Understanding seagrass seed microbiomes is crucial for developing microbial-mediated methods to improve germination in restoration efforts. Here, we used 16S rRNA gene and ITS2 amplicon sequencing to characterize the bacterial and fungal communities of seeds from the model seagrass, Zostera marina.

ABSTRACT Seagrass restoration practices are evolving to leverage microbiome applications, similar to agricultural systems that have demonstrated how targeted microbial communities enhance crop resilience in challenging environments. While adult seagrass microbiome research has expanded significantly, research on the seed microbiome remains critically understudied. This gap is important given that seeds represent a large portion of restoration efforts. Advancing seed microbiome research requires standardized experimental systems for controlled plant–microbe interaction studies, which are currently lacking in seagrass research. Here, we tested fabricated ecosystem devices (EcoFAB 2.0) as a standardized system for growing Zostera marina (eelgrass) seedlings, enabling a controlled study of aquatic plant–microbe interactions. Using these chambers, we addressed three key questions: (i) can we reliably grow eelgrass in a controlled laboratory setting, (ii) can we manipulate eelgrass microbiota assembly and its long-term trajectory, and (iii) can we detect shifts in the microbiota during plant development (host filtering)? Host morphology measurements and 16S rRNA gene amplicon sequencing were used to track microbiota assembly across three early developmental stages of the host. Because plants were grown in a sterile environment, surface sterilization of seeds (bleach and ethanol) removed epiphytes without disturbing the shared endophytic community, yet microbiota composition remained divergent at Stage 6 (143 differentially abundant ASVs), indicating that seed coat epiphytes make a lasting and distinct contribution to assembly trajectory. We also identified 26 stage-specific indicator ASVs across eelgrass development, suggesting stage-specific microbial associations during seedling establishment. This work demonstrates the potential for targeted manipulation of the microbiome in seagrass for restoration efforts. IMPORTANCE Using the Fabricated Ecosystem 2.0 (EcoFAB 2.0), we were able to successfully control the microbial environment of eelgrass, Zostera marina , resulting in the reduction of epiphytes and maintaining low microbial diversity across plants without compromising the morphology and growth of seedlings. Our findings advance the marine plant model system, Z. marina , by identifying taxonomic indicators across life stages. This work lays the foundation for a targeted understanding and application of microbiomes for seagrass restoration, bridging the critical knowledge gap between agricultural seed microbiome success and marine restoration applications.

Rhesus macaques are widely used model organisms for studying human biology, yet relatively few metagenome-assembled genomes (MAGs) are available from their microbiome. Here, we report 159 MAGs recovered from simian immunodeficiency virus-infected macaques, including those treated either with antiretroviral therapy or 10-hydroxystearic acid.

Background: Restrictions on the types of food available on land have repeatedly triggered the convergent evolution of herbivory in terrestrial animals. This pattern also holds true in many terrestrially adapted crabs, which have independently colonized land more than 17 times since the Cretaceous, and many clades are now almost completely herbivorous, standing in contrast to the ancestral pattern of detrivory. While many bacteria possess efficient pathways for degrading lignin and cellulose, the role of gut microbiomes in facilitating these dietary shifts in terrestrial crabs remains poorly understood. To explore the relationship between microbial community structure and the ability of land crabs to digest lignocellulose, we conducted read-based and assembly-based metagenomic analyses on feces collected from the guts of 14 crab species across six genera, representing a gradient of terrestriality from the lower intertidal zone to forested habitats. Results: We generated 129 metagenome-assembled genomes (MAGs) that represent key members of these gut microbial communities, establishing a foundational resource for future studies on crab-microbiome interactions. We found that host genus explained most of the variation in bacterial community composition, while degree of terrestrial adaptation (i.e. terrestrial grade) explained a smaller proportion. We also identified multiple bacterial genera that strikingly differed in relative abundance across terrestrial grades, crab genera, and diet type. Broad-scale functional analyses of general carbon metabolism across crabs revealed an absence of complete pathways in crabs from lower terrestrial grades, suggesting a functional divergence in gut communities linked to habitat transition. Fine-scale functional analyses of carbohydrate-active enzyme (CAZyme) domains allowed us to connect specific MAGs to lignocellulose degradation pathway genes, demonstrating that different crab genera harbor distinct microbial taxa that have similar CAZyme profiles in their guts. Conclusions: This work provides a foundational metagenomic resource for genomic exploration of microbial communities in terrestrial crab guts. These results suggest that the gut microbiomes of terrestrially adapted crabs are structured primarily by host identity and have convergently acquired microbes with similar functions to help perform lignocellulose degradation. Overall, different degrees of adaptation to terrestrial environments, including resulting dietary shifts, may be responsible for functional divergence in crab gut community assembly.

