Search Results (61)

Background: Salmonella is a bacterium that causes foodborne infections. This study characterized two strains isolated from cheese and beef in Brazil using whole-genome sequencing (WGS). Objectives: We evaluated their antimicrobial resistance profiles, virulence factors, plasmid content, serotypes and phylogenetic relationships. Methods: DNA was extracted and sequenced on the NovaSeq 6000 platform; the pangenome was assembled using the Roary tool; and the phylogenetic tree was constructed via IQ-TREE. Results and Discussion: For contextualization and comparison, 3493 Salmonella genomes of Brazilian origin from NCBI were analyzed. In our isolates, both strains carried the aac(6′)-Iaa_1 gene, while only Schwarzengrund harbored the qnrB19_1 gene and the Col440I_1 plasmid. Cerro presented the islands SPI-1, SPI-2, SPI-3, SPI-4, SPI-5 and SPI-9, while Schwarzengrund also possessed SPI-13 and SPI-14. Upon comparison with other Brazilian genomes, we observed that Cerro and Schwarzengrund represented only 0.40% and 2.03% of the national database, respectively. Furthermore, they revealed that Schwarzengrund presented higher levels of antimicrobial resistance, a finding supported by the higher frequency of plasmids in this serovar. Furthermore, national data corroborated our findings that SPI-13 and SPI-14 were absent in Cerro. A virulence analysis revealed distinct profiles: the cdtB and pltABC genes were present in the Schwarzengrund isolates, while the sseK and tldE1 family genes were exclusive to Cerro. The results indicated that the sequenced strains have pathogenic potential but exhibit low levels of antimicrobial resistance compared to national data. The greater diversity of SPIs in Schwarzengrund explains their prevalence and higher virulence potential. Conclusions: Finally, the serovars exhibit distinct virulence profiles, which results in different clinical outcomes. Full article

A backyard cat with symptoms of otitis was transferred to a veterinary clinic in Central Greece. A sample was obtained and P. aeruginosa was isolated. The strain exhibited an extensively drug-resistant (XDR) profile, as it was non-susceptible to all tested agents except colistin. DNA extraction and whole-genome sequencing (WGS) were performed using a robotic extractor and Ion Torrent technology, respectively. The genome was assembled and screened for resistance and virulence determinants. The isolate belonged to the high-risk clone ST308 with a total of 67 antibiotic resistance genes (ARGs) and 221 virulence factor-related genes being identified. No plasmids were detected. The metallo-beta-lactamase (MBL) bla_NDM-1 gene and 46 efflux pumps were included in the strain’s resistome. Both ARGs conferring tolerance to disinfecting agents and biofilm-related genes were identified, associated with the ability of this clone to adapt and persist in healthcare facilities. This case highlights the risk of relevant bacterial clones spreading in the community and even being transmitted to companion animals, causing challenging opportunistic infections to susceptible individuals, while others may become carriers, further spreading the clones to their owners, other animals and the environment. Full article

Brucella intermedia, a gram-negative, non-lactose-fermenting, aerobic, rod-shaped bacterium, is found in environmental sources (e.g., soil and water). In 2020, Ochrobactrum was reclassified as Brucella. We conducted a genomic analysis of B. intermedia from hospital wastewater samples in western Ghana. A hybrid genome assembly was constructed integrating short-read data from DNA Nanoball sequencing with long-read sequences generated by Oxford Nanopore MinION technology. Identification and antimicrobial susceptibility profiles were determined using MicroScan autoSCAN-4 based on Clinical and Laboratory Standard Institute documents. ResFinder and CARD Resistance Gene Identifier (RGI) were used to identify antimicrobial resistance (AMR) genes, and BLAST and VFDB datasets were used to identify virulence factor genes. The complete genome had two chromosomes, no plasmid, and a high average nucleotide identity value (98.05%) with B. intermedia. Resistance to trimethoprim-sulfamethoxazole was revealed, the first report in this species. CARD RGI revealed the presence of AMR genes, including ANT(9)-Ic and adeF. Local BLAST analysis revealed Cgs, a B. melitensis virulence factor. B. intermedia is an opportunistic human pathogen clinically isolated several times, suggesting the importance of accurately identifying multidrug resistance. B. intermedia may possess virulence factors similar to those of B. melitensis. Further study is needed to fully elucidate its pathogenesis. Full article

