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It has been known for decades that bacteriophages encode tRNA genes, but their function and the factors contributing to their acquisition and retention are unclear. Although tRNAs are found in a variety of phages infecting a variety of bacteria, many large-scale computational studies investigating tRNA acquisition and retention in phages are specific to Mycobacterium phages; however, these findings may not be representative of other phages or bacteria. This work uses a broader sampling of phages and hosts to investigate the relationships between codon usage bias, infection cycle, and tRNA gene numbers in phage genomes. We analyzed 154 phages infecting 7 host genera, including Gram-negative (Escherichia, Shigella, Salmonella) and Gram-positive (Bacillus, Lactobacillus, Staphylococcus, Mycobacterium) bacteria. Phages included temperate and virulent representatives, plus a range of tRNA numbers and morphologies. All phages and hosts were analyzed using four metrics: GC content, Effective Number of Codons, Relative Synonymous Codon Usage, and tRNA Adaptation Index. On a global scale, virulent phages with many tRNA genes show greater differences in codon usage and codon adaptation compared to their respective hosts. Gram-negative bacteria and their phages generally exhibit greater differences in codon usage compared to Gram-positive bacteria and their phages. Phages infecting Gram-negative hosts also tend to encode more tRNA genes. In nearly all genus-level comparisons, Mycobacterium phages were different from any other host and from global patterns. This suggests previous computational studies performed in Mycobacterium phages are likely not applicable on a global scale or to phages infecting other host genera. AUTHOR SUMMARYBacteriophages, or phages, are viruses infecting bacteria. They are abundant in all environments, yet how they interact with their bacterial hosts is still not well-understood. Like other viruses, phages must rely on the host translational components to replicate and form new phage particles; and similarly to other parasites, phages have genomes that differ significantly from their hosts in terms of composition. In this work, we explore the relationship between phage lifestyle, number of tRNA genes encoded, and genome differences from the host using a variety of phages and their associated hosts. Phages can be either virulent (do not integrate into the host genome) or temperate (capable of integrating into the host genome), with differences from the host genome more pronounced in virulent phages. There are many phages that also carry tRNA genes, and having higher numbers of tRNAs is associated with larger differences from the host genome. The findings here indicate that virulent phages carrying large numbers of tRNAs diverge the most from host genome composition.

Industrial waste containing hydrophobic pollutants, like oils and hydrocarbons, is toxic and difficult to degrade, posing both ecological and human health risks. Biosurfactants are eco-friendly surface-active compounds produced by microorganisms, known for their ability to lower surface and interfacial tension, enhancing the solubility and bioavailability of hydrophobic compounds, facilitating their breakdown. The current study focuses on isolating biosurfactant-producing bacteria from industrial waste sources near Dhaka, Bangladesh, and characterizing their properties, determining potential usage. Using diesel-enriched nutrient agar, bacterial strains were isolated and screened for biosurfactant production by oil displacement, emulsification index (E24%), and drop collapse assay. The most promising isolates were characterized according to their biochemical activities and 16S rRNA amplicon-based sequencing. Isolation and characterization of the surfactants have been carried out using chromatographic techniques. The identified bacteria passed the drop collapse and oil displacement tests. CTAB agar assay, indicates their anionic nature, showing an emulsification index ranging 10-41%. The potential biosurfactant producers belong to Bacillus, Pseudomonas, Acinetobacter, and Enterobacterium genera. The surfactants showed antibacterial, antifungal, and plant growth promotion activity and have been characterized in terms of pH stability, salinity, adhesion, and temperature tolerance. The study successfully identified and characterized potential biosurfactant-producing bacteria from industrial waste, highlighting their efficiency in breaking down hydrophobic pollutants and hydrocarbons. These microorganisms provide a green and economical substitute for synthetic surfactants due to their biodegradability and lower toxicity. Upon further research and scaling, these bacteria can be a good source of biosurfactants for potential applications in industrial, agricultural, and biomedical fields. IMPORTANCEThe study carries high significance as it creates multi-disciplinary scopes for utilizing these environmentally adapted biosurfactant-producing bacteria in industry, agriculture, and medicine. Since the bacterial isolates have hydrocarbon degradation ability, upon optimization for higher production, industrial usage in oil refinery and other industries can be adopted. Due to their biodegradable nature, usage in wound healing bandages and as antimicrobial agents in medicine will be noteworthy. The isolates have plant growth promotion ability and utilizing them as biofertilizer will reduce the dependency on chemical fertilizers. This is the first detailed report on biosurfactant-producing bacteria from this industrial waste-polluted Turag River of Dhaka City. Moreover, it compiles detailed screening protocols and methods for analyzing such environmentally friendly microbes. Such characterization also opens the scope for optimizing the production of the surfactant compounds on a large scale and utilizing them commercially.

