Probing the “Dark Matter” of the Human Gut Phageome: Culture Assisted Metagenomics Enables Rapid Discovery and Host-Linking for Novel Bacteriophages

Recent years have been marked by the growing interest towards virulent and temperate bacteriophage populations inhabiting the human lower gastrointestinal tract – the gut phageome. A number of studies demonstrated high levels of specificity and temporal stability of individual gut phageomes, as well as their specific alterations in disease cohorts, in parallel with changes in the bacteriome. It has been speculated that phages might have an active role in shaping the taxonomic composition and functional properties of the human gut bacteriome. An overwhelming majority of gut bacteriophages, however, remain uncultured, unclassified, and their specific hosts and infection strategies are still unknown. They are often referred to as “the viral dark matter”. A possible breakthrough in understanding of the phageome can only become possible when a significant proportion of the “the viral dark matter” is identified and linked to bacterial hosts. Here, we describe a method that enables rapid discovery and host-linking of novel bacteriophages in the gut via a combination of serial enrichment cultures and shotgun metagenomics of viral DNA. Using this approach dozens of novel and previously known bacteriophages were detected, including the ones infecting difficult-to-culture anaerobic bacteria. The majority of phages failed to produce lysis and propagate on host cultures in traditional assays. The newly identified phages include representatives of Siphoviridae, Myoviridae, Podoviridae, and crAss-like viruses, infecting diverse bacterial taxa of Bacteroidetes, Firmicutes, Actinobacteria, Verrucomicrobia and Proteobacteria phyla. The proposed new method has a potential for high-throughput screening applications for mass discovery of new phages in different environments.

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Distinct composition and metabolic functions of human gut microbiota are associated with cachexia in lung cancer patients

The ISME Journal ◽

10.1038/s41396-021-00998-8 ◽

2021 ◽

Author(s):

Yueqiong Ni ◽

Zoltan Lohinai ◽

Yoshitaro Heshiki ◽

Balazs Dome ◽

Judit Moldvay ◽

...

Keyword(s):

Lung Cancer ◽

Gut Microbiota ◽

Cancer Patients ◽

Gut Microbiome ◽

Human Gut ◽

Microbial Composition ◽

Shotgun Metagenomics ◽

Lung Cancer Patients ◽

Microbial Species ◽

Functional Pathways

AbstractCachexia is associated with decreased survival in cancer patients and has a prevalence of up to 80%. The etiology of cachexia is poorly understood, and limited treatment options exist. Here, we investigated the role of the human gut microbiome in cachexia by integrating shotgun metagenomics and plasma metabolomics of 31 lung cancer patients. The cachexia group showed significant differences in the gut microbial composition, functional pathways of the metagenome, and the related plasma metabolites compared to non-cachectic patients. Branched-chain amino acids (BCAAs), methylhistamine, and vitamins were significantly depleted in the plasma of cachexia patients, which was also reflected in the depletion of relevant gut microbiota functional pathways. The enrichment of BCAAs and 3-oxocholic acid in non-cachectic patients were positively correlated with gut microbial species Prevotella copri and Lactobacillus gasseri, respectively. Furthermore, the gut microbiota capacity for lipopolysaccharides biosynthesis was significantly enriched in cachectic patients. The involvement of the gut microbiome in cachexia was further observed in a high-performance machine learning model using solely gut microbial features. Our study demonstrates the links between cachectic host metabolism and specific gut microbial species and functions in a clinical setting, suggesting that the gut microbiota could have an influence on cachexia with possible therapeutic applications.

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The impact of sequencing depth on the inferred taxonomic composition and AMR gene content of metagenomic samples

10.1101/593301 ◽

2019 ◽

Author(s):

H. Soon Gweon ◽

Liam P. Shaw ◽

Jeremy Swann ◽

Nicola De Maio ◽

Manal AbuOun ◽

...

Keyword(s):

