Search Results (363)

The Bacillus anthracis pXO1 plasmid, encoding ~143 proteins, presents a compact model for exploring protein function and evolutionary patterns using protein language models. Due to the organism’s slow evolutionary rate, its limited amino acid variation enhances detection of physiologically relevant patterns in plasmid protein composition. In this study, we applied embedding-based analyses and machine learning methods to characterize pXO1 protein modules across diverse B. anthracis lineages. We generated protein sequence embeddings, constructed phylogenies, and compared plasmid content with whole genome variation. While whole genome and plasmid-based phylogenies diverge, the composition of proteins encoded along the pXO1 plasmid revealed lineage specific structure. Association rule mining combined with decision tree classification produced plasmid-encoded targets for assessing anthrax sublineage, which yielded functionally redundant protein modules that reflected geographic and phylogenetic patterns. A conserved DNA replication module exhibited both shared and B. anthracis lineage specific features. These results show that pXO1 plasmid protein modules contain biologically meaningful and evolutionarily informative signatures, exemplifying their value in phylogeographic characterizations of bacterial pathogens. This framework can be extended to study additional virulence plasmids across Bacillus and other environmental pathogens using scalable protein language model tools. Full article

The safety, genetic distinctiveness, and functional capabilities of Lactococcus sp. KTH0-1S, a strain isolated from Thai fermented shrimp (Kung-Som), were investigated to assess its potential as a next-generation probiotic and microbial cell factory. Whole-genome sequencing and multilocus sequence typing (MLST) analysis revealed that Lactococcus sp. KTH0-1S is a novel, phylogenetically distinct strain within the Lactococcus genus. Comprehensive in silico safety evaluation confirmed the absence of antimicrobial resistance genes and major virulence factors, supporting its suitability for food-grade applications. The genome encodes multiple probiotic-relevant traits, including stress tolerance (e.g., dnaK, clpP), adhesion and biofilm formation (e.g., gapA, luxS, glf2), and nutrient acquisition genes, enabling adaptation to gastrointestinal and fermentation environments. Notably, Lactococcus sp. KTH0-1S harbors a chromosomally encoded nisin Z biosynthesis gene cluster with auto-induction capability, providing a self-regulated and stable alternative to conventional plasmid-based NICE systems in Lactococcus lactis. The strain also exhibits nisin immunity, allowing tolerance to high nisin concentrations, thus supporting robust protein production. Genomic evidence and phenotypic assays confirmed a functional respiration metabolism activated by heme supplementation, enhancing biomass yield and culture stability. Furthermore, the presence of diverse CAZyme families (GHs, GTs, CEs) enables utilization of various carbohydrate substrates, including lignocellulosic and starchy agro-industrial residues. These properties collectively underscore Lactococcus sp. KTH0-1S as a safe, stable, and metabolically versatile candidate for probiotic applications and as a cost-effective, food-grade expression host for biotechnological production. Full article

Pediococcus pentosaceus is a lactic acid bacterium used inter alia for the fermentation of milk, meat, vegetables, fruits, and even for brewing beer. Several health-promoting effects, such as antibacterial and antifungal activities or microphage and immune system stimulation, have been attributed. Apart from refining foods during the fermentation process, P. pentosaceus strains are added to meat and meat products as protective cultures to improve food safety, while leaving the organoleptic properties untouched. Since knowledge on the latter issue is still limited, we investigated 32 isolates from milk samples and teat canal biofilms regarding their antibacterial efficacy as a prerequisite for possible application as protective cultures. P. pentosaceus strains were unequivocally identified by DNA sequencing of the rrnA gene encoding 16S rRNA. Binary matrices obtained from random amplification of polymorphic DNA experiments showed that all isolates differed by more than 5% and thus represented subspecies. The antibacterial profiles against eight food-borne pathogens and food spoilage bacteria were determined. They efficiently combatted, although to various extents, Gram-negative bacteria such as Pseudomonas aeruginosa or Salmonella enterica, and Gram-positive bacteria such as Staphylococcus aureus and Listeria monocytogenes. Interestingly, acid production was dependent on the presence of the challenged pathogen and did not correlate with the extent of inhibition. Bioinformatic analyses of the genomes of the three top-ranked isolates revealed a pronounced genomic plasticity with a core genome of 1460 genes and additional 91, 130, and 161 unique genes, respectively. Each strain included a set of three, five, or six plasmids and was equipped with different genes encoding bacteriocins. The data suggest that multiple strains of P. pentosaceus should be included in order to optimize the selection of a culture for food preservation. The approach could also be applicable to other bacterial species. Full article