A compact, in-house developed ultraviolet germicidal irradiation (UVGI) system adaptable to static, mobile, or robotic platforms was developed for the effective sterilization of bacteria and fungi using a wireless mode of operation. Under controlled laboratory conditions, its efficacy was evaluated against three representative biofilm-forming pathogens: Bacillus subtilis (Gram-positive, spore-forming, motile bacterium), Escherichia coli K12 (Gram-negative, non-spore-forming, non-motile bacterium), and Candida albicans M-207 (multi-drug-resistant, clinical yeast isolate). Microbial viability following UVGI exposure was assessed using colony-forming unit (CFU) and MTT assays, and morphological alterations were characterized by scanning electron microscopy (SEM). Cultures were exposed to UV-C radiation at distances of 1-5 m for 15-90 min. CFU assay demonstrated 100% kill of all tested organisms at 1 m and 15 min, corresponding to doses of 600.3, 576 & 697.5 mJ/cm{superscript 2}. Although MTT assays indicated residual metabolic activity under the same conditions, CFU results confirmed that surviving cells were unable to proliferate, highlighting the robustness of UV treatment for long-term inactivation. SEM confirmed distinct morphological alterations such as complete destruction of extracellular matrix & reduction in number of cells indicating cell death with increase in UV dose as compared to controls. A dose & time-dependent inactivation of biofilm-forming bacteria & fungi was observed on exposure to UVGI. Therefore, this pilot study validates the effectiveness of the newly developed UVGI surface sterilizer against biofilm-forming bacterial and fungal pathogens. Overall, the system demonstrates proof-of-concept efficacy under laboratory conditions and holds strong potential for future development and validation in hospitals and other contaminated public spaces. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/715580v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@150cefcorg.highwire.dtl.DTLVardef@450831org.highwire.dtl.DTLVardef@1cfd6borg.highwire.dtl.DTLVardef@1419ba8_HPS_FORMAT_FIGEXP M_FIG C_FIG IMPORTANCEMicroorganisms that form biofilms on surfaces are difficult to eliminate and contribute to the spread of infections in healthcare and indoor environments. There is a need for practical, easy-to-use disinfection technologies that can effectively reduce such contamination. In this study, we developed a compact, in-house, wireless UV-C disinfection system designed for flexible operation across different surface types. The system was evaluated under controlled laboratory conditions using representative biofilm-forming bacterial and fungal pathogens. Our findings show that the system can effectively reduce microbial contamination, demonstrating proof-of-concept efficacy. This work highlights the potential of accessible, non-chemical UV-based technologies and supports their further validation for applications in real-world disinfection settings.

BackgroundThere is currently no approved antiviral therapy against measles virus (MeV). Repurposing available compounds with broad antiviral activity may rapidly identify candidate drugs for clinical evaluation. Here we evaluated the antiviral activity of the clinically approved drugs azelastine hydrochloride and zafirlukast as well as the flavonoids quercetin and isoquercetin against MeV in preventative and therapeutic in vitro studies. MethodsCompounds were tested for antiviral activity against MeV in preventative (prophylactic and virucidal) and therapeutic (steady-state and persistent) assays in Vero/hSLAM cells. Viral loads and cell viability were measured 48h post-infection, and dose-response curves were used to calculate EC50 values. Flavonoids were also tested in the presence of 1 mM ascorbic acid. ResultsAzelastine hydrochloride did not show evidence of antiviral activity against MeV under these conditions, whereas zafirlukast, quercetin, and isoquercetin showed therapeutic activity against MeV. The addition of ascorbic acid enhanced the therapeutic potency of quercetin to 4.2-4.8 {micro}M and of isoquercetin to 10.7-10.9 {micro}M. Antiviral activity was dose-dependent when administered post-infection. ConclusionAmong the four compounds tested, quercetin showed the most potent therapeutic antiviral activity against MeV in vitro. Isoquercetin and zafirkulast also showed therapeutic activity. These findings support further evaluation of quercetin, isoquercetin, and zafirlukast as candidate antiviral drugs for MeV and highlight the utility of in vitro platforms for rapid antiviral drug screening.

