Search Results (586)

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

Liposomes are lipid bilayer vesicles that are highly biocompatible, able to interact with the cell membrane, and able to release their cargo easily. The improvement of the physicochemical properties of liposomes, such as surface charge, lipid composition, and functionalization, makes these vesicles eligible delivery nanosystems for the gene therapy of many pathological conditions. In the present study, pre-formulation analysis was conducted to develop liposomes that facilitate the delivery of nucleic acids to neuronal cells, with the aim of future delivery of a CRISPR/Cas9 system designed to silence genes responsible for autosomal dominant neurodegenerative disorders. To this aim, different nucleic acid cargo models, including λ phage DNA, plasmid DNA, and mRNA encoding GFP, were considered. Liposomes with varying lipid compositions were produced using the ethanol injection method and analyzed for their dimensional stability and ability to interact with DNA. The selected formulations were tested in vitro using a neuroblastoma cell line (SH-SY5Y) to evaluate their potential toxicity and the ability to transfect cells with a DNA encoding the green fluorescent protein (pCMV-GFP). Among all formulations, the one containing phosphatidylcholine, phosphatidylethanolamine, pegylated 1,2-distearoyl-sn-glycero-3-phosphethanolamine, cholesterol, and dioctadecyl-dimethyl ammonium chloride (in the molar ratio 1:2:4:2:2) demonstrated the highest efficiency in mRNA delivery. Although this study was designed with the goal of ultimately enabling the delivery of a CRISPR/Cas9 system for treating autosomal dominant neurodegenerative disorders such as polyglutamine spinocerebellar ataxias (SCAs), CRISPR/Cas9 components were not delivered in the present work, and their application remains the objective of future investigations. Full article

Tilapia Lake Virus (TiLV) is well known as a highly contagious viral infection in aquaculture, particularly affecting Tilapia worldwide. Until recently, various TiLV diagnostic methods have been used for rapid and accurate diagnostic procedures that are crucial for timely disease detection and reducing losses. In this study, we developed an alternative method for investigating TiLV diagnosis using Reverse Transcriptase Recombinase Polymerase Amplification (RT-RPA) assay combined with a lateral flow dipstick (LFD). The test was generated by specific anti-FITC and anti-Biotin capture antibodies that are compatible with the TiLV-specific primers tagged with FITC and Biotin. The test was conducted by the reverse transcriptase of target TiLV RNA and RPA amplification at 39 °C for 20 min. The products were then determined by a positive band signal via LFD. The RT-RPA-LFD assay detected the plasmid of TiLV (pTiLV) with a Limit of Detection (LOD) of 3.19 copies/µL, while the RT-PCR-LFD assay detected it with an LOD of 319 copies/µL. Our findings demonstrate that RT-RPA-LFD represents a possible alternative to RT-PCR for the rapid and sensitive detection of TiLV, especially in areas with limited infrastructure. 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

In molecular biology studies, suitable vectors are fundamental tools; however, most vectors can only express one target gene, which limits the ability to study multiple genes simultaneously within the same plant tissue. The traditional method for achieving multi-gene co-expression involves co-transferring multiple plasmids into plant tissues, but this approach is often inefficient due to the difficulty of successfully transforming multiple plasmids at once. To overcome this limitation, we have developed a series of vectors, called pMAGs (Multigene Assembly Genetic vectors), capable of simultaneously expressing or silencing two or three different genes in plants. These vectors not only provide an optimal solution for a wide range of biological experiments but also work effectively across numerous plant species. Full article