Objectives: The Taihe Black-Boned Silky Fowl (TBSF) is a unique indigenous chicken breed in China, characterized by widespread melanin deposition throughout its body. Fluoroquinolones (FQs) such as enrofloxacin can persist in TBSF for an extended period exceeding 100 days. The aim of this study was to examine the current status and development trends of FQ resistance within the TBSF breeding environment. Methods: Whole-genome sequencing was utilized to identify the molecular presence of quinolone resistance-determining region (QRDR) mutations and plasmid-mediated quinolone resistance (PMQR) genes in Escherichia coli isolates obtained from TBSF farms. Network inference based on strong Spearman correlations (ρ > 0.5) and statistically significant associations (p-value < 0.05) was applied to investigate the co-occurrence patterns among FQ residues, resistance phenotypes, and antibiotic resistance genes. Results: The results showed that FQ residues were identified as the primary contributor to FQ resistance in E. coli isolates. Mutations at QRDR sites were the predominant factor driving FQ resistance, rather than PMQR determinants. This study also reported the first identification of GyrA-S83Q mutation being associated with FQ resistance. Conclusions: It was concluded that E. coli strains in TBSF environments, where chickens have a long-term residual metabolic cycle of antimicrobials, may develop and evolve new mechanisms to adapt to this environment. Further research is warranted to investigate the evolution of FQ resistance in E. coli strains within TBSF environments. Full article

It is estimated that one in four people worldwide carries Mycobacterium tuberculosis bacteria. MPT64 is a protein exclusively secreted by Mycobacterium tuberculosis complex (MTC) bacteria. It serves as a crucial diagnostic marker and plays a role in the bacterium’s survival by modulating the host immune response. Consequently, the development of innovative diagnostic tools based on MPT64, as well as the production of high-purity MPT64 protein to support research on tuberculosis pathogenesis and the advancement of novel therapeutic strategies, is of great importance. In this study, optimization experiments were conducted to produce this protein in E. coli with high yield and purity. First, a gBlock was designed by codon optimization and then cloned into a plasmid vector using the LIC method. For more efficient production, E. coli BL21(DE3)-R3-pRARE2 strain, which carries rare tRNAs for rare codons, was used as the host. Five different culture media were tested to maximize protein production, with the highest yield obtained in eBHI medium. The resulting protein yield was 4.9 mg/L. To the best of our knowledge, this study provides the most detailed information on the recombinant production and characterization of MPT64 to date. Therefore, these results contribute important data for future studies on the MPT64 protein. Full article

Background: Sinorhizobium fredii HH103 is a fast-growing rhizobial strain capable of infecting a broad range of legumes, including plants forming determinate and indeterminate nodules, such as Glycine max (its natural host) and Glycyrrhiza uralensis, respectively. Previous studies reported the sequence and annotation of the genome of this strain (7.25 Mb), showing the most complex S. fredii genome sequenced to date. It comprises seven replicons: one chromosome and six plasmids. Among these plasmids, pSfHH103d, also known as the symbiotic plasmid pSymA, harbors most of the genes involved in symbiosis. Due to limitations of the sequencing technology used at the time and the presence of high number of clusters of transposable elements, this plasmid could only be partially assembled as four separated contigs. Methods: In this work, we have used a combination of PacBio and Illumina sequencing technologies to resolve these complex regions, obtaining an updated genome sequence (7.27 Mb). Results: This updated version includes an increase in size of the largest replicons (chromosome, pSfHH103d, and pSfHH103e) and a complete and closed symbiotic plasmid (pSfHH103d or pSymA). Additionally, we carried out a re-annotation of the updated genome, merging the previous annotation and the new one found in the remaining gaps. Notably, we found a high number of transposable elements in the HH103 genome, especially in three plasmids (pSfHH103b, pSfHH103c, and pSymA), a feature that is common among S. fredii strains. Conclusions: The combination of PacBio and Illumina sequencing technologies has allowed us to obtain a complete version of the HH103 pSymA. The presence of a high number of mobile elements seems to be a general characteristic among S. fredii strains, a fact that might be related to a high genome plasticity. Full article

