Search Results (401)

Microbial contamination in aquatic environments poses severe threats to aquaculture sustainability, ecological balance and public health. Traditional culture-based detection methods, while standardized, are time-consuming and labor-intensive, often failing to meet the urgent need for rapid on-site monitoring required to prevent disease outbreaks and manage water quality effectively. By integrating latest research advances (2020–2025), this study reviews advances in rapid detection technologies for aquatic microorganisms, including the evolution of nucleic acid amplification strategies, with a focused comparison of the analytical sensitivity and field deployability of quantitative polymerase chain reaction (qPCR) and mainstream isothermal amplification techniques (loop-mediated isothermal amplification, LAMP; recombinase polymerase amplification, RPA). Furthermore, this study reports on the emergence of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated protein (Cas) systems as next-generation diagnostic tools, highlighting their integration with microfluidic Lab-on-a-Chip (LOC) platforms to achieve attomolar sensitivity. We also consider the application of portable nanopore sequencing for real-time pathogen identification and the growing role of Artificial Intelligence (AI) in analyzing complex diagnostic datasets. Advanced molecular methods have achieved significant reductions in time consumption—from days to less than one hour—while challenges regarding sample preparation and environmental matrix inhibition remain. The future of aquatic monitoring lies in integrated, automated systems that combine the specificity of CRISPR-Cas diagnostics with the connectivity of IoT-enabled biosensors. Comparative analysis indicates that isothermal amplification methods (LAMP, RPA) coupled with CRISPR-Cas systems offer the optimal balance of sensitivity, speed, and field deployability for point-of-care aquaculture diagnostics, while qPCR/dPCR remain indispensable for quantitative regulatory applications. We propose a structured technology selection framework to guide researchers and practitioners in choosing appropriate detection modalities based on specific sensitivity, cost, throughput, and deployment requirements. Full article

Providencia stuartii (Ps) is a clinically significant opportunistic pathogen often associated with “difficult-to-treat resistance” (DTR) infections due to pan-resistance to first-line antimicrobials. We report the clinical diagnosis and rapid genomic characterization of strain Ps-CMC-4104, recovered from a human splenic abscess in a patient with infected necrotizing pancreatitis. To resolve the complex genetic architecture of this strain, we utilized hybrid sequencing combining Oxford Nanopore (long-read) and Illumina (short-read) technologies. Analysis revealed a 4,504,925 bp circular chromosome featuring a unique genomic resistance island (GRI) closely related to Salmonella SGI1. Notably, the PsGRI contains multiple copies of NDM-1 and PER-1 carbapenem-resistance and -inhibitor genes, a repetitive structure typically unresolvable by standard short-read methods. Additionally, a large 278,489 bp low-copy circular plasmid harbored single copies of these carbapenemase and extended-spectrum β-lactamase genes alongside other antimicrobial resistance determinants and ISCR1 insertion sequences. Nanopore technology allowed us to precisely identify the duplications, providing critical insights into the strain’s pan-resistant phenotype. This study serves as proof-of-concept for the importance of integrating long-read sequencing into clinical workflows to identify complex resistance mechanisms in DTR pathogens, facilitating targeted antimicrobial stewardship and infection control. Full article

Tick-borne encephalitis virus (TBEV) is primarily transmitted by Ixodes spp. and poses significant health risks, leading to morbidity and mortality in humans. Two of the five subtypes, Siberian and Far Eastern are known to circulate in Mongolia. In 2021, Ixodes persulcatus ticks were collected from Bulgan aimag (province) using flagging and dragging methods and subsequently screened for TBEV using PCR. Positive samples underwent sequencing using an Oxford Nanopore Technologies-based hybrid capture approach, resulting in two coding-complete TBEV genomes from separate tick pools. Phylogenetic analysis classified both genomes within the Siberian subtype, grouping them with other Mongolian sequences from I. persulcatus collected in 2014, 2020, 2021, and 2023. The study sequences, PX654173 and PX654174, showed high genetic similarity (99.9% and 99.8%, respectively) to the sequence PQ479142, obtained from I. persulcatus ticks in Selenge, Mongolia, in 2021. The estimated time to most recent common ancestor (TMRCA) of the Siberian genotype was approximately 981 CE (95% HPD: 646–1347) with the emergence of a distinct Mongolian clade of TBEV around 1888 CE (95% HPD: 1834–1934). These findings highlight the value of expanded whole-genome sequencing to improve our understanding of TBEV’s genetic diversity and evolutionary history in Central Asia. Full article