Plasmid DNA (pDNA) purification plays a key role in the development of vaccines and gene therapies. Affinity chromatography stands out as a promising method for plasmid purification, leveraging a range of biological and synthetic ligands to achieve selectivity. This study investigates the potential of a synthetic ligand library consisting of triazine-based bifunctional compounds designed to mimic the side chains of amino acids that are known to bind nucleic acids. A high-throughput screening method was employed to assess the binding ability of 158 ligands within the library to single-stranded, FITC-labeled homo-oligonucleotides (G and T), each comprising 20 nucleotides, under both hydrophilic and hydrophobic conditions. High-affinity ligands were identified for both T and G oligonucleotides. Follow-up microscale chromatographic screening uncovered some false positives from the initial FITC-based screening, narrowing the selection to 22 ligands for further investigation. In the next phase of the study, the binding affinity of these ligands towards double-stranded oligonucleotides (AT and CG) was assessed. Ligand 1/2, a mimic of Ala-Lys or Gly-Lys, and ligand 2/3, a mimic of Lys-Tyr, were chosen as initial candidates for evaluating plasmid DNA purification from an Escherichia coli crude extract. The results obtained with 0.4 M ammonium sulfate in 20 mM Tris-HCl (pH 8.0) as the binding buffer were similar to those observed when purifying plasmid DNA from E. coli clarified lysates by hydrophobic interaction chromatography. The affinity resins retained RNA, while the less hydrophobic plasmid DNA was excluded in the initial fractions. Future research will be directed towards exploring the potential of the most promising ligands to separate pDNA isoforms. Full article

SLC5A8 is a protein coded by the SLC5A8 gene, and has been proposed as a tumor suppressor and iodide transporter. Its expression is reduced in papillary thyroid carcinoma (PTC), yet the mechanisms underlying this phenomenon are largely unknown. We hypothesized that SLC5A8 expression in PTC is reduced by microRNAs and can be modulated by their inhibition. We used real-time PCR to analyze the expression of SLC5A8 and the microRNAs of interest in a set of 49 PTC/normal tissue pairs. We used an in silico approach to identify microRNAs upregulated in PTC and putatively binding to the SLC5A8 transcript. Luciferase assays were performed to confirm the direct binding of synthetic microRNAs to the 3′UTR of SLC5A8. Subsequently, using mir-expressing plasmids and microRNA sponges, including a microRNA sponge designed to simultaneously inhibit three selected microRNAs, we checked the impact of the modulation of microRNAs on endogenous SLC5A8. Finally, we investigated if modulation of SLC5A8 induces changes in transcriptomes. We confirmed the downregulation of SLC5A8 in PTC. In silico analysis revealed microRNAs potentially targeting SLC5A8. Luciferase assay confirmed direct binding between the 3′UTR of SLC5A8 and miR-181a-5p, miR-182-5p, and miR-494-3p. MiR-181a-5p and miR-182-5p were upregulated in PTC. In HEK293 cell lines, transfection with mir-181a- and mir-182-expressing plasmids decreased endogenous SLC5A8 mRNA, while silencing of miR-181a-5p, miR-182-5p, miR-494-3p, and all three microRNAs simultaneously increased SLC5A8 expression; however, only simultaneous inhibition was able to induce changes visible for SLC5A8 protein. Changes in SLC5A8 expression did not alter the whole transcriptome significantly. This study shows microRNA-dependent regulation of SLC5A8 expression and underlines the potential effectiveness of simultaneous inhibition of a few microRNAs to derepress their common target. Full article

Genomic islands (GIs) including integrative and conjugative elements (ICEs), prophages, and integrative plasmids are central drivers of horizontal gene transfer in bacterial plant pathogens. These elements often carry cargo genes encoding virulence factors, antibiotic and metal resistance determinants, and metabolic functions that enhance environmental adaptability. In plant-pathogenic species such as Pseudomonas syringae, GIs contribute to host specificity, immune evasion, and the emergence of novel pathogenic variants. ICEclc and its homologs represent integrative and mobilizable elements whose tightly regulated excision and transfer are driven by a specialized transcriptional cascade, while ICEs in P. syringae highlight the ecological impact of cargo genes on pathogen virulence and fitness. Pathogenicity islands further modulate virulence gene expression in response to in planta stimuli. Beyond P. syringae, GIs in genera such as Erwinia, Pectobacterium, and Ralstonia underpin critical traits like toxin biosynthesis, secretion system acquisition, and topoisomerase-mediated stability. Leveraging high-throughput genomics and structural biology will be essential to dissect GI regulation and develop targeted interventions to curb disease spread. This review synthesizes the current understanding of GIs in plant-pathogenic gammaproteobacteria and outlines future research priorities for translating mechanistic insights into sustainable disease control strategies. Full article

