International Journal of Food Microbiology

In many tropical countries, fermentation of palm sap into palm wine is an important fermented beverage contributing to local economies, tradition, and culture. Traditionally made in villages and families, palm sap is not inoculated with starter cultures and fermentation commences spontaneously. It is therefore possible that fermentation is influenced by multiple ecological factors, which affect microbial dynamics and thus flavour outcomes. Here, we studied microbial communities during fermentation of palm sap from three different palm tree species (palmyra, coconut, and sugar palm) on the island of Bali, Indonesia in both the wet and dry seasons. Our results suggest that season of collection has a strong influence on microbial dynamics and succession, and these changes positively correlate to metabolite concentration. The change of the season from the dry to wet season led to the loss of microbial diversity with lower richness in the dry season. The dominance of Saccharomyces cerevisiae was not affected by season and fermentation time and was dominant in all samples. Potential spoilage species, such as Candida tropicalis were negatively correlated to ester production and more abundant in the dry season. As microbial species varied in incidence and thus biochemical activity, the chemical groups of esters from their metabolism related to the change of season and fermentation time, while volatile compounds and small molecules were highly discriminated by season in the resultant wines. Ethyl octanoate was consistently different across all variables through comparison by three-way ANOVA and is proposed as a biomarker of seasonal variation in palm sap fermentation. These findings improve our understanding of microbial dynamics in palm sap fermentation, revealing flavour differentiation within season and suggests that strategies for microbial management, product development and quality assurance will elevate this traditional product into the future.

BackgroundReliable detection of Salmonella remains a major challenge for public health surveillance and food safety due to the growing diversity of circulating serovars and the limitations of existing molecular targets. This study aimed to identify an optimal molecular target and develop a TaqMan real-time PCR assay for the detection of Salmonella spp. MethodsBased on the results screening for Salmonella genes suitability as molecular markers, a TaqMan real-time PCR assay targeting the hilA gene was developed and validated. Analytical sensitivity, analytical specificity, and performance on bacterial isolates and artificially contaminated food samples were assessed. ResultsAmong all candidate targets, hilA demonstrated the broadest coverage and was detected in all tested Salmonella isolates, including representatives of rare serological groups, whereas invA conventionally used for this pathogen detection, was absent in a subset of strains. The assay exhibited a limit of detection of 100 bacterial cells/mL and 100 fg/L of genomic DNA. No cross-reactivity was observed with DNA from Shigella flexneri, Shigella sonnei, Yersinia pestis, Y. pseudotuberculosis, Y. enterocolitica, Y. kristensenii, Bacillus anthracis, Vibrio cholerae, or Francisella tularensis. The assay successfully detected Salmonella DNA in all artificially contaminated food samples tested. Evaluation using a collection of 25 bacterial isolates demonstrated positive amplification in all 24 confirmed Salmonella strains, while a strain initially identified by conventional bacteriology as Salmonella but subsequently confirmed by whole-genome sequencing as Proteus mirabilis yielded a negative result. ConclusionsThe hilA gene represents a highly conserved and reliable molecular target for the detection of Salmonella spp. The developed TaqMan real-time PCR assay demonstrated high analytical sensitivity, excellent specificity, and broad serovar coverage, supporting its application in laboratory detection of Salmonella, food safety monitoring, and epidemiological surveillance.