Phoronids are a phylum of animals with only ~12 described species, all of which are marine filter feeders that build external tubes for shelter and produce chemical deterrents against predators. Many tube-building invertebrates host distinct microbial communities and even have obligate symbionts for survival in sulfur-rich marine sediments. However, the microbiome of phoronids has only recently begun to be described. To address this, we surveyed the composition of the microbiome of the phoronid, Phoronopsis harmeri, using 16S rRNA gene amplicon and metagenomic sequencing. We found that the phoronid microbiome was dominated by members of the orders Campylobacterales, Desulfobulbales, and Desulfobacterales. We also found that the microbiomes of tubes and phoronids were less diverse than that of surrounding sediment, and that the microbiomes of phoronids, tubes and surrounding sediment were all distinctly structured. Based on analysis of metagenomic data, and even though we were only able to recover low quality MAGs of abundant taxa, we found preliminary evidence that taxa associated with phoronids and their tubes likely participate in sulfur cycling pathways. Future work should perform more robust metagenomic sequencing and chemical analysis to assess if there is a link between known phoronid chemical defenses and microorganisms. Overall, this study provides foundational insight into the microbial communities associated with phoronids and these initial findings suggest that these communities may play an important role in sulfur cycling in marine sediments.

The Microbial Dark Matter Symposium held on August 28-29, 2025, in Laguna Beach, Orange County, CA, convened a multidisciplinary group of scientists to address the vast unknowns in microbial life-from uncultured taxa and uncharacterized proteins to elusive viruses and spacefaring microbes. Set against a scenic coastal backdrop, the symposium highlighted advances in single-cell genomics, proximity ligation sequencing, and artificial intelligence-ready bioinformatics, while also probing the limits of microbial persistence, metabolism, and ecological distribution. Sessions explored microbial dark matter from multiple dimensions: cultivability, where new strategies are enabling recovery of elusive microbes; functional ambiguity, where metagenomic dark zones are illuminated by computational annotation; and genomic representation, where single-cell methods bridge gaps left by shotgun community sequencing. Researchers shared breakthroughs in identifying atmospheric microbiomes, "dark oxygen" production in groundwater ecosystems, and microbial survival on the International Space Station. The symposium emphasized integration of methods, disciplines, and ecosystems, advancing a collective push to illuminate the microbial dark matter on Earth and beyond. By highlighting emerging tools, pressing questions, and cross-domain insights, the symposium underscored the need for collaborative, open, and adaptive approaches to study the microbial unknown. The meeting marks a pivotal moment in microbiology, where cultivating knowledge of the uncultivated promises transformative understanding of life, everywhere.