Data Processing ◽

Open Source ◽

Open Source Software ◽

River Sediment ◽

Taxonomic Composition ◽

Sequencing Depth ◽

Gene Content ◽

Metagenomic Data ◽

Software Pipeline ◽

Shotgun Metagenomics

ABSTRACTBackgroundShotgun metagenomics is increasingly used to characterise microbial communities, particularly for the investigation of antimicrobial resistance (AMR) in different animal and environmental contexts. There are many different approaches for inferring the taxonomic composition and AMR gene content of complex community samples from shotgun metagenomic data, but there has been little work establishing the optimum sequencing depth, data processing and analysis methods for these samples. In this study we used shotgun metagenomics and sequencing of cultured isolates from the same samples to address these issues. We sampled three potential environmental AMR gene reservoirs (pig caeca, river sediment, effluent) and sequenced samples with shotgun metagenomics at high depth (∼200 million reads per sample). Alongside this, we cultured single-colony isolates ofEnterobacteriaceaefrom the same samples and used hybrid sequencing (short- and long-reads) to create high-quality assemblies for comparison to the metagenomic data. To automate data processing, we developed an open-source software pipeline, ‘ResPipe’.ResultsTaxonomic profiling was much more stable to sequencing depth than AMR gene content. 1 million reads per sample was sufficient to achieve <1% dissimilarity to the full taxonomic composition. However, at least 80 million reads per sample were required to recover the full richness of different AMR gene families present in the sample, and additional allelic diversity of AMR genes was still being discovered in effluent at 200 million reads per sample. Normalising the number of reads mapping to AMR genes using gene length and an exogenous spike ofThermus thermophilusDNA substantially changed the estimated gene abundance distributions. While the majority of genomic content from cultured isolates from effluent was recoverable using shotgun metagenomics, this was not the case for pig caeca or river sediment.ConclusionsSequencing depth and profiling method can critically affect the profiling of polymicrobial animal and environmental samples with shotgun metagenomics. Both sequencing of cultured isolates and shotgun metagenomics can recover substantial diversity that is not identified using the other methods. Particular consideration is required when inferring AMR gene content or presence by mapping metagenomic reads to a database. ResPipe, the open-source software pipeline we have developed, is freely available (https://gitlab.com/hsgweon/ResPipe).

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Baseline human gut microbiota profile in healthy people and standard reporting template

10.1101/445353 ◽

2018 ◽

Author(s):

Charles Hadley King ◽

Hiral Desai ◽

Allison C. Sylvetsky ◽

Jonathan LoTempio ◽

Shant Ayanyan ◽

...

Keyword(s):

Dark Matter ◽

Regulatory Agencies ◽

Fecal Transplant ◽

Human Gut ◽

Healthy Human ◽

Comprehensive Knowledge ◽

Analysis Workflow ◽

Standard Report ◽

Abundance Profile ◽

Microbiome Data

A comprehensive knowledge of the types and ratios of microbes that inhabit the healthy human gut is necessary before any kind of pre-clinical or clinical study can be performed that attempts to alter the microbiome to treat a condition or improve therapy outcome. To address this need we present an innovative scalable comprehensive analysis workflow, a healthy human reference microbiome list and abundance profile (GutFeelingKB), and a novel Fecal Biome Population Report (FecalBiome) with clinical applicability. GutFeelingKB provides a list of 157 organisms (8 phyla, 18 classes, 23 orders, 38 families, 59 genera and 109 species) that forms the baseline biome and therefore can be used as healthy controls for studies related to dysbiosis. The incorporation of microbiome science into routine clinical practice necessitates a standard report for comparison of an individual’s microbiome to the growing knowledgebase of “normal” microbiome data. The FecalBiome and the underlying technology of GutFeelingKB address this need. The knowledgebase can be useful to regulatory agencies for the assessment of fecal transplant and other microbiome products, as it contains a list of organisms from healthy individuals. In addition to the list of organisms and abundances the study also generated a list of contigs of metagenomics dark matter. In this study, metagenomic dark matter represents sequences that cannot be mapped to any known sequence but can be assembled into contigs of 10,000 nucleotides or higher. These sequences can be used to create primers to study potential novel organisms. All data is freely available from https://hive.biochemistry.gwu.edu/gfkb and NCBI’s Short Read Archive.

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High-throughput isolation and sorting of gut microbes reduce biases of traditional cultivation strategies

10.1101/759969 ◽

2019 ◽

Cited By ~ 1

Author(s):

William J. Watterson ◽

Melikhan Tanyeri ◽

Andrea R. Watson ◽

Candace M. Cham ◽

Yue Shan ◽

...

Keyword(s):

Ribosomal Rna ◽

Fold Increase ◽

Taxonomic Composition ◽

Biased Sampling ◽

Human Gut ◽

Rare Taxa ◽

Conventional Culture ◽

16S Ribosomal Rna Gene ◽

Slow Growing ◽

New Strategies

AbstractTraditional cultivation approaches in microbiology are labor-intensive, low-throughput, and often yield biased sampling of taxa due to ecological and evolutionary factors. New strategies are needed to enable ample representation of rare taxa and slow-growers that are outcompeted by fast-growing organisms. We developed a microfluidic platform that anaerobically isolates and cultivates microbial cells in millions of picoliter droplets and automatically sorts droplets based on colony density. We applied our strategy to mouse and human gut microbiomes and used 16S ribosomal RNA gene amplicons to characterize taxonomic composition of cells grown using different media. We found up to 4-fold increase in richness and larger representation of rare taxa among cells grown in droplets compared to conventional culture plates. Automated sorting of droplets for slow-growing colonies further enhanced the relative abundance of rare populations. Our method improves the cultivation and analysis of diverse microbiomes to gain deeper insights into microbial functioning and lifestyles.

