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In this perspective, we explore the transformative impact and inherent limitations of metagenomics and single-cell genomics on our understanding of microbial diversity and their integration into the Tree of Life. We delve into the key challenges associated with incorporating new microbial lineages into the Tree of Life through advanced phylogenomic approaches. Additionally, we shed light on enduring debates surrounding various aspects of the microbial Tree of Life, focusing on recent advances in some of its deepest nodes, such as the roots of bacteria, archaea, and eukaryotes. We also bring forth current limitations in genome recovery and phylogenomic methodology, as well as new avenues of research to uncover additional key microbial lineages and resolve the shape of the Tree of Life.

Antimicrobial resistance genes (ARG) are commonly found on acquired mobile genetic elements (MGEs) such as plasmids or transposons. Understanding the spread of resistance genes associated with mobile elements (mARGs) across different hosts and environments requires linking ARGs to the existing mobile reservoir within bacterial communities. However, reconstructing mARGs in metagenomic data from diverse ecosystems poses computational challenges, including genome fragment reconstruction (assembly), high-throughput annotation of MGEs, and identification of their association with ARGs. Recently, several bioinformatics tools have been developed to identify assembled fragments of plasmids, phages, and insertion sequence (IS) elements in metagenomic data. These methods can help in understanding the dissemination of mARGs. To streamline the process of identifying mARGs in multiple samples, we combined these tools in an automated high-throughput open-source pipeline, MetaMobilePicker, that identifies ARGs associated with plasmids, IS elements and phages, starting from short metagenomic sequencing reads. This pipeline was used to identify these three elements on a simplified simulated metagenome dataset, comprising whole genome sequences from seven clinically relevant bacterial species containing 55 ARGs, nine plasmids and five phages. The results demonstrated moderate precision for the identification of plasmids (0.57) and phages (0.71), and moderate sensitivity of identification of IS elements (0.58) and ARGs (0.70). In this study, we aim to assess the main causes of this moderate performance of the MGE prediction tools in a comprehensive manner. We conducted a systematic benchmark, considering metagenomic read coverage, contig length cutoffs and investigating the performance of the classification algorithms. Our analysis revealed that the metagenomic assembly process is the primary bottleneck when linking ARGs to identified MGEs in short-read metagenomics sequencing expe

Metagenomic sequencing has proven to be a powerful tool in the monitoring of antimicrobial resistance (AMR). Here, we provide a comparative analysis of the resistome from pigs, poultry, veal calves, turkey, and rainbow trout, for a total of 538 herds across nine European countries. We calculated the effects of per-farm management practices and antimicrobial usage (AMU) on the resistome in pigs, broilers, and veal calves. We also provide an in-depth study of the associations between bacterial diversity, resistome diversity, and AMR abundances as well as co-occurrence analysis of bacterial taxa and antimicrobial resistance genes (ARGs) and the universality of the latter. The resistomes of veal calves and pigs clustered together, as did those of avian origin, while the rainbow trout resistome was different. Moreover, we identified clear core resistomes for each specific food-producing animal species. We identified positive associations between bacterial alpha diversity and both resistome alpha diversity and abundance. Network analyses revealed very few taxa-ARG associations in pigs but a large number for the avian species. Using updated reference databases and optimized bioinformatics, previously reported significant associations between AMU, biosecurity, and AMR in pig and poultry farms were validated. AMU is an important driver for AMR; however, our integrated analyses suggest that factors contributing to increased bacterial diversity might also be associated with higher AMR load. We also found that dispersal limitations of ARGs are shaping livestock resistomes, and future efforts to fight AMR should continue to emphasize biosecurity measures.IMPORTANCEUnderstanding the occurrence, diversity, and drivers for antimicrobial resistance (AMR) is important to focus future control efforts. So far, almost all attempts to limit AMR in livestock have addressed antimicrobial consumption. We here performed an integrated analysis of the resistomes of five important farmed ani

Extremophiles and their products have been a major focus of research interest for over 40 years. Through this period, studies of these organisms have contributed hugely to many aspects of the fundamental and applied sciences, and to wider and more philosophical issues such as the origins of life and astrobiology. Our understanding of the cellular adaptations to extreme conditions (such as acid, temperature, pressure and more), of the mechanisms underpinning the stability of macromolecules, and of the subtleties, complexities and limits of fundamental biochemical processes has been informed by research on extremophiles. Extremophiles have also contributed numerous products and processes to the many fields of biotechnology, from diagnostics to bioremediation. Yet, after 40 years of dedicated research, there remains much to be discovered in this field. Fortunately, extremophiles remain an active and vibrant area of research. In the third decade of the twenty-first century, with decreasing global resources and a steadily increasing human population, the world’s attention has turned with increasing urgency to issues of sustainability. These global concerns were encapsulated and formalized by the United Nations with the adoption of the 2030 Agenda for Sustainable Development and the presentation of the seventeen Sustainable Development Goals (SDGs) in 2015. In the run-up to 2030, we consider the contributions that extremophiles have made, and will in the future make, to the SDGs.

Author contributions: MG: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Validation, Visualization, Writing – original draft. IN: Investigation, Resources, Writing – review & editing. L-JB: investigation, Resources, Writing – review & editing. JG: Data curation, Funding acquisition, Resources, Supervision, Writing – review & editing. SO-P: Data curation, Funding acquisition, Resources, Writing – review & editing. AR: Data curation, Resources, Writing – review & editing. SO: Data curation, Funding acquisition, Resources, Software, Writing – review & editing. VM: Funding acquisition, Resources, Writing – review & editing. BM: Resources, Writing – review & editing. BG-Z: Funding acquisition, Writing – review & editing. MB: Funding acquisition, Resources, Writing – review & editing. LM: Funding acquisition, Resources, Writing – review & editing. BB: Resources, Writing – review & editing. KV: Resources, Software, Writing – review & editing. NR: Funding acquisition, Resources, Writing – review & editing. MA: Funding acquisition, Project administration, Resources, Supervision, Writing – review & editing. SDK: Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Writing – original draft., Metagenomic sequencing is a promising method that has the potential to revolutionize the world of pathogen detection and antimicrobial resistance (AMR) surveillance in food-producing environments. However, the analysis of the huge amount of data obtained requires performant bioinformatics tools and databases, with intuitive and straightforward interpretation. In this study, based on long-read metagenomics data of chicken fecal samples with a spike-in mock community, we proposed confidence levels for taxonomic identification and AMR gene detection, with interpretation guidelines, to help with the analysis of the output data generated by KMA, a popular k-mer read alignment tool. Additionally, we demonstrated that the completeness and diversity of the genomes present in the reference databases are key parameters for accurate and easy interpretation of the sequencing data. Finally, we explored whether KMA, in a two-step procedure, can be used to link the detected AMR genes to their bacterial host chromosome, both detected within the same long-reads. The confidence levels were successfully tested on 28 metagenomics datasets which were obtained with sequencing of real and spiked samples from fecal (chicken, pig, and buffalo) or food (minced beef and food enzyme products) origin. The methodology proposed in this study will facilitate the analysis of metagenomics sequencing datasets for KMA users. Ultimately, this will contribute to improvements in the rapid diagnosis and surveillance of pathogens and AMR genes in food-producing environments, as prioritized by the EU., European Comission, Depto. de Sanidad Animal, Fac. de Veterinaria, TRUE, pub

Giant viruses (GVs) are key players in ecosystem functioning, biogeochemistry, and eukaryotic genome evolution. GV diversity and abundance in aquatic systems can exceed that of prokaryotes, but their diversity and ecology in lakes, especially polar ones, remain poorly understood. We conducted a comprehensive survey and meta-analysis of GV diversity across 20 lakes, spanning polar to temperate regions, combining our extensive lake metagenome database from the Canadian Arctic and subarctic with publicly available datasets. Leveraging a novel GV genome identification tool, we identified 3304 GV metagenome-assembled genomes, revealing lakes as untapped GV reservoirs. Phylogenomic analysis highlighted their dispersion across all Nucleocytoviricota orders. Strong GV population endemism emerged between lakes from similar regions and biomes (Antarctic and Arctic), but a polar/temperate barrier in lacustrine GV populations and differences in their gene content could be observed. Our study establishes a robust genomic reference for future investigations into lacustrine GV ecology in fast changing polar environments.

