Comparative analysis of Faecalibacterium prausnitzii genomes shows a high level of genome plasticity and warrants separation into new species-level taxa.

BACKGROUND: Faecalibacterium prausnitzii is a ubiquitous member of the human gut microbiome, constituting up to 15% of the total bacteria in the human gut. Substantial evidence connects decreased levels of F. prausnitzii with the onset and progression of certain forms of inflammatory bowel disease, which has been attributed to its anti-inflammatory potential. Two phylogroups of F. prausnitzii have been identified, with a decrease in phylogroup I being a more sensitive marker of intestinal inflammation. Much of the genomic and physiological data available to date was collected using phylogroup II strains. Little analysis of F. prausnitzii genomes has been performed so far and genetic differences between phylogroups I and II are poorly understood. RESULTS: In this study we sequenced 11 additional F. prausnitzii genomes and performed comparative genomics to investigate intraspecies diversity, functional gene complement and the mobilome of 31 high-quality draft and complete genomes. We reveal a very low level of average nucleotide identity among F. prausnitzii genomes and a high level of genome plasticity. Two genomogroups can be separated based on differences in functional gene complement, albeit that this division does not fully agree with separation based on conserved gene phylogeny, highlighting the importance of horizontal gene transfer in shaping F. prausnitzii genomes. The difference between the two genomogroups is mainly in the complement of genes associated with catabolism of carbohydrates (such as a predicted sialidase gene in genomogroup I) and amino acids, as well as defense mechanisms. CONCLUSIONS: Based on the combination of ANI of genomic sequences, phylogenetic analysis of core proteomes and functional differences we propose to separate the species F. prausnitzii into two new species level taxa: F. prausnitzii sensu stricto (neotype strain A2-165T = DSM 17677T = JCM 31915T) and F. moorei sp. nov. (type strain ATCC 27768T = NCIMB 13872T).

Interpersonal variability of the human gut virome confounds disease signal detection in IBD.

Viruses are increasingly recognised as important components of the human microbiome, fulfilling numerous ecological roles including bacterial predation, immune stimulation, genetic diversification, horizontal gene transfer, microbial interactions, and augmentation of metabolic functions. However, our current view of the human gut virome is tainted by previous sequencing requirements that necessitated the amplification of starting nucleic acids. In this study, we performed an original longitudinal analysis of 40 healthy control, 19 Crohn's disease, and 20 ulcerative colitis viromes over three time points without an amplification bias, which revealed and highlighted the interpersonal individuality of the human gut virome. In contrast to a 16 S rRNA gene analysis of matched samples, we show that α- and ß-diversity metrics of unamplified viromes are not as efficient at discerning controls from patients with inflammatory bowel disease. Additionally, we explored the intrinsic properties of unamplified gut viromes and show there is considerable interpersonal variability in viral taxa, infrequent longitudinal persistence of intrapersonal viruses, and vast fluctuations in the abundance of temporal viruses. Together, these properties of unamplified faecal viromes confound the ability to discern disease associations but significantly advance toward an unbiased and accurate representation of the human gut virome.

Stability of the human gut virome and effect of gluten-free diet.

The human gut microbiome consists of bacteria, archaea, eukaryotes, and viruses. The gut viruses are relatively underexplored. Here, we longitudinally analyzed the gut virome composition in 11 healthy adults: its stability, variation, and the effect of a gluten-free diet. Using viral enrichment and a de novo assembly-based approach, we demonstrate the quantitative dynamics of the gut virome, including dsDNA, ssDNA, dsRNA, and ssRNA viruses. We observe highly divergent individual viral communities, carrying on an average 2,143 viral genomes, 13.1% of which were present at all 3 time points. In contrast to previous reports, the Siphoviridae family dominates over Microviridae in studied individual viromes. We also show individual viromes to be stable at the family level but to vary substantially at the genera and species levels. Finally, we demonstrate that lower initial diversity of the human gut virome leads to a more pronounced effect of the dietary intervention on its composition.

Genomic characterization of three marine fungi, including Emericellopsis atlantica sp. nov. with signatures of a generalist lifestyle and marine biomass degradation.