Background: Fowl typhoid (FT), a septicemic infection caused by Salmonella Gallinarum (SG), and H9N2 avian influenza are two economically important diseases that significantly affect the global poultry industry. Methods: We exploited the live attenuated Salmonella Gallinarum (SG) mutant JOL3062 (SG: ∆lon ∆pagL ∆asd) as a delivery system for H9N2 antigens to induce an immunoprotective response against both H9N2 and FT. To enhance immune protection against H9N2, a prokaryotic and eukaryotic dual expression plasmid, pJHL270, was employed. The hemagglutinin (HA) consensus sequence from South Korean avian influenza A virus (AIV) was cloned under the Ptrc promoter for prokaryotic expression, and the B cell epitope of neuraminidase (NA) linked with matrix protein 2 (M2e) was placed for eukaryotic expression. In vitro and in vivo expressions of the H9N2 antigens were validated by qRT-PCR and Western blot, respectively. Results: Oral immunization with JOL3121 induced a significant increase in SG and H9N2-specific serum IgY and cloacal swab IgA antibodies, confirming humoral and mucosal immune responses. Furthermore, FACS analysis showed increased CD4+ and CD8+ T cell populations. On day 28 post-immunization, there was a substantial rise in the hemagglutination inhibition titer in the immunized birds, demonstrating neutralization capabilities of immunization. Both IFN-γ and IL-4 demonstrated a significant increase, indicating a balance of Th1 and Th2 responses. Intranasal challenge with the H9N2 Y280 strain resulted in minimal to no clinical signs with significantly lower lung viral titer in the JOL3121 group. Upon SG wildtype challenge, the immunized birds in the JOL3121 group yielded 20% mortality, while 80% mortality was recorded in the PBS control group. Additionally, bacterial load in the spleen and liver was significantly lower in the immunized birds. Conclusions: The current vaccine model, designed with a host-specific pathogen, SG, delivers a robust immune boost that could enhance dual protection against FT and H9N2 infection, both being significant diseases in poultry, as well as ensure public health. Full article

Electroporation-mediated gene delivery is a cornerstone of synthetic biology, offering several advantages over other methods: higher efficiencies, broader applicability, and simpler sample preparation. Yet, electroporation protocols are often challenging to integrate into highly multiplexed workflows, owing to limitations in their scalability and tunability. These challenges ultimately increase the time and cost per transformation. As a result, rapidly screening genetic libraries, exploring combinatorial designs, or optimizing electroporation parameters requires extensive iterations, consuming large quantities of expensive custom-made DNA and cell lines or primary cells. To address these limitations, we have developed a High-Throughput Microfluidic Electroporation (HTME) platform that includes a 384-well electroporation plate (E-Plate) and control electronics capable of rapidly electroporating all wells in under a minute with individual control of each well. Fabricated using scalable and cost-effective printed-circuit-board (PCB) technology, the E-Plate significantly reduces consumable costs and reagent consumption by operating on nano to microliter volumes. Furthermore, individually addressable wells facilitate rapid exploration of large sets of experimental conditions to optimize electroporation for different cell types and plasmid concentrations/types. Use of the standard 384-well footprint makes the platform easily integrable into automated workflows, thereby enabling end-to-end automation. We demonstrate transformation of E. coli with pUC19 to validate the HTME’s core functionality, achieving at least a single colony forming unit in more than 99% of wells and confirming the platform’s ability to rapidly perform hundreds of electroporations with customizable conditions. This work highlights the HTME’s potential to significantly accelerate synthetic biology Design-Build-Test-Learn (DBTL) cycles by mitigating the transformation/transfection bottleneck. Full article