Diagnostic testing of foot-and-mouth disease virus (FMDV) currently utilizes reverse transcription quantitative PCR (RT-qPCR) to detect the presence of viral RNA and double antibody sandwich ELISAs (DAS-ELISAs) to determine viral serotype. Serotype identification is critical to support informed vaccine selection to combat outbreaks. While DAS-ELISAs are capable of serotype identification, the test suffers from low sensitivity and requires a viral isolate for successful detection. In this study, we developed FMDV-ONTAPS: an Oxford Nanopore Technologies Amplicon P1 Sequencing protocol involving reverse transcription-PCR to amplify P1 of the FMDV genome, and Nanopore sequencing of the amplicons to provide genetic data for serotype and subtype/topotype identification. FMDV isolates representing all seven serotypes were successfully sequenced with this method. Additionally, the protocol successfully provided serotype identification from a variety of specimen matrices obtained from experimentally infected animals that included milk, serum, oral and nasal swabs, tissue suspensions, vesicular fluid, and oral fluid. The limit of detection for FMDV cell culture isolates was comparable for both sequencing and RT-qPCR detection. RT-qPCR Cq values for clinical samples evaluated ranged from 8 to 28.21. Sequencing was successful for all samples except for a single tissue suspension sample (Cq of 28.21). Identification of FMDV serotype in clinical samples is critical for effective outbreak response, and Nanopore sequencing offers a timelier and more sensitive alternative to DAS-ELISAs. Full article

Underutilised, nutrient-dense legumes in their sprouted form provide promising substrates for developing functional fermented foods capable of influencing gut microbial activity and metabolite production. This study evaluated the effects of probiotic lactic acid bacteria-fermented beverages derived from sprouted Bambara groundnut (Vigna subterranea) and cowpea (Vigna unguiculata) on gut microbiota composition and short-chain fatty acid (SCFA) production using an in vitro colonic fermentation model. The beverages were fermented with either Bifidobacterium animalis BB-12 (BCBF24) or Lactiplantibacillus plantarum 75 (BCL7524). During colonic fermentation, at 0, 12, 24, and 38 h, faecal slurries were collected for SCFA analysis using gas chromatography–mass spectrometry (GC-MS) and deoxyribonucleic acid (DNA) sequencing (Oxford Nanopore Technologies). Microbial diversity decreased, indicating selective enrichment of taxa. BCL7524 induced a major shift, significantly (p < 0.05) enriching Bacillota and driving Megasphaera to ~42% dominance within 24 h. This reflected cross-feeding from L. plantarum to lactate-utilising Megasphaera spp. Spearman correlation linked Megasphaera to a broad SCFA profile, including isobutyric, isovaleric, valeric, and hexanoic acids, with a significant (p < 0.05) positive correlation observed for hexanoic acid. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated proteolysis and mapped hexanoic acid to fatty acid biosynthesis pathways, suggesting chain-elongation activity contributing to hexanoate formation. In line with this, BCL7524 produced significantly (p < 0.05) higher levels of hexanoate (3–14 mM) and valerate (10–15 mM), supporting chain-elongation activity within the community. In contrast, BCBF24 enriched Actinomycetota and Bifidobacterium, correlating with acetate production (18–23 mM). This study demonstrates that specific synbiotic beverages can modulate gut microbial ecology and metabolic output under in vitro conditions. Full article

Kombucha fermentation is driven by a Symbiotic Culture of Bacteria and Yeast (SCOBY), a cellulose-rich biofilm that hosts a complex microbial consortium. While most kombucha studies focus on the liquid beverage, the SCOBY pellicle itself remains underexplored, particularly with respect to species-level microbial resolution and its intrinsic biological activities. In this study, a commercial kombucha SCOBY was characterized using full-length 16S rRNA gene and ITS amplicon sequencing based on Oxford Nanopore Technology, enabling species-level taxonomic resolution. In parallel, hydroalcoholic and aqueous extracts of dried SCOBY biomass were evaluated for in vitro antioxidant activity (DPPH and ABTS assays), antidiabetic-related enzyme inhibition (α-glucosidase and dipeptidyl peptidase-4, DPP4), and anti-aging-related enzyme inhibition (tyrosinase and elastase). The SCOBY bacterial community was strongly dominated by acetic acid bacteria, with Komagataeibacter saccharivorans and Acetobacter tropicalis accounting for more than 60% of total reads, reflecting a biofilm structure optimized for cellulose production and oxidative metabolism. The yeast community showed marked unevenness, with Brettanomyces bruxellensis representing over 80% of reads, consistent with its known role in ethanol production and stress tolerance within kombucha systems. In vitro assays revealed that hydroalcoholic SCOBY extracts consistently exhibited higher biological activity than aqueous extracts across all tested assays. However, both extracts showed substantially lower potency than purified reference compounds, indicating moderate but measurable bioactivity typical of complex fermented matrices. These findings support the potential valorization of SCOBY as a fermentation-derived biomaterial and functional ingredient while underscoring the need for further chemical characterization, mechanistic studies, and biological validation beyond enzyme-based assays. Full article

