Microbiology Resource Announcements

Compared with bacterial and archaeal extremophiles, single-celled eukaryotes living in extreme habitats are understudied and underrepresented in genomic databases. An exception is the obligately halophilic stramenopile Halocafeteria seosinensis strain EHF34. A transcriptome-focused analysis of this extremophilic protists revealed the importance of organic osmolyte regulation and transport in its adaptation to hypersaline environments. However, genomic resources for H. seosinensis are currently limited to a highly fragmented assembly generated by short-read sequencing, which has hindered further investigation of the genome biology and evolution of this fascinating organism. Here, we used long-read Oxford Nanopore sequencing to generate a highly contiguous, chromosome-scale genome assembly for H. seosinensis. The assembly is 38.8 megabase pairs (Mbp) in size and contains 60 nuclear contigs, making it the most contiguous genome for a member of the order Bicosoecida. Approximately 19% of the genome is comprised of transposable elements. Of the 11,684 predicted protein-coding genes, many appear to be associated with DNA mobility-related functions, and several may be linked to adaptation to a hypersaline environment. Analysis of the H. seosinensis long-read genome assembly presented herein will facilitate our understanding of the ways in which protists have adapted to extreme environments. SignificanceHalocafeteria seosinensis is an extremophilic protist adapted to hypersaline environments. Previous analyses of a transcriptome and short-read draft genome assembly for this organism provided insights into the molecular mechanisms underlying osmotic regulation, which facilitate its adaptation to high-salt conditions. However, the lack of contiguity and quality of the draft assembly prevented the characterization of complex genomic regions, including transposable elements and viral insertions, as well as genomic comparisons with related species. Here we present a highly contiguous, chromosome-scale genome assembly for H. seosinensis that enables accurate gene prediction, detailed analysis of repeat content, and comparative genomic analysis. This long-read genome assembly will serve as a valuable resource for studying one of the few tractable halophilic protists sequenced to date.

Entamoeba histolytica is a clinically important pathogenic eukaryote and the causative agent of amoebic dysentery. Entamoeba dispar, a nonpathogenic commensal species that resides in the human colon, is the closest sibling species, and serves as an appropriate comparator for genome-wide analysis. Although the genome of E. histolytica is approximately 26.9 Mb, and the largest known genome within the genus, that of E. invadens, is approximately 40.9 Mb, obtaining high-quality assemblies in this genus has remained challenging due to extensive repetitive regions, tRNA gene arrays, and aneuploidy. Here, we used PacBio HiFi sequencing to assemble the genomes of the pathogenic E. histolytica and the nonpathogenic E. dispar. We reconstructed all 36 chromosomes of E. histolytica and 35 chromosomes of E. dispar, assembling each as a single continuous DNA sequence (contig). The two species exhibited high genome-wide nucleotide similarity and conserved synteny at the amino acid level. At one end of each chromosome, we identified tRNA arrays, whereas the opposite end lacked such arrays, resulting in an asymmetric chromosomal architecture. Analysis of unique-read depth revealed widespread aneuploidy in both species: E. histolytica is predominantly tetraploid, whereas E. dispar is diploid, a conclusion further supported by SNP allele-frequency distributions. These assemblies provide a robust foundation for comparative genomics in Entamoeba and offer detailed insights into chromosome-end structure and ploidy.