Studies using 16S rRNA and shotgun metagenomics typically yield different results, usually attributed to PCR amplification biases. We introduce Greengenes2, a reference tree that unifies genomic and 16S rRNA databases in a consistent, integrated resource. By inserting sequences into a whole-genome phylogeny, we show that 16S rRNA and shotgun metagenomic data generated from the same samples agree in principal coordinates space, taxonomy and phenotype effect size when analyzed with the same tree.

Functional traits influence the assembly of microbial communities, but identifying these traits in the environment has remained challenging. We studied ectomycorrhizal fungal (EMF) communities inhabiting Populus trichocarpa roots distributed across a precipitation gradient in the Pacific Northwest, USA. We profiled these communities using taxonomic (meta-barcoding) and functional (metagenomic) approaches. We hypothesized that genes involved in fungal drought-stress tolerance and fungal mediated plant water uptake would be most abundant in drier soils. We were unable to detect support for this hypothesis; instead, the abundance of genes involved in melanin synthesis, hydrophobins, aquaporins, trehalose-synthases, and other gene families exhibited no significant shifts across the gradient. Finally, we studied variation in sequence homology for certain genes, finding that fungal communities in dry soils are composed of distinct aquaporin and hydrophobin gene sequences. Altogether, our results suggest that while EMF communities exhibit significant compositional shifts across this gradient, coupled functional turnover, at least as inferred using community metagenomics is limited. Accordingly, the consequences of these distinct EMF communities on plant water uptake remain critically unknown, and future studies targeting the expression of genes involved in drought stress tolerance are required.

Ticks are increasingly important vectors of human and agricultural diseases. While many studies have focused on tick-borne bacteria, far less is known about tick-associated viruses and their roles in public health or tick physiology. To address this, we investigated patterns of bacterial and viral communities across two field populations of western black-legged ticks (Ixodes pacificus). Through metatranscriptomic analysis of 100 individual ticks, we quantified taxon prevalence, abundance, and co-occurrence with other members of the tick microbiome. In addition to commonly found tick-associated microbes, we assembled 11 novel RNA virus genomes from Rhabdoviridae, Chuviridae, Picornaviridae, Phenuiviridae, Reoviridae, Solemovidiae, Narnaviridae and two highly divergent RNA virus genomes lacking sequence similarity to any known viral families. We experimentally verified the presence of these in I. pacificus ticks across several life stages. We also unexpectedly identified numerous virus-like transcripts that are likely encoded by tick genomic DNA, and which are distinct from known endogenous viral element-mediated immunity pathways in invertebrates. Taken together, our work reveals that I. pacificus ticks carry a greater diversity of viruses than previously appreciated, in some cases resulting in evolutionarily acquired virus-like transcripts. Our findings highlight how pervasive and intimate tick-virus interactions are, with major implications for both the fundamental biology and vectorial capacity of I. pacificus ticks.ImportanceTicks are increasingly important vectors of disease, particularly in the United States where expanding tick ranges and intrusion into previously wild areas has resulted in increasing human exposure to ticks. Emerging human pathogens have been identified in ticks at an increasing rate, and yet little is known about the full community of microbes circulating in various tick species, a crucial first step to understanding how they interact with e

The fields of Metagenomics and Metatranscriptomics involve the examination of complete nucleotide sequences, gene identification, and analysis of potential biological functions within diverse organisms or environmental samples. Despite the vast opportunities for discovery in metagenomics, the sheer volume and complexity of sequence data often present challenges in processing analysis and visualization. This article highlights the critical role of advanced visualization tools in enabling effective exploration, querying, and analysis of these complex datasets. Emphasizing the importance of accessibility, the article categorizes various visualizers based on their intended applications and highlights their utility in empowering bioinformaticians and non-bioinformaticians to interpret and derive insights from meta-omics data effectively.

Culture-based microbial natural product discovery strategies fail to realize the extraordinary biosynthetic potential detected across earth's microbiomes. Here we introduce Small Molecule In situ Resin Capture (SMIRC), a culture-independent method to obtain natural products directly from the environments in which they are produced. We use SMIRC to capture numerous compounds including two new carbon skeletons that were characterized using NMR and contain structural features that are, to the best of our knowledge, unprecedented among natural products. Applications across diverse marine habitats reveal biome-specific metabolomic signatures and levels of chemical diversity in concordance with sequence-based predictions. Expanded deployments, in situ cultivation, and metagenomics facilitate compound discovery, enhance yields, and link compounds to candidate producing organisms, although microbial community complexity creates challenges for the later. This compound-first approach to natural product discovery provides access to poorly explored chemical space and has implications for drug discovery and the detection of chemically mediated biotic interactions.

Historically neglected by microbial ecologists, soil viruses are now thought to be critical to global biogeochemical cycles. However, our understanding of their global distribution, activities and interactions with the soil microbiome remains limited. Here we present the Global Soil Virus Atlas, a comprehensive dataset compiled from 2,953 previously sequenced soil metagenomes and composed of 616,935 uncultivated viral genomes and 38,508 unique viral operational taxonomic units. Rarefaction curves from the Global Soil Virus Atlas indicate that most soil viral diversity remains unexplored, further underscored by high spatial turnover and low rates of shared viral operational taxonomic units across samples. By examining genes associated with biogeochemical functions, we also demonstrate the viral potential to impact soil carbon and nutrient cycling. This study represents an extensive characterization of soil viral diversity and provides a foundation for developing testable hypotheses regarding the role of the virosphere in the soil microbiome and global biogeochemistry.

Comparative analysis of (meta)genomes necessitates aggregation, integration, and synthesis of well-annotated data using standards. The Genomic Standards Consortium (GSC) collaborates with the research community to develop and maintain the Minimum Information about any (x) Sequence (MIxS) reporting standard for genomic data. To facilitate the use of the GSC's MIxS reporting standard, we provide a description of the structure and terminology, how to navigate ontologies for required terms in MIxS, and demonstrate practical usage through a soil metagenome example.

BACKGROUND: Chronic exposure to microorganisms inside homes can impact respiratory health. Few studies have used advanced sequencing methods to examine adult respiratory outcomes, especially continuous measures. We aimed to identify metagenomic profiles in house dust related to the quantitative traits of pulmonary function and airway inflammation in adults. Microbial communities, 1264 species (389 genera), in vacuumed bedroom dust from 779 homes in a US cohort were characterized by whole metagenome shotgun sequencing. We examined two overall microbial diversity measures: richness (the number of individual microbial species) and Shannon index (reflecting both richness and relative abundance). To identify specific differentially abundant genera, we applied the Lasso estimator with high-dimensional inference methods, a novel framework for analyzing microbiome data in relation to continuous traits after accounting for all taxa examined together. RESULTS: Pulmonary function measures (forced expiratory volume in one second (FEV1), forced vital capacity (FVC), and FEV1/FVC ratio) were not associated with overall dust microbial diversity. However, many individual microbial genera were differentially abundant (p-value < 0.05 controlling for all other microbial taxa examined) in relation to FEV1, FVC, or FEV1/FVC. Similarly, fractional exhaled nitric oxide (FeNO), a marker of airway inflammation, was unrelated to overall microbial diversity but associated with differential abundance for many individual genera. Several genera, including Limosilactobacillus, were associated with a pulmonary function measure and FeNO, while others, including Moraxella to FEV1/FVC and Stenotrophomonas to FeNO, were associated with a single trait. CONCLUSIONS: Using state-of-the-art metagenomic sequencing, we identified specific microorganisms in indoor dust related to pulmonary function and airway inflammation. Some were previously associated with respiratory conditions; others were novel, sug