Staphylococcal cassette chromosome (SCC) elements are mobile genetic elements that integrate at the rlmH gene and are predominantly responsible for methicillin resistance in staphylococci. Although SCCmec typing tools exist, none can extract the element sequence itself or explicitly classify SCC elements that lack methicillin resistance genes. Here we present SCCmecExtractor, a lightweight Python toolkit that identifies SCC element boundaries through degenerate attachment site (att) pattern matching, extracts complete elements from whole-genome assemblies and characterises their mec and ccr gene content. Benchmarking on 7,297 genomes spanning 70 species across Staphylococcus and Mammaliicoccus demonstrated 100% typing concordance with the sccmec tool1 on 1,454 S. aureus genomes. The tool extracted 1,562 SCC elements, from 1,454 S. aureus, 5,295 non-aureus Staphylococcus and 548 Mammaliicoccus genomes, achieving effective extraction rates (excluding assembly-limited genomes and those lacking valid ccr pairs) of 87.3% for S. aureus, 58.8% for non-aureus Staphylococcus, and 61.9% for Mammaliicoccus. Notably, 616 of the 1,562 extracted elements (39.4%) were non-mec SCC elements lacking methicillin resistance genes, a class of mobile element often overlooked. Non-mec SCC prevalence increased from 12.2% in S. aureus to 55.6% in non-aureus Staphylococcus and 76.0% in Mammaliicoccus, revealing a substantial reservoir of SCC diversity beyond methicillin resistance. SCCmecExtractor is freely available via PyPI, Docker and Singularity under an MIT licence. Impact StatementStaphylococcal cassette chromosome (SCC) elements are mobile genetic elements responsible for methicillin resistance in staphylococci and are central to methicillin resistant Staphylococcus aureus (MRSA) epidemiology. Existing tools focus on typing SCCmec from assemblies but cannot extract the element itself, limiting our ability to comprehensively monitor and examine these elements. SCCmecExtractor is a lightweight, portable tool that detects the attachment sites, required by SCC elements to integrate into the genome, extracts the SCC element, both mec gene carrying and not, and characterises their gene content. Applied across 7,297 genomes spanning two genera, we demonstrate that non-mec SCC elements are the dominant SCC class outside S. aureus, a finding enabled by systematic extraction and classification of SCC elements regardless of mec gene content. SCCmecExtractor provides the research community with an accessible, confidence-first approach (based on biology) to SCC element analysis across all staphylococci and mammaliicocci species. Data SummaryThe code for this pipeline is available at: https://github.com/AlisonMacFadyen/SCCmecExtractor, with a Docker image available at: https://hub.docker.com/repository/docker/alisonmacfadyen/sccmecextractor and PyPi package at: https://pypi.org/project/sccmecextractor/. All reference databases are bundled with the tool. Benchmarking genome accessions: 1,454 S. aureus, 5,295 non-aureus Staphylococcus, and 548 Mammaliicoccus genomes from NCBI. A complete list of genome accessions is provided as supplementary data (Supplementary Table S1). Extracted SCC elements can be obtained from Zenodo: 10.5281/zenodo.19355206

This study investigated the antiplasmodial and antibacterial activities of fractionated extracts of Moringa oleifera seeds, focusing on the influence of solvent polarity on bioactivity. The results revealed a polarity-dependent distribution of activity. Polar aqueous extracts (crude and residual fractions) exhibited the most pronounced antiplasmodial effects against Plasmodium falciparum (3D7 strain), with IC values of 107-135 {micro}g/mL. Time-dependent analyses of the crude and residual fractions showed that parasitaemia declined steadily over time, and consequently, percentage inhibition increased with time, with both extracts reaching 70-80% inhibition by 48 hours at higher concentrations. In contrast, moderately polar organic fractions, notably ethyl acetate and dichloromethane, demonstrated strong antibacterial activity against both Gram-positive and Gram-negative clinical isolates, including resistant strains such as MRSA and ESBL-producing Escherichia coli. Minimum inhibitory concentrations (MICs) ranged from 6.3 to 25 mg/mL for the ethyl acetate fraction, and all active fractions exhibited bactericidal properties (MBC/MIC [≤] 4). Comparative analysis showed that while antiplasmodial activity was moderate relative to the standard drug (chloroquine), antibacterial activity was robust and clinically promising. Fractionation revealed that distinct phytochemical classes underlie the two activities: polar compounds appear responsible for antiplasmodial effects, whereas moderately polar compounds drive antibacterial potency. The moderate antiplasmodial activity is significant in the context of adjunctive therapy and resistance management, while the strong antibacterial activity is highly relevant to the global challenge of antimicrobial resistance. Together, the results position Moringa oleifera as a promising source of phytochemicals for integrated infectious disease management, particularly in regions where malaria and bacterial co-infections are prevalent.

