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Brucella anthropi is an emerging opportunistic pathogen characterized by intrinsic resistance to most $\beta$-lactams. However, the acquisition of carbapenem resistance in this species has rarely been documented in environmental, animal, or clinical settings. In this study, a multidrug-resistant strain, SBA01, was isolated from wastewater-irrigated soil. SBA01 exhibited phenotypic resistance to carbapenems and colistin, the latter being independent of mcr genes. Genomic analysis localized bla_IMP-1 on a stable 21 kb plasmid maintained by a Type II toxin–antitoxin system. While non-self-transmissible, this plasmid was mobilized to Escherichia coli and Klebsiella pneumoniae via an unclassified 50 kb helper plasmid. Additionally, a 217 kb prophage-bearing megaplasmid was identified, enhancing genomic plasticity. Genomic screening identified 32 putative virulence determinants, including markers associated with host interaction. Risk profiling indicated an elevated hazard index for SBA01, driven by the convergence of multidrug resistance, cryptic mobilization capacity, and opportunistic survival traits. These findings position B. anthropi as a resilient environmental reservoir for clinically relevant carbapenemases. Expanding surveillance frameworks to include such adaptive hosts is necessary to better evaluate potential occupational exposures at the wastewater–soil interface. Full article

The isolation of locally adapted rhizobial strains with high symbiotic activity represents an effective strategy for increasing soybean yield under extreme environmental conditions. In this study, seven novel strains were isolated from nodules of soybeans grown in a greenhouse using field soil from the Lower Volga region. Five genomes were assembled into complete circular chromosomes, whereas two strains yielded near-complete chromosomes containing single repeat-mediated junctions. All strains had putative plasmids that were independently validated as circular by long-read mapping and confirmed by the presence of characteristic replication and conjugation-associated genes. Genome sequences of strains were about 11 Mb, and GC contents were 63.1–63.3%. Comparative genome analyses demonstrated that all strains had average nucleotide identity values of 95.4% with Bradyrhizobium japonicum USDA 6^T and 96.3% with Bradyrhizobium barranii 144S4^T, forming a distinct cluster in phylogenetic trees. No significant differences were detected between B. japonicum and B. barranii that would explain the species boundary. Therefore, it is proposed to unite all novel strains into the subspecies Bradyrhizobium japonicum subsp. saratovii subsp. nov., and all other strains of B. japonicum and B. barranii we suggest dividing into four subspecies: Bradyrhizobium japonicum subsp. japonicum subsp. nov., Bradyrhizobium japonicum subsp. barranii comb. nov., Bradyrhizobium japonicum subsp. apii comb. nov., and Bradyrhizobium japonicum subsp. saratovii subsp. nov. The proposed taxonomic framework expands current knowledge of the biodiversity of soybean symbiotic bacteria and contributes to a better understanding of the distribution and the evolution of bacteria Bradyrhizobium spp. in previously unexplored regions. Full article

Fecal microbiota are shaped by upstream digestive processes and reflect the outcome of host–microbe interactions, including the resistant microbial fraction that survives to be excreted. This is particularly crucial for assessing zoonotic risks and environmental contamination, as feces are the primary source of dissemination, which is considered an emerging One Health threat. Therefore, we conducted a pilot study to obtain the exploratory findings regarding the cattle GIT microbial composition, potential resistome, and their transmission drivers, such as plasmids, using metagenomic analysis from different districts in Khyber Pakhtunkhwa (KP) province, Pakistan. For this purpose, a total of 150 fecal samples (50 from each district) of healthy cattle were collected from various farms in Mardan (FC1), Peshawar (FC2), and Dera Ismail Khan (FC3) districts. Total DNA from each sample was extracted, pooled (FC1, FC2, and FC3), and sequenced via the Illumina platform. Bacteria were the highly abundant kingdom, while Pseudomonadota and Bacillota were dominant phyla in all samples. Caryophanon latum and Escherichia coli were highly abundant at the species level. A large resistome (40–49 genes), including critical genes, such as tet(X), bla_OXA-427, and plasmidomes (16–22), such as IncF, was detected in the samples. The prominence of certain commensal or opportunistic pathogens in the fecal microbiota may indicate the presence of sub-clinical gastrointestinal disruptions or disease that may affect cattle herds. The fecal resistome is extensive, identifying dairy cattle in these regions as important reservoirs for AMR genes capable of spreading via HGT. This pilot study establishes that the fecal microbiota of dairy cattle in this region are not merely a waste product but a complex ecosystem, rich in microbiota of One Health significance. Full article