Colorectal cancer (CRC), the third most common cancer globally, has shown links to disturbed gut microbiota. While significant efforts have been made to establish a microbial signature indicative of CRC using shotgun metagenomic sequencing, the challenge lies in validating this signature with 16S ribosomal RNA (16S) gene sequencing. The primary obstacle is reconciling the differing outputs of these two methodologies, which often lead to divergent statistical models and conclusions. In this study, we introduce an algorithm designed to bridge this gap by mapping shotgun-derived taxa to their 16S counterparts. This mapping enables us to assess the predictive performance of a shotgun-based microbiome signature using 16S data. Our results demonstrate a reduction in performance when applying the 16S-mapped taxa in the shotgun prediction model, though it retains statistical significance. This suggests that while an exact match between shotgun and 16S data may not yet be feasible, our approach provides a viable method for comparative analysis and validation in the context of CRC-associated microbiome research, This research was funded by Instituto de Salud Carlos III, co-funded by FEDER funds–a way to build Europe–grants PI17-00092 and PI20-01439; Spanish Association Against Cancer (AECC) Scientific Foundation—grant GCTRA18022MORE; Consortium for Biomedical Research in Epidemiology and Public Health (CIBERESP) action Genrisk. E.R. has funding from Fundació Marató TV3—grant 875/C/2021. D.B-C. is supported by Instituto de Salud Carlos III Sara Borrell—grant CD21/00094. O.K-L is supported by the Formación de profesorado universitario (FPU) program from the Spanish Ministerio de Universidades—grant FPU2020-02907. T.G. group acknowledges support from the Spanish Ministry of Science and Innovation for grants PID2021-126067NB-I00, CPP2021-008552, PCI2022-135066-2, and PDC2022-133266-I00, cofounded by ERDF “A way of making Europe”; from the Catalan Research Agency (AGAUR) SGR01551; from the European Union’s Horizon 2020 research and innovation programme (ERC-2016-724173); from the Gordon and Betty Moore Foundation (Grant GBMF9742); from the “La Caixa” foundation (Grant LCF/PR/HR21/00737), and from the Instituto de Salud Carlos III (IMPACT Grant IMP/00019 and CIBERINFEC CB21/13/00061—ISCIII-SGEFI/ERDF). Sample collection of this work was supported by the Plataforma Biobancos (PT17/0015/0024) and ICOBIOBANC, sponsored by the Catalan Institute of Oncology., Peer Reviewed, "Article signat per 12 autors/es: Elies Ramon, Mireia Obón-Santacana, Olfat Khannous-Lleiffe, Ester Saus,Toni Gabaldón, Elisabet Guinó, David Bars-Cortina, Gemma Ibáñez-Sanz, Lorena Rodríguez-Alonso, Alfredo Mata, Ana García-Rodrígue and Victor Moreno ", Postprint (published version)

Public health is intrinsically linked to population lifestyle, prompting significant global investments annually to foster its improvement. In alignment with this global trend, obesity, smoking, and alcohol abuse have been recognized as three predominant health risk factors in the Australian society. During 2017-2018, approximately 67% of Australians were overweight, with 31% classified as obese. Obesity now is the second leading risk of ill health and death in Australia. This trend, still increasing, has escalated the prevalence of type 2 diabetes. At the same time, alcohol and tobacco are major burdens of preventable diseases and death. As of 2016, the prevalence of smoking in individuals aged 18 and over was approximately 14%, and the risky alcohol users associated with diseases or injury was 17%. Annually, smoking is responsible for nearly 20,500 deaths, whereas alcohol causes about 1,500 deaths. Despite a yearly decline in the number of Australians who smoke and abuse alcohol, the substantial impact of alcohol and tobacco on public health, social safety, and the economy continues. Traditional monitoring methods of these major public health risk factors, such as self-reporting and hospital surveillance, often fall short of providing timely and accurate data, in addition to concerns over privacy and increased financial costs. To address the problems, this study explored a novel approach of surveying the use and health impact of tobacco, alcohol and obesity rate in communities through analyzing the collective human gut microbiome in wastewater samples collected from the service catchments. The gut microbiome, known to reflect human health, from the community population is mixed in municipal wastewater, which thus contains extensive data related to human health of the community population. This study employed sequencing method to analyze the gut microbiome present in wastewater to identify potential microbial biomarkers that could differentiate different levels of

Microbial lipids, used as taxonomic markers and physiological indicators, have mainly been studied through cultivation. However, this approach is limited due to the scarcity of cultures of environmental microbes, thereby restricting insights into the diversity of lipids and their ecological roles. Addressing this limitation, here we apply metalipidomics combined with metagenomics in the Black Sea, classifying and tentatively identifying 1623 lipid-like species across 18 lipid classes. We discovered over 200 novel, abundant, and structurally diverse sphingolipids in euxinic waters, including unique 1-deoxysphingolipids with long-chain fatty acids and sulfur-containing groups. Sphingolipids were thought to be rare in bacteria and their molecular and ecological functions in bacterial membranes remain elusive. However, genomic analysis focused on sphingolipid biosynthesis genes revealed that members of 38 bacterial phyla in the Black Sea can synthesize sphingolipids, representing a 4-fold increase from previously known capabilities and accounting for up to 25% of the microbial community. These sphingolipids appear to be involved in oxidative stress response, cell wall remodeling, and are associated with the metabolism of nitrogen-containing molecules. Our findings underscore the effectiveness of multi-omics approaches in exploring microbial chemical ecology.

Although long-read sequencing has enabled obtaining high-quality and complete genomes from metagenomes, many challenges still remain to completely decompose a metagenome into its constituent prokaryotic and viral genomes. This study focuses on decomposing an estuarine metagenome to obtain a more accurate estimate of microbial diversity. To achieve this, we developed a new bead-based DNA extraction method, a novel bin refinement method, and obtained 150 Gbp of Nanopore sequencing. We estimate that there are ~500 bacterial and archaeal species in our sample and obtained 68 high-quality bins (>90% complete, <5% contamination, ≤5 contigs, contig length of >100 kbp, and all ribosomal and tRNA genes). We also obtained many contigs of picoeukaryotes, environmental DNA of larger eukaryotes such as mammals, and complete mitochondrial and chloroplast genomes and detected ~40,000 viral populations. Our analysis indicates that there are only a few strains that comprise most of the species abundances. Ocean and estuarine microbiomes play critical roles in global element cycling and ecosystem function. Despite the importance of these microbial communities, many species still have not been cultured in the lab. Environmental sequencing is the primary way the function and population dynamics of these communities can be studied. Long-read sequencing provides an avenue to overcome limitations of short-read technologies to obtain complete microbial genomes but comes with its own technical challenges, such as needed sequencing depth and obtaining high-quality DNA. We present here new sampling and bioinformatics methods to attempt decomposing an estuarine microbiome into its constituent genomes. Our results suggest there are only a few strains that comprise most of the species abundances from viruses to picoeukaryotes, and to fully decompose a metagenome of this diversity requires 1 Tbp of long-read sequencing. We anticipate that as long-read sequencing technologies continue to

Intraspecific genomic diversity brings the potential for an unreported and diverse reservoir of cryptic antibiotic resistance genes in pathogens, as cryptic resistance can occur without major mutations and horizontal transmission. Here, we predicted the differences in the types of antibiotics and genes that induce cryptic and latent resistance between micro-diverse Escherichia coli strains. For example, we hypothesize that known resistance genes will be the culprit of latent resistance within clinical strains. We used a modified functional metagenomics method to induce expression in eight E. coli strains. We found a total of 66 individual genes conferring phenotypic resistance to 11 out of 16 antibiotics. A total of 14 known antibiotic resistance genes comprised 21% of total identified genes, whereas the majority (52 genes) were unclassified cryptic resistance genes. Between the eight strains, 1.2% of core orthologous genes were positive (conferred resistance in at least one strain). Sixty-four percent of positive orthologous genes conferred resistance to only one strain, demonstrating high intraspecific variability of latent resistance genes. Cryptic resistance genes comprised most resistance genes among laboratory and clinical strains as well as natural, semisynthetic, and synthetic antibiotics. Known antibiotic resistance genes primarily conferred resistance to multiple antibiotics from varying origins and within multiple strains. Hence, it is uncommon for E. coli to develop cross-cryptic resistance to antibiotics from multiple origins or within multiple strains. We have uncovered prospective and previously unknown resistance genes as well as antibiotics that have the potential to trigger latent antibiotic resistance in E. coli strains from varying origins.IMPORTANCEIntraspecific genomic diversity may be a driving force in the emergence of adaptive antibiotic resistance. Adaptive antibiotic resistance enables sensitive bacterial cells to acquire temporary antibio

Modern broiler breeds allow for high feed efficiency and rapid growth, which come at a cost of increased susceptibility to pathogens and disease. Broiler growth rate, feed efficiency, and health are affected by the composition of the gut microbiota, which in turn is influenced by diet. In this study, we therefore assessed how diet composition can affect the broiler jejunal gut microbiota. A total of 96 broiler chickens were divided into four diet groups: control, coated butyrate supplementation, medium-chain fatty acid supplementation, or a high-fibre low-protein content. Diet groups were sub-divided into age groups (4, 12 and 33 days of age) resulting in groups of 8 broilers per diet per age. The jejunum content was used for metagenomic shotgun sequencing to determine the microbiota taxonomic composition at species level. The composed diets resulted in a total of 104 differentially abundant bacterial species. Most notably were the butyrate-induced changes in the jejunal microbiota of broilers 4 days post-hatch, resulting in the reduced relative abundance of mainly Enterococcus faecium (-1.8 l2fc, Padj = 9.9E-05) and the opportunistic pathogen Enterococcus hirae (-2.9 l2fc, Padj = 2.7E-08), when compared to the control diet. This effect takes place during early broiler development, which is critical for broiler health, thus exemplifying the importance of how diet can influence the microbiota composition in relation to broiler health. Future studies should therefore elucidate how diet can be used to promote a beneficial microbiota in the early stages of broiler development.