Antibiotic resistance to Klebsiella pneumoniae poses a major public health threat, particularly in intensive care unit (ICU) settings. The emergence of extensively drug-resistant (XDR) strains complicates treatment options, requiring a deeper understanding of their genetic makeup and potential therapeutic targets. This research delineated an extensively drug-resistant (XDR) Klebsiella pneumoniae strain obtained from an ICU patient and telomeric temperate phage derived from hospital effluent. The bacteria showed strong resistance to multiple antibiotics, including penicillin (≥16 μg/mL), ceftriaxone (≥32 μg/mL), and meropenem (≥8 μg/mL), which was caused by SHV-11 beta-lactamase, NDM-1 carbapenemase, and porin mutations (OmpK37, MdtQ). The strain was categorized as K46 and O2a types and carried virulence genes involved in iron acquisition, adhesion, and immune evasion, as well as plasmids (IncHI1B_1_pNDM-MAR, IncFIB) and eleven prophage regions, reflecting its genetic adaptability and resistance dissemination. The 172,025 bp linear genome and 46.3% GC content of the N-15-like phage showed strong genomic similarities to phages of the Sugarlandvirus genus, especially those that infect K. pneumoniae. There were structural proteins (11.8%), DNA replication and repair enzymes (9.3%), and a toxin–antitoxin system (0.4%) encoded by the phage genome. A protelomerase and ParA/B partitioning proteins indicate that the phage is replicating and maintaining itself in a manner similar to the N15 phage, which is renowned for maintaining a linear plasmid prophage throughout lysogeny. Understanding the dynamics of antibiotic resistance and pathogen development requires knowledge of phages like this one, which are known for their temperate nature and their function in altering bacterial virulence and resistance profiles. The regulatory and structural proteins of the phage also provide a model for research into the biology of temperate phages and their effects on microbial communities. The importance of temperate phages in bacterial genomes and their function in the larger framework of microbial ecology and evolution is emphasized in this research. Full article

The isolation and characterization of novel probiotics from dairy products, fermented foods, and the gut have gained significant attention. In particular, Streptococcus salivarius shows promise for use in oral probiotic preparations. In this study, we isolated two strains of S. salivarius—S.82.15 and S.82.20—from the oral cavity of a healthy individual. These strains exhibited distinct antimicrobial profiles. We thoroughly assessed the morphology and growth patterns of both strains and confirmed auto-aggregation and hemolytic activity. Through comprehensive genomic analysis, we found notable strain differences within the same bacterial species isolated from the same individual. Notably, the presence or absence of plasmids varied between the two strains. The genome of S.82.15 spans 2,175,688 bps and contains 1994 coding DNA sequences (CDSs), while S.82.20 has a genome size of 2,414,610 bps, a GC content of 40.62%, and 2276 annotated CDSs. Both strains demonstrated antibacterial activity against Group A Streptococcus (GAS), Micrococcus. luteus, and Porphyromonas gingivalis. To investigate the antibacterial properties further, we identified a gene cluster of salivaricin 9 on the plasmid of S.82.20 and a blp gene family on the chromosomes of both S.82.15 and S.82.20. Moreover, the gene expression of the blp family was upregulated when the isolated strains were co-cultured with GAS. Full article