Background: Human activities such as mining generally lead to increased heavy metal concentrations in the environment. While traditional remediation techniques are often costly, the use of fungi as bioremediators, known as mycoremediation, is increasingly gaining attention as a sustainable approach for removal of heavy metals. Here, we evaluated heavy metal levels inside the Kiirunavaara iron ore mine in Northern Sweden and analysed fungal responses to various metal concentrations by comparing growth and metal uptake in mine-derived isolates and closely related control isolates. Results: Sediments inside the mine were enriched in heavy metals compared to those from the outlet of the mine to natural lakes. Six Fusarium isolates were recovered from contaminated mining environments: five isolates from inside the mine were identified as Fusarium oxysporum, and one isolate from the outlet was identified as Fusarium tricinctum. Isolates from the mine and outlet showed overall higher survival and biomass production in presence of copper, iron, and zinc across a range of concentrations (up to 1000 mg/L) compared to control isolates. At the same time, these isolates often exhibited reduced relative metal uptake. As a result, mycoremediation potential, assessed as total uptake in the grown mycelium, was isolate-dependent. Conclusions: Based on these results, we conclude that Fusarium isolates from the Kiirunavaara mine show increased growth in media enriched with heavy metals compared to closely related control isolates. We additionally show that mycoremediation potential is not necessarily associated with environmental origin. Instead, mycoremediation potential should be evaluated on a case-by-case basis for each isolate and based on specific needs for mycoremediation.

It was recently reported that Methylobacterium extorquens AM1 produces the citrate-hydroxamate siderophore N-deoxyschizokinen A, identified by LC-HRMS. Multiple properties were inconsistent with the assignment: the feature eluted far later than the other schizokinen derivatives (17 min versus 6-8 min), a reversed-phase shift larger than a single-hydroxyl difference in a molecule can explain, further its accurate mass deviated from the calculated one by 28 ppm, well outside the error on the co-analyzed standards and its diagnostic m/z 105 and 77 fragments suggest a molecule with an aromatic moiety. A replicate comparison of identical samples in plastic versus glass autosampler vials was decisive: the m/z 387 feature was reproducibly present with plastic vials and absent with glass. We therefore conclude that the reported detection of N-deoxyschizokinen A in M. extorquens AM1 is an artifact, and recommend glass-vial and solvent-blank controls, an explicit accurate-mass threshold, and narrow MS/MS isolation when assigning trace siderophore-like features from complex extracts.

Nutrient-rich food-processing wastewaters represent valuable yet under-utilised side streams for sustainable protein production in the form of microbial biomass. Here we present an integrated dual-loop bioprocess that converts soybean-processing wastewater into microbial single-cell protein (SCP) while achieving substantial nutrient removal and product refinement. In the first loop, previously enriched microbial consortia were inoculated and cultivated in four parallel sequencing batch reactors (SBRs) for 44days at a hydraulic retention time (HRT) of 3days. This bioprocess configuration demonstrated features that support future scale-up while maintaining process stability, achieving a protein content of 33.3{+/-}3.2%, doubling the protein yield (15.32{+/-}3.49g dry weight per g soluble TKN) and quadrupling the production rate (0.29{+/-}0.06g dry weight L-1 d-1) compared to operating reactors without inoculation (HRT: 7.2days). Effluent treatment was stable, with 84% carbon and 78% nitrogen removal efficiencies, demonstrating efficient nutrient recovery. The SCP biomass was enriched in functional taxa, including Acidipropionibacterium, Lactococcus, Megasphaera, and Azospirillum, suggesting that reactor conditions and inoculum selection promoted a stable, protein-productive microbial community with potential probiotic benefits. In the second loop, bioreactor effluent was reused as aqueous matrix for heat treatment (60{degrees}C) of the SCP biomass, reducing the RNA content from 8.6% to 2.6%, with a 39% biomass loss accompanied by a 30% increase in total amino acid concentration. Hence, our valorisation approach integrates microbial biomass production, effluent reuse, and product refinement within a circular framework. The system provides a resource-efficient pathway for converting food-sector side streams into high-quality microbial community-based SCP, highlighting its potential scalability for sustainable nutrient and water management.