Porcine epidemic diarrhea virus (PEDV) is a highly contagious coronavirus that causes severe diarrhea, dehydration, and high mortality in piglets, leading to significant economic losses in the swine industry. The spike (S) protein of PEDV is the primary target for neutralizing antibodies and is critical for vaccine development. In this study, the pUC57-S01 and pUC57-S02 plasmids carrying the codon-optimized truncated S gene sequence were constructed. The mRNA S01 showed higher protein expression in vitro than mRNA S02, as confirmed by Western blotting. The safety and immunogenicity of mRNA S01 were evaluated in animal experiments. The results indicated that the mRNA S01 vaccine was safe for piglets and pregnant sows. Immunogenicity was assessed by a neutralization assay, which revealed that encapsulated mRNA S01 induced high levels of neutralizing antibody titers in pigs. Challenge protection efficiency tests showed that the mRNA S01 vaccine conferred immunity to newborn piglets, protecting them from a homologous PEDV strain challenge. This study provides a foundation for the clinical application of PEDV mRNA vaccines and offers a reference for the development of novel vaccines against PEDV. Full article

Background: Bovine mastitis (BM) is a major disease affecting dairy herds (DHs), with Serratia marcescens (S. marcescens) being increasingly implicated as a causative agent. The growing concern over antimicrobial resistance (AMR) extends to BM-associated S. marcescens isolates, where resistance patterns are emerging. Methods: Here, four BM Gram-negative isolates were investigated: 1-DH1, 2-DH1, 3-DH2, and 4-DH3. Phenotypic characterization was performed using the Neg-Urine-Combo98 panel on a MicroScan WalkAway Plus system. Whole-genome sequencing (WGS) was performed to characterize and identify AMR and virulence factors (VF) genes and plasmids in isolates 1-DH1, 3-DH2, and 4-DH3, and phylogenomic analyses were conducted for a visual comparison of the genomes. Results: Phenotypically, isolates 1-DH1, 2-DH1, and 4-DH3 were identified as S. marcescens, and 3-DH2 as Serratia odorifera (confirmed as S. marcescens by WGS). A 28.00% (n = 25) prevalence of phenotypic AMR for isolates 1-DH1, 2-DH1, and 4-DH3 against Aug-E, AM, To, Cfx, Crm, Cl, and Fd was shown, and 24.00% (n = 25) for isolate 3-DH2 against Aug-E, AM, To, Crm, Cl, and Fd. The AMR genes AAC(6′)-Ic, aac(6′)-Ic_1, aac(6′)-Ial, H-NS, SRT-2, oqxB, oqxB_1, oqxB25, mexI, CRP, and blaSST-1, and flgH, fliP, fliM, and fliG VF genes were identified in the whole genome of the S. marcescens sequenced isolates 1-DH1, 2-DH1, and 4-DH3. In addition, a phylogenomic analysis of these three isolates revealed that WGS genomes are more closely related to S. marcescens prevenient from environmental sources. Conclusions: This study reports, for the first time, AMR resistance to tobramycin, cefuroxime, colistin, and nitrofurantoin in BM S. marcescens isolates. Genomic analysis revealed the presence of multiple AMR and VF genes, further highlighting the pathogenic potential of these isolates. Phylogenomic analysis revealed that the genome of the three BM S. marcescens isolates is more closely related to environmental S. marcescens strains. Full article

The aim of this study was to evaluate the haemostatic potential and biocompatibility of a newly developed composite material for its use in blood-contacting applications. Based on promising reports on polylactide (PLA), sodium alginate (ALG), and bioactive additives such as hardystonite (HT) and zinc sulphide (ZnS), a melt-blown PLA nonwoven was modified via dip-coating using an ALG solution as a matrix for incorporating HT and ZnS particles, resulting in the PLA-ALG-ZnS-HT composite. The material was characterised in terms of surface morphology, specific surface area, pore volume, average pore size, and zeta potential (pH~7.4). Haemostatic activity was assessed by measuring blood coagulation parameters, while biocompatibility was evaluated through the viability of human peripheral blood mononuclear (PBM) cells and human foreskin fibroblasts (Hs68). Genotoxicity was analysed using the comet assay and plasmid relaxation test. Results confirmed a uniform alginate coating with dispersed HT and ZnS particles on PLA fibres. The modification increased the surface area and pore volume and caused a shift toward less negative zeta potential. Haemostatic testing showed prolonged activated partial thromboplastin time (aPTT), likely due to Zn²⁺ interactions with clotting factors. Biocompatibility tests showed high cell viability and no genotoxic effects. Our findings suggest that the PLA-ALG-ZnS-HT composite is safe for blood and skin cells and may serve as an anticoagulant material. Full article

