Search Results (61)

Biocontainment laboratories often have limited access to a range of instruments required for conducting standard assays on infected materials. Consequently, some of the protocols involving infected samples are conducted outside a biocontainment facility. To be compliant with regulatory requirements and minimize health and safety risks for scientific personnel, it is imperative to test procedures rigorously for safely removing infected samples from biocontainment areas. This study validated the chemical inactivation of Nipah virus (NiV), a representative member of the Henipavirus genus, in animal tissues and serum. Importantly, this work demonstrated successful NiV-spiking of non-human primate (NHP) tissues and their subsequent inactivation. This is important because NHP tissues contain unpredictable amounts of infectious virus. The primary objective was to establish standardized protocols that are compliant with regulations to permit safe retrieval of infected biological samples with high NiV infectious virus content from ABSL-4 laboratories for subsequent downstream processing under lower biocontainment conditions. Full article

Toxin–antitoxin (TA) modules represent sophisticated regulatory networks that have evolved from simple plasmid maintenance factors into multifunctional genetic modules orchestrating bacterial stress responses, pathogenesis, and ecological adaptation. This review highlights a compelling correlation between the abundance of toxin–antitoxin (TA) modules and bacterial pathogenicity, as exemplified by Mycobacterium tuberculosis (M.tb), which encodes 118 TA loci—significantly more than the fewer than 10 found in closely related saprophytic species. The clinical significance of TA modules extends beyond traditional stress response roles to encompass antimicrobial persistence, where systems like VapBC and MazEF facilitate dormant subpopulations that survive antibiotic therapy while maintaining chronic infections. Recent discoveries have revealed TA modules as sophisticated bacterial defense mechanisms against bacteriophage infection, with DarTG and ToxIN systems representing novel antiviral immunity components that complement CRISPR-Cas and restriction–modification systems. The immunomodulatory capacity of TA modules demonstrates their role in host–pathogen interactions, where systems such as VapC12 in M.tb promote macrophage polarization toward permissive M2 phenotypes while inducing anti-inflammatory cytokine production. Large-scale genomic analyses reveal that TA modules function as drivers of horizontal gene transfer networks, with their signatures enabling accurate prediction of plasmid community membership and serving as determinants of microbial community structure. The biotechnological applications of TA modules have expanded to include genetic circuit stabilization, biocontainment device construction, and multi-species microbial community engineering, while therapeutic strategies focus on developing multi-target inhibitors against conserved TA protein families as promising approaches for combating drug-resistant bacterial infections. The evolutionary conservation of TA modules across diverse bacterial lineages underscores their fundamental importance as central organizing principles in bacterial adaptation strategies, where their multifunctional nature reflects complex selective pressures operating across environmental niches and host-associated ecosystems. This review provides an integrated perspective on TA modules as dynamic regulatory elements that support bacterial persistence, immune evasion, and ecological versatility, establishing them as genetic elements with truly “many faces and functions” in prokaryotic biology. Full article

The MTBseq pipeline, published in 2018, was designed to address bioinformatics challenges in tuberculosis (TB) research using whole-genome sequencing (WGS) data. It was the first publicly available tool on GitHub to perform full analysis of WGS data for Mycobacterium tuberculosis complex (MTBC) encompassing quality control through mapping, variant calling for lineage classification, drug resistance prediction, and phylogenetic inference. However, the pipeline’s architecture is not optimal for analyses on high-performance computing or cloud computing environments that often involve large datasets. To overcome this limitation, we developed MTBseq-nf, a Nextflow wrapper that provides parallelization for faster execution speeds in addition to several other significant enhancements. The MTBseq-nf wrapper can run several instances of the same step in parallel, fully utilizing the available resources, unlike the linear, batched analysis of samples in the TBfull step of the MTBseq pipeline. For evaluation of scalability and reproducibility, we used 90 M. tuberculosis genomes (European Nucleotide Archive—ENA accession PRJEB7727) for the benchmarking analysis on a dedicated computational server. In our benchmarks, MTBseq-nf in its parallel mode is at least twice as fast as the standard MTBseq pipeline for cohorts exceeding 20 samples. Through integration with the best practices of nf-core, Bioconda, and Biocontainers projects MTBseq-nf ensures reproducibility and platform independence, providing a scalable and efficient solution for TB genomic surveillance. Full article