Course-based undergraduate research experiences (CUREs) are widely recognized as a high-impact practice in biology education, yet most existing CURE frameworks treat the research organism as an interchangeable teaching prop rather than a genuine scientific contribution. We argue that place-based, community-embedded CUREs - in which students isolate, characterize, and publicly deploy a locally meaningful wild organism - constitute a qualitatively distinct model warranting broader adoption. As proof of concept, we present the Declaration of Fermentation project at Indiana University Bloomington: graduate researchers isolated a wild Saccharomyces cerevisiae strain from the bark of a campus landmark tree, confirmed its wild provenance by whole-genome sequencing and phylogenomics, and partnered with local craft breweries to produce a colonial-era inspired ale released publicly for the 250th anniversary of the Declaration of Independence. Volunteer sensory panels at two independent public tasting events (combined n = 33-34 per attribute) confirmed a fruity-funky profile consistent with wild-strain fermentation, with no significant differences between events (Mann-Whitney U, Benjamini-Hochberg-corrected p > 0.05 for all 11 attributes). We describe three design principles - genomically confirmed strain identity, mandatory community partnership, and place-based historical narrative - that distinguish this model from prior wild yeast brewing CUREs, discuss how these principles generalize to other institutions and fermentation vehicles, and identify next steps for formal learning assessment. Complete implementation protocols are provided as supplemental Appendices 1-6, and the bioinformatics pipeline is freely available at https://doi.org/10.5281/zenodo.20679384.

From Brazilian limestone caves, we isolated 29 bacteria utilizing phenol (23 bacteria), toluene (all bacteria), and/or benzene (all bacteria) as sole carbon sources. One isolate showed phosphate solubilization, while lipase/esterase activity occurred in two isolates; no amylase activity was detected, but 16 isolates ([~]55%) exhibited protease activity. Among them, Nocardioides sp. SF1 was selected for whole-genome sequencing due to its aromatic compound tolerance and protease activity. Additionally, catechol cleavage assays yielded unexpected purple pigmentation, suggesting non-canonical aromatic metabolism. Its high-quality draft genome (4.25 Mbp, 16 contigs, N50 of 887 kb) lacks canonical phenol hydroxylase but encodes alternative oxidation systems, phenylacetyl-CoA pathway, besides, desferrioxamine siderophore, biosurfactants, and phosphate solubilization, key adaptations for oligotrophic caves and biotechnologically interesting activities. Whole-genome comparisons (TYGS/GGDC, OrthoANI and k-mer) suggest potential new species. Lacks acquired antimicrobial resistance genes (ResFinder) and pathogenicity potential (PathogenFinder). Nocardioides sp. SF1 emerges as a non-pathogenic candidate for aromatic bioremediation and plant growth promotion in contaminated, nutrient-poor environments, highlighting cave actinobacterias unexplored biotechnological potential.

The COVID-19 pandemic has imposed a reevaluation of safety protocols across various sectors, including the arts. This study addresses a critical gap in understanding SARS-CoV-2 persistence on materials commonly associated with musical instruments and scores, such as alloys, varnishes, reeds, and paper. While previous research has explored viral survival on various surfaces, limited data exists for materials specific to musical contexts. In this work, we investigate the efficacy of quarantine as a non-destructive method for inactivating SARS-CoV-2 on 16 materials, including brass, silver plating, ABS plastic, ebonite, and various varnishes and paper types. Results revealed significant variability in viral persistence across materials. Non-porous surfaces like metals and ABS plastic cleared infectivity within 3 days, while porous materials such as reeds and music scores required up to 7 days. Gold-plated brass and certain varnishes showed intermediate persistence, with infectivity clearing after 4 days. These findings are in agreement with prior studies indicating that SARS-CoV-2 survival is highly dependent on surface composition, with porous and organic-coated materials retaining viable virus longer due to reduced environmental stress. Our results highlight the feasibility of stratified quarantine protocols based on material type, offering practical guidelines for musicians and institutions and provides critical insights for mitigating SARS-CoV-2 transmission risks in musical settings.