BackgroundDNA polymerase activity assays are required for enzyme quality control in biotechnology and diagnostics, but standard methods rely on specialist reagents, radioactivity and other hazardous materials, or real-time PCR instruments that are not widely accessible in resource-limited settings. This constrains local production of high quality, validated reagents and increases dependence on imported enzymes. MethodsBased on experiences derived from partnerships with scientists in several low and middle-income countries (LMICs) and stakeholder consultations, we adapted a commercial EvaGreen-based fluorometric DNA polymerase activity assay for isothermal operation using minimal equipment. Assay conditions were optimized using Design of Experiments (DOE) methodology, varying temperature, reaction volume, and MgCl2 concentration. To address reagent cost and supply-chain constraints, we developed detailed protocols for in-house synthesis of the off-patent AOAO-12 DNA dye (sold commercially as EvaGreen) and generation of single-stranded DNA templates via asymmetric PCR. ResultsOptimized isothermal assay conditions (40{degrees}C, 7.75 mM MgCl2) reliably quantified activity across multiple DNA polymerase families. In-house synthesized AOAO-12 dye exhibited comparable DNA-binding performance to commercial alternatives (R{superscript 2} = 0.95), reducing costs by more than an order of magnitude when normalized to working concentrations, enabling assay costs of approximately {pound}0.001 per reaction. The assay is effective across multiple polymerases (Bst-LF, OpenVent, Taq, Q5) and is compatible with both plate readers and qByte, a low-cost, open-source fluorometric device. ConclusionsThis stakeholder-informed assay provides an accessible, cost-effective solution for DNA polymerase quality control in resource-limited settings. The combination of optimized commercial protocols and in-house reagent synthesis offers flexibility for different resource contexts, potentially improving access to molecular biology tools globally.

BackgroundGlobally, seagrass ecosystems are threatened by anthropogenic activities that are leading to increased levels of eutrophication, coastal pollution and thermal conditions. Consequently, there is a growing need to develop new approaches that work to mitigate these stressors and enhance restoration efforts in seagrass meadows. One promising strategy is to identify, isolate and characterize microbial consortia that are likely to support seagrass productivity. However, our current understanding of key microbial functions that support plant growth in marine systems is limited. Based on evidence from terrestrial plant-microbe systems, seagrass-associated bacteria are expected to provide the plant with nitrogen and phosphorus resources while detoxifying sulfur and producing phytohormones. Here, we sequenced 61 bacterial cultures isolated from the rhizosphere, rhizoplane, and endosphere of the seagrass, Zostera marina to identify a consortium of six putative plant growth promoting (PGP) candidates. ResultsOur cultivation approach using plant-based media allowed us to isolate 201 bacteria from Z. marina, which reflected 18% of the total microbial diversity of the starting inoculum. Genomic and phenotypic analyses of the 61-sequenced pure-cultures revealed that most of the sequenced taxa were able to mobilize nitrogen primarily through catabolic pathways, including denitrification (51%), dissimilatory nitrate reduction to ammonia (71%), and C-N bond cleavage (83%). Six of the isolates, which represent new lineages of Agarivorans, coded for the nitrogenase gene cassette. Additionally, 52% of the genomes had genes for sulfur and/or thiosulfate oxidation, 88.5% for phosphorus solubilization, and 60.5% for IAA production. Genomic analysis also revealed that some pathways, including denitrification and dissimilatory nitrite to ammonia DNRA, required cross-species cooperation as no one taxa contained all the genes needed to complete these metabolic pathways. Based on draft genome models and results from phenotypic assays, isolates Streptomyces sp. (Iso23 and Iso384), Mesobacillus sp (Iso127), Roseibuim sp. (Iso195), Peribacillus sp. (Iso49), and Agarivorans sp. (Iso311) represent a minimal microbial community that is likely to promote seagrass growth and enhance restoration efforts. ConclusionOur work provides a detailed genomic and phenotypic analysis of bacteria isolated from Z. marina and identifies a minimal microbial community with complementary PGP traits. Isolating, identifying and characterizing bacteria that promote seagrass growth is critical towards enhancing restoration efforts of seagrass meadows.

Leaf diseases pose a serious threat to faba bean production. Leaf blotch of faba bean, caused by Alternaria spp., has become increasingly widespread and destructive in several countries. Leaf diseases pose a serious threat to faba bean production. The infection of plant by pathogens can be influenced by various factors associated with the host plant, environmental conditions and presence of other microorganisms. The phyllosphere and endosphere play a critical role in plant health and disease development. This study aimed to evaluate the factors shaping the structure and diversity of fungal communities associated with faba beans. Plant samples were collected in 2004 from two intensively managed faba bean production fields in the central region of Latvia. Fungal assemblages were characterized using an ITS region metabarcoding approach based on Illumina MiSeq sequencing. Among the assigned amplicon sequence variant (AVS), 65% belonged to the phylum Ascomycota, while approximately 4% were classified as Basidiomycota. Alternaria and Cladosporium were the dominant genera across samples. The alfa and beta diversities of fungal communities was higher during flowering of faba beans to compare with ripening. The higher abundance of Basidiomycota yeasts were observed during flowering, in contrast, Cladosporium genus was significantly more abundant during ripening. Alternaria DNA was found on leaves that showed no symptoms of the disease. The diversity and composition of fungal communities were significantly influenced by sampling time and presence of leaf blotch, caused by Alternaria spp.