Due to the barriers imposed by the restriction–modification (RM) system, Nisin-producing industrial strains of Lactococcus lactis often encounter low transformation efficiency, which seriously hinders the widespread application of genetic engineering in non-model L. lactis. Herein, we present a novel pre-modification strategy (PMS) coupled with optimized plasmid delivery systems designed to systematically evade RM barriers and substantially improve Nisin biosynthesis in L. lactis. Through the use of engineered Escherichia coli strains with methylation profiles specifically optimized for L. lactis C20, we have effectively evaded RM barriers, thereby facilitating the efficient introduction of large Nisin biosynthetic gene clusters into L. lactis. The PMS tools, which significantly improve the transformation efficiency (~10³ transformants per microgram of DNA), have been further improved in combination with a Rolling Circle Amplification, resulting in a higher enhancement in transformation efficiency (~10⁴ transformants per microgram of DNA). Using this strategy, large Nisin biosynthetic gene clusters and the expression regulation of all genes within the cluster were introduced and analyzed in L. lactis, leading to a highest Nisin titer of 11,052.9 IU/mL through a fed-batch fermentation in a 5 L bioreactor. This is the first systematic report on the expression regulation and application of a complete Nisin biosynthesis gene cluster in L. lactis. Taken together, our studies provide a versatile and efficient strategy for systematic evasion and enhancement of RM barriers and Nisin biosynthesis, thereby paving the way for genetic modification and metabolic engineering in L. lactis. Full article

Ciprofloxacin (CPL) is an effective antibiotic against Pseudomonas aeruginosa. However, its use is limited by the emergence of multi-resistant strains. In this study, 8–15 nm manganese ferrite (MnFe₂O₄) nanoparticles, aminated and/or PEGylated, have been used as drug-delivery systems of CPL. The magnetic nanoparticles (MNPs) were prepared in the presence of the aliphatic amines octadecylamine (ODA), oleylamine (OAm), or PEG8000 to achieve the appropriate surface chemistry for the direct conjugation of CPL and drug loading into the PEG matrix, respectively. The primary MNPs proved to be biocompatible in calf thymus (CT)-DNA interaction studies, with binding constant values K_b in the range of 4.43–6.5 × 10⁴ (g/mL)⁻¹. ODA as a coater gave rise to MnFe₂O₄ MNPs, with a high percentage of free amines that further allowed for the conjugation of 90.9% CPL, which gradually released via a non-Fickian anomalous transport motif. The 25.1% CPL that loaded in the PEGylated MNPs led to a partial transformation of the nanoflowers into more aggregated forms. The release profile, although steeper, is described by the same model. The isolated magnetic nanocarrier with a high content of CPL was evaluated for its antimicrobial activity against a multi-resistant strain of P. aeruginosa using an automated industrial instrument (BacT/ALERT^®3D), and its molecular profile was outlined by studying its interaction with plasmid DNA (pDNA). The prototype use of BacT/ALERT^®3D allows for the simultaneous screening of multiple samples, while it foreshadows the transition to a preclinical phase. Full article

A novel Gram-negative, orange-colored, rod-shaped, oxidase and catalase-positive, non-spore-forming bacterium, designated as zp-37^T, was isolated from the rhizosphere soil of Phragmites australis (Cav.) Trin. ex Steud in Kashgar County, Xinjiang, China. The phylogenetic analysis, based on the 16S rRNA genes, revealed that strain zp-37^T belongs to the genus Halomonas. Growth of strain zp-37^T was observed at 10–43 °C, pH 6.0–11.0, and 0–20% NaCl (w/v). The principal fatty acids of strain zp-37^T were summed feature 8 (C_18:1ω7c and/or C_18:1ω6c, 55.67%) and summed feature 3 (C_16:1ω7c and/or C_16:1ω6c, 20.16%). The polar lipid profile contained diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), unidentified phospholipids (UPL 1–3), unidentified aminophospholipids (UAPL 1–2), and unidentified lipid (UL). Its main respiratory quinone was ubiquinone Q-9 (100%). The genome of strain zp-37^T was 3,489,967 bp in size, containing two plasmids with lengths of 18,112 bp and 4364 bp, respectively. The genomic DNA G+C content of strain zp-37^T was 59.3%. By the genome annotation, various genes related to the function of saline-alkaline stress tolerance and plant growth promotion were predicted. The average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values between strain zp-37^T and its five closely related strains were 72.64–75.59% and 19.70–20.40%, respectively, which were lower than the threshold for species delineation (ANI: 95–96%, dDDH: 70%). Based on the phylogenetic, phenotypic, and chemotaxonomic analyses and genomic comparisons, strain zp-37^T was suggested to represent a novel species within the genus Halomonas, for which the name Halomonas kashgarensis sp. nov. is proposed. The strain type was designated zp-37^T (=CGMCC 1.62213^T = JCM 37305^T). Full article