Marine photosynthetic microbes contribute substantially to global primary production, yet many algal lineages still lack reference genomes with the continuity and annotation quality required for fine-scale structural, regulatory and comparative analyses. Ostreococcus tauri, one of the smallest known free-living photosynthetic eukaryotes, has been a model marine picoeukaryote for over two decades. Despite successive improvements to its historical reference genome, previous assemblies retained hundreds of gaps and incomplete genes, hampering high-resolution genomic analyses. Here, we present O. tauri RCC4221 genome version 2026, a telomere-to-telomere assembly of all 20 chromosomes spanning 13.34 Mb with no gaps. This assembly combines PacBio long-read sequencing, Illumina short-read polishing, correction of unresolved regions guided by independent Nanopore-based assemblies. The updated reference supports a curated annotation comprising 7,683 protein-coding genes, 48 tRNA genes, 3 rRNA operons, 116 medium-sized noncoding RNAs, one signal recognition particle RNA and 138 small nucleolar RNAs. It also improves gene-model integrity and recovers candidate coding loci absent from the 2014 reference. Structural analyses resolved the organization of the two atypical low-GC chromosome 2 and 19 that contain duplicated regions that were collapsed or misrepresented in previous assemblies. Finally, bisulfite sequencing and PacBio SMRT sequencing revealed a dual DNA methylation landscape, with CG-context cytosine methylation concentrated in gene bodies and N6-methyladenosine (m6A) enriched at the start codon. The updated O. tauri 2026 assembly provides a complete and curated reference resource for chromosome biology, comparative genomics, epigenomics and RNA biology in a model marine picoeukaryote.

Syngas fermentation by carbon monoxide (CO)-utilising acetogens offers a sustainable route for converting gasified waste materials into value-added chemicals. In this study, we isolated a novel thermophilic CO-utilising bacterium, strain AZ2, from marine hydrothermal sediment collected on the island of Sao Miguel, Azores, Portugal. Strain AZ2 is an obligately anaerobic, spore-forming bacterium. Average nucleotide identity (ANI; 78.4-86.7%) and digital DNA-DNA hybridization (dDDH; 23.4-32.5 %) analyses indicate that strain AZ2 represents a novel species within a previously uncharacterised lineage represented by the GTDB placeholder genus UBA2545 in the Neomoorellaceae family. Strain AZ2 was able to grow fermentatively on CO, producing acetate. We further demonstrated that its closest isolated relatives - Thermanaeromonas toyohensis, T. burensis, and Thermanaeromonas sp. strain 9S - are capable of growing on CO, producing either acetate or hydrogen gas (H2). Additionally, we unveiled the genomic potential for CO utilisation within other members of the GTDB placeholder class DSM-521 (previously Moorellia) to which our isolate belongs, expanding the list of possible thermophilic CO-utilising acetogens. We propose that strain AZ2T represents the type strain of a novel genus and species, named Thermobium azorense gen. nov., sp. nov. (= DSM 121889T = JCM 39698T).

Background Following the cessation of COVID-19 travel restrictions in July 2022, concerns about a delayed dengue outbreak prompted the Solomon Islands Ministry of Health to establish enhanced genomic surveillance of circulating dengue virus (DENV) strains. Methods We performed amplicon-based whole genome sequencing (WGS) on PCR-positive serum samples collected at the National Referral Hospital, Honiara, between January 2023 and March 2025 (n = 63). Genomes were compared with publicly available sequences, and maximum-likelihood phylogenies were used to explore regional transmission dynamics. Findings We generated the first whole genome sequences from Solomon Islands (n = 45), with high recovery rates from acute infections (90%, Ct 17-44, mean coverage > 80%). Co-circulation of DENV-1, DENV-2, and DENV-4 was observed, with evidence of a serotype shift emerging in 2024. Phylogeographic analyses suggest ancestral introductions from Papua New Guinea for DENV-1 and DENV-2. Interpretation This study demonstrates the feasibility of whole genome sequencing for dengue surveillance in the Solomon Islands through a referral sequencing model that provides a pathway for progressive local capacity-building. By addressing technical challenges and critical gaps in regional genomic representation, our findings strengthen the evidence base needed for equitable and sustainable implementation of pathogen genomics across Pacific Island countries and territories.

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.

Oligella otitidis MSHR-50489EDL strain (ATCC: TSD462; DSMZ: DSM118617) is a new species of the genus Oligella that was isolated from a middle ear discharge swab from a child with chronic suppurative otitis media (CSOM). This Gram-negative coccobacillus produces small, circular, smooth, whitish-opaque and occasionally mucoid colonies. It grows in aerobic conditions at a temperature range from 25-42oC. Phylogenetic analysis demonstrates a relationship to other species of the genera Oligella and average nucleotide identity and digital DNA/DNA hybridization values indicate a distinct species in comparison to other Oligella species. Thus far, the majority of isolates exhibit resistance to ciprofloxacin, the first line treatment for CSOM.