This study was aimed at characterizing the physicochemical analysis of stingless bees honey (SBH) in the Wonchi district, Southwest Shewa Zone, Ethiopia. In this study, a total of 30 stingless bees honey samples were collected from Damu Dagele, Fite Wato, and Warabu Messe sites from underground soils through an excavation of natural nests. Physicochemical characterization of properties and proximate analysis of the honey were performed. The result showed a total mean of 20.12{+/-}1.14% moisture content, 8.62{+/-}2.73 meq./kg free acidity, 1.8{+/-}0.52 mS/cm electrical conductivity, 3.39{+/-}0.32 pH, 40.52{+/-}6.61 mg/kg HMF, 0.83{+/-}0.33% ash, 0.56{+/-}0.25% protein, 0.56{+/-}0.24% fat, and 0.59{+/-}0.23% WISC for physicochemical properties of stingless bees honey. Among sugar profiles of SBH, fructose constituted the highest proportion at 18.87 g per 100 g (53.87%), while sucrose exhibited the lowest concentration at 5 g per 100 g (14.33%). The result showed that the highest constituted mean of mineral composition was observed with potassium (K) of 16.64{+/-}0.257 mg/kg, while magnesium (Mg) showed the lowest concentration of 3.48{+/-}0.17 mg/kg. A substantial correlation was observed between K and Mg, with a correlation coefficient of 0.72 and 0.72, and similarly between K and Calcium (Ca); the correlation was highly significant, exhibiting a correlation coefficient of 0.65. Furthermore, the correlation between fatty and other physicochemical and proximate analyses showed very insignificant correlations. In general, this study showed that the SBH produced in the current study area has good physicochemical properties and moisture and contains high-quality honey, which may help its traditional medicinal uses. The findings of the study further suggests the potentiality of the area for quality honey, and to easily locate priority areas for stingless bee conservation, further detailed studies of other stingless species honey medicinal values are recommended.

Long-term exposure to high-energy visible (HEV) blue light and infrared-A (IR-A) radiation accelerates oxidative stress, inflammation, and transepidermal water loss (TEWL), leading to photoaging and damage to the skin barrier. In this study, we developed Raybloc(R), a marine bioactive silica microsponge formulation, and evaluated its protective effects against combined high-energy visible (HEV; 410-480 nm) and infrared-A (IR-A; 700-1400 nm) exposure in a preclinical model. We divided 36 nude BALB/c-nu/nu mice into six groups: one that didnt get any treatment, one that got Raybloc(R) (no radiation), one that got Raybloc(R) 5%, one that got Raybloc(R) 8%, one that got HA 0.5%, and one that got HA 0.8%. Animals underwent topical treatment for 14 days under regulated exposure to HEV (410-480 nm, 100 J/cm2/day) and IR-A (700-1400 nm, 30 mW/cm2). We examined transepidermal water loss (TEWL), skin hydration, oxidative stress, inflammatory cytokines (IL-1{beta}, IL-6, TNF-, IL-10), and histological indicators of collagen preservation through biophysical, biochemical, and histopathological techniques. In the Raybloc(R) 8% group, TEWL dropped by 48.3 {+/-} 4.6% (p < 0.001), and skin hydration went up by 62.7 {+/-} 5.1%. The levels of ROS and MMP-1 expression decreased by 63.4% and 57.2%, respectively, while collagen I increased by 2.1 times compared to HA 0.8%. There was a big drop in the pro-inflammatory cytokines IL-1{beta}, IL-6, and TNF- (-54%, -49%, and -46%), and a big rise in IL-10 (+38%). Histological analysis demonstrated well-preserved epidermal integrity and dense collagen bundles in Raybloc(R)-treated mice, whereas irradiated controls exhibited dermal disorganization and inflammatory infiltration. Raybloc(R) showed better photoprotective, antioxidant, and moisturizing effects than HA-based products. It also helped reduce oxidative and inflammatory skin damage caused by blue light and IR-A. These results support Raybloc(R) as a next-generation multifunctional dermocosmetic that can help stop photoaging caused by digital and solar radiation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/713389v1_ufig1.gif" ALT="Figure 1"> View larger version (70K): org.highwire.dtl.DTLVardef@54e046org.highwire.dtl.DTLVardef@502f87org.highwire.dtl.DTLVardef@6088daorg.highwire.dtl.DTLVardef@1b8c241_HPS_FORMAT_FIGEXP M_FIG C_FIG

