA positive plasmid, pHNGXN23C145Ea-1, also carried multiple resistance genes, including tet(S), fexB, erm(B), ant(6)-Ia, aph(3′)-III. Furthermore, the poxtA gene was flanked by IS1216E mobile elements. The optrA bearing plasmid, pHNGXN23C145Ea-2, harbours a common genetic array of ‘IS1216E fexA-optrA-erm(A)-IS1216E’. Conjugation experiments indicated that neither the poxtA- nor the optrA-bearing plasmid was transferred to recipient strains, which was consistent with sequence analysis showing that both plasmids lacked intact conjugative transfer regions. Stability assays confirmed that poxtA and optrA remained highly stable in the absence of selective pressure. Notably, this discovery was made in a livestock sample, despite the non-use of linezolid in food animals, suggesting that such niches may act as silent reservoirs for resistance genes, which could persist and potentially transfer to clinically relevant MDR pathogens. Full article

Antimicrobial resistance (AMR) represents a global health crisis, driven largely by the mobility of resistance determinants through mobile genetic elements (MGEs). These include plasmids, integrons, insertion sequences, transposons, integrative and conjugative elements (ICEs), and prophages, which together facilitate horizontal gene transfer (HGT) across bacterial species and ecosystems. This review aims to provide a comprehensive synthesis of current knowledge on the types, mechanisms, ecological drivers, and impacts of MGEs in the dissemination of antibiotic resistance genes (ARGs). Methods involved critical evaluation of recent genomic, epidemiological, and ecological studies, alongside case studies of clinically significant resistance outbreaks. Findings highlight how MGEs function as hubs for ARG capture, recombination, and stabilization, enabling the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) pathogens. We also explored their interactions with ecological pressures such as antibiotics, heavy metals, and biocides, as well as their role in One Health transmission pathways. The significance of this study lies in linking molecular insights with applied strategies, including genomic surveillance, MGE-targeted inhibitors, phage therapy, and CRISPR-based interventions. Understanding MGEs is essential for designing effective interventions to mitigate AMR and protect global health. Full article

Soybean (Glycine max) is relatively recalcitrant to genetic manipulations; hence, it is often interrogated with transient means such as virus-induced gene silencing (VIGS). We earlier modified cowpea severe mosaic virus (CPSMV) to develop a soybean-friendly VIGS system referred to as QUIN-FZ. Here we report additional calibrations of this system. We enhanced the intra-bacterial stability of plasmid QUIN, which contained a CPSMV RNA1 cDNA embedded with four introns, by adding a fifth intron, resulting in PENTIN. We separately upgraded the plasmid FZ, which contained a modified CPSMV RNA2 cDNA with a cloning site in the middle of the viral polyprotein, by creating another cloning site within the 3′ untranslated region, leading to ZY. We next used the new PENTIN-ZY system to investigate a putative soybean protein kinase designated QL18. Virus-mediated overexpression of two allelic, 147-amino-acid (aa) protein fragments, derived from two different QL18 orthologs, elicited drastically different responses in soybeans. While the fragment derived from soybean accession OX20-8 prevented the cognate virus from infecting top young leaves in at least 50% of inoculated seedlings, its allelic counterpart derived from soybean accession PI427105B elicited apical necrosis in 100% of soybean seedlings. By examining progeny viruses as well as viruses encoding chimeras of the two fragments, we identified more than a dozen mutations that abrogated these unique phenotypes. Our findings establish the PENTIN-ZY system as a versatile tool for overexpressing small proteins and protein fragments, accelerating their functional characterization. Full article

Biologically relevant primary cell samples are inherently heterogeneous and often require selective enrichment prior to genetic manipulation. We previously demonstrated a vortex-assisted microfluidic platform that integrates size-selective cell trapping with electroporation; however, its limited processing capacity constrained applications requiring larger sample volumes. Here, we present a scaled version of this integrated system achieved through electrode array redesign and electrical optimization. The updated architecture increases processing capacity while preserving size-selective trapping behavior, electric field uniformity, and device stability. Systematic optimization of electrical and buffer conditions enables efficient delivery of plasmid DNA and in vitro-transcribed mRNA into primary human cells, with performance approaching benchmark chemical transfection methods. By scaling an integrated trapping–electroporation workflow without compromising delivery performance, this platform advances microfluidic cell engineering toward practical processing of heterogeneous primary cell samples. Full article