Hemp (Cannabis sativa L.) is an increasingly important crop with applications spanning fibre, seed oil and bioactive cannabinoid production, yet the development of reliable tissue culture systems for this species remains a significant challenge. The establishment of axenic seedling cultures is a prerequisite for hypocotyl-based regeneration and future genetic transformation pipelines, but hemp seeds harbour diverse endophytic microbial communities that frequently overwhelm standard surface sterilisation protocols. Here, we present a systematic comparative evaluation of seed sterilisation strategies across seven industrial hemp accessions, examining the effects of sterilisation chemistry, seed provenance and accession identity on both contamination outcome and the subsequent morphogenic competence of hypocotyl explants. Across all treatments and accessions, in-house glasshouse-harvested seeds achieved higher sterility rates than commercially sourced material regardless of protocol applied. This provenance effect, combined with considerable batch-to-batch variation within seed sources, indicates that contamination load is a primary determinant of successful hemp seed sterilisation. Among the sterilisation treatments evaluated, a baseline of 75% ethanol combined with sequential 1% hydrogen peroxide incubation performed consistently well for low-load seed batches, while supplementation with Plant Preservative Mixture (PPMTM) might be necessary to achieve acceptable rates of non-contaminated seedlings from high contamination load batches. Beyond their effect on contamination, sterilisation treatments influenced the morphogenic fate of hypocotyl explants independently of sterility outcomes. Notably, seedling treatment with the Prochloraz-based fungicide Octave promoted shoot and root co-regeneration in the absence of exogenous plant growth regulators in some cases. Hormone-free organogenesis from hypocotyl explants was achievable across multiple hemp accessions, demonstrating that this developmental capacity is broadly distributed within hemp, though its frequency and consistency varied with accession identity and protocol conditions. Together, these findings provide a practical framework for axenic hemp seed culture that can be used as starting point requiring local adaptation based on seed source, batch history and the intended downstream application.

Compact poultry raising has turned poultry litter into an environmental problem, as it may all be packed with heavy metals and drug-resistant germs. Of all the metals, chromium contamination not only disturbs the general environment but is also a source of concern for public health. Poultry litters were taken from 14 farms in different places, and the bacteria characters from different places were tested for their capacity to tolerate Cr(VI). A total of 31 bacterial isolates were initially screened, and three of them (AH-2, AZ-1, and AMF-3) appeared to be very resistant to chromium. The isolates were able to survive at the highest concentration, 800 mg/L of the Cr(VI); however, AH-2 was the most resistant one (MIC: 900 mg/L; MBC: 1000 mg/L). Chromium reduction tests showed that AMF-3 at high concentration showed the maximum chromium reduction, while AH-2 achieved higher chromium reduction at medium concentration. Phenotypic and biochemical analysis showed that the isolates were Staphylococcus spp., which was confirmed by 16S rRNA gene sequencing as S. cohnii, S. saprophyticus, and S. gallinarum. Moreover, chromium was detected at higher levels in poultry litter compared to the feed, with the highest accumulation in AZ farm litter (4464.0 {micro}g/kg). The highlighting feature of our article is the presence of chromium-tolerant and reducing bacteria in poultry environments. Besides that, the level of chromium in poultry litter is really high, and it points to the need for better waste management.