Here, we describe three endosymbiotic bacterial strains isolated from the gills of the shipworm, Bankia setacea (Teredinidae: Bivalvia). These strains, designated as Bs08(T), Bs12(T) and Bsc2(T), are Gram- stain-negative, microaerobic, gammaproteobacteria that grow on cellulose and a variety of substrates derived from lignocellulose. Phenotypic characterization, phylogeny based on 16S rRNA gene and whole genome sequence data, amino acid identity and percentage of conserved proteins analyses, show that these strains are novel and may be assigned to the genus Teredinibacter. The three strains may be differentiated and distinguished from other previously described Teredinibacter species based on a combination of four characteristics: colony colour (Bs12(T), purple; others beige to brown), marine salt requirement (Bs12(T), Bsc2(T) and Teredinibacter turnerae strains), the capacity for nitrogen fixation (Bs08(T) and T. turnerae strains) and the ability to respire nitrate (Bs08(T)). Based on these findings, we propose the names Teredinibacter haidensis sp. nov. (type strain Bs08(T)=ATCC TSD-121(T)=KCTC 62964(T)), Teredinibacter purpureus sp. nov. (type strain Bs12(T)=ATCC TSD-122(T)=KCTC 62965(T)) and Teredinibacter franksiae sp. nov. (type strain Bsc2(T)=ATCC TSD-123(T)=KCTC 62966(T)).

Fungi play pivotal roles in terrestrial ecosystems as decomposers, pathogens, and endophytes, yet their significance in marine environments is often understudied. Seagrasses, as globally distributed marine flowering plants, have critical ecological functions, but knowledge about their associated fungal communities remains relatively limited. Previous amplicon surveys of the fungal community associated with the seagrass, Zostera marina have revealed an abundance of potentially novel chytrids. In this study, we employed deep metagenomic sequencing to extract metagenome-assembled genomes (MAGs) from these chytrids and other microbial eukaryotes associated with Z. marina leaves. Our efforts resulted in the recovery of five eukaryotic MAGs, including a single fungal MAG in the order Loubulomycetales (65% BUSCO completeness), three MAGs representing diatoms in the family Bacillariaceae (93%, 70% and 31% BUSCO completeness) and a single MAG representing a haptophyte algae in the genus Prymnesium (40% BUSCO completeness). Whole-genome phylogenomic assessment of these MAGs suggests they all largely represent under sequenced, and possibly novel eukaryotic lineages. Of particular interest, the chytrid MAG was placed within the order Lobulomycetales, consistent with the identity of the dominant chytrid from previous Z. marina amplicon survey results. Annotation of this MAG yielded 5,650 gene models of which 77% shared homology to current databases. With-in these gene models, we predicted 121 carbohydrate-active enzymes and 393 secreted proteins (103 cytoplasmic effectors, 30 apoplastic effectors). Exploration of orthologs between the Lobulomycetales MAG and existing Chytridiomycota genomes have revealed a landscape of high-copy gene families related to host recognition and interaction. Further machine learning analyses based on carbohydrate-active enzyme composition predict that this MAG is a symbiont. Overall, these five eukaryotic MAGs represent substantial genomic novelty and valuable community resources, contributing to a deeper understanding of the roles of fungi and other microbial eukaryotes in the larger seagrass ecosystem. ### Competing Interest Statement Jonathan A. Eisen is on the Scientific Advisory Board of Zymo Research, Inc. Jason E. Stajich is a scientific consultant for Michroma, Inc.

IntroductionThe amphibian skin microbiome is an important line of defense against pathogens including the deadly chytrid fungus, Batrachochytrium dendrobatidis (Bd). Bd is known to preferentially infect ventral skin surfaces and feet of host amphibians, often leaving dorsal surfaces like the back uninfected. Within-individual variation in infection distribution across the skin, therefore, may relate to differences in microbiomes among skin regions. However, microbiome heterogeneity within amphibian individuals remains poorly characterized.MethodsWe utilized 16S rRNA gene amplicon sequencing to compare microbiomes of 10 body regions from nine captive Rana sierrae individuals and their tank environments. These individuals were naive to Bd, allowing us to assess whether microbiomes differed among body regions prior to any impacts that may be caused by infection.ResultsWe found that frog skin and tank environments harbored distinct microbial communities. On frog skin, the bacterial families Burkholderiaceae (phylum Proteobacteria) and Rubritaleaceae (phylum Verrucomicrobia) were dominant, driven in large part by relative abundances of undescribed members of these families that were significantly higher on frogs than in their environment. Within individuals, we detected differences between microbiomes of body regions where Bd infection would be expected compared to regions that infrequently experience infection. Notably, putative Bd-inhibitory relative abundance was significantly higher on body regions where Bd infection is often localized.DiscussionThese findings suggest that microbiomes in certain skin regions may be predisposed for interactions with Bd. Further, our results highlight the importance of considering intraindividual heterogeneities, which could provide insights relevant to predicting localized interactions with pathogens.