Komagataeibacter strains are important bacterial cellulose producers, yet closely related isolates can differ in cellulose yield, pellicle properties, and genetic stability during propagation. Such variability suggests that lineage structure and mobile genetic elements both contribute to strain-level genomic divergence. Here, complete genome comparisons were used to integrate vertical relatedness, gene-content structure, cellulose-associated signatures, and mobilome heterogeneity across 22 closed Komagataeibacter assemblies. A maximum likelihood phylogeny inferred from 642 single copy core genes provided the lineage scaffold. An anvi’o pangenome analysis defined a constant core gene cluster component across genomes and a noncore fraction that accounted for most of the genome differences in gene content. Targeted features linked to cellulose biosynthesis and local c-di-GMP-associated context were extracted from each genome. These features captured differences in bcs neighborhood composition and the presence of nearby GGDEF and EAL domain signals. The resulting feature matrix was projected by principal component analysis to summarize between-genome variation. Mobilome profiles were strongly strain dependent. Plasmid homology clustering identified 12 clusters comprising 36 plasmids from 13 genomes, including two dominant clusters of seven and six plasmids. Mash-based distance summaries further distinguished clusters consistent with conserved backbones from clusters consistent with heterogeneous, module-driven relationships. Prophage sequences, assessed as VIBRANT-predicted regions, were widespread but sparse per genome and dominated by medium length fragments. Insertion sequence burden ranged from 50 to 181 elements per genome, indicating substantial differences in transposition-associated sequence content. Pairwise association tests did not support robust cross module covariation beyond expected relationships among pangenome composition metrics at the current sampling depth. Overall, these results provide a complete genome reference framework linking lineage structure and mobilome heterogeneity, and they define reusable resources for comparative studies in bacterial cellulose biotechnology. Full article

Background/Objectives: Plasmids are key vehicles for the dissemination of antimicrobial resistance (AMR), yet their contribution to the global resistome architecture of Escherichia coli remains poorly resolved. This study aimed to quantify how plasmid backbones shape the distribution, mobility, and stabilization of resistance genes across diverse phylogenetic backgrounds. Methods: We analyze 9700 high-quality genomes spanning major phylogroups and sequence types. Plasmidome reconstruction was integrated with lineage-resolved antimicrobial resistance gene (ARG) mapping to characterize plasmid–ARG associations and evolutionary patterns. Results: Although most antimicrobial resistance genes (ARGs) are chromosomal, plasmids disproportionately encode clinically important determinants including bla_NDM-5, mcr-1.1, and multiple bla_CTX-M alleles that show strong, recurrent associations with a restricted set of backbone families, most notably IncX3, IncX4, IncI, and IncF. These conserved plasmid–gene modules recur across phylogenetic backgrounds and continental scales. We identify a marked divergence in evolutionary strategies: generalist phylogroups (A, B1, D) maintain plasmid-rich and highly diverse resistomes, whereas globally dominant Extraintestinal Pathogenic E. coli (ExPEC) clones such as ST131 and ST410 exhibit reduced plasmid dependency and frequent chromosomal integration of extended-spectrum β-lactamase (ESBL) genes, particularly bla_CTX-M-15, consistent with a shift toward vertically stabilized resistomes. By integrating plasmidome reconstruction with lineage-resolved ARG mapping, this study delivers the most extensive plasmid-focused resistome analysis to date, revealing highly modular plasmid–ARG networks structured around a small number of high-risk backbone types. These backbones account for the majority of globally relevant ARGs, including 64.6% of bla_NDM-5 and 76.4% of mcr-1.1 detections. Conclusions: Together, our findings establish plasmid lineages rather than individual genes or clones as central units of AMR dissemination and critical targets for future genomic surveillance and intervention strategies. Full article

The rapid spread of antibiotic resistance through plasmid-mediated conjugation remains a primary global health concern. Despite its critical role in horizontal gene transfer, no approved drugs currently target this process, leaving a critical therapeutic gap. Integration Host Factor (IHF), a DNA-binding protein essential for plasmid replication and mobilization, emerges as a promising yet underexplored target for anti-conjugation strategies. This work aimed to develop a predictive computational model and identify small molecules that disrupt IHF function, thereby reducing plasmid transfer and limiting resistance gene dissemination. A curated dataset of 65 compounds with reported anti-plasmid activity was analyzed using a 3D-QSAR model based on algebraic descriptors computed with QuBiLS-MIDAS. The model was validated through leave-one-out cross-validation (Q² = 0.82), Tropsha’s criteria, and Y-scrambling. Representative compounds were selected via pharmacophore clustering and evaluated through molecular docking at both the DNA-binding site and a predicted allosteric pocket of IHF. The most promising complexes underwent 200 ns molecular dynamics simulations to assess stability and interaction patterns. The QSAR model demonstrated strong predictive performance (R² = 0.90). Docking simulations revealed more favorable binding energies at the allosteric site (up to −12.15 kcal/mol) compared to the DNA-binding site. Molecular dynamics confirmed the stability of these interactions, with allosteric complexes showing lower RMSD fluctuations and consistent binding energy profiles. Dynamic cross-correlation analysis revealed that allosteric ligand binding induces conformational changes in key catalytic residues, including Pro65, Pro61, and Leu66. These alterations may compromise DNA recognition and disrupt the initiation of replication. To our knowledge, this is the first computational study proposing allosteric inhibition of IHF as an anti-conjugation strategy. These findings provide a foundation for experimental validation and the development of novel agents to prevent horizontal gene transfer, offering a promising approach to restoring antibiotic efficacy against multidrug-resistant pathogens. Full article