Snakebite is a critical global public health issue, causing substantial mortality and morbidity, particularly in tropical and subtropical regions. The development of innovative antivenoms targeting snake venom toxins is therefore of paramount importance. In this study, we adopted an epitope-directed approach to design three degenerate 15-mer peptides based on amino acid sequence alignments of snake venom phospholipase A₂s (PLA₂s) and snake venom serine proteases (SVSPs) from snake (Crotalus atrox). By leveraging their immunogenic and inhibitory profiles, these peptides were specifically designed to target the Asp49 and Lys49 variants of PLA₂ and SVSP toxins. Groups of five mice were immunized with each peptide, and IgG mRNA was subsequently extracted from peripheral blood mononuclear cells (PBMCs) and spleen lymphocytes of the top three responders. The extracted mRNA was reverse-transcribed into complementary DNA (cDNA), and the variable regions of the IgG heavy and kappa chains were amplified using polymerase chain reaction (PCR). These amplified regions were then linked with a 66-nucleotide spacer to construct single-chain variable fragments (scFvs). Sequence analysis of 48 randomly selected plasmids from each PLA₂ and SVSP scFv library revealed that over 80% contained scFv sequences with notable diversity observed in the complementarity-determining regions (CDRs), particularly CDR3. Enzyme-linked immunosorbent assay (ELISA) results demonstrated that the SP peptide elicited a broader immune response in mice compared to the Asp49 peptide, implying the strong immunogenicity of the SP peptide. These scFvs represent a promising foundation for the development of recombinant human monoclonal antibodies targeting snake PLA₂ and SVSP toxins, providing a potential therapeutic strategy for the treatment of snakebites. Full article

Background/Objectives: The nucleic acid-based product (NAP) portfolio is expanding continuously and provides safer curative options for many disease indications. Nucleic acid-based products offer several advantages compared to proteins and virus-based products. They represent an emerging field; thus, their quality control and regulatory landscape is evolving to ensure adequate quality and safety. Next-Generation Sequencing (NGS) is mostly recommended for NAP identity testing, and we are leveraging its application for impurity profiling. Methods: We proposed a workflow for the purity assessment of NAPs through short-read Illumina NGS followed by data analysis of mRNA vaccine and pDNA samples. We determined the sequence identity, DNA and RNA contamination, off-target RNA contamination, and poly-A count with the proposed workflow. Results: Our workflow predicted most of the critical quality controls of mRNA vaccine and plasmid DNA samples, especially focusing on the identity and the nucleotide-based impurities. Additionally, NGS data interpretation also assisted in strategic decisions for NAP manufacturing process optimizations. Conclusions: We recommend the adaptation of incremental NGS data by regulatory agencies to identify nucleotide-based impurities in NAPs. Perhaps NGS adaptation under cGMP compliance needs to be deliberated with the regulatory bodies, especially focusing on the methods qualification and validation part, starting from the sample collection, NGS library preparation, NGS run, and its data analysis pipeline. Full article