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 rat tapeworm, Hymenolepis diminuta, is an important laboratory model for uncovering molecular processes that underly the success of tapeworms as parasites. Despite its importance, a high-quality reference genome for this species is lacking. Here we present a highly contiguous and effectively complete genome of H. diminuta assembled from PacBio HiFi long-read sequencing data. Our primary assembly consists of 7 scaffolds (N50=29.25 Mb) with total length of 186.53 Mb, has only 7 gaps, and contains 95.7% complete Lophotrochozoan BUSCOs. Our assembly allows us to confirm aspects of Hymenolepis genome organization, such as high repeat content and unusual chromosomal ends, and to show that Hymenolepis genomes encode [~]10,000 genes. Together with annotations of nuclear tRNAs, mtDNA protein coding genes, and mtDNA tRNAs, our assembly currently provides one of the most complete genome resources for a tapeworm species and will enable research on parasitism, animal regeneration, development, and evolution.

Plant-associated bacteria can promote plant growth under saline conditions, but salinity-dependent changes in bacterial physiological traits remain insufficiently understood. Here, we isolated bacteria from seedlings of quinoa (Chenopodium quinoa Willd.) lines maintained under laboratory propagation for more than 30 years and evaluated their activity under saline conditions. A quinoa-associated Pantoea isolate, strain 6PN, promoted primary root elongation and whole-plant dry weight of Arabidopsis thaliana under salt stress, whereas no significant effect was observed under non-saline conditions. Comparative analyses with reference Pantoea agglomerans strains showed that strain 6PN exhibited salinity-responsive indole-3-acetic acid (IAA) production. Genome analysis identified a putative ipdC gene and additional genes related to stress responses, nutrient acquisition, polysaccharide biosynthesis and export, flagellar biosynthesis, and chemotaxis. Phylogenomic analysis indicated that strain 6PN was genomically distinct from representative Pantoea species examined here. In an Arabidopsis trench-plate assay, GFP-labeled strain 6PN was recovered from spatially separated plant tissues at higher levels than a GFP-labeled reference strain under saline conditions. These results identify strain 6PN as a quinoa-associated Pantoea isolate with salinity-responsive IAA production and plant growth-promoting activity under defined salt-stress conditions.

Wastewater treatment relies on complex microbial communities, yet existing genome-resolved references for this essential engineered ecosystem remain dominated by short-read assemblies, limiting genome contiguity and linkage between taxonomic and metabolic function. We applied long-read sequencing to activated sludge from 83 globally distributed plants, reconstructing 53,501 metagenome-assembled genomes to establish the Microbial Database of Activated Sludge (MiDAS) global genome catalog. The catalog encompasses high-quality genomes for 12,047 prokaryotic species, 82% of which are not represented in GTDB release 226, and provides a median of 32 high-quality genomes for each of the 250 core prokaryotic genera previously defined in our MiDAS global 16S rRNA gene survey. This enables analyses of predicted functional traits and their ecological context, for example, we identified two sparsely represented Nitrospiraceae genera with conserved nitrite-oxidation genes that are abundant in higher-temperature wastewater treatment plants. In summary, the MiDAS genome catalog provides a framework for linking taxonomy, metabolism and ecological roles in wastewater treatment systems globally.