Background: Oxidative stress arises from an imbalance in redox homeostasis, leading to the accumulation of reactive oxygen species. This condition is associated with premature aging, as well as the progression of several chronic noncommunicable diseases. Among the natural products, geopropolis stands out as a source of molecules with different biological properties. Despite reports of its therapeutic potential, data on the effects on biomolecules and lifespan remains unexplored. Objectives: In this context, we investigated the effects of hydroethanolic geopropolis extracts of Melipona orbignyi and Melipona quadrifasciata anthidioides on in vitro and in vivo protection against oxidative stress, as well as their toxicity and effects on lifespan. Methods: Firstly, we assessed the effect on protein integrity under AAPH-induced oxidative stress and on DNA stability following exposure to hydrogen peroxide and UV radiation. Furthermore, we evaluated the extracts toxicity, protection against juglone-induced oxidative stress and thermal stress, and effects on longevity in a Caenorhabditis elegans preclinical model. Results: In vitro, both extracts protected bovine serum albumin (BSA) from AAPH-induced oxidation, with maximum BSA integrity reaching 98.2 ± 1.8% (HGMO) and 91.7 ± 3.0% (HGMQ). In a UV/H₂O₂ plasmid assay, both extracts protected against oxidative DNA fragmentation across the tested range, achieving 100% protection (fully preserved DNA integrity) at the highest evaluated concentrations. In vivo, HGMO and HGMQ showed no acute toxicity (24–48 h), with survival comparable to controls, and increased survival under juglone-induced oxidative stress (80 µM, 24 h), with maximum viability gains of 37.3% (HGMO) and 23.9% (HGMQ). Both extracts extended lifespan, increasing maximum lifespan from 24 to 32 days (+33%). Conclusions: Overall, these findings support geopropolis extracts as promising candidates for biotechnological products targeting oxidative stress and healthy aging. Full article

Background/Objectives: Sebaceous carcinomas (SebCAs) of the ocular adnexa, primarily arising from the Meibomian glands, are locally aggressive eyelid tumors with metastatic potential. Upregulation of the oncogene MYC has been demonstrated in SebCA, suggesting a role in tumor initiation and progression. In other epithelial tumors, the insulin and insulin-like growth factor (IGF) signaling (IIS) pathway has been implicated in stem cell renewal via MYC activation and stabilization. This study aimed to evaluate the effects of pharmacologic and genetic modulation of the IIS pathway on MYC expression in human Meibomian gland epithelial cells (HMGECs) and meibocytes of adult C57B6 mice. Methods: HMGECs were incubated with either IIS activators or inhibitors or were subject to transfection with either an IGF1R plasmid or siRNA before assessments of viability, proliferation, immunostaining, and MYC quantification were performed. Murine eyelids were treated topically with small-molecule IIS modulators prior to tissue harvest for histology, immunolabeling, and qPCR. Results: HMGECs treated with IIS activators demonstrated downregulated IGF1R and upregulated MYC expression, increased viability and proliferation, and reduced autophagy, while treatment with inhibitors yielded the inverse effects. Incubation with the selective insulin receptor agonist, demethylasterriquinone B1, yielded the most phenotypic variability. IGF1R-overexpressing HMGECs exhibited relative upregulation of both Akt and MYC. Murine eyelids treated with an IIS agonist demonstrated a more mesenchymal phenotype and significantly induced MYC expression. Conclusions: Collectively, these results suggest that the IIS pathway may represent a novel approach for regulating high MYC expression in SebCA. Full article