The emergence of extensively drug-resistant (XDR) foodborne pathogens poses grave threats to food safety. This study characterizes the genome of an XDR Salmonella Kentucky isolate (Sal23C1) co-harboring cfr, mcr-1 and tet(A) from Shanghai chicken meat in 2022, which was the only isolate co-harboring these three key resistance genes among 502 screened Salmonella isolates. Genomic analysis revealed that the multidrug resistance gene cfr, which confers resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins and streptogramin A, was identified within a Tn3-IS6-cfr-IS6 structure on the transferable plasmid p3Sal23C1 (32,387 bp), showing high similarity to the Citrobacter braakii plasmid pCE32-2 (99% coverage, 99.98% identity). Concurrently, the mcr-1 gene resided in a pap2-mcr-1 structure on the transferable IncI2 plasmid p2Sal23C1 (63,103 bp). Notably, both genes could be co-transferred to recipient bacteria via conjugative plasmids at frequencies of (1.15 ± 0.98) × 10⁻⁶. Furthermore, a novel ~79 kb multidrug resistance region (MRR) chromosomally inserted at the bcfH locus was identified, carrying fosA3, mph(A), rmtB, qnrS1 and bla_CTX-M-55. Additionally, a novel Salmonella Genomic Island 1 variant (SGI1-KI) harbored aadA7, qacEΔ1, sul1 and the tet(A) variant. The acquisition of these antibiotic resistance genes in this isolate enhanced bacterial resistance to 21 antimicrobials, including resistance to the critical last-resort antibiotics tigecycline and colistin, which left virtually no treatment options for potential infections. Taken together, this is the first comprehensive genomic report of an XDR poultry-derived Salmonella Kentucky isolate co-harboring cfr, mcr-1 and the tet(A) variant. The mobility of these resistance genes, facilitated by IS6 elements and conjugative plasmids, underscores significant public health risks associated with such isolates in the food chain. Full article

Funded during the emergency phase of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, messenger RNA (mRNA) vaccines are single-stranded, 5′-capped mRNAs produced using a cell-free in vitro transcription from the corresponding plasmid DNA (pDNA) templates, encoding the viral spike (S) protein of SARS-CoV-2 [...] Full article

Dehydroshikimate (DHS) dehydratase (DSD) catalyzes the conversion of DHS into 3,4-dihydroxybenzoic acid (3,4-DHBA), a compound with promising applications across various industries. The DSD from Podospora anserina (DSD_Pa) was characterized and its catalytic properties were compared with those of previously investigated enzymes, AsbF (Bacillus thuringiensis), Qa-4 (Neurospora crassa), and QsuB (Corynebacterium glutamicum), both in vitro and in vivo using tube fermentation. Escherichia coli and C. glutamicum were used as platforms to construct model 3,4-DHBA producers. To increase DHS availability in both hosts, shikimate dehydrogenase AroE was inactivated, and the plasmid pVS7-aroG4, encoding 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (E. coli), was introduced. In E. coli, heterologous 3,4-DHBA synthesis was achieved through chromosomal integration of dsd genes. The fungal genes were codon-optimized for this bacterium. The same genes were cloned into the pVK9 vector and introduced into C. glutamicum, where 3,4-DHBA degradation was disrupted (ΔpcaHG). AsbF (k_cat ~ 1 s⁻¹) showed poor 3,4-DHBA accumulation in both hosts (1–1.5 g/L). The enzymes with better catalytic characteristics, QsuB (k_cat ~ 60 s⁻¹), DSD_Pa (k_cat ~ 125 s⁻¹), and Qa-4 (k_cat ~ 220 s⁻¹), provided 5 g/L 3,4-DHBA in E. coli and 3 g/L 3,4-DHBA in C. glutamicum, except for Qa-4. The low production (~1.5 g/L) observed for Qa-4 in C. glutamicum might be attributed to a non-optimal nucleotide sequence rich in codons rare for C. glutamicum. Full article