Metagenomic sequencing has proven to be a powerful tool in the monitoring of antimicrobial resistance (AMR). Here, we provide a comparative analysis of the resistome from pigs, poultry, veal calves, turkey, and rainbow trout, for a total of 538 herds across nine European countries. We calculated the effects of per-farm management practices and antimicrobial usage (AMU) on the resistome in pigs, broilers, and veal calves. We also provide an in-depth study of the associations between bacterial diversity, resistome diversity, and AMR abundances as well as co-occurrence analysis of bacterial taxa and antimicrobial resistance genes (ARGs) and the universality of the latter. The resistomes of veal calves and pigs clustered together, as did those of avian origin, while the rainbow trout resistome was different. Moreover, we identified clear core resistomes for each specific food-producing animal species. We identified positive associations between bacterial alpha diversity and both resistome alpha diversity and abundance. Network analyses revealed very few taxa–ARG associations in pigs but a large number for the avian species. Using updated reference databases and optimized bioinformatics, previously reported significant associations between AMU, biosecurity, and AMR in pig and poultry farms were validated. AMU is an important driver for AMR; however, our integrated analyses suggest that factors contributing to increased bacterial diversity might also be associated with higher AMR load. We also found that dispersal limitations of ARGs are shaping livestock resistomes, and future efforts to fight AMR should continue to emphasize biosecurity measures. IMPORTANCE Understanding the occurrence, diversity, and drivers for antimicrobial resistance (AMR) is important to focus future control efforts. So far, almost all attempts to limit AMR in livestock have addressed antimicrobial consumption. We here performed an integrated analysis of the resistomes of five important farmed anim

Metagenomic sequencing is a promising method that has the potential to revolutionize the world of pathogen detection and antimicrobial resistance (AMR) surveillance in food-producing environments. However, the analysis of the huge amount of data obtained requires performant bioinformatics tools and databases, with intuitive and straightforward interpretation. In this study, based on long-read metagenomics data of chicken fecal samples with a spike-in mock community, we proposed confidence levels for taxonomic identification and AMR gene detection, with interpretation guidelines, to help with the analysis of the output data generated by KMA, a popular k-mer read alignment tool. Additionally, we demonstrated that the completeness and diversity of the genomes present in the reference databases are key parameters for accurate and easy interpretation of the sequencing data. Finally, we explored whether KMA, in a two-step procedure, can be used to link the detected AMR genes to their bacterial host chromosome, both detected within the same long-reads. The confidence levels were successfully tested on 28 metagenomics datasets which were obtained with sequencing of real and spiked samples from fecal (chicken, pig, and buffalo) or food (minced beef and food enzyme products) origin. The methodology proposed in this study will facilitate the analysis of metagenomics sequencing datasets for KMA users. Ultimately, this will contribute to improvements in the rapid diagnosis and surveillance of pathogens and AMR genes in food-producing environments, as prioritized by the EU.

Human activities are a significant contributor to the spread of antibiotic resistance genes (ARGs), which pose a serious threat to human health. These ARGs can be transmitted through various pathways, including air, within the context of One Health. This study used metagenomics to monitor the resistomes in urban air from two critical locations: a wastewater treatment plant and a hospital, both indoor and outdoor. The presence of cell-like structures was confirmed through fluorescence microscopy. The metagenomic analysis revealed a wide variety of ARGs and a high diversity of antibiotic-resistant bacteria in the airborne particles collected. The wastewater treatment plant showed higher relative abundances with 32 ARG hits per Gb and m3, followed by the main entrance of the hospital (indoor) with ≈5 ARG hits per Gb and m3. The hospital entrance exhibited the highest ARG richness, with a total of 152 different ARGs classified into nine categories of antibiotic resistance. Common commensal and pathogenic bacteria carrying ARGs, such as Moraxella, Staphylococcus and Micrococcus, were detected in the indoor airborne particles of the hospital. Interestingly, no ARGs were shared among all the samples analysed, indicating a highly variable dynamic of airborne resistomes. Furthermore, the study found no ARGs in the airborne viral fractions analysed, suggesting that airborne viruses play a negligible role in the dissemination of ARGs.

Background Anthropogenic activities significantly contribute to the dissemination of antibiotic resistance genes (ARGs), posing a substantial threat to humankind. The development of methods that allow robust ARG surveillance is a long-standing challenge. Here, we use city-scale monitoring of ARGs by using two of the most promising cutting-edge technologies, digital PCR (dPCR) and metagenomics. Methods ARG hot-spots were sampled from the urban water and wastewater distribution systems. Metagenomics was used to provide a broad view of ARG relative abundance and richness in the prokaryotic and viral fractions. From the city-core ARGs in all samples, the worldwide dispersed sul2 and tetW conferring resistance to sulfonamide and tetracycline, respectively, were monitored by dPCR and metagenomics. Results The largest relative overall ARG abundance and richness were detected in the hospital wastewater and the WWTP inlet (up to ≈6,000 ARGs/Gb metagenome) with a large fraction of unclassified resistant bacteria. The abundance of ARGs in DNA and RNA contigs classified as viruses was notably lower, demonstrating a reduction of up to three orders of magnitude compared to contigs associated to prokaryotes. By metagenomics and dPCR, a similar abundance tendency of sul2 and tetW was obtained, with higher abundances in hospital wastewater and WWTP input (≈125–225 ARGs/Gb metagenome). dPCR absolute abundances were between 6,000 and 18,600 copies per ng of sewage DNA (≈105–7 copies/mL) and 6.8 copies/mL in seawater near the WWTP discharging point. Conclusions dPCR was more sensitive and accurate, while metagenomics provided broader coverage of ARG detection. While desirable, a reliable correlation of dPCR absolute abundance units into metagenomic relative abundance units was not obtained here (r2 < 0.4) suggesting methodological factors that introduce variability. Evolutionary pressure does not significantly select the targeted ARGs in natural aquatic environments.

The signs of climate change are undeniable, and the impact of these changes on ecosystem function heavily depends on the response of microbes that underpin the food web. Antarctic ice shelf is a massive mass of floating ice that extends from the continent into the ocean, exerting a profound influence on global carbon cycles. Beneath Antarctic ice shelves, marine ice stores valuable genetic information, where marine microbial communities before the industrial revolution are archived. Here, in this proof-of-concept, by employing a combination of single-cell technologiesand metagenomics, we have been able to sequence frozen microbial DNA (≈300 years old) stored in the marine ice core B15 collected from the Filchnner-Ronne Ice Shelf. Metagenomic data indicated that Proteobacteria and Thaumarchaeota (e.g., Nitrosopumilus spp.), followed by Actinobacteria (e.g., Actinomarinales), were abundant. Remarkably, our data allow us to “travel to the past” and calibrate genomic and genetic evolutionary changes for ecologically relevant microbes and functions, such as Nitrosopumilus spp., preserved in the marine ice (≈300 years old) with those collected recently in seawater under an ice shelf (year 2017). The evolutionary divergence for the ammonia monooxygenase gene amoA involved in chemolithoautotrophy was about 0.88 amino acid and 2.8 nucleotide substitution rate per 100 sites in a century, while the accumulated rate of genomic SNPs was 2,467 per 1 Mb of genome and 100 years. Whether these evolutionary changes remained constant over the last 300 years or accelerated during post-industrial periods remains an open question that will be further elucidated.