Aspergillus fijiensis is an industrially important filamentous fungus, whose genetic analysis has been limited by the absence of species-specific tools. This study establishes an optimized CRISPR–Cas9 genome editing platform for A. fijiensis, from protoplast preparation to DNA repair pathway engineering. Antibiotic screening first identified hygromycin B and 5-FOA (5-fluoroorotic acid) as effective positive and counter-selection markers. A high-efficiency protoplast regeneration protocol was developed depending on specific osmotic stabilization and mycelial competence. Evaluation of a plasmid-based CRISPR system revealed that while autonomous replication was feasible, gene editing was constrained by low efficiency and a predominant bias toward NHEJ (non-homologous end joining). We implemented a Cas9–sgRNA RNP (ribonucleoprotein) delivery approach, with RNP delivery alone producing frequent indels. However, targeted integration remained inefficient when using conventional MMEJ (Microhomology-mediated end joining) donors. By employing donors containing short (5 bp) microhomology arms between cleavage sites, we effectively engaged the MMEJ pathway, enabling precise insertions and large-fragment deletions in 92% of the analyzed transformants. Donor templates containing minimal 5 bp microhomology sequences could effectively shift the predominant repair pathway from NHEJ to MMEJ. These findings demonstrate that MMEJ is the superior pathway with a unique mechanism for genome engineering in A. fijiensis, providing a versatile toolkit for unlocking the biotechnological potential of this recalcitrant species and a successful paradigm for establishing genetic systems in other species. Full article

Monochamus alternatus larvae, as concealed trunk-boring pests, evade conventional insecticide contact due to their cryptic feeding niche. To overcome this limitation, previous studies have engineered strains of the naturally entomopathogenic bacterium Yersinia entomophaga. The lethality of these strains against M. alternatus was enhanced by incorporating extracellular secretion systems and enriching insecticidal proteins within the larval midgut. However, plasmid loss occurs during serial subculturing. Here, we established an engineered strain that expresses the red fluorescent protein gene mCherry to explore the applicability of bacterial conjugation transfer to Yersinia. We then constructed a chromosomally integrated strain (CSLH88-pCHSW) that incorporates extracellular secretion systems. The results of stability assays demonstrated 100% retention of the mCherry and Cry3Aa-T-HasA genes over 78 generations. SDS-PAGE and Western blot analyses confirmed the extracellular secretion of the Cry3Aa-T protein in the CSLH88-pCHSW strain. Bioassays revealed that the CSLH88-pCHSW strain was significantly more virulent against M. alternatus larvae than both the wild-type strain (CSLH88) and the plasmid-transformed strain (CSLH88-pCHKW), and exhibited markedly faster insecticidal kinetics. Our study reveals the application of bacterial conjugation transfer technology for constructing biocontrol strains. This genomically stabilized Yersinia strain eliminates the risks of failure associated with plasmid loss in the field, enabling the sustainable control of M. alternatus. Full article

In this work, we designed non-viral gene delivery vector systems based on three poly(2-ethyl-2-oxazoline)-ran-poly[2-(3-butenyl)-2-oxazoline] copolymers functionalized by primary, secondary, and tertiary amino groups. The impact of copolymer structure and composition was sought through the examination of basic physicochemical and biological parameters. The complexation ability of copolymers with plasmid DNA was studied by ethidium bromide quenching assay. The polyplex particles size and ζ-potential were determined by dynamic and electrophoretic light scattering. The release ability of copolymers was assessed by competitive displacement of DNA using dextran sulfate. The biological performance of amino-functionalized poly(2-ethyl-2-oxazoline)-ran-poly[2-(3-butenyl)-2-oxazoline] based gene delivery systems was evaluated, and their behavior under various environmental conditions, such as pH and ionic strength, was investigated. Cytotoxicity was assessed in two human lung-derived cell lines, and the ability of the copolymers to mediate plasmid DNA delivery and expression was examined. The resulting polyplex nanoparticles exhibited the ability to release DNA molecules and sensitivity to alterations in pH and ionic strength. All systems showed high biocompatibility and were able to mediate plasmid DNA delivery, resulting in detectable EGFP expression in vitro. The vector properties were found to be driven by a multifactorial interplay among hydrophobic character, thermoresponsive behavior, polymer mobility, charge accessibility, intracellular environmental responsiveness, secondary structure effects, etc. The copolymer bearing primary amino groups displayed a distinct balance between DNA binding and release, characterized by moderate complex stability and enhanced sensitivity to environmental changes. These findings provide mechanistic insight into how amino functionality and polymer structure influence the structure–property–behavior relationships of polyoxazoline-based non-viral gene delivery systems. Full article