Marine fungi remain poorly covered in global genome sequencing campaigns; the 1000 fungal genomes (1KFG) project attempts to shed light on the diversity, ecology and potential industrial use of overlooked and poorly resolved fungal taxa. This study characterizes the genomes of three marine fungi: Emericellopsis sp. TS7, wood-associated Amylocarpus encephaloides and algae-associated Calycina marina. These species were genome sequenced to study their genomic features, biosynthetic potential and phylogenetic placement using multilocus data. Amylocarpus encephaloides and C. marina were placed in the Helotiaceae and Pezizellaceae (Helotiales), respectively, based on a 15-gene phylogenetic analysis. These two genomes had fewer biosynthetic gene clusters (BGCs) and carbohydrate active enzymes (CAZymes) than Emericellopsis sp. TS7 isolate. Emericellopsis sp. TS7 (Hypocreales, Ascomycota) was isolated from the sponge Stelletta normani. A six-gene phylogenetic analysis placed the isolate in the marine Emericellopsis clade and morphological examination confirmed that the isolate represents a new species, which is described here as E. atlantica. Analysis of its CAZyme repertoire and a culturing experiment on three marine and one terrestrial substrates indicated that E. atlantica is a psychrotrophic generalist fungus that is able to degrade several types of marine biomass. FungiSMASH analysis revealed the presence of 35 BGCs including, eight non-ribosomal peptide synthases (NRPSs), six NRPS-like, six polyketide synthases, nine terpenes and six hybrid, mixed or other clusters. Of these BGCs, only five were homologous with characterized BGCs. The presence of unknown BGCs sets and large CAZyme repertoire set stage for further investigations of E. atlantica. The Pezizellaceae genome and the genome of the monotypic Amylocarpus genus represent the first published genomes of filamentous fungi that are restricted in their occurrence to the marine habitat and form thus a valuable resource for the community that can be used in studying ecological adaptions of fungi using comparative genomics.

Gut Bacteriophage: Current Understanding and Challenges.

The gut microbiome is widely accepted to have a significant impact on human health yet, despite years of research on this complex ecosystem, the contributions of different forces driving microbial population structure remain to be fully elucidated. The viral component of the human gut microbiome is dominated by bacteriophage, which are known to play crucial roles in shaping microbial composition, driving bacterial diversity, and facilitating horizontal gene transfer. Bacteriophage are also one of the most poorly understood components of the human gut microbiome, with the vast majority of viral sequences sharing little to no homology to reference databases. If we are to understand the dynamics of bacteriophage populations, their interaction with the human microbiome and ultimately their influence on human health, we will depend heavily on sequence based approaches and in silico tools. This is complicated by the fact that, as with any research field in its infancy, methods of analyses vary and this can impede our ability to compare the outputs of different studies. Here, we discuss the major findings to date regarding the human virome and reflect on our current understanding of how gut bacteriophage shape the microbiome. We consider whether or not the virome field is built on unstable foundations and if so, how can we provide a solid basis for future experimentation. The virome is a challenging yet crucial piece of the human microbiome puzzle. In order to develop our understanding, we will discuss the need to underpin future studies with robust research methods and suggest some solutions to existing challenges.

Choice of assembly software has a critical impact on virome characterisation.

BACKGROUND: The viral component of microbial communities plays a vital role in driving bacterial diversity, facilitating nutrient turnover and shaping community composition. Despite their importance, the vast majority of viral sequences are poorly annotated and share little or no homology to reference databases. As a result, investigation of the viral metagenome (virome) relies heavily on de novo assembly of short sequencing reads to recover compositional and functional information. Metagenomic assembly is particularly challenging for virome data, often resulting in fragmented assemblies and poor recovery of viral community members. Despite the essential role of assembly in virome analysis and difficulties posed by these data, current assembly comparisons have been limited to subsections of virome studies or bacterial datasets. DESIGN: This study presents the most comprehensive virome assembly comparison to date, featuring 16 metagenomic assembly approaches which have featured in human virome studies. Assemblers were assessed using four independent virome datasets, namely, simulated reads, two mock communities, viromes spiked with a known phage and human gut viromes. RESULTS: Assembly performance varied significantly across all test datasets, with SPAdes (meta) performing consistently well. Performance of MIRA and VICUNA varied, highlighting the importance of using a range of datasets when comparing assembly programs. It was also found that while some assemblers addressed the challenges of virome data better than others, all assemblers had limitations. Low read coverage and genomic repeats resulted in assemblies with poor genome recovery, high degrees of fragmentation and low-accuracy contigs across all assemblers. These limitations must be considered when setting thresholds for downstream analysis and when drawing conclusions from virome data.

Whole-Virome Analysis Sheds Light on Viral Dark Matter in Inflammatory Bowel Disease.