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Baryonic Dark Halos: a model with MACHOS and cold gas globules

Symposium - International Astronomical Union ◽

10.1017/s0074180900232300 ◽

1996 ◽

Vol 171 ◽

pp. 167-170

Author(s):

Ortwin Gerhard ◽

Joseph Silk

Keyword(s):

Dark Matter ◽

Galaxy Formation ◽

Active Role ◽

Galaxy Formation And Evolution ◽

Substellar Objects ◽

Formation And Evolution ◽

Cold Gas

The dark matter in the halos of galaxies may well be baryonic, and much of the mass within them could be in the form of clusters of substellar objects within which are embedded cold gas globules. Such halos might play an active role in galaxy formation and evolution.

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Antibiotics create a shift from mutualism to competition in human gut communities with a longer-lasting impact on fungi than bacteria

Microbiome ◽

10.1186/s40168-020-00899-6 ◽

2020 ◽

Vol 8 (1) ◽

Author(s):

Bastian Seelbinder ◽

Jiarui Chen ◽

Sascha Brunke ◽

Ruben Vazquez-Uribe ◽

Rakesh Santhaman ◽

...

Keyword(s):

Fungal Community ◽

Fungal Infections ◽

Cell Damage ◽

Fungal Colonization ◽

Antibiotic Administration ◽

Bacterial Strains ◽

Human Gut ◽

Healthy Human ◽

Shotgun Metagenomics ◽

Fungal Interactions

Abstract Background Antibiotic treatment has a well-established detrimental effect on the gut bacterial composition, but effects on the fungal community are less clear. Bacteria in the lumen of the gastrointestinal tract may limit fungal colonization and invasion. Antibiotic drugs targeting bacteria are therefore seen as an important risk factor for fungal infections and induced allergies. However, antibiotic effects on gut bacterial-fungal interactions, including disruption and resilience of fungal community compositions, were not investigated in humans. We analysed stool samples collected from 14 healthy human participants over 3 months following a 6-day antibiotic administration. We integrated data from shotgun metagenomics, metatranscriptomics, metabolomics, and fungal ITS2 sequencing. Results While the bacterial community recovered mostly over 3 months post treatment, the fungal community was shifted from mutualism at baseline to competition. Half of the bacterial-fungal interactions present before drug intervention had disappeared 3 months later. During treatment, fungal abundances were associated with the expression of bacterial genes with functions for cell growth and repair. By extending the metagenomic species approach, we revealed bacterial strains inhibiting the opportunistic fungal pathogen Candida albicans. We demonstrated in vitro how C. albicans pathogenicity and host cell damage might be controlled naturally in the human gut by bacterial metabolites such as propionate or 5-dodecenoate. Conclusions We demonstrated that antibacterial drugs have long-term influence on the human gut mycobiome. While bacterial communities recovered mostly 30-days post antibacterial treatment, the fungal community was shifted from mutualism towards competition.

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The impact of human-facilitated selection on the gut microbiota of domesticated mammals

FEMS Microbiology Ecology ◽

10.1093/femsec/fiz121 ◽

2019 ◽

Vol 95 (9) ◽

Cited By ~ 8

Author(s):

Giulia Alessandri ◽

Christian Milani ◽

Leonardo Mancabelli ◽

Marta Mangifesta ◽

Gabriele Andrea Lugli ◽

...

Keyword(s):

Gut Microbiota ◽

Horizontal Transmission ◽

Taxonomic Composition ◽

Functional Adaptation ◽

Rrna Gene ◽

Feed Conversion ◽

Shotgun Metagenomics ◽

Domesticated Animals ◽

And Behavior ◽

The Impact

ABSTRACT Domestication is the process by which anthropogenic forces shape lifestyle and behavior of wild species to accommodate human needs. The impact of domestication on animal physiology and behavior has been extensively studied, whereas its effect on the gut microbiota is still largely unexplored. For this reason, 16S rRNA gene-based and internal transcribed spacer-mediated bifidobacterial profiling, together with shotgun metagenomics, was employed to investigate the taxonomic composition and metabolic repertoire of 146 mammalian fecal samples, corresponding to 12 domesticated–feral dyads. Our results revealed that changes induced by domestication have extensively shaped the taxonomic composition of the mammalian gut microbiota. In this context, the selection of microbial taxa linked to a more efficient feed conversion into body mass and putative horizontal transmission of certain bacterial genera from humans were observed in the fecal microbiota of domesticated animals when compared to their feral relatives and to humans. In addition, profiling of the metabolic arsenal through metagenomics highlighted extensive functional adaptation of the fecal microbial community of domesticated mammals to changes induced by domestication. Remarkably, domesticated animals showed, when compared to their feral relatives, increased abundance of specific glycosyl hydrolases, possibly due to the higher intake of complex plant carbohydrates typical of commercial animal feeds.