Dogs, especially as pets but also an increasing number of stray dogs, share environments with humans, facilitating the transfer of antibiotic resistance genes (ARGs) between genetic compartments, with zoonotic and public health implications that must be addressed within One Health. In this cross-sectional comparative study, we explored the distribution of seven selected clinically relevant ARGs in both genomic DNA (gDNA) and plasmid DNA (pDNA), and the phenotypic resistance profile of the cultivable microbiota, between pet dogs (PeDs, n = 12) and free-roaming dogs (FRDs, n = 10) in Mexico. Tetracycline resistance genes (tetQ, tetW, and tetM) predominated in both compartments (40% to 100%), suggesting the presence of a core tetracycline-associated resistome. In contrast, plasmid-associated differences were group-specific: in pDNA cfxA was enriched in FRDs (90%) and tetK in PeDs (42%), whereas blaTEM-1 and ermC were absent in two dog populations. Cultivable bacteria from both groups exhibited phenotypic multidrug resistance, particularly by β-lactams, macrolides, lincosamides, and tetracyclines. FRDs also harbored pathogenic–zoonotic bacteria such as Yersinia enterocolitica, Campylobacter jejuni, and Enterococcus faecalis. Our findings indicated that FRDs and PeDs harbor substantial resistomes, with differences in plasmid-associated ARGs, revealing a transfer potential related to environmental exposure. 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

Although the replication mechanism of the F plasmid and its regulatory strategies have been addressed in several studies, a comprehensive understanding of these processes remains incomplete. In this work, we present new observations that contribute to refining the current model of F plasmid replication control. In this work, the results indicate that plasmid copy number control in both the F plasmid and its derivatives is consistent with two previously proposed mechanisms: the titration model and the loop formation model. In both cases, the intracellular concentration and functional state of the RepE protein appear to play a central role. Consistent with earlier reports, the data of this study support the conclusion that the RepE monomer functions as the active replication initiator. Importantly, the transcriptional analyses suggest that not only RepE dimers but also monomers contribute to autoregulatory control of repE expression. These findings support a model in which the monomer–dimer equilibrium of RepE shapes both replication initiation and transcriptional autoregulation of the F plasmid. 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

Listeria innocua is a bacterium frequently detected in food and food production plants (FPPs). Understanding the heterogeneity of L. innocua food isolates is essential for predicting potential food safety threats and developing preventive and control measures. This study aimed to characterize L. innocua isolated from raw drinking milk by investigating the genomic features related to virulence, antimicrobial resistance, and persistence using whole-genome sequencing (WGS), along with phenotypic antimicrobial susceptibility testing using the disk diffusion method. All ten isolates analyzed in this study belonged to sequence type (ST) 492 and were distantly related to the reference strain. A total of 80 virulence-associated genes were identified, including the complete Listeria Pathogenicity Islands-3 (LIPI-3) and LIPI-4 clusters typically found in virulent L. monocytogenes clones, as well as 66 additional genes involved in adhesion, invasion, motility, post-translational modification, regulation, immune modulation, and stress survival. Stress survival islet 2 (SSI-2) and genes encoding the Clp protease complex (clpC, clpE, clpP), which support both persistence and virulence, were also detected, whereas LIPI-1 and internalin genes were not detected. The antimicrobial resistance determinants included fosX, lin, norB, sul, and three multidrug efflux pumps (lde, mdrL and mdrM). Mobile genetic elements (plasmids, prophages, or transposons) were not detected. All isolates were phenotypically susceptible to benzylpenicillin, ampicillin, meropenem, erythromycin, and trimethoprim–sulfamethoxazole. These findings underscore the importance of ongoing genomic surveillance of L. innocua in food environments and highlight the need to assess the potential risk posed by specific lineages, such as ST492, to food safety. 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