Antimicrobial resistance represents a continuing threat to clinical infection management, with methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Escherichia coli identified by the World Health Organization as priority pathogens. This study evaluated the antimicrobial activity, synergistic potential, and chemical composition of six plant-derived preparations (three ethanolic extracts: nettle, thyme, rosemary; and three essential oils: lavender, lemongrass, doTERRA Peace blend) against MRSA, methicillin-sensitive S. aureus (MSSA), and E. coli K-12 by disc diffusion, broth microdilution, post-exposure culturability, antimicrobial interactions assessed by checkerboard assay, and GC-MS profiling. Disc diffusion produced no interpretable zones of inhibition for any plant preparation tested; however, broth microdilution revealed reproducible inhibitory activity within published ranges across the panel. Three essential oils achieved a median Minimum Inhibitory Concentration (MIC) of 0.39 mg/mL against MRSA despite presenting compositionally distinct chemotypes: lavender was linalool-dominated (61% combined), lemongrass was citral-dominated (76%), and the doTERRA blend was sesquiterpene-rich. Rosemary ethanolic extract achieved the same potency (0.39 mg/mL) against MSSA. No preparation produced a bactericidal reduction (>=3 log10 CFU/mL) at any timepoint, with all reductions transient and recovering by 24 hours. Checkerboard combinations of plant preparations with vancomycin and ciprofloxacin were uniformly classified, according to the Fractional Inhibitory Concentration Index (FICI), as indifference/no interaction, attributable in part to inoculum-mediated effects on vancomycin MIC. To analyse the relationship between chemical composition and antimicrobial outcomes, we introduce a Chemotype Similarity Index (CSI), a chemometric framework quantifying pairwise compositional similarity between essential oils by Pearson correlation and relating it to log2-MIC differences across strains. CSI revealed a strain-dependent chemistry-activity relationship, convergent against MRSA, monotonic against MSSA, and absent against E. coli, indicating that compositional similarity predicts antimicrobial outcomes on a strain-specific basis. The convergence of three chemotypically divergent essential oils with the same anti-MRSA potency suggested a shared membrane-disrupting mechanism operating through distinct chemical routes. Although exploratory at this scale, the CSI framework provides a reusable analytical scaffold for linking phytochemical composition to antimicrobial activity, and identifies the MRSA convergence as a specific direction for mechanistic investigation into the development of plant-derived antimicrobial adjuncts.

Biological control strategies are increasingly being explored as sustainable alternatives for managing rice blast disease caused by Magnaporthe oryzae. In this study, we characterized three Bacillus pumilus isolates (DC01, DC09, and DC13) and evaluated their antifungal and plant-beneficial properties against M. oryzae. Whole genome sequencing revealed multiple biosynthetic gene clusters associated with the production of antimicrobial metabolites. All three isolates inhibited fungal growth in dual-culture assays, whereas heat-stable diffusible antifungal activity was primarily associated with the cell-free supernatants of DC09 and DC13. Exposure to bacterial supernatants disrupted fungal development, inducing abnormal hyphal morphology characterized by bulbous swelling, altered polarity, and increased branching in M. oryzae. Volatile organic compound assays further revealed that the DC isolates suppress fungal growth in the absence of physical contact. The isolates additionally inhibited the growth of other phytopathogenic fungi and selected human bacterial pathogens. All strains exhibited plant growth-promoting traits, including indole-3-acetic acid production and osmotic stress tolerance, whereas DC09 also displayed phosphate-solubilizing activity. Importantly, root inoculation with the DC isolates significantly reduced rice blast disease severity and induced expression of defense-associated genes involved in jasmonic acid/ethylene signaling and immune priming. Collectively, these findings identify the DC isolates, particularly DC09 and DC13, as promising multi-mechanistic biological control agents for sustainable rice blast management.