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The impact of sequencing depth on the inferred taxonomic composition and AMR gene content of metagenomic samples

Environmental Microbiome ◽

10.1186/s40793-019-0347-1 ◽

2019 ◽

Vol 14 (1) ◽

Cited By ~ 12

Author(s):

H. Soon Gweon ◽

◽

Liam P. Shaw ◽

Jeremy Swann ◽

Nicola De Maio ◽

...

Keyword(s):

Data Processing ◽

Open Source ◽

Open Source Software ◽

River Sediment ◽

Taxonomic Composition ◽

Sequencing Depth ◽

Gene Content ◽

Metagenomic Data ◽

Software Pipeline ◽

Shotgun Metagenomics

Abstract Background Shotgun metagenomics is increasingly used to characterise microbial communities, particularly for the investigation of antimicrobial resistance (AMR) in different animal and environmental contexts. There are many different approaches for inferring the taxonomic composition and AMR gene content of complex community samples from shotgun metagenomic data, but there has been little work establishing the optimum sequencing depth, data processing and analysis methods for these samples. In this study we used shotgun metagenomics and sequencing of cultured isolates from the same samples to address these issues. We sampled three potential environmental AMR gene reservoirs (pig caeca, river sediment, effluent) and sequenced samples with shotgun metagenomics at high depth (~ 200 million reads per sample). Alongside this, we cultured single-colony isolates of Enterobacteriaceae from the same samples and used hybrid sequencing (short- and long-reads) to create high-quality assemblies for comparison to the metagenomic data. To automate data processing, we developed an open-source software pipeline, ‘ResPipe’. Results Taxonomic profiling was much more stable to sequencing depth than AMR gene content. 1 million reads per sample was sufficient to achieve < 1% dissimilarity to the full taxonomic composition. However, at least 80 million reads per sample were required to recover the full richness of different AMR gene families present in the sample, and additional allelic diversity of AMR genes was still being discovered in effluent at 200 million reads per sample. Normalising the number of reads mapping to AMR genes using gene length and an exogenous spike of Thermus thermophilus DNA substantially changed the estimated gene abundance distributions. While the majority of genomic content from cultured isolates from effluent was recoverable using shotgun metagenomics, this was not the case for pig caeca or river sediment. Conclusions Sequencing depth and profiling method can critically affect the profiling of polymicrobial animal and environmental samples with shotgun metagenomics. Both sequencing of cultured isolates and shotgun metagenomics can recover substantial diversity that is not identified using the other methods. Particular consideration is required when inferring AMR gene content or presence by mapping metagenomic reads to a database. ResPipe, the open-source software pipeline we have developed, is freely available (https://gitlab.com/hsgweon/ResPipe).

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Ecological Structuring of Temperate Bacteriophages in the Inflammatory Bowel Disease-Affected Gut

Microorganisms ◽

10.3390/microorganisms8111663 ◽

2020 ◽

Vol 8 (11) ◽

pp. 1663

Author(s):

Hiroki Nishiyama ◽

Hisashi Endo ◽

Romain Blanc-Mathieu ◽

Hiroyuki Ogata

Keyword(s):

Inflammatory Bowel Disease ◽

Bowel Disease ◽

Large Scale ◽

Sequence Data ◽

Vital Role ◽

Temperate Bacteriophage ◽

Human Gut ◽

Potential Association ◽

Wide Range ◽

Inflammatory Bowel

The aim of this study was to elucidate the ecological structure of the human gut temperate bacteriophage community and its role in inflammatory bowel disease (IBD). Temperate bacteriophages make up a large proportion of the human gut microbiota and are likely to play a role in IBD pathogenesis. However, many of these bacteriophages await characterization in reference databases. Therefore, we conducted a large-scale reconstruction of temperate bacteriophage and bacterial genomes from the whole-metagenome sequence data generated by the IBD Multi’omics Database project. By associating phages with their hosts via genome comparisons, we found that temperate bacteriophages infect a phylogenetically wide range of bacteria. The majority of variance in bacteriophage community composition was explained by variation among individuals, but differences in the abundance of temperate bacteriophages were identified between IBD and non-IBD patients. Of note, in active ulcerative colitis patients, temperate bacteriophages infecting Bacteroides uniformis and Bacteroides thetaiotaomicron—two species experimentally proven to be beneficial to gut homeostasis—were over-represented, whereas their hosts were under-represented in comparison with non-IBD patients. Supporting the mounting evidence that gut viral community plays a vital role in IBD, our results show potential association between temperate bacteriophages and IBD pathogenesis.

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