Canine distemper virus (CDV) affects both domestic and wild carnivores and is associated with a high mortality rate. The virus can cross species barriers, infecting a wide range of mammals, which raises concerns for both wildlife conservation and domestic animal health. During our study, we processed a total of n = 552 oral and rectal swab samples from n = 260 red foxes (Vulpes vulpes) and n = 16 golden jackals (Canis aureus). The samples were collected by the National Food Chain Safety Office (NÉBIH) as part of a Rabies monitoring programme from Hungary in 2024. We performed a Real-Time RT-PCR, followed by a CDV-specific amplicon-based sequencing method using Oxford Nanopore Technologies to obtain the complete genome. All golden jackal samples tested negative, while both oral and rectal samples of one red fox tested positive for viral RNA. From this positive sample, we were able to sequence a partial CDV genome. Based on phylogenetic analysis of the haemagglutinin gene, our CDV sequence was assigned to the Europe lineage, one of the endemic lineages in the continent, infecting both threatened and common animals. This finding highlights the ongoing presence of CDV in wildlife populations and illustrates the value of integrated monitoring systems. Full article

Solid-state nanopore sequencing, a key third-generation sequencing technology, offers considerable potential for genomics and diagnostics due to its long read lengths, real-time detection, and amplification-free operation. The technology identifies DNA sequences by measuring characteristic changes in ionic current as single-stranded DNA translocates through a nanoscale pore. However, its practical development faces challenges including limited spatiotemporal resolution, pore clogging from nonspecific adsorption, and significant electrical noise. This review systematically examines strategies developed to address these limitations. We discuss the use of ultrathin two-dimensional materials such as graphene and molybdenum disulfide to improve spatial resolution, and methods to modulate DNA translocation through optimized solution conditions, pore geometry, surface charge engineering, and bio-solid hybrid pore designs. Furthermore, we detail noise suppression strategies targeting key sources like thermal noise, 1/f noise, and dielectric noise. These approaches encompass careful material selection, surface coatings, innovations in chip and amplifier design, and machine learning–based signal processing. The review also outlines surface functionalization techniques that reduce clogging and enhance analytical specificity. While challenges remain, continued convergence of materials science, nanofabrication, and data science is advancing solid-state nanopore technology toward reliable, high-precision sequencing platforms, promising to significantly impact personalized medicine and biological research. Full article

Chrysomya megacephala is a synanthropic fly with a high potential to act as a mechanical vector of pathogenic bacteria, surpassing Musca domestica in both bacterial load and diversity. Native to Asia and Africa, it has become a cosmopolitan species, successfully adapting to a wide range of environments, including natural ecosystems. In Colombia, studies on its role as a vector are limited and have largely relied on traditional culturing methods. This study aimed to characterize the pathogenic bacterial microbiota associated with C. megacephala using 16S rRNA gene sequencing in urban, rural, and forest settings of a coastal tourist city. Flies were collected using Van Someren Rydon traps with attractants and sterile materials. Bacterial identification was performed through Oxford Nanopore MinION sequencing (Manufactured by Oxford Nanopore Technologies, Oxford, UK). A total of 49 bacterial species were identified, with urban environments showing the highest taxonomic richness. The forest environment was characterized by a highly dominant community structure, led by Vagococcus carniphilus. Notably, 20 bacterial species of public health relevance were detected, including Clostridium botulinum, Clostridium perfringens, Ignatzschineria ureiclastica, Escherichia coli, and Streptococcus agalactiae. These findings indicate that bacterial community composition varies by environment and underscore the potential role of C. megacephala as a mechanical vector, highlighting the importance of surveillance for its public health implications. Full article