Objectives: Severe early childhood caries (S-ECC) is highly prevalent, affecting children's oral health. S-ECC development is closely associated with the complex oral microbial microbiome and its microorganism interactions, such as the imbalance of bacteriophages and bacteria. Till now, little is known about oral phageome on S-ECC. Therefore, this study aimed to investigate the potential role of the oral phageome in the pathogenesis of S-ECC. Methods: Unstimulated saliva (2 mL) was collected from 20 children with and without S-ECC for metagenomics analysis. Metagenomics sequencing and bioinformatic analysis were performed to determine the two groups’ phageome diversity, taxonomic and functional annotations. Statistical analysis and visualization were performed with R and SPSS Statistics software. Results: 85.7 % of the extracted viral sequences were predicted from phages, in which most phages were classified into Myoviridae, Siphoviridae, and Podoviridae. Alpha diversity decreased, and Beta diversity increased in the S-ECC phageome compared to the healthy group. The abundance of Podoviridae phages increased, and the abundance of Inoviridae, Herelleviridae, and Streptococcus phages decreased in the S-ECC group. Functional annotation revealed increased annotation on glycoside hydrolases and nucleotide metabolism, decreased glycosyl transferases, carbohydrate-binding modules, and biogenic metabolism in the S-ECC phageome. Conclusions: Metagenomic analysis revealed reduced Streptococcus phages and significant changes in functional annotations within the S-ECC phageome. These findings suggest a potential weakening of the regulatory influence of oral bacteria, which may indicate the development of innovative prevention and treatment strategies for S-ECC. These implications deserve further investigation and hold promise for advancing our understanding and management of S-ECC. Clinical significance: The findings of this study indicate that oral phageomes are associated with

Anaerobic digestion (AD) has shown the potential to reduce the abundance of antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs) in animal manures. It stands as a promising option to reduce the risk of the spread of antimicrobial resistance (AMR) due to livestock production and manure applications. However, the underlying mechanisms driving these changes still need to be fully understood. This multidisciplinary study aimed to utilize metagenomics to investigate the molecular and microbial mechanisms associated with the evolution of ARGs and MGEs during the anaerobic digestion (AD) of bovine dairy manure. The research focused on three main aspects: 1) examining the long-term effects of mesophilic (MAD) and thermophilic (TAD) anaerobic digestion on the entire set of ARGs (resistome) and MGEs (mobilome) in manure; 2) comparing the impact of alternative manure treatments, such as storage and solid-liquid separation, on resistomes and mobilomes; 3) identifying microbial groups potentially associated with ARGs and MGEs and microbial shifts potentially driving changes in resistomes and mobilomes. A meta-analysis conducted early in this research informed the primary focus of this research. Two anaerobic digesters operating at mesophilic (36 °C) and the other at thermophilic (55 °C) temperatures were set up, operated, monitored, and studied for 4 and 2 years, respectively. Metagenomics analyses were used to evaluate resistomes, mobilomes, and microbiomes in the mesophilic and thermophilic digesters operating under steady state and in the bovine manure used as substrate. The results indicated that MAD and TAD lowered ARG levels in fresh cattle manure by over 50% and MGEs by over 65%. Surprisingly, TAD did not outperform MAD at reducing ARGs and MGEs. Co-occurrence analysis indicated a strong association between microbial groups from the phyla Bacillota (e.g., Jeotgalicoccus, Streptococcus, Enterococcus), Actinomycetota (e.g., Brevibacterium, Rhodococcus)

Marine ecosystems harbour a vast microbial diversity which play a crucial role in ecosystemfunctioning. Advancements in DNA sequencing technologies have transformed our ability to analyse microbial populations comprehensively. Metagenomic sequencing has emerged as a pivotal tool for characterising microbial communities across various environments. Bioinformatics, an interdisciplinary field, facilitates the analysis and interpretation of large biological datasets, including microbiome data. This thesis aims to enhance bioinformatics approaches for analysing marine microbiomes. It comprises four papers covering bioinformatic developments and genomic data analysis across multiple topics, including metagenomics, pangenomics, comparative genomics and population genomics: Paper I evaluated three assembly strategies for constructing gene catalogues from metagenomic samples: individual sample assembly with gene clustering, co-assembly of all samples, and a new hybrid approach, mix assembly. The efficacy of the mix-assembly approach was highlighted for maximising information extraction from metagenomic samples, offering opportunities for further exploration in microbial ecology and environmental genomics. Using the mix-assembly approach, we conducted a comprehensive analysis of 124 metagenomic samples sourced from the Baltic Sea, resulting in the refinement of the Baltic Sea Gene Set (BAGS v1.1), which now encompasses 66.53 million genes annotated for both functionality and taxonomy. In Paper II, we introduced an open-access initiative that provided the mix-assembly pipeline code. We also developed the BAGS-Shiny web application to facilitate user interaction with this extensive gene catalogue. Paper III focused on whole-genome sequencing and assembly of 82 environmental V. vulnificus strains from the Baltic Sea, enabling comprehensive comparative genomic analysis. I developed the PhyloBOTL pipeline, which uses a phylogeny-based approach to identify genes associated with pat, Havsmiljöer hyser en enorm mikrobiell mångfald som spelar en avgörande roll för ekosystemens funktion. Framsteg inom DNA-sekvenseringstekniker har revolutionerat vår förmåga att analysera den mikrobiella populationen på ett omfattande sätt. Metagenomisk sekvensering har framträtt som ett centralt verktyg för att karakterisera mikrobiella samhällen i olika miljöer. Bioinformatik, ett tvärvetenskapligt fält, underlättar analys och tolkning av stora biologiska dataset, inklusive mikrobiomdata. Den här avhandlingen syftar till att förbättra bioinformatiska metoder för att analysera marina mikrobiom. Den består av fyra artiklar som täcker bioinformatisk utveckling och analys av genomdata inom flera områden, inklusive metagenomik, pangenom, jämförande genomik och populationsgenetik: Artikel I utvärderade tre monteringsstrategier för att konstruera genkataloger från metagenomiska prover: montering av enskilda prover med genglustering, sammontering av alla prover och en ny hybridmetod, mixmontering. Effektiviteten hos mixmonteringsmetoden lyftes fram för att maximera informationsutvinning från metagenomiska prover, vilket öppnar för vidare utforskning inom mikrobiell ekologi och miljögenomik. Med hjälp av mixmonteringsmetoden genomförde vi en omfattande analys av 124 metagenomiska prover från Östersjön, vilket resulterade i förfiningen av Östersjöns gensets (BAGS v1.1), som nu omfattar 66,53 miljoner gener annoterade för både funktion och taxonomi. I artikel II introducerade vi ett öppet initiativ som tillhandahöll koden för mixmonterings-pipeline. Vi utvecklade också BAGS-Shiny webbapplikationen för att underlätta användarinteraktion med denna omfattande gensetskatalog. Artikel III fokuserade på helgenomsekvensering och montering av 82 miljörelaterade V. vulnificus-stammar från Östersjön, vilket möjliggjorde omfattande jämförande genomisk analys. Jag utvecklade PhyloBOTL-pipelinen, som använder en fylogenibaserad metod för att identifiera gener associerade med patogenicite, QC 2024-05-15

A joint archaeogenetic perspective integrates diverse lines of evidence from archaeology and paleogenetics to advance our understanding of human prehistory. In this thesis I contribute to the corpus of archaeogenetic data by producing and analysing ancient genomic data from osseous and masticated material. Masticated birch bark pitch (also called resin) is presented as a novel source for ancient DNA that contains an abundance of data from different species. The masticated lumps analysed in this work, which come from a Mesolithic site in western Scandinavia, encapsulated human genetic information (individual genomes and oral microbiomes) and environmental aDNA from species (plants and animals) consumed or used as raw material at the site. Ancient DNA from this archaeological material transpires as a direct link between a practised archaeological culture and the genotype of the practitioners, in this case a particular lithic technology (the eastern pressure blade concept) and Scandinavian hunter-gatherers (SHG). The paleopathological information obtained from the microbiomes from this masticated material suggests poor oral health in the Early Mesolithic population of Scandinavia. I use the genomic information obtained from various sources to examine hunter-gatherer populations in two opposite parts of northern Eurasia, the northeast Asia and the Scandinavian Peninsula. While the Mesolithic period in Europe has been subjected to intensive study, the paleogenetic history of northern Asia has remained only partially explored. I provide an overview of population dynamics in areas of northeast Asia and around Lake Baikal (Sakha republic, Cis-Baikal and Transbaikal) starting from the end of the Last Glacial Maximum (LGM). Additionally, the most northeastern occurrence of an early form of plague bacteria gets recorded, which is correlated with the population decline in the area during the Late Neolithic / Early Bronze Age periods. While this part of the world was occupied b

Environmental DNA (eDNA) is becoming an increasingly important tool in diverse scientific fields from ecological biomonitoring to wastewater surveillance of viruses. The fundamental challenge in eDNA analyses has been the bioinformatical assignment of reads to taxonomic groups. It has long been known that full probabilistic methods for phylogenetic assignment are preferable, but unfortunately, such methods are computationally intensive and are typically inapplicable to modern next-generation sequencing data. We present a fast approximate likelihood method for phylogenetic assignment of DNA sequences. Applying the new method to several mock communities and simulated datasets, we show that it identifies more reads at both high and low taxonomic levels more accurately than other leading methods. The advantage of the method is particularly apparent in the presence of polymorphisms and/or sequencing errors and when the true species is not represented in the reference database.