Accurate metagenomic classification relies on comprehensive, up-to-date, and validated reference databases. While the NCBI BLAST Nucleotide (nt) database, encompassing a vast collection of sequences from all domains of life, represents an invaluable resource, its massive size-currently exceeding 1012 nucleotides-and exponential growth pose significant challenges for researchers seeking to maintain current nt-based indices for metagenomic classification. Recognizing that no current nt-based indices exist for the widely used Centrifuge classifier, and the last public version currently available was released in 2018, we addressed this critical gap by leveraging advanced high-performance computing resources. We present new Centrifuge-compatible nt databases, meticulously constructed using a novel pipeline incorporating different quality control measures, including reference decontamination and filtering. These measures demonstrably reduce spurious classifications, as shown through our reanalysis of published metagenomic data where Plasmodium annotations were dramatically reduced using our decontaminated database, highlighting how database quality can significantly impact research conclusions. Through temporal comparisons, we also reveal how our approach minimizes inconsistencies in taxonomic assignments stemming from asynchronous updates between public sequence and taxonomy databases. These discrepancies are particularly evident in taxa such as Listeria monocytogenes and Naegleria fowleri, where classification accuracy varied significantly across database versions. These new databases, made available as pre-built Centrifuge indexes, respond to the need for an open, robust, nt-based pipeline for taxonomic classification in metagenomics. Applications such as environmental metagenomics, forensics, and clinical metagenomics, which require comprehensive taxonomic coverage, will benefit from this resource. Our work highlights the importance of treating reference databases as dynamic entities, subject to ongoing quality control and validation akin to software development best practices. This approach is crucial for ensuring accuracy and reliability of metagenomic analysis, especially as databases continue to expand in size and complexity. IMPORTANCE:Accurately identifying the diverse microbes present in a sample, whether from the human gut, a soil sample, or a crime scene, is crucial for fields ranging from medicine to environmental science. Researchers rely on comprehensive DNA databases to match sequenced DNA fragments to known microbial species. However, the widely used NCBI nt database, while vast, poses significant challenges. Its massive size makes it difficult for many researchers to use effectively with taxonomic classifiers, and inconsistencies and contamination within the database can impact the accuracy of microbial identification. This work addresses these challenges by providing cleaned, updated, and validated nt-based databases specifically optimized for the widely used Centrifuge classification tool. This new resource demonstrably reduces errors and improves the reliability of microbial identification across diverse taxonomic groups. Moreover, by providing readily usable indexes, we overcome the size barrier, enabling researchers to leverage the full potential of the nt database for metagenomic analysis. Our findings underscore the need to treat reference databases as dynamic entities, emphasizing continuous quality control and versioning as essential practices for robust and reproducible metagenomics research.