Antibiotic misuse accelerates resistance dissemination via plasmid conjugation, but quorum sensing (QS) regulatory mechanisms remain undefined. Using Escherichia coli (E. coli) MG1655 conjugation models (RP4-7/EC600 plasmids), we demonstrate that long-chain acyl-homoserine lactones (C10/C12-HSL) enhance transfer frequency by up to 7.7-fold (200 μM C12-HSL; p < 0.001), while quorum-quenching by sub-inhibitory vanillin suppressed this effect by 95% (p < 0.0001). C12-HSL compromised membrane integrity via ompF upregulation (4-fold; p < 0.01) and conjugative pore assembly (trbBp upregulated by 1.38-fold; p < 0.05), coinciding with ROS accumulation (1.5-fold; p < 0.0001) and SOS response activation (recA upregulated by 1.68-fold; p < 0.001). Crucially, rpoS and rmf deletion mutants reduced conjugation by 65.5% and 55.8%, respectively (p < 0.001), exhibiting attenuated membrane permeability (≤65.5% reduced NPN influx; p < 0.0001), suppressed ROS (≤54% downregulated; p < 0.0001), and abolished transcriptional induction of conjugation/stress genes. Reciprocal RpoS–RMF (ribosomal hibernation factor) crosstalk was essential for AHL responsiveness, with deletions mutually suppressing expression (≤65.9% downregulated; p < 0.05). We establish a hierarchical mechanism wherein long-chain AHLs drive resistance dissemination through integrated membrane restructuring, stress adaptation, and RpoS–RMF-mediated genetic plasticity, positioning QS signaling as a viable target for curbing resistance spread. Full article

Antioxidative neuroprotection is effective at preventing ischemic stroke (IS). Ferulic acid (FA) offers benefits in the treatment of many diseases, mostly due to its antioxidant activities. In this study, a glycosylated ferulic acid conjugate (FA-Glu), with 1,2,3-triazole as a linker and bioisostere between glucose at the C6 position and FA at the C4 position, was designed and synthesized. The hydrophilicity and chemical stability of FA-Glu were tested. FA-Glu’s protection against DNA oxidative cleavage was tested using pBR322 plasmid DNA under the Fenton reaction. The cytotoxicity of FA-Glu was examined via the PC12 cell and bEnd.3 cell tests. Antioxidative neuroprotection was evaluated, in vitro, via a H₂O₂-induced PC12 cell test, measuring cell viability and ROS levels. Antioxidative alleviation of cerebral ischemia–reperfusion injury (CIRI), in vivo, was evaluated using a rat middle cerebral artery occlusion (MCAO) model. The results indicated that FA-Glu was water-soluble (LogP −1.16 ± 0.01) and chemically stable. FA-Glu prevented pBR322 plasmid DNA cleavage induced via •OH radicals (SC% 88.00%). It was a non-toxic agent based on PC12 cell and bEnd.3 cell tests results. FA-Glu significantly protected against H₂O₂-induced oxidative damage in the PC12 cell (cell viability 88.12%, 100 μM) and inhibited excessive cell ROS generation (45.67% at 100 μM). FA-Glu significantly reduced the infarcted brain areas measured using TTC stain observation, quantification (FA-Glu 21.79%, FA 28.49%, I/R model 43.42%), and H&E stain histological observation. It sharply reduced the MDA level (3.26 nmol/mg protein) and significantly increased the GSH level (139.6 nmol/mg protein) and SOD level (265.19 U/mg protein). With superior performance to FA, FA-Glu is a safe agent with effective antioxidative DNA and neuronal protective actions and an ability to alleviate CIRI, which should help in the prevention of IS. Full article

Proteus mirabilis is a zoonotic pathogen that poses a growing threat to both animal and human health due to rising antimicrobial resistance (AMR). It is widely found in animals, including China’s nationally protected captive giant and red pandas. This study isolated Proteus mirabilis from panda feces to assess AMR and virulence traits, and used whole-genome sequencing (WGS) to evaluate the spread of resistance genes (ARGs) and virulence genes (VAGs). In this study, 37 isolates were obtained, 20 from red pandas and 17 from giant pandas. Multidrug-resistant (MDR) strains were present in both hosts. Giant panda isolates showed the highest resistance to ampicillin and cefazolin (58.8%), while red panda isolates were most resistant to trimethoprim/sulfamethoxazole (65%) and imipenem (55%). Giant panda-derived strains also exhibited stronger biofilm formation and swarming motility. WGS identified 31 ARGs and 73 VAGs, many linked to mobile genetic elements (MGEs) such as plasmids, integrons, and ICEs. In addition, we found frequent co-localization of drug resistance genes/VAGs with MGEs, indicating a high possibility of horizontal gene transfer (HGT). This study provides crucial insights into AMR and virulence risks in P. mirabilis from captive pandas, supporting targeted surveillance and control strategies. Full article