Soil microbial diversity is a vital factor for the progression of vegetation and biogeochemical cycles in an ecosystem. It is affected by the chemical composition, soil microenvironment and anthropogenic activities. The present study investigated the effect of environmental conditions and anthropogenic activities on the bacterial diversity of three distantly related soils in India. The multiple soil samples were collected from Hospital waste sites (BTC2) with extensive anthropogenic activity, Himalayan forest soil (RM1) with low anthropogenic activity and Tso Kar Lake sediment soil samples with negligible anthropogenic activity with environmental factors such as soil pH, temperature and altitude on the bacterial diversity. The soil samples were analyzed for physico-chemical properties that suggest significant variations in pH, electrical conductivity (EC), total dissolved solids (TDS), temperature and altitude. The SEM-EDS (Scanning electron microscopy with energy-dispersive X-ray spectroscopy) analysis revealed the elemental composition of the soil samples. The bacterial diversity of three different soil samples was studied using 16S rRNA sequencing-based metagenomic analysis. The results suggested that the bacterial diversity in Hospital waste site soil samples was higher than in the other two soil samples based on chao1 (richness and evenness) analysis. The Phyla Firmicutes were more abundant in Hospital waste site soil, and Himalayan forest soil showed a higher abundance of phylum Proteobacteria (28.86%) and Actinobacteria (26.70%). Tso Kar Lake sediment soil samples showed the most abundant phylum as Proteobacteria (46.28%). The study suggests that increased anthropogenic activity increases soil bacterial diversity. It may eventually help to develop new approaches for sustainable land-use management practices.

Methylmercury (MeHg) is a hazardous neurotoxin that bioaccumulates throughout the food web, which is converted from inorganic mercury (Hg) by microorganisms in oxygen-deprived (hypoxic, or anoxic) conditions. Climate change challenges the ability to limit Hg methylation in the environment since increasing temperatures are predicted to increase the prevalence of hypoxic and anoxic conditions in water columns. My Master thesis project aimed to investigate the diversity, abundance, composition, and structure of Hg-methylating communities to tie together the influence of environmental conditions. To do so, I used bioinformatic tools to analyze a dataset containing freshwater environmental DNA sequences from a broad diversity of metagenomes. The dataset was collected in tandem with environmental metadata (e.g., oxygen) which was used to compare the abundance of one of the two Hg-methylating genes; hgcA. I found that the majority of hgcA genes came from metagenomes collected in hypoxic and anoxic water layers. Interestingly, certain metagenomes in hypoxic and anoxic water layers did not contain any hgcA genes and thus potentially no Hg methylators. Some hgcA genes were found in metagenomes from oxic water layers, which could be explained by settling particles originating from the sediment-water interface acting as oxygen deprived niches. These findings are crucial as they imply that the anticipated changes in freshwater oxygenation, resulting from climate change, will lead to increased niches for Hg-methylating microorganisms. Particularly, increasing algal blooms and persistent lake stratification leads to more deoxygenated water columns, which explains why MeHg can become an increasing hazard to animal and human well-being.

A joint archaeogenetic perspective integrates diverse lines of evidence from archaeology and paleogenetics to advance our understanding of human prehistory. In this thesis I contribute to the corpus of archaeogenetic data by producing and analysing ancient genomic data from osseous and masticated material. Masticated birch bark pitch (also called resin) is presented as a novel source for ancient DNA that contains an abundance of data from different species. The masticated lumps analysed in this work, which come from a Mesolithic site in western Scandinavia, encapsulated human genetic information (individual genomes and oral microbiomes) and environmental aDNA from species (plants and animals) consumed or used as raw material at the site. Ancient DNA from this archaeological material transpires as a direct link between a practised archaeological culture and the genotype of the practitioners, in this case a particular lithic technology (the eastern pressure blade concept) and Scandinavian hunter-gatherers (SHG). The paleopathological information obtained from the microbiomes from this masticated material suggests poor oral health in the Early Mesolithic population of Scandinavia. I use the genomic information obtained from various sources to examine hunter-gatherer populations in two opposite parts of northern Eurasia, the northeast Asia and the Scandinavian Peninsula. While the Mesolithic period in Europe has been subjected to intensive study, the paleogenetic history of northern Asia has remained only partially explored. I provide an overview of population dynamics in areas of northeast Asia and around Lake Baikal (Sakha republic, Cis-Baikal and Transbaikal) starting from the end of the Last Glacial Maximum (LGM). Additionally, the most northeastern occurrence of an early form of plague bacteria gets recorded, which is correlated with the population decline in the area during the Late Neolithic / Early Bronze Age periods. While this part of the world was occupied b

In nature, secondary metabolites mediate interactions between microorganisms residing in complex microbial communities. However, the degree to which community dynamics can be linked to secondary metabolite potential remains largely unknown. In this study, we address the relationship between community succession and secondary metabolism variation. We used 16S and 18S rRNA gene and adenylation domain amplicon sequencing, genome-resolved metagenomics, and untargeted metabolomics to track the taxons, biosynthetic gene clusters, and metabolome dynamics in situ of microorganisms during marine biofilm succession over 113 days. Two phases were identified during the community succession, with a clear shift around Day 29, where the alkaloid secondary metabolites, pseudanes, were also detected. The microbial secondary metabolite potential changed between the phases, and only a few community members, including Myxococotta spp., were responsible for the majority of the biosynthetic gene cluster potential in the early succession phase. In the late phase, bryozoans and benthic copepods were detected, and the microbial nonribosomal peptide potential drastically decreased in association with a reduction in the relative abundance of the prolific secondary metabolite producers. Conclusively, this study provides evidence that the early succession of the marine biofilm community favors prokaryotes with high nonribosomal peptide synthetase potential. In contrast, the late succession is dominated by multicellular eukaryotes and a reduction in bacterial nonribosomal peptide synthetase potential.

Background: The possibility of recovering metagenome-assembled genomes (MAGs) from sequence reads allows for further insights into microbial communities and their members, possibly even analyzing such sequences with tools designed for single-isolate genomes. As result quality depends on sequence quality, performance of tools for single-isolate genomes on MAGs should be tested beforehand. Bioinformatics can be leveraged to quickly create varied synthetic test sets with known composition for this purpose. Results: We present MAGICIAN, a flexible, user-friendly pipeline for the simulation of MAGs. MAGICIAN combines a synthetic metagenome simulator with a metagenomic assembly and binning pipeline to simulate MAGs based on user-supplied input genomes, allowing users to test performance of tools on MAGs while having a ground truth to compare results to. Using MAGICIAN, we found that even very slight (1%) changes in depth of coverage can drastically affect whether a genome can be recovered. We also demonstrate the use of simulated MAGs by evaluating the suitability of such genomes obtained with MAGICIAN's current default pipeline for analysis with the antimicrobial resistance gene identification tool ResFinder. Conclusions: Using MAGICIAN, it is possible to simulate MAGs which, while generally high in quality, reflect issues encountered with real-world data, thus providing realistic best-case data. Evaluating the results of ResFinder analysis of these genomes revealed a risk for plausible-looking false positives, which underlines the need for pipeline validation so that researchers are aware of the potential issues when interpreting real-world data. Furthermore, the effects of fluctuations in depth of coverage on genome recovery in our simulated "random sequencing" warrant further investigation and indicate random subsampling of reads may affect discovery of more genomes.

Analysis of flanking region sequences can be useful when comparing mobile prokaryotic sequences from different bacterial isolates or metagenomes. Here, we report a new bioinformatics tool, a pipeline called Flankophile, which can analyze flanking regions and sequence variants. The main feature of Flankophile is that it visualizes flanking region synteny and sequence variants in publication-ready plots with distance trees, gene annotations, and metadata. Antimicrobial resistance is a significant threat to human health, and surveillance of the spread of antimicrobial resistance is essential. The study of acquired antimicrobial resistance genes (ARGs) is an obvious use case for Flankophile due to the typically diverse genetic context of the genes. Flanking sequences offer a stronger epidemiological signal than the gene sequence alone. To demonstrate Flankophile, we applied it to 2,057 sequenced bacterial isolates and 273 metagenomes from humans and pigs in Denmark and compared the ARGs found in each host reservoir. Gene variant results showed that just ca. 4% of all unique ARG variants found (N = 1,052) were detected in samples from both humans and pigs. Flanking region analysis of ARG variants frequently detected in this study showed multiple examples of genes where the entire 3,000-base pair flanking region was identical in samples from both hosts but also cases where no overlap was observed, and the clustering showed a complete separation of the samples from pigs and the samples from humans.