Endophytic biocontrol agents may contribute to grapevine health, but their ability to establish, persist, and move within woody tissues remains poorly understood. In this study, a stem injection method was developed to introduce Pseudomonas chlororaphis M71 and Trichoderma atroviride SC1 into rooted and grafted grapevine plants, and their spatial and temporal colonization patterns were compared with the movement of a dye tracer. The dye tracer moved rapidly through xylem tissues, whereas both microorganisms showed more restricted early distribution. Over time, M71 and SC1 displayed distinct colonization patterns. M71 persisted after injection, but remained localized near the inoculation site, with limited movement toward roots or distal aerial tissues. In grafted plants, M71 recovery depended on the injection site and declined more markedly after rootstock injection than after scion injection. In contrast, SC1 showed broader and more persistent colonization. In rooted cuttings, SC1 was recovered from stem and root tissues up to 56 days post-injection, and in grafted plants it was recovered across the graft union, particularly after scion injection. Microscopy supported internal localization of both microorganisms. GFP-labelled M71 and SC1 hyphae were observed mainly within xylem vessels, and viable microorganisms were recovered from corresponding wood tissues. No contamination was observed in control plants. These results show that beneficial microorganisms can be introduced into grapevine tissues by stem injection and that bacterial and fungal biocontrol agents differ markedly in their internal movement and persistence. IMPORTANCEIntroducing beneficial microorganisms directly into plant tissues could help in establishing protective endophytic populations, but little is known about how such microorganisms move and persist inside grapevine. This study shows that stem injection can deliver Pseudomonas chlororaphis M71 and Trichoderma atroviride SC1 into grapevine tissues without visible phytotoxicity. The two microorganisms followed different colonization patterns. M71 remained mostly localized near the injection site, whereas SC1 spread more broadly and persisted in both rooted and grafted plants. These findings provide a basis for developing targeted endophytic biocontrol strategies in grapevine propagation and early plant establishment. This approach may be particularly relevant for grapevine trunk diseases and other vascular disorders, in which pathogens colonize internal woody tissues.

As extreme heat events increase in frequency and intensity worldwide, understanding how woody perennial crops respond to higher maximum temperatures is critical. Perennials face distinct challenges, persisting across many seasons under increasingly variable and extreme conditions, and heat waves threaten the viability of wine grape cultivars through impacts on yield, wine quality, and long-term vine health. To test whether irrigation practices before and during heat waves affect grapevine carbon (C) storage and health, we experimentally manipulated irrigation regimes surrounding heat waves from 2019-2021 in a commercial Cabernet Sauvignon vineyard in the Lodi AVA of Californias Central Valley. Vine physiological traits and yield were measured throughout, and whole-vine nonstructural carbohydrate (NSC) concentrations were quantified after three growing seasons. Although lower supplemental irrigation reduced photosynthesis, stomatal conductance, and fruit yield, whole-vine NSCs did not differ significantly in any perennial organ by the experiments end, indicating that reproductive output and final NSC status responded to irrigation on different timescales. These results suggest that moderate supplemental irrigation during heat events is sufficient to mitigate negative impacts on yield and quality while supporting recovery of NSC reserves, though longer-term monitoring is needed to confirm that this short-term resilience persists.

Pre-harvest litter management is a key determinant of broiler production conditions, influencing NH3; generation, pathogen exposure, nutrient retention, and microbial reservoirs that accumulate across production cycles. Conventional chemical and physical management strategies can support flock health, but their effects on pathogen-associated bacterial populations are often transient and may not account for the microbial interactions that govern persistence, exclusion, and community succession. Here, we evaluated an alternative litter management strategy combining IndigoLT pre-/postbiotic with reduced-rate NaHSO4; across two broiler growouts, with litter sampled at the end of each flock to determine impact on prokaryotic microbiome structure, physicochemistry, and Enterococcus abundance. Alternative management influenced observed richness, phylogenetic diversity, community composition, and co-occurrence network structure while reducing the relative abundance of Enterococcus, including E. cecorum and E. hirae. Digital PCR corroborated sequencing-based Enterococcus abundance patterns, although 16S-based treatment effects were not always reflected as lower absolute copy number at terminal sampling, consistent with reduced proportional dominance rather than sustained absolute suppression. Complementary biofilm- and growth-inhibition assays performed with IndigoLT demonstrated context-dependent antibiofilm and bacteriostatic activity against reference and poultry-derived Enterococcus isolates, with stronger responses for E. cecorum than E. hirae and bactericidal-level reductions in viable recovery at higher exposure levels. These findings demonstrate that biologic-based litter management can alter microbiome structure and pathogen-associated taxa under commercial production conditions, providing a basis for microbiome-informed amendment strategies aimed at reducing pathogen load and supporting broiler health.