Precise control of protein translocation is essential for synthetic biology and protein engineering. Here, we present a temperature-responsive system using elastin-like polypeptides (ELPs) to regulate the translocation of a conditionally lethal enzyme in Escherichia coli. The enzyme, levansucrase, whose activity becomes lethal in the presence of sucrose, was engineered with an N-terminal signal peptide and a C-terminal ELP tag. At 37 °C, the ELP tag induced intracellular aggregation of the fusion protein, preventing its secretion and allowing cell survival, as indicated by translucent colony formation. In contrast, at 16 °C, the ELP remained soluble, permitting levansucrase secretion into the medium. The resulting conversion of sucrose into levan by the secreted enzyme led to host cell death. These findings highlight ELP-mediated aggregation as a reversible and tunable strategy for regulating protein localization and secretion in E. coli, with potential applications in synthetic biology, metabolic engineering, and biocontainment systems. Full article

Background/Objectives: Typically, studies aiming to assess the ability of canine parvovirus (CPV) vaccines to immunise puppies with maternally derived antibody (MDA) are undertaken using group-housed puppies. Since live attenuated vaccine virus is invariably shed in the faeces, this can result in repeated oral re-exposure and puppies which failed to respond to the initial vaccination may respond instead to shed vaccine virus in the environment, thus artificially enhancing the efficacy of the vaccine. This problem can be avoided by adopting a pair-housed study design where one vaccinated pup is housed with one unvaccinated sentinel. Using this design, we examine the capability of four commercially available canine parvovirus vaccines to immunise MDA-positive pups. Methods: Thirty-four 6-week-old puppies born to vaccinated dams were divided into four vaccine groups with similar MDA ranges. Within each group puppies were paired based on matching MDA titres, and each pair was housed in separate biocontainment accommodation. In each pair, the pup with the highest MDA was vaccinated and the other left as an unvaccinated sentinel. All vaccinates were given a single dose of one of the vaccines. Vaccinates and sentinels were then bled every 2–4 days and CPV antibody was measured. Daily rectal swabs were also collected from all pups to identify any shed vaccinal CPV. Results: All the pups vaccinated with Nobivac DP PLUS seroconverted, with significantly higher antibody titres compared to the pups in other vaccine groups, all shed vaccine virus, and all bar one of the sentinel pups seroconverted. In the other groups, only vaccinated pups with lower levels of MDA seroconverted and shed vaccine virus but none of the sentinel pups seroconverted. Conclusions: Different canine parvovirus vaccines differ in their ability to replicate in and immunise puppies with MDA, the levels of which may vary widely between individuals. The shedding of vaccinal CPV is an important consideration when designing studies to demonstrate efficacy in MDA-positive puppies. Full article

Bacteriophages (phages), the most abundant biological entities on Earth, have long served as both model systems and therapeutic tools. Recent advances in synthetic biology and genetic engineering have revolutionized the capacity to tailor phages with enhanced functionality beyond their natural capabilities. This review outlines the current landscape of synthetic and functional engineering of phages, encompassing both in-vivo and in-vitro strategies. We describe in-vivo approaches such as phage recombineering systems, CRISPR-Cas-assisted editing, and bacterial retron-based methods, as well as synthetic assembly platforms including yeast-based artificial chromosomes, Gibson, Golden Gate, and iPac assemblies. In addition, we explore in-vitro rebooting using TXTL (transcription–translation) systems, which offer a flexible alternative to cell-based rebooting but are less effective for large genomes or structurally complex phages. Special focus is given to the design of customized phages for targeted applications, including host range expansion via receptor-binding protein modifications, delivery of antimicrobial proteins or CRISPR payloads, and the construction of biocontained, non-replicative capsid systems for safe clinical use. Through illustrative examples, we highlight how these technologies enable the transformation of phages into programmable bactericidal agents, precision diagnostic tools, and drug delivery vehicles. Together, these advances establish a powerful foundation for next-generation antimicrobial platforms and synthetic microbiology. Full article