Background/Objective: The present study focused on the analysis of the Spanish clone belonging to the successful 4,[5],12:i:- monophasic variant of Salmonella enterica serovar Typhimurium. Methods: All isolates of the clone recovered in a Spanish region from human clinical samples between 2008 and 2018 (N = 14) were investigated using microbiological approaches and genome sequence analysis. In addition, they were compared with isolates from the years 2000 to 2003 (N = 21), which were previously characterized but had not yet been sequenced. Results: Phylogenetic analyses indicate that all isolates are closely related (differing by 1 to 103 SNPs) but belong to two clades termed A and B. With few exceptions, clade A comprised isolates of the first period, also including two “older” control strains, LSP 389/97 and LSP 272/98. Clade B only contained isolates from the second period. Isolates from both periods were resistant to antibiotics and biocides, with almost all resistance genes located on large IncC plasmids, additionally carrying pSLT-derived virulence genes. The number of resistance genes was highly variable, resulting in a total of 22 ABR (antibiotic biocide resistance) profiles. The number of antibiotic resistance genes, but not that of biocide resistance genes, was considerably lower in isolates from the second than from the first period (with averages of 5.5 versus 9.6 genes). Importantly, IS26, which resides in multiple copies within these plasmids, appears to be playing a crucial role in the evolution of resistance, and it was also responsible for the monophasic phenotype, which was associated with four different deletions eliminating the fljAB region. Conclusions: the observed reduction in the number of antibiotic resistance genes could correlate with the loss of adaptive advantage originating from the ban on the use of antibiotics as feed additives implemented in the European Union since 2006, facilitated by the intrinsic instability of the IncC plasmids. Two consecutive IS26 transposition events, which can explain both the clonal relationship of the isolates and their variability, may account for the observed fljAB deletions. Full article

Acinetobacter baumannii is listed by the World Health Organization as an emerging bacterial priority pathogen, the prevalence and multidrug resistance of which have been increasing. This functional genomics study aimed to understand the drug-resistance mechanisms of an extensively drug-resistant (XDR) A. baumannii strain (IRMCBCU95U) isolated from a transtracheal aspirate sample from a female patient with end-stage renal disease in Saudi Arabia. The whole genome of IRMCBCU95U (4.3 Mbp) was sequenced using Oxford Nanopore long-read sequencing to identify and compare the antibiotic-resistance profile and genomic features of A. baumannii IRMCBCU95U. The antibiogram of A. baumannii IRMCBCU95U revealed resistance to multiple antibiotics, including cefepime, ceftazidime, ciprofloxacin, imipenem, meropenem and piperacillin/tazobactam. A comparative genomic analysis between IRMCBCU95U and A. baumannii K09-14 and ATCC 19606 identified significant genetic heterogeneity and mosaicism among the strains. This analysis also demonstrated the hybrid nature of the genome of IRMCBCU95U and indicates that horizontal gene transfer may have occurred between these strains. The IRMCBCU95U genome has a diverse range of genes associated with antimicrobial resistance and mobile genetic elements (ISAba1 and IS26) associated with the spread of multidrug resistance. The presence of virulence-associated genes that are linked to iron acquisition, motility and transcriptional regulation confirmed that IRMCBCU95U is a priority human pathogen. The plasmid fragment IncFIB(pNDM-Mar) observed in the strain is homologous to the plasmid in Klebsiella pneumoniae (439 bp; similarity: 99.09%), which supports its antimicrobial resistance. From these observations, it can be concluded that the clinical A. baumannii IRMCBCU95U isolate is an emerging extensively drug-resistant human pathogen with a novel combination of resistance genes and a plasmid fragment. The complex resistome of IRMCBCU95U highlights the urgent need for genomic surveillance in hospital settings in Saudi Arabia to fight against the spread of extensively drug-resistant A. baumannii. Full article

In the present research, metal–organic framework-5 (MOF-5) was synthesized and loaded with zerumbone (ZER@MOF-5), followed by the evaluation of its anticancer, antibacterial, antifungal, DNA-binding, and free radical scavenging potentials. The synthesized nanoparticles were characterized using X-ray diffraction, ultraviolet–visible spectroscopy, Fourier-transform infrared spectroscopy, energy-dispersive X-ray spectroscopy, and scanning electron microscopy. The in vitro anticancer activity of ZER@MOF-5 was studied in a human breast cancer cell line (MCF-7) using the CCK-8 assay. The interaction of ZER@MOF-5 with pBR322 plasmid DNA was assessed by gel electrophoresis. The antimicrobial effect of ZER@MOF-5 was examined in gram-positive and gram-negative bacterial strains and yeast strains using the microdilution method. The free radical scavenging activity was assessed using the DPPH assay. Cytotoxicity assay revealed a notable enhancement in the anticancer activity of zerumbone upon its encapsulation into MOF-5. The IC₅₀ value for ZER@MOF-5 was found to be 57.33 µg/mL, which was lower than that of free zerumbone (IC₅₀: 89.58 µg/mL). The results of the DNA-binding experiment indicate that ZER@MOF-5 can bind to target DNA and cause a conformational change in DNA. The results of the antibacterial activity experiment showed that the antibacterial ability of ZER@MOF-5 was limited compared to free zerumbone. The results of the DPPH assay demonstrated that the antioxidant activity of free zerumbone was higher than that of ZER@MOF-5. MOFs encapsulate compounds within their porous crystalline structure, which leads to prolonged circulation time compared to single ligands. Although the unique structure of MOFs may limit their antibacterial and antioxidant activity in the short term, it may increase therapeutic efficacy in the long term. However, to fully understand the long-term antibacterial and antioxidant effects of the ZER@MOF-5, further comprehensive in vitro and in vivo experiments are necessary. This finding indicates that the MOF-5 could potentially be an impressive carrier for the oral administration of zerumbone. Full article