The negative ecological impact of industrialization, which involves the use of petroleum products and dyes in the environment, has prompted research into effective, sustainable, and economically beneficial green technologies. For green remediation primarily based on active microbial metabolites, these microbes are typically from relevant sources. Active microbial metabolite production and genetic systems involved in xenobiotic degradation provide these microbes with the advantage of survival and proliferation in polluted ecological niches. In this study, we evaluated the ability of wheat root-associated L. fusiformis MGMM7 to degrade xenobiotic contaminants such as crude oil, phenol, and azo dyes. We sequenced the whole genome of MGMM7 and provided insights into the genomic structure of related strains isolated from contaminated sources. The results revealed that influenced by its isolation source, L. fusiformis MGMM7 demonstrated remediation and plant growth-promoting abilities in soil polluted with crude oil. Lysinibacillus fusiformis MGMM7 degraded up to 44.55 ± 5.47% crude oil and reduced its toxicity in contaminated soil experiments with garden cress (Lepidium sativum L.). Additionally, L. fusiformis MGMM7 demonstrated a significant ability to degrade Congo Red azo dye (200 mg/L), reducing its concentration by over 60% under both static and shaking cultivation conditions. However, the highest degradation efficiency was observed under shaking conditions. Genomic comparison among L. fusiformis strains revealed almost identical genomic profiles associated with xenobiotic assimilation. Genomic relatedness using Average Nucleotide Identity (ANI) and digital DNA–DNA hybridization (DDH) revealed that MGMM7 is distantly related to TZA38, Cu-15, and HJ.T1. Furthermore, subsystem distribution and pangenome analysis emphasized the distinctive features of MGMM7, including functional genes in its chromosome and plasmid, as well as the presence of unique genes involved in PAH assimilation, such as phnC/T/E, which is involved in phosphonate biodegradation, and nemA, which is involved in benzoate degradation and reductive degradation of N-ethylmaleimide. These findings highlight the potential properties of petroleum-degrading microorganisms isolated from non-contaminated rhizospheres and offer genomic insights into their functional diversity for xenobiotic remediation. Full article

Background/Objectives: Hemophilia B is a hereditary bleeding disorder due to the production of liver malfunctional factor IX (FIX). Gene therapy with viral vectors offers a cure. However, applications are limited due to pre-existing antibodies, eligibility for children under 12 years of age, hepatotoxicity, and excessive costs. Lipid nanoparticles are a potential alternative owing to their biocompatibility, scalability, and non-immunogenicity. However, their therapeutic applications are still elusive due to the poor transfection efficiencies in delivering plasmid DNA into primary cells and target organs in vivo. To develop efficient liver-targeted lipid nanoparticles, we explored galactosylated lipids to target asialoglycoprotein receptors (ASGPRs) abundantly expressed on hepatocytes. Methods: We developed 12 novel liposomal formulations varying the galactose lipid Gal-LNC 5, cationic lipid MeOH16, DOPE, and cholesterol. We evaluated their physicochemical properties, toxicity profiles, and transfection efficiencies in hepatic cell lines. Among the formulations, Gal-LNC 5 could efficiently transfect the reporter plasmid eGFP in hepatic cell lines and specifically distribute into the liver in vivo. Toward developing functional factor IX, we cloned Padua mutant FIX-L in a CpG-free backbone to enhance the expression and duration. Results: We demonstrated superior expression of FIX with our galactosylated lipid nanoparticle system. Conclusions: The current research presents a specialized lipid nanoparticle system viz. Gal-LNC which is a specialized lipid nanoparticle system for liver-targeted gene therapy in hemophilia B patients that has potential for clinical use. The Gal-LNC successfully delivers a CpG-free Padua FIX gene to liver cells, producing therapeutically relevant levels of FIX protein. Among its benefits are the ideal qualities of stability, targeting the liver specifically, and maximizing efficiency of transfection. Optimization of liver-targeting lipid nanoparticle systems and function FIX plasmids will pave the way for novel lipid nanoparticle-based gene therapy products for hemophilia B and other monogenic liver disorders. Full article