Auxotrophic strains of Agrobacterium tumefaciens have been developed to address the Agrobacterium overgrowth issue in plant genetic transformation; however, their application remains limited. Here, we generated novel histidine and leucine auxotrophic strains of A. tumefaciens EHA105, namely EHA105hisD− and EHA105leuA−, as well as a dual auxotrophic strain EHA105hisD−leuA−, through gene deletion. The transient expression efficiency and survival rate of these three auxotrophic strains in Nicotiana benthamiana were significantly impaired but could be restored to wild-type EHA105 levels by supplementation with appropriate concentrations of the corresponding amino acids (CAAs). The use of these three auxotrophic strains for the genetic transformation of N. benthamiana resulted in a significant reduction in Agrobacterium overgrowth and achieved transformation efficiency comparable to wild-type EHA105, when appropriate exogenous concentrations of the CAAs were supplied during the co-cultivation stage. Furthermore, through incubation experiments on various plants and soil, it was confirmed that the incidence of surviving cells from these three auxotrophic strains was much lower than that observed for the wild-type EHA105. In summary, this study reports on the characteristics of the novel auxotrophic strains of A. tumefaciens along with the effective use of such auxotrophic A. tumefaciens strains in plant genetic transformation. Full article

Microbial engineering has made a significant breakthrough in pharmaceutical biotechnology, greatly expanding the production of biologically active compounds, therapeutic proteins, and novel drug candidates. Recent advancements in genetic engineering, synthetic biology, and adaptive evolution have contributed to the optimization of microbial strains for pharmaceutical applications, playing a crucial role in enhancing their productivity and stability. The CRISPR-Cas system is widely utilized as a precise genome modification tool, enabling the enhancement of metabolite biosynthesis and the activation of synthetic biological pathways. Additionally, synthetic biology approaches allow for the targeted design of microorganisms with improved metabolic efficiency and therapeutic potential, thereby accelerating the development of new pharmaceutical products. The integration of artificial intelligence (AI) and machine learning (ML) plays a vital role in further advancing microbial engineering by predicting metabolic network interactions, optimizing bioprocesses, and accelerating the drug discovery process. However, challenges such as the efficient optimization of metabolic pathways, ensuring sustainable industrial-scale production, and meeting international regulatory requirements remain critical barriers in the field. Furthermore, to mitigate potential risks, it is essential to develop stringent biocontainment strategies and implement appropriate regulatory oversight. This review comprehensively examines recent innovations in microbial engineering, analyzing key technological advancements, regulatory challenges, and future development perspectives. Full article

Cancer vaccines, aimed at evolving the human immune system to eliminate tumor cells, have long been explored as a method of cancer treatment with significant clinical potential. Traditional delivery systems face significant challenges in directly targeting tumor cells and delivering adequate amounts of antigen due to the hostile tumor microenvironment. Emerging evidence suggests that certain bacteria naturally home in on tumors and modulate antitumor immunity, making bacterial vectors a promising vehicle for precision cancer vaccines. Live bacterial vehicles offer several advantages, including tumor colonization, precise drug delivery, and immune stimulation, making them a compelling option for cancer immunotherapy. In this review, we explore the mechanisms of action behind living bacteria-based vaccines, recent progress in popular bacterial chassis, and strategies for specific payload delivery and biocontainment to ensure safety. These approaches will lay the foundation for developing an affordable, widely applicable cancer vaccine delivery system. This review also discusses the challenges and future opportunities in harnessing bacterial-based vaccines for enhanced therapeutic outcomes in cancer treatment. Full article

Salmonella is diffused worldwide, and Salmonella enterica subsp. enterica is spread worldwide with many serovars associated with the infection of domestic bovines. The most spread are S. Dublin, S. Typhimurium and S. Infantis. S. Napoli is, however very rarely reported in domestic ruminants. Here, we report an outbreak of S. Napoli on a dairy cow farm in Northern Italy (Piacenza). A total of 18 S. Napoli isolates were recovered from aborted fetuses, feces, tissues and environmental samples. Whole genome sequencing suggested that all isolates belonged to the same cluster. After the application of stringent biocontainment and biosecurity measures, no further cases were reported. However, four months after the first case, the serovar was still isolated in environmental samples, underlying the importance of adopting the correct biosecurity and biocontainment measures in order to prevent the circulation and transmission of Salmonella within the farm. Full article

The prevention of disease introduction into swine herds requires the practice of science-based protocols of biosecurity that have been validated to reduce the risk of the entry of targeted pathogens. The fundamental pillars of biosecurity include bio-exclusion, biocontainment, and bio-management. Biosecurity protocols must be science-based, a way of life, continuously validated, cost-effective, and benchmarked over time. This paper will review these concepts, the direct and indirect routes of transmission of porcine reproductive and respiratory syndrome virus (PRRSV), and the interventions that have been designed and validated to prevent infection of the breeding herd. It will close with a review of Next Generation Biosecurity, describing how a science-based approach is being used to prevent PRRSV infection in breeding herds from a large commercial pork production system in the US. Full article