Background: Genetic stability of live-attenuated infectious laryngotracheitis virus (ILTV) vaccines is essential for consistent efficacy and safety; however, marker-based assessments targeting partial genes are often insufficient given the virus’s large, structurally complex genome. The ILTV genome contains long internal inverted repeats (IRs) that can give rise to genomic isomers, complicating short-read assembly and accurate resolution of genome structure. Methods: To overcome these limitations, we used a hybrid whole-genome sequencing (WGS) strategy, combining Oxford Nanopore Technologies (ONT) long reads to improve assembly contiguity with Illumina short reads for high-accuracy polishing at the single-nucleotide level. Using this approach, we generated complete de novo genome assemblies for the commercial Serva and Salsbury #146 vaccine strains. Results: The assemblies showed high sequence concordance with targeted regions validated by Sanger sequencing. Whole-genome analysis further enabled detection and independent validation of a structural inversion in the unique short (US) region of the Salsbury strain, consistent with herpesvirus genome isomerization. To enable phylogenetic inference despite structural variability, we performed a pangenome-based analysis to define a conserved core-genome dataset that robustly resolved vaccine-associated lineages, separating Serva- and Salsbury-derived strains. Conclusions: Collectively, these findings show that a hybrid WGS workflow can generate high-confidence genome assemblies for the specific commercial ILTV vaccine vials analyzed and can support QC-relevant detection of major structural variations. Because this study is cross-sectional (two strains; single lot/vial per strain), it cannot distinguish potential biological lot-to-lot variation from methodological differences, and a comprehensive genetic stability evaluation will require applying this workflow across defined passage levels and/or multiple production lots. Full article

In this study, we comparatively assessed short-read (Illumina), long-read (Oxford Nanopore Technologies, ONT), and hybrid (Illumina + ONT) sequencing strategies for bacterial genome analysis using the aquaculture-derived isolate 160P. Genomic DNA was extracted and sequenced on Illumina paired-end and ONT long-read platforms, and de novo assemblies were generated using SPAdes, Canu, Flye, and Unicycler under short-read-only, long-read-only, and hybrid workflows, followed by evaluation with QUAST assembly metrics. Among the tested approaches, the hybrid Unicycler assembly provided the highest contiguity, yielding seven contigs and a dominant 4.55 Mb contig consistent with near-complete chromosomal representation. Downstream analyses included functional genome annotation and in silico screening of antimicrobial resistance determinants (CARD), virulence-associated genes (VFDB), and secondary metabolite biosynthetic gene clusters (antiSMASH). Comparative genomic relatedness based on Average Nucleotide Identity (ANI) and digital DNA–DNA Hybridization (dDDH) indicated that 160P is most closely related to Aeromonas sobria CECT 4245^T yet falls below commonly applied species-level thresholds, supporting its placement as a genomically distinct lineage warranting further taxonomic investigation. Collectively, these findings underscore the value of hybrid sequencing for improving assembly continuity, enhancing annotation completeness, and strengthening taxonomic resolution in bacterial pathogen genomics. Full article

Background/Objectives: Dietary composition and physical activity are major determinants of gut microbiome structure, and dysbiosis is strongly associated with metabolic disorders. While both diet and exercise independently influence the gut microbiome, their interactive effects—particularly across different exercise modalities—remain incompletely understood. This study investigated the combined effects of diet type (normal chow [NC] vs. high-fat diet [HFD]) and exercise modality (control [C], voluntary [V], and forced [F]) on gut microbiota composition in rats. Methods: Sixty-three Wistar rats were randomized into six groups according to diet and exercise status. Fecal samples were collected and analyzed using full-length 16S rRNA gene sequencing (Oxford Nanopore Technologies). Alpha and beta diversity metrics were calculated, and taxonomic composition was assessed at phylum and genus levels. Results: HFD groups exhibited significantly higher alpha diversity than NC groups (Shannon index: 3.47–3.63 vs. 2.76–2.94, p < 0.001), with forced exercise associated with a greater diversity than voluntary exercise. Beta-diversity analysis confirmed diet as the dominant factor influencing microbial structure (PERMANOVA p = 0.001), with exercise providing an additional modulatory effect. Firmicutes, Bacteroidota, Deferribacterota, and Proteobacteria predominated, with Firmicutes decreasing under HFD. Forced exercise significantly enriched beneficial genera, including Akkermansia (detected exclusively in exercised HFD groups; p = 0.03), Blautia, Coprococcus, and Roseburia. Akkermansia abundance correlated positively with exercise distance (p < 0.001) and negatively with body weight (p < 0.01). Conclusions: Structured exercise, particularly forced treadmill training, attenuates HFD-associated dysbiosis and promotes the beneficial gut bacteria that is associated with metabolic health. These findings highlight exercise modality as a critical factor in dietary strategies targeting gut microbiome modulations. Full article