Antimicrobial resistance (AMR) constitutes one of the most severe and pressing threats to global public health, food security, and environmental integrity. This review synthesizes current evidence across interconnected One Health domains—humans, animals, food, and the environment—to delineate the scope, mechanisms, and drivers of AMR transmission. Our analysis reveals three principal findings. First, the scope of AMR is alarmingly extensive, with antibiotic-resistant bacteria (ARB) and genes (ARGs) now pervasive across all four ecological compartments, transcending traditional clinical boundaries. Second, this widespread distribution is critically facilitated by horizontal gene transfer mechanisms, particularly via mobile genetic elements such as plasmids, which enable ARGs to disseminate rapidly between diverse bacterial populations across different ecosystems. Third, we identify multiple interconnected drivers that actively promote this cross-ecosystem spread, encompassing both evolutionary and transmission drivers. By characterizing these critical transmission pathways and underlying drivers, this review provides an integrated framework to identify critical transmission risks and inform integrated strategies for mitigating antimicrobial resistance across One Health domains. 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

Objective: Endometrial cancer (EC) remains a significant clinical challenge, particularly for patients with advanced or recurrent disease. This study aims to investigate the effects of Sanguinarine Chloride (S.C), a natural benzophenanthridine alkaloid with broad anti-tumor properties, on EC cell growth and invasion, and to elucidate its underlying molecular mechanisms. Methods: S.C’s effects on EC cell viability, proliferation, invasion, and apoptosis were evaluated using CCK-8, EdU, colony formation, 3D matrigel drop assay, FACS and Western blotting (WB). To evaluate its effects on ferroptosis, malondialdehyde (MDA) assay kits, DCFH-DA and the C11 BODIPY581/591 probe, were employed. The molecular mechanisms through which S.C regulates FTO-ACSL4 axis were investigated using plasmid transfection and WB. Additionally, a mouse xenograft model derived from EC cells was established to evaluate the in vivo effects of S.C and its molecular mechanisms, utilizing hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC) and WB. Results: S.C significantly inhibited EC cell growth and invasion. It induced cell death primarily through ferroptosis, as inhibitors (Ferrostatin-1, Deferoxamine) reversed this effect. S.C downregulated the RNA demethylase FTO, leading to increased ACSL4 expression, enhanced lipid peroxidation, suppression of the NRF2-GPX4 axis, and activated NCOA4-mediated ferritinophagy. Knocking down or pharmacologically inhibiting ACSL4 reduced S.C-induced ferroptosis. Furthermore, in a murine xenograft model, S.C significantly suppressed tumor growth, which was associated with consistent alterations in these ferroptosis-related markers in vivo. Conclusions: Our findings reveal that S.C triggers ferroptosis in EC via the novel FTO-ACSL4 axis, highlighting its potential as a therapeutic agent and identifying the FTO-ACSL4 pathway as a promising target for endometrial cancer treatment. Full article

Objective: Rare-earth elements are extensively employed across diverse industrial sectors, increasingly raising concerns about their potential health hazards in both occupational and environmental contexts. Samarium oxide (Sm₂O₃), a routinely processed rare-earth product, reproducibly precipitates pulmonary fibrosis in experimental models, yet the molecular circuitry that transduces its fibrogenic signal remains almost entirely unmapped. This study aims to elucidate the role of miR-214-3p in Sm₂O₃-induced pulmonary fibrosis and to investigate its regulatory mechanism at the molecular level. Methods: A murine model of pulmonary fibrosis was established via intratracheal instillation of Sm₂O₃, and histopathological changes were assessed using hematoxylin and eosin (H&E) and Masson’s trichrome staining. RNA sequencing was performed on lung tissues to identify differentially expressed mRNAs. Leveraging our previously generated miRNA landscape of Sm₂O₃-exposed lungs, we subjected the dataset to Gene Ontology and KEGG enrichment analyses, which convergently identified miR-214-3p as the top-ranking candidate regulator of the fibrogenic MAPK axis. The direct targeting of MAP2K3 by miR-214-3p was validated using a dual-luciferase reporter assay. Expression levels of fibrotic markers (α-SMA, Collagen I) and key components of the MAPK signaling pathway (MAP2K3, p-MAPK14, MST1) were quantified in both in vivo and in vitro models using qRT-PCR and Western blotting. Gain- and loss-of-function studies, complemented by rescue assays, were performed in human embryonic lung fibroblasts (HELFs) via transient transfection of miR-214-3p mimics, inhibitors, or MAP2K3-overexpression plasmids. Cell proliferation was evaluated using the EdU assay, and TGF-β1 secretion was measured by ELISA. Results: Sm₂O₃ exposure induced significant pulmonary fibrosis in mice, accompanied by marked downregulation of miR-214-3p and upregulation of MAP2K3 in lung tissues. Overexpression of miR-214-3p or silencing of MAP2K3 effectively suppressed Sm₂O₃-induced fibroblast activation, including reduced cell proliferation, decreased expression of α-SMA and Collagen I, and inhibition of p38 MAPK phosphorylation. Notably, ectopic overexpression of MAP2K3 reversed the protective effects conferred by miR-214-3p, confirming a functional rescue. Conclusions: miR-214-3p directly silences MAP2K3, thereby blunting p38 MAPK-driven fibrogenesis after Sm₂O₃ exposure. Our data unveil a miR-214-3p–MAP2K3–p38 MAPK axis that constitutes a readily druggable target for rare-earth-element-induced pulmonary fibrosis. Full article