Background/Objectives: Clostridioides difficile is a “One Health” pathogen and a cause of antibiotics-associated diarrhea and pseudomembranous colitis. Mobile genetic elements (MGEs) have been documented in the genomes of clinical C. difficile strains; however, the presence of MGEs in environmental strains remains poorly characterized. Thus, the present study was conducted with the objective of identifying the prevalence of MGEs, including mobilizable transposons (MTns), conjugative transposons (CTns), plasmids, and insertion sequences, in whole genome sequences (WGSs) of environmental C. difficile isolates. Methods: The analysis of MGEs was conducted using 166 WGSs obtained from C. difficile strains isolated from various environmental sources contaminated with feces. The MGEs were identified using bioinformatic tools. Results: A total of 48.2% (80/166) of the studied genomes were identified to harbor nine transposons, including Tn916, Tn6194-like, Tn5397, Tn6215, Tn4001, Tn6073, Tn6110, Tn6107, or Tn5801-like. The majority of MTns and CTns could be found within C. difficile sequence types ST11, ST3, and ST35. The results demonstrated close genetic relatedness among the studied genomes, the array of antimicrobial resistance (AMR) genes, such as tetM, ermB, and aac(6′)-aph(2″), and the presence of CTns. Furthermore, the analysis revealed that 24.7% (41/166) of the genome sequences of isolates were associated with various predominant plasmid groups, including pCD6, pCD-ECE4-6, pCD-WTSI1-4, pCDBI1, and pCd1_3, which belonged to 16 different sequence types. Furthermore, several plasmids were identified as harboring the prophage phiCDHM19. Conclusions: The results of the current study suggest that the identified plasmids are abundant and may encode functions that are relevant to C. difficile physiology. The genomes of C. difficile strains examined contain closely related CTns, suggesting that horizontal transfer of AMR is important in this species or other bacterial species. Further research is required to ascertain the effect of these genetic elements and their transferability on the biology of C. difficile. Full article

Most large-scale crude oil spills occur in marine environments. We screened easily propagable/maintainable halophiles to develop agents for the bioremediation of marine spills. A bacterial strain isolated from a polluted region of the Great Salt Lake was characterized and tested for its ability to degrade crude oil. The strain (Salinivibrio costicola) is motile, catalase- and lipase-positive, a facultative anaerobe, and an obligate halophile. Its growth optimum and tolerance ranges are: NaCl (5%, 1.25–10%), pH (8, 6–10), and temperature (22 °C, 4–45 °C). Its genome (3,166,267 bp) consists of two circular chromosomes and a plasmid, containing 3197 genes, including some genes potentially relevant to hydrocarbon metabolism. The strain forms a biofilm but is considered nonpathogenic and is sensitive to some common antibiotics. Lytic bacteriophages infecting the strain are rare in the water samples we tested. The strain survived on desiccated agar media at room temperature for a year, grew optimally in complex media containing 0.1–1% crude oil, but failed to reduce total recoverable petroleum hydrocarbons from crude oil. Thus, a recalcitrant halophile may endure crude oil without mineralizing. Due to some of their advantageous attributes, such strains can be considered for genetic manipulation to develop improved agents for bioremediation. Full article