Space-based platforms currently represent the most accurate means to experimentally assess the influence of the space environment on biological systems. However, performing such experiments remains technically challenging and requires highly specialized instrumentation. This study describes the current development and hardware qualification of ExocubeBio, a miniaturized experimental platform for in-situ biological space exposure. This experiment is scheduled for installation on the exterior of the International Space Station in 2027, as part of Exobio, the European Space Agencys new generation exobiology exposure facility. ExocubeBio will expose live microbial samples to the low Earth orbit environment, and combine autonomous in-situ optical density and fluorescence measurements, with the capacity to return preserved samples to Earth. Achieving these experimental goals requires a specialized, robust and reliable hardware system. The ExocubeBio hardware testing described here includes assessment of material biocompatibility and durability, functional validation of the miniaturized fluidic system, and optimization of the integrated optical subsystem for optical density and fluorescence measurements. These results demonstrate that the ExocubeBio experimental hardware components can each execute their core functional and operational requirements; subsystems allow for sample exposure, in-situ measurements of microbial cultures, and the chemical preservation of samples for post-flight analysis. As ExocubeBio transitions from hardware development to mission readiness, the results presented here validate the overall design and engineering approaches utilized. By combining the strengths of in-situ monitoring and sample return, ExocubeBio represents a significant advancement in space-based experimentation, and will provide new insights into microbial responses to the space environment.

Antimicrobial resistance (AMR) is a significant and growing threat to human, plant and animal health, the global economy, and food security. The One Health approach to AMR recognises the role of the environment in the evolution, emergence, and dissemination of AMR. In part, this is due to anthropogenic pollution that releases AMR organisms alongside cocktails of compounds that may select for AMR in situ, which then pose an exposure risk to humans and animals. This has spurred growing interest from cross-sectoral stakeholders in environmental risk assessment (ERA) of antibiotics, with regards to their selective potential. Many different experimental and modelling approaches have been used to determine the lowest concentration of an antibiotic that may select for AMR. Debates continue regarding which individual approach, if any, may be best for determining concentrations of antibiotics that may select for AMR, for ERA purposes. This paper contributes to this ongoing discourse by refining and using a previously published method SELECT (SELection Endpoints in Communities of bacTeria) to rapidly generate predicted no effect concentrations for resistance (PNECRs) for 32 antibiotics on the premise that reduction in growth of complex community of bacteria correlates with selection for AMR resistance genes. The database of PNECRs of antibiotics presented here is the largest generated using a single experimental, empirical approach that will aid future efforts towards creating a standardised test. PNECR data were used to conduct ERAs using measured environmental concentrations of antibiotics to rank antibiotics by potential selection risk in different environments. The experimental approach and statistical code have been made open access, with online tutorials available to facilitate other laboratories using the SELECT 2.0 method. Finally, we discuss the limitations of this approach and how these could be addressed in future studies.

AimsSponge microbiomes have been extensively studied, in part due to their potential as sources of novel antimicrobials and other biologics, with most research focusing on Demosponges. Here, we investigate the Hexactinellid sponge Pheronema carpenteri, previously identified as a promising source of antibiotic-producing bacteria. MethodsUsing next-generation sequencing of bacterial 16S rRNA genes and a single sponge metagenome, we examined the composition of bacterial communities of P. carpenteri sponges recovered from the Porcupine Seabight, along with local water and sediment samples. ResultsOur results show that P. carpenteri harbours a microbiome abundant in Proteobacteria (47.1-59.4%) and Actinobacteria (11.5-27.5%), with consistent intra-aggregation similarities and structured intra-sponge communities. A metagenomic analysis revealed the presence of several nitrogen cycling genes (nirK, nosZ, nirS homologues of proteobacterial origin), supporting a suggestion that these sponges may play a role in nitrogen cycling, while biosynthetic gene clusters (BGCs) were limited (4 complete clusters). Notably, bacterial community structures within P. carpenteri aggregations resemble those observed in both low and high microbial abundance (LMA/HMA) sponges. ConclusionsHexactinellids are traditionally considered LMA sponges, so identifying species that deviate from this dichotomy provides new insights into sponge microbiome ecology. Integrating Hexactinellids into both culture-dependent and culture-independent studies will advance our broader understanding of sponge-associated microbial diversity and could inform biodiscovery programmes in marine environments. Impact StatementOur findings support the suggestion that a combination of culture-based and molecular analyses is required to generate a comprehensive picture of the biosynthetic potential of P. carpenteri sponges. We also reveal insights into the ecosystem services that sponge microbiomes may contribute towards. These observations could facilitate a deeper understanding of the biotechnological and environmental value of key marine resources.

Fusarium oxysporum FO12 was originally isolated from cork oak (Quercus suber L.) and has been characterised as a highly effective biological control agent of wilt diseases on different crops. FO12 endophytically colonises roots and basal stems of plants, reducing the establishment of the soil-borne pathogen Verticillium dahliae and triggering plant defence-related genes. Here, we report a chromosome-level genome assembly of FO12 using Nanopore and Hi-C data. The 57.60 Mb assembly comprises 14 chromosome-scale scaffolds with centromeres resolved and telomeric repeats detected at 4 of 28 chromosome ends. This high-quality reference genome provides a valuable resource for further research into the use of FO12 in agriculture as a biocontrol agent.