Climate-driven increases in sea surface temperature have been associated with the expansion of Vibrio species and a corresponding rise in vibriosis cases in both human populations and aquaculture systems. Coastal waters across the south of England are increasingly becoming suitable for the growth and establishment of both human- and aquaculture-associated Vibrio species, potentially increasing vulnerability to the types of infections and disease outbreaks already reported elsewhere in the world. In this study, we report the presence of a diverse and well-established Vibrio community within the Teign Estuary, (Southwest, UK), including the human-pathogenic species V. parahaemolyticus, V. cholerae (non-O1/non-O139), V. alginolyticus, and V. diabolicus, as well as the important aquaculture pathogens V. jasicida, V. aestuarianus, and V. anguillarum. We identified V. diabolicus, a species that was indistinguishable from V. alginolyticus using conventional biochemical identification methods and could only be accurately resolved by whole-genome sequencing and developed novel PCR targets to differentiate these species in the lab. Using the insect infection model Galleria mellonella, we demonstrate that environmental isolates of V. cholerae (non-O1/non-O139), V. parahaemolyticus, and V. alginolyticus possess virulence potential. We also investigated the effects of sewage effluent on the growth of Vibrio isolates from the Teign Estuary and found that sewage can preferentially promote the growth of Vibrio species. Furthermore, several Vibrio isolates were multidrug resistant and carried antimicrobial resistance genes, highlighting the potential role of environmental Vibrio populations in the Teign Estuary as reservoirs of antimicrobial resistance. Together, these findings demonstrate how rising sea surface temperatures and sewage pollution may influence the emergence, persistence, and public health and aquaculture significance of Vibrio species in UK coastal waters.

This study investigated the genomic and biochemical characteristics of two amylolytic microbial strains, NATIVEDY160T (= JE7B6T = NRRL B-68523T) and NATIVEDY161T (= JL13D9T, = NRRL B-68524T) isolated from the rumen of healthy Holstein dairy cattle. Both strains are obligately anaerobic, non-motile, Gram positive, catalase-negative, and oxidase-negative. Morphologically, NATIVEDY160T grows in long coccoid chains while NATIVEDY161T grows in short chains or pairs. NATIVEDY160T can catabolize amygdalin, esculin/ferric citrate, and starch, compared to NATIVEDY161T which utilizes amygdalin, arbutin, esculin/ferric citrate, glycogen, and D-maltose as determined by API 50 CH carbon panels. Starch degradation ability was verified for both strains, but neither showed cellulolytic activity as confirmed by starch agar and Congo red agar assays, respectively. HPLC analysis revealed that lactate was the primary end product of both strains carbohydrate fermentation, while strain NATIVEDY161T also produced small amounts of acetate. 16S rRNA sequences from both strains cluster with the Oscillospiraceae (formerly Ruminococcaceae) lineage Ruminococcus species, but average nucleotide identity of either strain compared to closely related Ruminococcus members was under the species threshold (95%). Genomic, phylogenetic, and phenotypic interrogation support NATIVEDY160T and NATIVEDY161T as novel species. Each strain was isolated from the rumen of dairy cows located within the central valley of southern California, which has a rich history of Dutch and Basque dairy farm ownership and is still the case today in the region. In recognition of the contributions and heritage of the central and southern California dairy industry, the names Ruminococcus hollandia and Ruminococcus vasco are proposed with NATIVEDY160T and NATIVEDY161T as their respective type strains.