Seagrass restoration practices are evolving to leverage microbiome applications, similar to agricultural systems that have demonstrated how targeted microbial communities enhance crop resilience in challenging environments. While adult seagrass microbiome research has expanded significantly, research on the seed microbiome remains critically understudied. This gap is particularly important given that seeds represent a large portion of restoration efforts. Advancing seed microbiome research requires standardized experimental systems for controlled plant-microbe interaction studies, which are currently lacking in seagrass research. Here, we tested fabricated ecosystem devices (EcoFAB 2.0) as a standardized system for growing seedlings of Zostera marina (eelgrass), enabling a controlled study of aquatic plant-microbe interactions. Using these chambers, we addressed three key questions: (i) Can we reliably grow eelgrass in a controlled laboratory setting? (ii) Can we manipulate eelgrass microbiome assembly and its long-term trajectory? and (iii) Can we detect shifts in the microbiome during plant development (host filtering)? Host morphology measurements and 16S rRNA gene amplicon sequencing were used to track microbiome assembly across three early developmental stages of the host. We identified 143 ASVs that differed between plants from surface-sterilized versus intact seeds, highlighting the strong influence of epiphytic bacteria on microbiome assembly. We also identified 26 stage-specific indicator ASVs across eelgrass development, suggesting stage-specific microbial associations during seedling establishment. This work demonstrates the potential for targeted manipulation of the microbiome in seagrass for restoration efforts. Importance Using the Fabricated Ecosystem 2.0 (EcoFAB 2.0), we were able to successfully control the microbial environment of Z. marina , resulting in the removal of epiphytes and maintaining low microbial diversity across plants without compromising the growth dynamics of the seedlings. Our findings advance the marine plant model system, Zostera marina , by identifying taxonomic indicators across life stages. This work lays the foundation for a targeted understanding and application of microbiomes for seagrass restoration, bridging the critical knowledge gap between agricultural seed microbiome success and marine restoration applications. ### Competing Interest Statement P.F.A. and T.R.N. are inventors on patent US11510376B2 held by University of California that covers Ecosystem device for determining plant-microbe interactions. T.R.N. and B.P.B. are scientific co-founder and hold equity in Datacodec with prior approval from LBNL. U.S. National Science Foundation, NSF BioOce 2311577 Gordon and Betty Moore Foundation, Marine Model Systems #9359 Joint Genome Institute, CSP New Investigator Proposal 509268 Department of Energy and Environment, Contract No. DE-AC02-05CH11231

Bacterial symbionts, with their shorter generation times and capacity for horizontal gene transfer (HGT), play a critical role in allowing marine organisms to cope with environmental change. The closure of the Isthmus of Panama created distinct environmental conditions in the Tropical Eastern Pacific (TEP) and Caribbean, offering a “natural experiment” for studying how closely related animals evolve and adapt under environmental change. However, the role of bacterial symbionts in this process is often overlooked. We sequenced the genomes of endosymbiotic bacteria in two sets of sister species of chemosymbiotic bivalves from the genera Codakia and Ctena (family Lucinidae) collected on either side of the Isthmus, to investigate how differing environmental conditions have influenced the selection of symbionts and their metabolic capabilities. The lucinid sister species hosted different Candidatus Thiodiazotropha symbionts and only those from the Caribbean had the genetic potential for nitrogen fixation, while those from the TEP did not. Interestingly, this nitrogen-fixing ability did not correspond to symbiont phylogeny, suggesting convergent evolution of nitrogen fixation potential under nutrient-poor conditions. Reconstructing the evolutionary history of the nifHDKT operon by including other lucinid symbiont genomes from around the world further revealed that the last common ancestor (LCA) of Ca. Thiodiazotropha lacked nif genes, and populations in oligotrophic habitats later re-acquired the nif operon through HGT from the Sedimenticola symbiont lineage. Our study suggests that HGT of the nif operon has facilitated niche diversification of the globally distributed Ca. Thiodiazotropha endolucinida species clade. It highlights the importance of nitrogen availability in driving the ecological diversification of chemosynthetic symbiont species and the role that bacterial symbionts may play in the adaptation of marine organisms to changing environmental conditions.