Gastrointestinal disorder caused by the ingestion of (oo)cysts of Cryptosporidium and Giardia is one of the major health problems in developing countries. Fruits and vegetables that are usually consumed unpeeled, poorly washed and or cooked and are the major modes of transmission. Frequent large-scale screening of the food samples is necessary to prevent outbreaks but screening of vegetables for such microbes is limited in Nepal. In this study, we used a smartphone microscopy system to study prevalence and quantification of (oo)cysts of Cryptosporidium and Giardia in 651 vegetable samples collected from nine major vegetable collection sites across Nepal. The overall prevalence rate of vegetable samples was 37.5% with at least with one of the parasites. We found that 23.2% samples were contaminated with Giardia and 33.3% samples were contaminated with Cryptosporidium. Among eight vegetable types, the prevalence rate was lowest in carrot (20%) and highest in spinach (48%). The prevalence rate of vegetable samples at different sites ranged from 13% in Dhading to 61% in Dhangadi. The contamination rate was 28% for winter, 43% for summer and 33% for monsoon seasons in samples collected from Kathmandu. These vegetables should be considered as a potential source of parasitic contamination in people. These vegetables can cause infection if consumed poorly washed and or cooked, posing a potential source of parasitic contamination in people.

Background HIV remains a major public health challenge in South Africa, with gaps in early diagnosis and linkage to care driving onward transmission. Adolescent girls and young women face barriers to timely care, including stigma, privacy concerns, and limited clinic access, while healthcare providers work in resource-constrained settings with high client volumes. We evaluated the Self-Care from Anywhere (SCFA) toolkit, an AI-enabled intervention comprising an AI Companion for AGYW and a provider-facing Clinical Portal to support HIV prevention, testing, and linkage to care. The AI Companion is designed to complement and extend human-delivered services, particularly in resource constrained settings, rather than replace in-person counselling. Methods We conducted an exploratory study to assess the usability, feasibility, and acceptability of the SCFA toolkit in Gauteng Province (November 2024-May 2025). AGYW engaged with the AI Companion, and a subset completed a simulated HIV self-testing activity with AI-delivered counselling. Pre and post-intervention surveys, including the System Usability Scale (SUS), were administered. Usability testing of the Clinical Portal involved healthcare providers using the toolkit without formal training to capture first impressions. A subset of AGYW and healthcare providers participated in separate focus group discussions or in-depth interviews. Quantitative data were analysed using descriptive statistics, and qualitative data were analysed thematically. Results A total of 97 AGYW were enrolled; 75.3% had completed high school and 91.8% were unemployed or full time students. Most participants (85.6%) self-reported HIV-negative status, and 63.9% reported sexual activity in the past 12 months. The AI Companion demonstrated high usability (mean SUS 87.7, SD 12.7) and was perceived as acceptable and useful, particularly for its personalisation and confidentiality features. Healthcare providers had a mean age of 34 years (SD 6.5), with about half serving as HIV testing and screening counsellors. Most providers rated the Clinical Portal ease of use, comprehension, and client support as positive to very positive, though 23% expressed concerns regarding workflow efficiency and their ability to manage additional client volume. Providers also highlighted the Clinical Portal value for case management. Conclusion AI-powered digital health tools, such as the SCFA toolkit, show potential to enhance user engagement and support care delivery, with high usability and acceptability demonstrated among AGYW and healthcare providers. Continued user-centred refinement is essential to ensure these tools remain responsive to the evolving needs and care contexts of diverse user groups.

BACKGROUNDAutomated antimicrobial susceptibility testing (AST) systems are crucial for accurate, timely detection of drug-resistant microbial isolates. This meta-analysis assessed the performance of the BD Phoenix ("Phoenix", BD Diagnostic Solutions), Vitek(R) 2 ("Vitek 2", bioMerieux), and DxM MicroScan WalkAway ("MicroScan", Beckman Coulter, Inc.) AST systems relative to common reference methodology. METHODSA systematic literature search in Ovid (MEDLINE and Embase) yielded 275 unique (not duplicated) records, with 44 additional records retrieved from handsearching; 39 studies met inclusion criteria. Categorical agreement (CA), essential agreement (EA), very major errors (VMEs), and major errors (MEs) for the three instruments were compared to a common reference method. Ratios of proportions were analyzed using random-effect meta-regression. RESULTSThe instruments did not differ significantly in CA, EA, or ME. Vitek 2 showed a higher overall VME rate than Phoenix ([~]44% higher; Vitek 2-to-Phoenix ratio = 1.44; p=0.062 [approaching significance]) and MicroScan (74% higher; ratio = 1.74; p=0.045). No appreciable difference was observed for VME between Phoenix and MicroScan. Subgroup analyses should be interpreted cautiously due to limited overall significance indicating varying performance across systems. Vitek 2 generally had higher relative VMEs for gram-negative organisms and lower relative VMEs for gram-positive organisms, whereas Phoenix showed the opposite pattern. MicroScan had relatively low VMEs when stratified by Clinical and Laboratory Standards Institute (CLSI) criteria; no differences in VMEs were observed using European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. CONCLUSIONAlthough some VME differences were noted, overall performance of the three systems was comparable. Organism- and drug-specific VME patterns--and updates to CLSI criteria over time--highlight the importance of continued monitoring of current breakpoints for all three instruments.