The broad range of applications offered by synthetic biology and bioengineering has revolutionized the ability to design and redesign microorganisms to express specific functions, overcoming the limitations of natural biological systems. This advancement has been achieved through the use of mathematical models and genetic circuits, enabling the precise design of synthetic microbial communities. These are defined as artificially created communities through co-cultures of selected species that share similar characteristics and environments. Reprogramming an organism is carried out by inserting synthetic genetic circuits, which are designed in a controlled manner to obtain biotechnological products beneficial to humans, their health, and the environment. The potential applications in medicine, bioremediation, industry, and pharmaceuticals make the research of synthetic microbial communities a promising field for the future. However, the implementation of synthetic microbial communities carries potential risks, such as horizontal gene transfer and possible environmental impacts. It is crucial to carefully evaluate these functions and risks, considering biocontainment and the associated ethical and ecological implications. Full article

The potential for infectious salmon anemia virus (ISAV)—an internationally regulated pathogen of salmon—to transmit vertically from parent to offspring is currently unclear. While the highly virulent ISAV phenotype known as ISAV-HPRΔ has been observed intra-ova, evidence for vertical transmission of the avirulent ISAV phenotype known as ISAV-HPR0 is lacking. In this study, we identified ISAV-HPR0-infected Atlantic salmon broodstock during spawning within a government research recirculating aquaculture facility using qPCR. Eggs and milt from infected brood were used to initiate 16 unique family dam-sire crosses from which 29–60 fertilized eggs per cross were screened for ISAV using qPCR (limit of detection ~100 virus genome copies/egg). A portion of eggs (~300) from one family cross was hatched and further reared in biosecure containment and periodically screened for ISAV by gill clipping over a 2-year period. ISAV was not detected in any of the 781 eggs screened from 16 family crosses generated by infected brood, nor in 870 gill clips periodically sampled from the single-family cohort raised for 2 years in biocontainment. Based on these findings, we conclude that ISAV-HPR0 has a limited likelihood for vertical parent-to-offspring transmission in cultured Atlantic salmon. Full article

Since the development of recombinant DNA technologies, the need to establish biosafety and biosecurity measures to control genetically modified organisms has been clear. Auxotrophies, or conditional suicide switches, have been used as firewalls to avoid horizontal or vertical gene transfer, but their efficacy has important limitations. The use of xenobiological systems has been proposed as the ultimate biosafety tool to circumvent biosafety problems in genetically modified organisms. Xenobiology is a subfield of Synthetic Biology that aims to construct orthogonal biological systems based on alternative biochemistries. Establishing true orthogonality in cell-based or cell-free systems promises to improve and assure that we can progress in synthetic biology safely. Although a wide array of strategies for orthogonal genetic systems have been tested, the construction of a host harboring fully orthogonal genetic system, with all parts operating in an orchestrated, integrated, and controlled manner, still poses an extraordinary challenge for researchers. In this study, we have performed a thorough review of the current literature to present the main advances in the use of xenobiology as a strategy for biocontainment, expanding on the opportunities and challenges of this field of research. Full article

Infections acquired in research laboratories and unintentional pathogen escapes from breaches in biocontainment pose risks to humans and the environment, necessitating the need for effective biosafety and biosecurity management frameworks in biocontainment research animal facilities (BRAFs). We examine key biosafety issues associated with BRAFs, including inadequate decontamination procedures for wastewater and experimental samples, handling high biosafety level pathogens in lower-level laboratories, risks of animal bites and sharps injuries, contamination of bedding and enrichment materials, and improper management and transportation of biohazard samples. Additionally, we discuss the role of veterinarians in research animal facilities and the challenges they encounter in maintaining biocontainment standards. We emphasise the importance of routine monitoring of effluent water to detect possible disease outbreaks. We recommend a thorough investigation of disease outbreaks to identify potential sources of pathogen release from BRAFs, which could serve as hotspots for future disease outbreaks. Findings from such investigations will inform the development of policies aimed at safeguarding human populations from future pandemics and preventing BRAFs from becoming sources of infectious disease outbreaks. Full article