Norovirus is a major cause of acute viral gastroenteritis in humans. Molecular biology-based detection methods play a pivotal role in ensuring accurate and specific diagnosis. The inclusion of Qβ phage particles as armored positive controls in these assays can further enhance their reliability and specificity. Herein, we discuss rational design strategies to improve the stability of Qβ bacteriophage capsid proteins armored with RNA using Discovery Studio 2019 protein design software. Amino acid mutation sites were deter-mined based on changes in folding free energy differences (ΔΔGmut). These single-site mutations were subsequently evaluated using molecular dynamics simulations. Wild-type and mutant recombinant expression plasmids were constructed and transformed into Escherichia coli BL21 (DE3) for cloning and expression. The stability of Qβ virus-like particles (VLPs) was assessed using real-time fluorescence RT-qPCR. The results showed that structurally intact and uniformly distributed wild-type and single-site mutant VLPs were successfully obtained. Stability analyses indicated that at 4 °C, 25 °C, 37 °C, 45 °C, and 60 °C, the single-site mutant exhibited a significantly lower rate of degradation than the wild-type. In conclusion, rational design enables the generation of single-site mutant VLPs with enhanced stability, providing a safer and more stable standard reference material for the molecular detection of foodborne viruses. Full article

The rise in antimicrobial resistance (AMR) among the most clinically significant bacteria presents a global threat. The coexistence of resistance mechanisms to both carbapenems and colistin is particularly concerning, as these are last-line treatments, specifically reserved for the most challenging infections caused by clinically multidrug-resistant Enterobacterales. Natural aquatic environments have become environmental reservoirs for the transmission of AMR, particularly concerning mechanisms against these two types of critically important drugs. The crucial role of environmental settings as a driving force for the spread and evolution of AMR associated with these drugs is underestimated, and scientific knowledge on this topic is limited. This review aims to fill an important gap in the scientific literature and comprehensively consolidate the available data on carbapenem- and colistin-associated AMR in the aquatic environment. This study provides a comprehensive synthesis of the current knowledge by integrating bibliographic data with a detailed genomic analysis of 278 bacterial genomes sourced from natural waters. It explores the distribution of carbapenemase and mobile colistin resistance (mcr) genes, identifying their hosts, geographical spread, and complex gene–plasmid–host associations. This review distinguishes two critical host groups for genes that provide resistance to last-resort drugs, Enterobacterales and autochthonous aquatic microbiota, highlighting both confirmed and potential interactions between them. Crucially, genomic analysis highlights the alarming co-occurrence of carbapenem and colistin resistance in single cells and on single plasmids, contributing to the spread of multidrug resistance phenotypes. These findings clearly indicate that aquatic environments are not merely passive recipients but active, evolving hubs for high-risk AMR determinants. Future research should focus on the interplay between allochthonous vectors and autochthonous microbiota to better understand the long-term stabilization of carbapenemase and mcr genes. Such efforts, combined with advanced sequencing technologies, are essential to ensure that carbapenems and colistin remain viable treatment options in clinical settings. Full article

Background/Objectives: Cervical cancer is a leading cause of cancer-related mortality among women, primarily driven by persistent infections with high-risk human papillomavirus (HPV), particularly HPV-16. Vaccines based on plasmid DNA encoding the viral oncogenes E6 and E7 represent a promising immunotherapeutic strategy, but their efficacy remains limited due to poor cellular uptake. Cell-penetrating peptides such as RALA improve intracellular delivery, and functionalization with octa-arginine peptide conjugated to mannose (R8M) further enhances targeting of antigen-presenting cells (APCs). This study aimed to obtain the minicircle DNA (mcDNA) encoding mutant HPV-16 E6 and/or E7 antigens, and optimize its complexation with mannosylated RALA-based nanoparticles to improve vector delivery and consequently antigen presentation. Methods: Nanoparticles were formulated at different concentrations of RALA, with and without R8M functionalization. Their characterization included hydrodynamic diameter, polydispersity index, zeta potential, complexation efficiency (CE), stability, morphology, and Fourier-Transform Infrared Spectroscopy. In vitro assays in JAWS II dendritic cells (DCs) assessed biocompatibility, transfection efficiency and target gene expression. Results: Optimal conditions were obtained at 72.5 µg/mL of RALA, producing nanoparticles smaller than 150 nm with high CE (>97%) and uniform size distribution. Functionalization with R8M at 58 µg/mL preserved these characteristics when complexed with all mcDNA vectors. The formulations were biocompatible and effectively transfected DCs. Mannosylated formulations enhanced antigenic expression compared to non-mannosylated counterparts, evidencing a mannose-receptor-mediated uptake, while increasing the production of pro-inflammatory cytokines. Conclusions: Nanoparticles based on the RALA peptide and functionalized with R8M significantly improved mcDNA transfection and gene expression in APCs. These findings support further investigation of this system as a targeted DNA vector delivery platform against HPV-16. Full article