BackgroundHerpesviruses are a group of double-stranded DNA viruses known to infect a wide range of vertebrates and establish life-long latent infections. While bats serve as natural reservoir hosts for numerous viral families, relatively few bat herpesviruses have been successfully isolated. In this study, we report the isolation and characterization of two novel alphaherpesvirus strains obtained from Rousettus aegyptiacus bats in Coastal Kenya. MethodsThe samples of oral and rectal swabs were collected from three different species of bats from coastal Kenya between October 2024 and April 2025; the bat species collected include Hipposideros spp., Coleura afra, and Rousettus aegyptiacus. Virus isolation was performed by inoculation of samples in Vero E6 cells and subsequent monitoring for cytopathic effects (CPE). Total nucleic acids were extracted from CPE positive cultures and subjected to library preparation to enable unbiased detection of both RNA and DNA viruses. The libraries were sequenced using next-generation sequencing with Illumina MiSeq platform. Subsequently, bioinformatic analysis was carried out to identify the virus, generate consensus genomes as well as phylogenetic analysis to determine the placement of identified viruses. ResultsTwo samples from R. aegyptiacus (KIK_460_O and KIK_465_O) induced typical CPE within five days. Sequencing and assembly yielded partial consensus sequences of approximately 60 kb (KIK_460_O) and 70 kb (KIK_465_O), representing extended genomic data for a bat-associated alphaherpesvirus. This virus has a genome of about 140kb, indicating that our partial assemblies account for about 43-50% of the total genome. Both isolates were found to be closely related to Dzifa herpesvirus, an alphaherpesvirus previously identified in Kilifi, Kenya. Alphaherpesvirus was identified based on partial sequencing of UL19 (3,787bp) and UL30 (2,846bp) genes. The two isolates were found to be identical at the UL19 gene, showing that they belonged to the same virus strain. Phylogenetic analysis showed that the novel alphaherpesvirus belongs to primate alphaherpesviruses under the subfamily Alphaherpesvirinae. ConclusionThis study reports the isolation and genomic characterization of a novel fruit bat alphaherpesvirus from Kenyan Rousettus aegyptiacus bats. The partial genome assembly (60-70 kb) represent the first extended genomic data for this virus, covering approximately 43-50% of the estimated 140 kb complete genome. The phylogenetic placement of this alphaherpesvirus near primate viruses, especially Pteropodid alphaherpesvirus 1, suggests bat-association and needs further investigation into its zoonotic potential.

We present an update to the PIGEON (Phages and Integrated Genomes Encapsidated or Not) reference database of DNA viral sequences (vOTUs, mostly dsDNA bacteriophages) from global ecosystems. To reflect the inclusion of only virus size-fractionated metagenome- (virome-)derived vOTUs, we reintroduce the database as PEONY (Phages Encapsidated ONlY) and present new data summarizing the utility of this database.

Microbial fuel cells (MFCs) technology offers sustainable electricity production. Current research largely focuses on few select model organisms, therefore the true prevalence of exoelectrogenesis amongst bacteria remaining largely unknown. We present a broad-scale survey of monomicrobial electricity production among environmental bacterial isolates inoculated in MFCs, using model organism Shewanella oneidensis MR-1 as a benchmark. Of the assessed taxa, 11-22% displayed exoelectrogenic activity, exceeding current predictions and identifying a further three novel exoelectrogenic species. Phylogenetic analysis based on the 16S sequences enabled the evolutionary relationship between isolates to be visualised, revealing that exoelectrogenesis is non-randomly distributed and phylogenetically conserved. Polarisation studies were implemented, revealing that numerous electron transfer mechanism were being utilised to perform exoelectrogenesis. The results of this study imply that bacterial electricity production is more widespread amongst culturable bacteria than previously estimated, with implications for bioprospecting novel exoelectrogens and predicting electrogenic activity in diverse microbial communities.

Fusarium oxysporum MRL8996 is a fungal strain isolated from a severe case of contact lens-associated keratitis. Here we report a near-complete genome assembly of this isolate using a hybrid Nanopore and Hi-C scaffolding approach. The assembly resolves the genome into 16 distinct chromosomes, including 11 core and 5 lineage-specific chromosomes. This high-quality reference genome provides an unprecedented tool for investigating the large-scale structural variations and evolutionary mechanisms driving adaptation in this highly versatile fungal lineage.

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.