AAV integration has become an important safety consideration in gene therapy. However, accurately determining integration sites remains challenging due to biases introduced by library preparation methods, sequencing technologies, and bioinformatic pipelines. In this study, we developed a PCR-free amplification based on a CRISPR-Cas9 cleavage strategy for AAV DNA that overcomes the limitations of PCR amplification imposed by the ITR structure. When combined with long-read nanopore sequencing, this CRISPR-Cas9-based workflow preserves native AAV integration states and enables unbiased detection of integration junctions. We used AAV-transduced HeLa single-cell clones to evaluate the performance of this approach. To confirm integration site identification, AAV integration junctions were also detected using a probe hybridization capture strategy followed by Illumina short-read sequencing. Integration junctions identified by both methods were further confirmed by PCR. The results showed strong consistency between the two approaches in accurately identifying AAV integration sites in each clone. Overall, these findings demonstrate that the CRISPR-Cas9-enabled, PCR-free long-read sequencing workflow provides a promising tool for characterizing AAV integration events. Full article

Long-read sequencing technologies, particularly those developed by Oxford Nanopore Technologies (ONT), have transformed genome sequencing by enabling high-resolution analysis of complex microbial communities. Among ONT devices, the MinION remains affordable and scalable for low-resource settings. However, its limited onboard computing power constrains high-accuracy basecalling and limits its ability to address inherent sequencing errors effectively. To overcome these constraints, we assembled a streamlined in-house workflow that integrates at least five MinION devices with a GPU-powered workstation running Ubuntu 20 and MinKNOW. Rather than a new sequencing platform, this “home-made GridION” represents a practical integration of existing ONT devices with dedicated computing resources. At its core is a live basecalling pipeline capable of handling both FAST5 and POD5 file formats. The system supports high-throughput basecalling using Guppy on FAST5 files as well as Dorado on POD5 files, ensuring compatibility with both legacy and current ONT data standards. File monitoring is automated via inotifywait, enabling immediate detection of new files, real-time basecalling, and organized output of FASTQ batches. Beyond basecalling, we implemented an automated downstream pipeline for metagenomic analysis, enabling taxonomic profiling and detection of antimicrobial resistance genes (ARG). Tested on 10 hospital wastewater samples, the workflow generated at least 500,000 reads per sample within six hours, which were analysed for antimicrobial resistance gene abundance. This demonstrates its potential as an open, scalable hardware/software platform that extends the utility of MinION sequencing for microbial genomics in resource-limited environments. The setup can channel as many MinIONs as available USB ports, with a ratio of 1 MK1D for 1 TB of storage capacity on the associated computer. Full article

Background/Objectives: The primary outcome of this 8-week randomized, controlled, parallel trial was to assess longitudinal shifts in gut microbiota structure and predicted metabolic potential in 45 elite football players following protein supplementation. Methods: Participants combined resistance training with daily intake (30 g) of whey protein concentrate (WPC), pea protein isolate (PPI), rice protein isolate (RPI), or a plant-protein blend (MIX). For the acquisition of prokaryotic metataxonomic data, the V3–V8 region of the 16S rRNA gene was sequenced using Oxford Nanopore Technology (ONT). Functional potential was inferred through the MACADAM database and STAMP software. Strict dietary monitoring and gravimetric adherence checks were performed to isolate the intervention effect. Results: While microbial alpha-diversity indices (Chao1, Shannon, Simpson) remained stable across all groups, significant source-specific shifts in taxonomic structure and predicted metabolic activity were identified. Whey protein concentrate (WPC) was associated with an increase in Bacteroidetes abundance and greater balance within the microbial community structure, whereas pea protein isolate (PPI) and the MIX correlated with reduced fermentative bacteria and elevated taxa potentially involved in cadaverine biosynthesis. Rice protein isolate (RPI) supplementation was associated with a higher predicted representation of taxa involved in succinate-to-butyrate fermentation pathways. These functional markers and differential responses of selected bacterial groups to particular protein types were observed. Conclusions: The data indicate complex interactions between supplement type, exposure duration, and microbiome response, underscoring the necessity for individualized dietary recommendations and supplementation strategies to optimize gut health and training adaptation in professional football players. Full article