The core clock gene Period2 (PER2) is associated with mammary gland development and lipid synthesis in rodents and has recently been found to have a diurnal variation in the process of lactation, but has not yet been demonstrated in bovine mammary epithelial cells (BMECs). To explore the regulatory function of PER2 on milk fat synthesis in bovine mammary epithelial cells, we initially assessed the expression of clock genes and milk fat metabolism genes for 24 h using real-time quantitative PCR and fitted the data to a cosine function curve. Subsequently, we overexpressed the PER2 in BMECs using plasmid vector (pcDNA3.1-PER2), with empty vector pcDNA3.1-myc as the control. After transfecting BMECs for 48 h, we assessed the protein abundance related to milk fat synthesis by Western blot, the expression of genes coding for these proteins using real time-quantitative PCR, the production of triacylglycerol, and the fatty acid profile. The findings indicated that a total of nine clock genes (PER1/2, CRY1/2, REV-ERBα, BMAL1, NCOR1, NR2F2, FBXW11), seven fatty acid metabolism genes (CD36, ACSS2, ACACA, SCD, FADS1, DGAT1, ADFP), and six nuclear receptor-related genes (INSIG1, SCAP, SREBF1, C/EBP, PPARG, LXR) exhibited oscillation with a period close to 24 h in non-transfected BMECs (R² ≥ 0.7). Compared to the control group (transfected with empty pcDNA3.1-myc), the triglyceride content significantly increased in the PER2 overexpression group (p < 0.05). The lipogenic genes for fatty acid transport and triglyceride synthesis (ACACA, SCD, LPIN1, DGAT1, and SREBF1) were upregulated after PER2 overexpression, along with the upregulation of related protein abundance (p < 0.05). The contents and ratios of palmitic acid (C16:0), oleic acid (C18:1n9c), and trans-oleic acid (C18:1n9t) were significantly increased in the overexpression group (p < 0.05). Overall, the data supported that PER2 participated in the process of milk fat metabolism and is potentially involved in the de novo synthesis and desaturation of fatty acid in bovine mammary epithelial cells. Full article

Morbillivirus canis (canine distemper virus (CDV)) is recognized as a multihost pathogen responsible for a transmissible disease affecting both domestic and wild animals. A considerable portion of wildlife populations remain unvaccinated due to a lack of safety and immunogenicity data on existing vaccines for the prevention of CDV infection in these species. This review aimed to assess the current state of CDV vaccination research for both domestic and wild animals and to explore novel vaccine candidates through in vivo studies. It also sought to synthesize the scattered information from the extensive scientific literature on CDV vaccine research, identify key researchers in the field, and highlight areas where research on CDV vaccination is lacking. A scoping review was conducted across four databases following the PRISMA-ScR protocol, with information analyzed using absolute and relative frequencies and 95% confidence intervals (CIs) for study number proportions. Among the 2321 articles retrieved, 68 met the inclusion criteria and focused on CDV vaccines in various animal species, such as dogs, ferrets, minks, and mice. Most of the scientific community involved in this research was in the USA, Canada, France, and Denmark. Various vaccine types, including MLV CDV, recombinant virus, DNA plasmids, inactivated CDV, and MLV measles virus (MeV), were identified, along with diverse immunization routes and schedules employed in experimental and commercial vaccines. Safety and efficacy data were summarized. Notably, 37 studies reported postimmunization CDV challenge, primarily in dogs, revealing the survival rates of vaccinated animals. In summary, CDV vaccines generally demonstrate an acceptable safety profile in dogs and show promise as a means of controlling CDV. However, significant gaps in vaccine research persist, particularly concerning wildlife reservoirs, indicating the need for further investigation. Full article

Therapeutic HPV vaccines that induce potent HPV-specific cellular immunity and eliminate pre-existing infections remain elusive. Among various candidates under development, those based on DNA constructs are considered promising because of their safety profile, stability, and efficacy. However, the use of electroporation (EP) as a main delivery method for such vaccines is notorious for adverse effects like pain and potentially irreversible muscle damage. Moreover, the requirement for specialized equipment adds to the complexity and cost of clinical applications. As an alternative to EP, lipid nanoparticles (LNPs) that are already commercially available for delivering mRNA and siRNA vaccines are likely to be feasible. Here, we have compared three intramuscular delivery systems in a preclinical setting. In terms of HPV-specific cellular immune responses, mice receiving therapeutic HPV DNA vaccines encapsulated with LNP demonstrated superior outcomes when compared to EP administration, while the naked plasmid vaccine showed negligible responses, as expected. In addition, SM-102 LNP M exhibited the most promising results in delivering candidate DNA vaccines. Thus, LNP proves to be a feasible delivery method in vivo, offering improved immunogenicity over traditional approaches. Full article