Meat analogues play an increasing role in meeting global nutritional needs. However, while it is well known that meat possesses inherent characteristics that create favourable conditions for the growth of various pathogenic bacteria, much less is known about meat analogues. This study aimed to compare the growth and survival of Escherichia coli HEHA16, Listeria monocytogenes, Salmonella enterica Typhi, Cronobacter sakazakii, and a cocktail of these bacteria in sterile juices from minced chicken, pig, and beef, as well as pea-based and soy-based minced meat. Traditional microbiology and next-generation sequencing of those metagenomes were employed to analyse the pathogen variability, abundance, and survival after an incubation period. Our findings show that all the meat juices provided favourable conditions for the growth and proliferation of the studied bacteria, with the exception of E. coli HEHA16, which showed lower survival rates in the chicken matrix. Meat analogue juice mainly supported L. monocytogenes survival, with C. sakazakii survival supported to a lesser extent. A correlation was observed between the traditional culturing and metagenomic analysis results, suggesting that further work is needed to compare these technologies in foodborne setups. Our results indicate that plant-based meats could serve as vectors for the transmission of certain, but likely not all, foodborne pathogens, using two accurate detection methods. This warrants the need for additional research to better understand and characterise their safety implications, including their potential association with additional pathogens.

Antimicrobial resistance (AMR) is a major global health concern, further complicated by its spread via the microbiome bacterial members. While mathematical models discuss AMR transmission through the symbiotic microbiome, experimental studies are scarce. Herein, we used a gregarious cockroach, Pycnoscelus surinamensis, as an in vivo animal model for AMR transmission investigations. We explored whether the effect of antimicrobial treatment is detectable with metagenomic sequencing, and whether AMR genes can be spread and established in unchallenged (not treated with antibiotics) individuals following contact with treated donors, and under various frequencies of interaction. Gut and soil substrate microbiomes were investigated by metagenomic sequencing for bacterial community composition and resistome profiling. We found that tetracycline treatment altered the treated gut microbiome by decreasing bacterial diversity and increasing the abundance of tetracycline resistance genes. Untreated cockroaches that interacted with treated donors also had elevated tetracycline resistance. The levels of resistance differed depending on the magnitude and frequency of donor transfer. Additionally, treated donors showed signs of microbiome recovery due to their interaction with the untreated ones. Similar patterns were also recorded in the soil substrate microbiomes. Our results shed light on how interacting microbiomes facilitate AMR gene transmission to previously unchallenged hosts, a dynamic influenced by the interaction frequencies, using an in vivo model to validate theoretical AMR transmission models. IMPORTANCE Antimicrobial resistance is a rising threat to human and animal health. The spread of resistance through the transmission of the symbiotic gut microbiome is of concern and has been explored in theoretical modeling studies. In this study, we employ gregarious insect populations to examine the emergence and transmission of antimicrobial resista

Interest in fermented foods, especially plant-based ones, has increased considerably in the last decade. Miso—a Japanese paste traditionally fermented with soybeans, salt, and kōji (Aspergillus oryzae grown on grains or beans)—has gained attention among chefs for its rich flavour and versatility. Some chefs have even been experimenting with making novel misos with untraditional substrates to create new flavours. Such novel fermented foods also offer new scientific opportunities. To explore the microbial diversity of these new traditional foods, we sampled six misos made by the team at a leading restaurant called Noma in Copenhagen (Denmark), using yellow peas (including a nixtamalised treatment), lupin seeds, Swedish Vreta peas, grey peas, and Gotland lentils as substrates. All misos were made with the same recipe and fermented for 3 months at 28 °C. Samples were collected at the end of fermentation for subsequent shotgun metagenomic sequencing and a genome-resolved metagenomic analysis. The taxonomic profile of the samples revealed the presence of kōji mould (A. oryzae) and Bacillus amyloliquefaciens in all misos. Various species of the genera Latilactobacillus, Lactiplantibacillus, Pediococcus and Staphylococcus were also detected. The Metagenome-Assembled Genomes (MAGs) revealed genomic sequences belonging to 12 different species and functional analyses of these MAGs were performed. Notably, we detected the presence of Exiguobacterium—the first reported instance of the genus in miso—and Average Nucleotide Identity (ANI) analyses suggest a potentially new species. We hope these results will improve the scientific literature on misos and contribute to developing novel fermented plant-based foods.

Many environmentally relevant micro-organisms cannot be cultured, and even with the latest metagenomic approaches, achieving complete genomes for specific target organisms of interest remains a challenge. Cable bacteria provide a prominent example of a microbial ecosystem engineer that is currently unculturable. They occur in low abundance in natural sediments, but due to their capability for long-distance electron transport, they exert a disproportionately large impact on the biogeochemistry of their environment. Current available genomes of marine cable bacteria are highly fragmented and incomplete, hampering the elucidation of their unique electrogenic physiology. Here, we present a metagenomic pipeline that combines Nanopore long-read and Illumina short-read shotgun sequencing. Starting from a clonal enrichment of a cable bacterium, we recovered a circular metagenome-assembled genome (5.09 Mbp in size), which represents a novel cable bacterium species with the proposed name Candidatus Electrothrix scaldis. The closed genome contains 1109 novel identified genes, including key metabolic enzymes not previously described in incomplete genomes of cable bacteria. We examined in detail the factors leading to genome closure. Foremost, native, non-amplified long reads are crucial to resolve the many repetitive regions within the genome of cable bacteria, and by analysing the whole metagenomic assembly, we found that low strain diversity is key for achieving genome closure. The insights and approaches presented here could help achieve genome closure for other keystone micro-organisms present in complex environmental samples at low abundance., BT/Environmental Biotechnology

Hadal sediments are hotspots of microbial activity in the deep-sea and exhibit strong biogeochemical gradients. But while these gradients are widely assumed to exert selective forces on hadal microbial communities, the actual relationship between biogeochemistry, functional traits, and microbial community structure remains poorly understood. We tested whether the biogeochemical conditions in hadal sediments select for microbes based on their genomic capacity for respiration and carbohydrate utilization via a metagenomic analysis of over 153 samples from the Atacama Trench region (max. Depth 8085 m). The obtained 1357 non-redundant microbial genomes were affiliated with about one third of all known microbial phyla, with more than half belonging to unknown genera. This indicated that the capability to withstand extreme hydrostatic pressure is a phylogenetically widespread trait and that hadal sediments are inhabited by diverse microbial lineages. While community composition changed gradually over sediment depth, these changes were not driven by selection for respiratory or carbohydrate degradation capability in the oxic and nitrogenous zones, except in the case of anammox bacteria and nitrifying archaea. However, selection based on respiration and carbohydrate degradation capacity did structure the communities of the ferruginous zone, where aerobic and nitrogen respiring microbes declined exponentially (half-life 125–419 years) and were replaced by subsurface communities. These results highlight a delayed response of microbial community composition to selective pressure imposed by redox zonation and indicated that gradual changes in microbial composition are shaped by the high-resilience and slow growth of microbes in the seafloor.

Editorial on the Research Topic Community series-extremophiles: microbial genomics and taxogenomics, volume II

This research investigates the effects of phytogenic feed additives (PFAs) on the growth performance, gut microbial community, and microbial metabolic functions in weaned piglets via a combined 16S rRNA gene amplicon and shotgun metagenomics approach. A controlled trial was conducted using 200 pigs to highlight the significant influence of PFAs on gut microbiota dynamics. Notably, the treatment group revealed an increased gut microbiota diversity, as measured with the Shannon and Simpson indices. The increase in diversity is accompanied by an increase in beneficial bacterial taxa, such as Roseburia, Faecalibacterium, and Prevotella, and a decline in potential pathogens like Clostridium sensu stricto 1 and Campylobacter. Shotgun sequencing at the species level confirmed these findings. This modification in microbial profile was coupled with an altered profile of microbial metabolic pathways, suggesting a reconfiguration of microbial function under PFA influence. Significant shifts in overall microbial community structure by week 8 demonstrate PFA treatment’s temporal impact. Histomorphological examination unveiled improved gut structure in PFA-treated piglets. The results of this study indicate that the use of PFAs as dietary supplements can be an effective strategy, augmenting gut microbiota diversity, reshaping microbial function, enhancing gut structure, and optimising intestinal health of weaned piglets providing valuable implications for swine production. Key points • PFAs significantly diversify the gut microbiota in weaned piglets, aiding balance. • Changes in gut structure due to PFAs indicate improved resistance to weaning stress. • PFAs show potential to ease weaning stress, offering a substitute for antibiotics in piglet diets.