Colletotrichum spp. have a complicated history of association with land plants. Perhaps most well-known as plant pathogens for the devastating effect they can have on agricultural crops, some Colletotrichum spp. have been reported as beneficial plant endophytes. However, there have been only a handful of reports of Colletotrichum spp. isolated from aquatic plant hosts and their ecological role in the marine ecosystem is underexplored. To address this, we present the draft genome and annotation of Colletotrichum sp. CLE4, previously isolated from rhizome tissue from the seagrass Zostera marina. This genome (48.03 Mbp in length) is highly complete (BUSCO ascomycota: 98.8%) and encodes 12,015 genes, of which 5.7% are carbohydrate-active enzymes (CAZymes) and 12.6% are predicted secreted proteins. Phylogenetic placement puts Colletotrichum sp. CLE4 within the C. acutatum complex, closely related to C. godetiae. We found a 8.69% smaller genome size, 21.90% smaller gene count, and the absence of 591 conserved gene families in Colletotrichum sp. CLE4 relative to other members of the C. acutatum complex, suggesting a streamlined genome possibly linked to its specialized ecological niche in the marine ecosystem. Machine learning analyses using CATAStrophy on CAZyme domains predict this isolate to be a hemibiotroph, such that it has a biotrophic phase where the plant is kept alive during optimal environmental conditions followed by a necrotrophic phase where the fungi actively serves a pathogen. While future work is still needed to definitively tease apart the lifestyle strategy of Colletotrichum sp. CLE4, this study provides foundational insight and a high-quality genomic resource for starting to understand the evolutionary trajectory and ecological adaptations of marine-plant associated fungi. ### Competing Interest Statement Jonathan A. Eisen is on the Scientific Advisory Board of Zymo Research, Inc. Jason E. Stajich is a scientific consultant for Michroma, Inc.

Tramway Ridge, a geothermal Antarctic Specially Protected Area (elevation 3340 m) located near the summit of Mount Erebus, is home to a unique community composed of cosmopolitan surface-associated micro-organisms and abundant, poorly understood subsurface-associated microorganisms. Here, we use shotgun metagenomics to compare the functional capabilities of this community to those found elsewhere on Earth and to infer in situ diversity and metabolic capabilities of abundant subsurface taxa. We found that the functional potential in this community is most similar to that found in terrestrial hydrothermal environments (hot springs, sediments) and that the two dominant organisms in the subsurface carry high rates of in situ diversity which was taken as evidence of potential endemicity. They were found to be facultative anaerobic heterotrophs that likely share a pool of nitrogenous organic compounds while specializing in different carbon compounds. Metagenomic insights have provided a detailed understanding of the microbe-based ecosystem found in geothermally heated fumaroles at Tramway Ridge. This approach enabled us to compare Tramway Ridge with other microbial systems, identify potentially endemic taxa and elucidate the key metabolic pathways that may enable specific organisms to dominate the ecosystem.

The design of two overlapping genes in a microbial genome is an emerging technique for adding more reliable control mechanisms in engineered organisms for increased stability. The design of functional overlapping gene pairs is a challenging procedure, and computational design tools are used to improve the efficiency to deploy successful designs in genetically engineered systems. GENTANGLE (Gene Tuples ArraNGed in overLapping Elements) is a high-performance containerized pipeline for the computational design of two overlapping genes translated in different reading frames of the genome. This new software package can be used to design and test gene entanglements for microbial engineering projects using arbitrary sets of user-specified gene pairs.