Marek’s disease (MD), caused by the oncogenic Marek’s disease virus (MDV), is a highly contagious avian infection that induces lymphoproliferative tumors. The RNA-binding protein ELAVL1 is known to regulate tumor cell proliferation and apoptosis, but its role in MDV-induced oncogenesis remains unclear. This study investigated whether ELAVL1 modulates proliferation and apoptosis in the MDV-transformed MSB1 cell line and whether its effects involve the cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) pathway. MSB1 cells were transiently transfected with ELAVL1-overexpressing plasmids (pEGFP-C-ELAVL1) or ELAVL1-specific siRNA, with expression confirmed by real-time PCR (qRT-PCR). Cell proliferation was assessed using the CCK-8 assay, while cell cycle distribution and apoptosis rates were analyzed by flow cytometry. COX-2 and PGE2 expression levels were determined by qRT-PCR, Western blotting, and ELISA. Overexpression of ELAVL1 significantly promoted the proliferation of MSB1 cells, decreased transition into the G1 phase, increased the proportions of S and G2 phase cells, and suppressed apoptosis. Correspondingly, both mRNA and protein levels of COX-2 and PGE2 were significantly elevated. Conversely, ELAVL1 knockdown significantly inhibited proliferation, induced G1 phase arrest, decreased S phase cells, and significantly decreased COX-2 and PGE2 expression. These findings indicate that ELAVL1 promotes proliferation and inhibits apoptosis in MDV-transformed MSB1 cells, potentially via the COX-2/PGE2 signaling pathway. Full article

The cause of chronic neurological effects associated with Lyme disease (LD) remains unclear. We propose that bacterial extracellular vesicles (BEVs) released by Borrelia burgdorferi, the causative agent of LD, exacerbate spirochete-induced damage and serve as a persistent source of antigenic stimulation. We showed that, over a 10-day period, in vitro cultures of B. burgdorferi B31 produced 38,000 BEVs per spirochete with a distinctive double-membrane structure and median diameter of 143.3 nm. BEVs contained known immunogenic and immunomodulatory molecules such as peptidoglycan, p66, flagellar filament protein (FlaB), basic membrane proteins A/B/D, BdrV, GroEL, CRASP-1, ErpA8, glycerophosphodiester phosphodiesterase, p37, OMS28, p13, OspA/B/C, VlsE, and outer membrane glycolipids (e.g., cholesteryl 6-O acyl beta D galactopyranoside). Chromosome-encoded 16S ribosomal RNA and cp32 plasmid-encoded OspE and terminase genes were also detected in the BEVs. Of the 45 Borrelia proteins identified in the urine of a C3H/HeJ murine model of Lyme disease, 14 were associated with BEVs. In human urine samples, 31 of 289 spirochete proteins detected in patients with either acute Lyme disease or persistent borreliosis post-treatment symptoms, including p66 and FlaB, were also BEV-associated. BEV treatment of HMC3 human microglial cells reduced phagocytic activity and triggered aberrant activation of inflammatory and immunometabolic pathways, including upregulation of interferon-alpha (IFN-α), aconitate decarboxylase 1 (Acod1), and Toll-like receptor 2 (TLR2) gene expression. BEVs also induced NRF2 nuclear translocation. In conclusion, these findings support that BEVs can amplify spirochete-induced damage and act as antigenic debris, driving dampened phagocytic activity and dysregulated inflammation, with implications for diagnostics and therapeutics targeting vesicle-mediated pathology. Full article