During mycological surveys in Yunnan Province, China, specimens of a fungus producing massive, upright stromata up to 50 cm high and individually 2.2 Kg in weight were sampled. Through an integrative taxonomic approach combining detailed morphology, multilocus phylogeny (ITS, LSU, RPB2, TUB2), and phylogenomic analyses, this fungus is proposed as the new species Dianjunus rex gen. et sp. nov., the type of the new family Dianjunaceae (Xylariales). Phylogenetic analyses robustly place Dianjunaceae as a distinct sister clade to Graphostromataceae. Divergence time estimation dates the origin of this family to the early Paleocene (~65 Mya), coinciding with the post-K-Pg extinction period, when an estimated 75% of all plant and animal species went extinct, and a significant ecological reorganization of life on earth happened. The stromata of D. rex represent the largest fructifications documented within the Ascomycota, significantly expanding the known morphological range of the Xylariales. The study provides a comprehensive description, including a nodulisporium-like anamorph with periconiella-like branching patterns, and discusses the taxon's phylogenetic placement, and distinctive morphology. This discovery highlights the unexplored fungal diversity in East Asian forests.

Rabies remains a significantly underreported fatal zoonosis in India, where the Arctic-like 1a (AL1a) lineage predominates in dog populations. While atypical clinical presentations in dogs can delay diagnosis and increase human exposure risk, genomic and clinical data on neonatal canine rabies remain limited. This study reports an exceptional case of rabies in a 54-day old unvaccinated German shepherd puppy which presented with severe pruritus and self-biting behaviour. The puppy was euthanized due to poor clinical response. Post-mortem testing revealed viral antigen (by Direct Fluorescent Antibody Test) and viral RNA (by real-time RTPCR) in the brain tissue. Whole-genome sequencing recovered a near-complete rabies virus genome (11,947 nucleotides; 99.5% genome coverage), classified within the AL1a_A1.1 sublineage. Phylogenetic analysis revealed close genetic relatedness to contemporary Indian rabies virus strains. Comparative genomic analysis identified 4, 3, 6, and 8 non-synonymous substitutions in the phosphoprotein, matrix, glycoprotein, and polymerase genes, respectively. This case is one of the youngest documented cases of canine rabies with atypical manifestations, caused by the AL1a viral clade. Our findings highlight the risks associated with neonatal canine rabies, the need for heightened clinical suspicion in atypical cases, and the importance of genomic surveillance to monitor evolving rabies virus lineages in endemic regions.

We describe a chromosome-level, haplotype-resolved genome assembly from a male European moose (Alces alces alces). The assembly comprises two pseudo-haplotypes of 3,148 Mb and 3,112 Mb, with sex chromosomes in haplotype one, and 33 autosomes in each haplotype (68 in total). Assembly completeness is high (BUSCO 98.3% and 95.7%), with 21,496 and 20,498 annotated protein-coding genes for haplotypes one and two, respectively. This genome assembly is the most complete so far generated for European moose.

Rodents are an important reservoir of zoonotic viruses and are ubiquitously present in densely populated urban areas. Betacoronaviruses in the Embecovirus lineage are well known to infect both humans and animals and have established rodent reservoirs. Here three Betacoronavirus gravedinis genomes were sequenced and characterized in white footed mice (Peromyscus leucopus, commonly white footed mice) collected in New York City, the second most populous city in North America. The genomes were distinct from mouse hepatitis virus (MHV), the prototype mouse betacoronavirus, and highly similar and identical in one case to previously characterized B. gravedinis sequences from white footed mice in Connecticut. Codon aware evolutionary models were used to identify specific sites under positive selection within the spike protein of B. gravedinis. A novel method was developed to predict the probable geographic distribution of the virus using publicly available data from the Global Biodiversity Information Facility to generate a weighted distribution map highlighting overlapping potential host ranges based on the evolutionary distance using a high resolution cytocrome B (CYTB) phylogeny of rodent species with potentially overlapping ranges. Our models predict three current hotspots of circulation in North America under different possible transmission regimes, and an additional fourth hotspot was predicted to arise in a warming future. This study highlights the continued need for biodiversity-informed surveillance of potential zoonotic pathogens in rodents.