Journal Article Patient-derived organoids for prediction of treatment response in oesophageal adenocarcinoma Get access Jarlath C Bolger, Jarlath C Bolger Division of Thoracic Surgery, Toronto General Hospital, University Health Network, Toronto, Ontario, CanadaDepartment of Surgery, Royal College of Surgeons in Ireland, Dublin, Ireland https://orcid.org/0000-0002-0796-633X Search for other works by this author on: Oxford Academic Google Scholar Jonathan Allen, Jonathan Allen Latner Thoracic Surgery Research Laboratories, Princess Margaret Cancer Research Tower, University Health Network, Toronto, Ontario, Canada Search for other works by this author on: Oxford Academic Google Scholar Nikolina Radulovich, Nikolina Radulovich Princess Margaret Living Biobank, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada Search for other works by this author on: Oxford Academic Google Scholar Christine Ng, Christine Ng Princess Margaret Living Biobank, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada Search for other works by this author on: Oxford Academic Google Scholar Mathieu Derouet, Mathieu Derouet Latner Thoracic Surgery Research Laboratories, Princess Margaret Cancer Research Tower, University Health Network, Toronto, Ontario, Canada Search for other works by this author on: Oxford Academic Google Scholar Premalatha Shathasivam, Premalatha Shathasivam Latner Thoracic Surgery Research Laboratories, Princess Margaret Cancer Research Tower, University Health Network, Toronto, Ontario, Canada Search for other works by this author on: Oxford Academic Google Scholar Gavin W Wilson, Gavin W Wilson Latner Thoracic Surgery Research Laboratories, Princess Margaret Cancer Research Tower, University Health Network, Toronto, Ontario, Canada Search for other works by this author on: Oxford Academic Google Scholar Ming-Sound Tsao, Ming-Sound Tsao Department of Laboratory Medicine and Pathobiology, University Health Network, Toronto, Ontario, Canada Search for other works by this author on: Oxford Academic Google Scholar Elena Elimova, Elena Elimova Division of Medical Oncology, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada Search for other works by this author on: Oxford Academic Google Scholar Gail E Darling, Gail E Darling Division of Thoracic Surgery, Toronto General Hospital, University Health Network, Toronto, Ontario, CanadaLatner Thoracic Surgery Research Laboratories, Princess Margaret Cancer Research Tower, University Health Network, Toronto, Ontario, Canada Search for other works by this author on: Oxford Academic Google Scholar ... Show more Jonathan C Yeung Jonathan C Yeung Division of Thoracic Surgery, Toronto General Hospital, University Health Network, Toronto, Ontario, CanadaLatner Thoracic Surgery Research Laboratories, Princess Margaret Cancer Research Tower, University Health Network, Toronto, Ontario, Canada Correspondence to: Jonathan Yeung, Department of Surgery, Toronto General Hospital, 200 Elizabeth Street, 9N-949, Toronto, ON M5G 2C4, Canada (e-mail: jonathan.yeung@uhn.ca) https://orcid.org/0000-0001-5759-3028 Search for other works by this author on: Oxford Academic Google Scholar British Journal of Surgery, Volume 111, Issue 1, January 2024, znad408, https://doi.org/10.1093/bjs/znad408 Published: 29 January 2024 Article history Received: 07 September 2023 Revision received: 07 November 2023 Accepted: 19 November 2023 Published: 29 January 2024

Violacein is a water-insoluble violet pigment produced by various Gram-negative bacteria. The compound and the bacteria that produce it have been gaining attention due to the antimicrobial and proposed antitumour properties of violacein and the possibility that strains producing it may have broad industrial uses. Bacteria that produce violacein have been isolated from diverse environments including fresh and ocean waters, glaciers, tropical soils, trees, fish and the skin of amphibians. We report here the isolation and characterization of six violacein-producing bacterial strains and three non- violacein-producing close relatives, each isolated from either an aquatic environment or moist food materials in northern California, USA. For each isolate, we characterized traditional phenotypes, generated and analysed draft genome sequences, and carried out multiple types of taxonomic, phylogenetic and phylogenomic analyses. Based on these analyses we assign putative identifications to the nine isolates, which include representatives of the genera Chromobacterium, Aquitalea, Iodobacter, Duganella, Massilia and Janthinobacterium. In addition, we discuss the utility of various metrics for taxonomic assignment in these groups including average nucleotide identity, whole genome phylogenetic analysis and extent of recent homologous recombination using the software program PopCOGenT.