The human gut virome is thought to significantly impact the microbiome and human health. However, most virome analyses have been performed on a limited fraction of known viruses. Using whole-virome analysis on a published keystone inflammatory bowel disease (IBD) cohort and an in-house ulcerative colitis dataset, we shed light on the composition of the human gut virome in IBD beyond this identifiable minority. We observe IBD-specific changes to the virome and increased numbers of temperate phage sequences in individuals with Crohn's disease. Unlike prior database-dependent methods, no changes in viral richness were observed. Among IBD subjects, the changes in virome composition reflected alterations in bacterial composition. Furthermore, incorporating both bacteriome and virome composition offered greater classification power between health and disease. This approach to analyzing whole virome across cohorts highlights significant IBD signals, which may be crucial for developing future biomarkers and therapeutics.

The Human Gut Virome Is Highly Diverse, Stable, and Individual Specific.

The human gut contains a vast array of viruses, mostly bacteriophages. The majority remain uncharacterized, and their roles in shaping the gut microbiome and in impacting on human health remain poorly understood. We performed longitudinal metagenomic analysis of fecal viruses in healthy adults that reveal high temporal stability, individual specificity, and correlation with the bacterial microbiome. Using a database-independent approach that uses most of the sequencing data, we uncovered the existence of a stable, numerically predominant individual-specific persistent personal virome. Clustering of viral genomes and de novo taxonomic annotation identified several groups of crAss-like and Microviridae bacteriophages as the most stable colonizers of the human gut. CRISPR-based host prediction highlighted connections between these stable viral communities and highly predominant gut bacterial taxa such as Bacteroides, Prevotella, and Faecalibacterium. This study provides insights into the structure of the human gut virome and serves as an important baseline for hypothesis-driven research.

Biology and Taxonomy of crAss-like Bacteriophages, the Most Abundant Virus in the Human Gut.

CrAssphages represent the most abundant virus in the human gut microbiota, but the lack of available genome sequences for comparison has kept them enigmatic. Recently, sequence-based classification of distantly related crAss-like phages from multiple environments was reported, leading to a proposed familial-level taxonomic group. Here, we assembled the metagenomic sequencing reads from 702 human fecal virome/phageome samples and analyzed 99 complete circular crAss-like phage genomes and 150 contigs ≥70 kb. In silico comparative genomics and taxonomic analysis enabled a classification scheme of crAss-like phages from human fecal microbiomes into four candidate subfamilies composed of ten candidate genera. Laboratory analysis was performed on fecal samples from an individual harboring seven distinct crAss-like phages. We achieved crAss-like phage propagation in ex vivo human fecal fermentations and visualized short-tailed podoviruses by electron microscopy. Mass spectrometry of a crAss-like phage capsid protein could be linked to metagenomic sequencing data, confirming crAss-like phage structural annotations.

Reproducible protocols for metagenomic analysis of human faecal phageomes.

BACKGROUND: Recent studies have demonstrated that the human gut is populated by complex, highly individual and stable communities of viruses, the majority of which are bacteriophages. While disease-specific alterations in the gut phageome have been observed in IBD, AIDS and acute malnutrition, the human gut phageome remains poorly characterised. One important obstacle in metagenomic studies of the human gut phageome is a high level of discrepancy between results obtained by different research groups. This is often due to the use of different protocols for enriching virus-like particles, nucleic acid purification and sequencing. The goal of the present study is to develop a relatively simple, reproducible and cost-efficient protocol for the extraction of viral nucleic acids from human faecal samples, suitable for high-throughput studies. We also analyse the effect of certain potential confounding factors, such as storage conditions, repeated freeze-thaw cycles, and operator bias on the resultant phageome profile. Additionally, spiking of faecal samples with an exogenous phage standard was employed to quantitatively analyse phageomes following metagenomic sequencing. Comparative analysis of phageome profiles to bacteriome profiles was also performed following 16S rRNA amplicon sequencing. RESULTS: Faecal phageome profiles exhibit an overall greater individual specificity when compared to bacteriome profiles. The phageome and bacteriome both exhibited moderate change when stored at + 4 °C or room temperature. Phageome profiles were less impacted by multiple freeze-thaw cycles than bacteriome profiles, but there was a greater chance for operator effect in phageome processing. The successful spiking of faecal samples with exogenous bacteriophage demonstrated large variations in the total viral load between individual samples. CONCLUSIONS: The faecal phageome sequencing protocol developed in this study provides a valuable additional view of the human gut microbiota that is complementary to 16S amplicon sequencing and/or metagenomic sequencing of total faecal DNA. The protocol was optimised for several confounding factors that are encountered while processing faecal samples, to reduce discrepancies observed within and between research groups studying the human gut phageome. Rapid storage, limited freeze-thaw cycling and spiking of faecal samples with an exogenous phage standard are recommended for optimum results.