The Bacillus genus comprises physiologically versatile, endospore-forming bacteria widely distributed in natural environments. In this study, we report the isolation and genomic characterization of strain Bva_UNVM-123, recovered from agricultural soil in Pergamino, Argentina. Whole-genome sequencing using Illumina technology yielded a 5.1 Mbp draft genome assembled in 67 contigs with a GC content of 36%. Comparative genomic analyses using the TYGS server and digital DNADNA hybridization (dDDH) values supported its classification as a potentially novel species within the Bacillus sensu lato (s.l.) group. Genome annotation revealed 4,866 protein-coding genes, including multiple determinants conferring resistance to antibiotics (e.g., fosfomycin, tetracycline, beta-lactams) and toxic heavy metals (e.g., arsenic, cadmium, mercury), supporting its potential application in bioremediation. Additionally, PathogenFinder predicted a low probability of human pathogenicity (0.207), reinforcing its safety for environmental use. Functional classification based on Swiss-Prot further supported a metabolically versatile profile and revealed the presence of resistance-related categories associated with environmental adaptation. This study adds to the growing knowledge of environmental Bacillus species and their biotechnological potential

Antimicrobial resistance (AMR) presents a pressing need to ensure that the right antimicrobials are used to target the right microbes at the right time. Ideally, the appropriate antimicrobial is selected after patient samples have been cultured and assessed with antimicrobial sensitivity testing (AST). However, the time needed for culture-based diagnosis leads to immediate empirical treatment, often with broad-spectrum and/or high-tier antimicrobials. Direct nanopore metagenomic whole genome sequencing to identify pathogens and predict their antimicrobial resistance is a rapid and patient-side alternative. A limitation of this approach is potential inconsistencies in in silico predicted AMR phenotypes. Here, we benchmarked the current performance of in silico AMR prediction strategies for nanopore-generated long read data. Using nanopore data paired with AST phenotyping for 201 samples, we assessed the impact of basecalling mode, data volume, and assembly strategy, and compared the performance of eight in silico AMR prediction tools with seven AMR databases. We found that basecalling accuracy mode does not affect the overall accuracy of in silico AMR predictions, but assembly strategy and data volume both do. Prediction tools using the ResFinder database scored best for balanced accuracy (0.80 {+/-} 0.02 for both ResFinder and ABRicate), whilst DeepARG scored best for sensitivity (0.65 {+/-} 0.03). However, even the best performing in silico AMR prediction strategy missed some resistance identified by lab-based AST. In silico AMR prediction can therefore supplement lab-based AST, but cannot yet replace it. Impact statementAntimicrobial resistance (AMR) is threatening modern standards of human and veterinary healthcare. Rapid and patient-side diagnostic tests are needed to diagnose bacterial infections and allow clinicians to select effective antibiotics. Current tests based on bacterial cultures take several days, which may delay diagnosis and treatment, or lead to inappropriate "just in case" treatment while waiting for the results. In contrast, nanopore metagenomic whole genome sequencing can identify bacterial infections and predict which antibiotics will be effective in minutes to hours. However, the accuracy of these tests is uncertain. We therefore compared the performance of eight AMR prediction tools and seven databases of AMR determinants, using 201 bacterial samples with known antibiotic susceptibility and resistance. We found that the sensitivity (i.e. false negative rate), specificity (i.e. false positive rare) and overall accuracy of the tools and databases varied. In particular, even the best performing AMR prediction methods missed some AMR. Therefore, while these tools are useful for rapid and patient-side diagnosis and treatment decisions, they still have limitations and should be used alongside bacterial cultures and antibiotic sensitivity testing. Data summarySequencing data for the samples sequenced for this study are available at the NCBI under BioProject ID PRJNA1292816 (SRA accessions for all datasets used here are available in Supplementary Table S1). All commands and code used can be found at: https://github.com/nataliering/nanopore_AMR_tools/ The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.