BackgroundGroundbreaking studies have linked the gut microbiome with immune homeostasis and antitumor immune responses. Mounting evidence has also demonstrated an intratumoral microbiome, including in soft tissue sarcomas (STS), although detailed characterization of the STS intratumoral microbiome is limited. We sought to characterize the intratumoral microbiome in patients with STS undergoing preoperative radiotherapy and surgery, hypothesizing the presence of a distinct intratumoral microbiome with potentially clinically significant microbial signatures.MethodsWe prospectively obtained tumor and stool samples from adult patients with non-metastatic STS using a strict sterile collection protocol to minimize contamination. Metagenomic classification was used to estimate abundance using genus and species taxonomic levels across all classified organisms, and data were analyzed with respect to clinicopathologic factors.ResultsFifteen patients were enrolled. Most tumors were located at an extremity (67%) and were histologic grade 3 (87%). 40% were well-differentiated/dedifferentiated liposarcoma histology. With a median follow-up of 24 months, 4 (27%) patients developed metastases, and 3 (20%) died. Despite overwhelming human DNA (>99%) intratumorally, we detected a small but consistent proportion of bacterial DNA (0.02–0.03%) in all tumors, includingProteobacteria, Bacteroidetes,andFirmicutes, as well as viral species. In the tumor microenvironment, we observed a strong positive correlation between viral relative abundance and natural killer (NK) infiltration, and higher NK infiltration was associated with superior metastasis-free and overall survival by immunohistochemical, flow cytometry, and multiplex immunofluorescence analyses.ConclusionsWe prospectively demonstrate the presence of a distinct and measurable intratumoral microbiome in patients with STS at multiple time points. Our data suggest that the STS tumor microbiome has prognostic significance with viral relative abundance associated with NK infiltration and oncologic outcome. Additional studies are warranted to further assess the clinical impact of these findings.

Whether the human fetus and the prenatal intrauterine environment (amniotic fluid and placenta) are stably colonized by microbial communities in a healthy pregnancy remains a subject of debate. Here we evaluate recent studies that characterized microbial populations in human fetuses from the perspectives of reproductive biology, microbial ecology, bioinformatics, immunology, clinical microbiology and gnotobiology, and assess possible mechanisms by which the fetus might interact with microorganisms. Our analysis indicates that the detected microbial signals are likely the result of contamination during the clinical procedures to obtain fetal samples or during DNA extraction and DNA sequencing. Furthermore, the existence of live and replicating microbial populations in healthy fetal tissues is not compatible with fundamental concepts of immunology, clinical microbiology and the derivation of germ-free mammals. These conclusions are important to our understanding of human immune development and illustrate common pitfalls in the microbial analyses of many other low-biomass environments. The pursuit of a fetal microbiome serves as a cautionary example of the challenges of sequence-based microbiome studies when biomass is low or absent, and emphasizes the need for a trans-disciplinary approach that goes beyond contamination controls by also incorporating biological, ecological and mechanistic concepts.

Molecular biology methods and technologies have advanced substantially over the past decade. These new molecular methods should be incorporated among the standard tools of planetary protection (PP) and could be validated for incorporation by 2026. To address the feasibility of applying modern molecular techniques to such an application, NASA conducted a technology workshop with private industry partners, academics, and government agency stakeholders, along with NASA staff and contractors. The technical discussions and presentations of the Multi-Mission Metagenomics Technology Development Workshop focused on modernizing and supplementing the current PP assays. The goals of the workshop were to assess the state of metagenomics and other advanced molecular techniques in the context of providing a validated framework to supplement the bacterial endospore-based NASA Standard Assay and to identify knowledge and technology gaps. In particular, workshop participants were tasked with discussing metagenomics as a stand-alone technology to provide rapid and comprehensive analysis of total nucleic acids and viable microorganisms on spacecraft surfaces, thereby allowing for the development of tailored and cost-effective microbial reduction plans for each hardware item on a spacecraft. Workshop participants recommended metagenomics approaches as the only data source that can adequately feed into quantitative microbial risk assessment models for evaluating the risk of forward (exploring extraterrestrial planet) and back (Earth harmful biological) contamination. Participants were unanimous that a metagenomics workflow, in tandem with rapid targeted quantitative (digital) PCR, represents a revolutionary advance over existing methods for the assessment of microbial bioburden on spacecraft surfaces. The workshop highlighted low biomass sampling, reagent contamination, and inconsistent bioinformatics data analysis as key areas for technology development. Finally, it was concluded that implementing metagenomics as an additional workflow for addressing concerns of NASA's robotic mission will represent a dramatic improvement in technology advancement for PP and will benefit future missions where mission success is affected by backward and forward contamination.