Antibiotic resistance genes (ARGs) are emerging pollutants that enter the farm and surrounding environment via the manure of antibiotic-treated animals. Pretreatment of livestock manure by composting decreases ARGs abundance, but how antibiotic residues affect ARGs removal efficiency remains poorly understood. Here, we explored the fate of the resistome under different doxycycline residue levels during aerobic swine manure composting. Metagenomic sequencing showed that the presence of high levels of doxycycline generally had a higher abundance of tetracycline ARGs, and their dominant host bacteria of Firmicutes, especially Clostridium and Streptococcus, also had limited elimination in composting under high levels of doxycycline stress. Moreover, high levels of doxycycline impaired the removal of the total ARGs number in finished composts, with a removal rate of 51.74 % compared to 63.70 % and 71.52 % for the control and low-level doxycycline manure, respectively. Horizontal gene transfer and strengthened correlations among the bacterial community fostered ARGs preservation at high doxycycline levels during composting. In addition, ARGs carried by both plasmids and chromosomes, such as multidrug ARGs, showed wide host characteristics and rebound during compost maturation. Compared with chromosomes, a greater variety of ARGs on plasmids suggested that the majority of ARGs were characterized by horizontal mobility during composting, and the cross-host characteristics of ARGs during composting deserve further attention. This study provided deep insight into the fate of ARGs under residual antibiotic stress during manure composting and reminded the associated risk for environmental and public health.

Interest in fermented foods, especially plant-based ones, has increased considerably in the last decade. Miso—a Japanese paste traditionally fermented with soybeans, salt, and kōji (Aspergillus oryzae grown on grains or beans)—has gained attention among chefs for its rich flavour and versatility. Some chefs have even been experimenting with making novel misos with untraditional substrates to create new flavours. Such novel fermented foods also offer new scientific opportunities. To explore the microbial diversity of these new traditional foods, we sampled six misos made by the team at a leading restaurant called Noma in Copenhagen (Denmark), using yellow peas (including a nixtamalised treatment), lupin seeds, Swedish Vreta peas, grey peas, and Gotland lentils as substrates. All misos were made with the same recipe and fermented for 3 months at 28 °C. Samples were collected at the end of fermentation for subsequent shotgun metagenomic sequencing and a genome-resolved metagenomic analysis. The taxonomic profile of the samples revealed the presence of kōji mould (A. oryzae) and Bacillus amyloliquefaciens in all misos. Various species of the genera Latilactobacillus, Lactiplantibacillus, Pediococcus and Staphylococcus were also detected. The Metagenome-Assembled Genomes (MAGs) revealed genomic sequences belonging to 12 different species and functional analyses of these MAGs were performed. Notably, we detected the presence of Exiguobacterium—the first reported instance of the genus in miso—and Average Nucleotide Identity (ANI) analyses suggest a potentially new species. We hope these results will improve the scientific literature on misos and contribute to developing novel fermented plant-based foods.

Ecological role of the viral community on the fate of antibiotic resistance genes (ARGs) (reduction vs proliferation) remains unclear in anaerobic digestion (AD). Metagenomics revealed a dominance of Siphoviridae and Podoviridae among 13,895 identified viral operational taxonomic units (vOTUs) within AD, and only 21 of the vOTUs carried ARGs, which only accounted for 0.57 ± 0.43% of AD antibiotic resistome. Conversely, ARGs locating on plasmids and integrative and conjugative elements accounted for above 61.0%, indicating a substantial potential for conjugation in driving horizontal gene transfer of ARGs within AD. Virus–host prediction based on CRISPR spacer, tRNA, and homology matches indicated that most viruses (80.2%) could not infect across genera. Among 480 high-quality metagenome assembly genomes, 95 carried ARGs and were considered as putative antibiotic-resistant bacteria (pARB). Furthermore, lytic phages of 66 pARBs were identified and devoid of ARGs, and virus/host abundance ratios with an average value of 71.7 indicated extensive viral activity and lysis. The infectivity of lytic phage was also elucidated through laboratory experiments concerning changes of the phage-to-host ratio, pH, and temperature. Although metagenomic evidence for dissemination of ARGs by phage transduction was found, the higher proportion of lytic phages infecting pARBs suggested that the viral community played a greater role in reducing ARB numbers than spreading ARGs in AD.

Metagenomic binning is an essential technique for genome-resolved characterization of uncultured microorganisms in various ecosystems but hampered by the low efficiency of binning tools in adequately recovering metagenome-assembled genomes (MAGs). Here, we introduce BASALT (Binning Across a Series of Assemblies Toolkit) for binning and refinement of short- and long-read sequencing data. BASALT employs multiple binners with multiple thresholds to produce initial bins, then utilizes neural networks to identify core sequences to remove redundant bins and refine non-redundant bins. Using the same assemblies generated from Critical Assessment of Metagenome Interpretation (CAMI) datasets, BASALT produces up to twice as many MAGs as VAMB, DASTool, or metaWRAP. Processing assemblies from a lake sediment dataset, BASALT produces ~30% more MAGs than metaWRAP, including 21 unique class-level prokaryotic lineages. Functional annotations reveal that BASALT can retrieve 47.6% more non-redundant opening-reading frames than metaWRAP. These results highlight the robust handling of metagenomic sequencing data of BASALT.

MOTIVATION: Taxonomic classification of short reads and taxonomic profiling of metagenomic samples are well-studied yet challenging problems. The presence of species belonging to groups without close representation in a reference dataset is particularly challenging. While k-mer-based methods have performed well in terms of running time and accuracy, they tend to have reduced accuracy for such novel species. Thus, there is a growing need for methods that combine the scalability of k-mers with increased sensitivity. RESULTS: Here, we show that using locality-sensitive hashing (LSH) can increase the sensitivity of the k-mer-based search. Our method, which combines LSH with several heuristics techniques including soft lowest common ancestor labeling and voting, is more accurate than alternatives in both taxonomic classification of individual reads and abundance profiling. AVAILABILITY AND IMPLEMENTATION: CONSULT-II is implemented in C++, and the software, together with reference libraries, is publicly available on GitHub https://github.com/bo1929/CONSULT-II.

Anaerobic digestion (AD) has shown the potential to reduce the abundance of antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs) in animal manures. It stands as a promising option to reduce the risk of the spread of antimicrobial resistance (AMR) due to livestock production and manure applications. However, the underlying mechanisms driving these changes still need to be fully understood. This multidisciplinary study aimed to utilize metagenomics to investigate the molecular and microbial mechanisms associated with the evolution of ARGs and MGEs during the anaerobic digestion (AD) of bovine dairy manure. The research focused on three main aspects: 1) examining the long-term effects of mesophilic (MAD) and thermophilic (TAD) anaerobic digestion on the entire set of ARGs (resistome) and MGEs (mobilome) in manure; 2) comparing the impact of alternative manure treatments, such as storage and solid-liquid separation, on resistomes and mobilomes; 3) identifying microbial groups potentially associated with ARGs and MGEs and microbial shifts potentially driving changes in resistomes and mobilomes. A meta-analysis conducted early in this research informed the primary focus of this research. Two anaerobic digesters operating at mesophilic (36 °C) and the other at thermophilic (55 °C) temperatures were set up, operated, monitored, and studied for 4 and 2 years, respectively. Metagenomics analyses were used to evaluate resistomes, mobilomes, and microbiomes in the mesophilic and thermophilic digesters operating under steady state and in the bovine manure used as substrate. The results indicated that MAD and TAD lowered ARG levels in fresh cattle manure by over 50% and MGEs by over 65%. Surprisingly, TAD did not outperform MAD at reducing ARGs and MGEs. Co-occurrence analysis indicated a strong association between microbial groups from the phyla Bacillota (e.g., Jeotgalicoccus, Streptococcus, Enterococcus), Actinomycetota (e.g., Brevibacterium, Rhodococcus)