Background: Antimicrobial resistance (AMR) is a global health crisis, necessitating alternative antibacterial strategies. Bacteriophages (phages) offer a promising solution, and their use as a therapeutic agent relies on stringent bioinformatic characterisation using whole genome sequencing (WGS) technologies. However, phages are highly diverse, with no clear consensus on best practices concerning phage DNA extraction or sequencing platform. Efficient and repeatable DNA extraction, sequencing, and bioinformatics processes are critical for safety assessments but remain poorly defined. Additionally, the impact of sequencing platform choice and DNA extraction methods on downstream genomic analyses is not well understood. Methods: We evaluated multiple DNA extraction, library preparation, and sequencing approaches using a diverse collection of Pseudomonas phages from the PhageWA biobank. Column-based and precipitation-based DNA extraction methods were compared for DNA yield and recovery efficiency. Genome sequencing was performed using short-read (Illumina) and long-read (Oxford Nanopore Technologies) platforms, incorporating multiple library preparation kits and Nanopore basecalling models. Assemblies were assessed for completeness, quality, and sequence concordance using standardised bioinformatics pipelines, with hybrid Illumina-Nanopore assemblies used as references for comparison. Results: DNA extraction efficiency varied substantially between protocols, with the Puregene precipitation-based method yielding significantly higher DNA recovery than column-based approaches when normalised to phage titre. Illumina sequencing consistently generated complete genome assemblies, although assembly fragmentation was observed for several jumbo phages when using the SeqWell ExpressPlex 2.0 library preparation method. For Nanopore sequencing, ligation-based native barcoding libraries produced longer reads than rapid barcoding libraries, while selection of the Dorado v5.0.0 basecalling model significantly improved read quality. Genome assembly success was dependent on phage genus; native Nanopore sequencing failed to assemble several Pbunavirus genomes, likely due to modified DNA bases, but an amplification-based library preparation successfully resolved these genomes. Across successfully assembled samples, Illumina and Nanopore platforms produced highly concordant genomes with comparable completeness scores, and hybrid polishing identified only minor sequence differences. Conclusions: DNA extraction methodology, sequencing chemistry, and basecalling model selection significantly influence phage WGS outcomes. Precipitation-based DNA extraction improved DNA recovery, while both Illumina and Nanopore sequencing generated high-quality phage genomes suitable for therapeutic characterisation. Nanopore sequencing provided assemblies comparable to Illumina with minimal benefit from hybrid polishing, supporting its routine use for phage genomics. These findings provide practical guidance for phage genome characterisation workflows and contribute to the development of standardised, regulatory-grade approaches for therapeutic phage assessment.

Microbial succession can be used to estimate the postmortem interval (PMI); however, the impact of spatial variability within the cadaver decomposition island (CDI) is not well understood. This study examined spatial variation in necrobiome communities where soil samples were collected over time and across spatial locations from the CDIs of two human body donors. Microbial communities were characterized using 16S rRNA sequencing and statistical modelling of variation and PMI were conducted. Necrobiome community metrics showed no significant differences across anatomical sampling sites within the CDI at a single timepoint. Temporal modelling identified 11 taxa with significant relationships to PMI in one donor, with spatial sampling having a minimal impact on the PMI relationships. Non-linear approaches also identified taxa with likely PMI signals in the second donor. These findings demonstrate that opportunistic sampling can capture robust linear and non-linear PMI signals in later decomposition stages.

Animal cell-cultured ingredients are entering the EU and UK pet food markets under frameworks that do not require pre-market, ingredient-level safety assessments, creating an ethical need for transparent safety disclosure. We present the first public safety dossier for this sector, describing the proprietary mouse embryonic stem cell line PE25 and its derived, non-viable cellular and conditioned media ingredient produced in food and feed-grade media. PE25 characterization confirmed Mus musculus identity, sterility, absence of mycoplasma and replication-competent retroviruses, and stable growth. Doxorubicin-induced p53 stress testing, CD44/BMI1 profiling, and soft agar assays showed no cancer-like traits and a non-tumorigenic profile; the final ingredient contains no viable cells. Independent OECD TG 471 and 487 assays confirmed non-genotoxicity. Heavy metals, biogenic amines, solvents, and chemical residues were below regulatory limits. Given process variability, we recommend case-by-case safety evaluation and propose this dossier as a model for responsible commercialization.