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Despite recent advances in sequencing and culturing, a deep knowledge of the wiring and functioning of the human gut ecosystem and its microbiota as a community is still missing. A holistic mechanistic understanding will require study of the gut microbiota as an interactive and spatially organized biological system, which is difficult to do in complex natural communities. Synthetic gut microbial ecosystems can function as model systems to further current understanding of the composition, stability and functional activities of the microbiota. In this Review, we provide an overview of the current synthetic ecology strategies that can be used towards a more comprehensive understanding of the human gut ecosystem. Such approaches that integrate in vitro experiments using cultured isolates with mathematical modelling will enable the ultimate goal: translating mechanistic and ecological knowledge into novel and effective therapies.

Background Autism spectrum disorders (ASD) are complex neurobiological disorders that impair social interactions and communication and lead to restricted, repetitive, and stereotyped patterns of behavior, interests, and activities. The causes of these disorders remain poorly understood, but gut microbiota, the 10 13 bacteria in the human intestines, have been implicated because children with ASD often suffer gastrointestinal (GI) problems that correlate with ASD severity. Several previous studies have reported abnormal gut bacteria in children with ASD. The gut microbiome-ASD connection has been tested in a mouse model of ASD, where the microbiome was mechanistically linked to abnormal metabolites and behavior. Similarly, a study of children with ASD found that oral non-absorbable antibiotic treatment improved GI and ASD symptoms, albeit temporarily. Here, a small open-label clinical trial evaluated the impact of Microbiota Transfer Therapy (MTT) on gut microbiota composition and GI and ASD symptoms of 18 ASD-diagnosed children. Results MTT involved a 2-week antibiotic treatment, a bowel cleanse, and then an extended fecal microbiota transplant (FMT) using a high initial dose followed by daily and lower maintenance doses for 7–8 weeks. The Gastrointestinal Symptom Rating Scale revealed an approximately 80% reduction of GI symptoms at the end of treatment, including significant improvements in symptoms of constipation, diarrhea, indigestion, and abdominal pain. Improvements persisted 8 weeks after treatment. Similarly, clinical assessments showed that behavioral ASD symptoms improved significantly and remained improved 8 weeks after treatment ended. Bacterial and phagedeep sequencing analyses revealed successful partial engraftment of donor microbiota and beneficial changes in the gut environment. Specifically, overall bacterial diversity and the abundance of Bifidobacterium , Prevotella , and Desulfovibrio among other taxa increased following MTT, and these changes persisted after treatment stopped (followed for 8 weeks). Conclusions This exploratory, extended-duration treatment protocol thus appears to be a promising approach to alter the gut microbiome and virome and improve GI and behavioral symptoms of ASD. Improvements in GI symptoms, ASD symptoms, and the microbiome all persisted for at least 8 weeks after treatment ended, suggesting a long-term impact. Trial registration This trial was registered on the ClinicalTrials.gov, with the registration number  NCT02504554

Understanding the pathology of COVID-19 is a global research priority. Early evidence suggests that the respiratory microbiome may be playing a role in disease progression, yet current studies report contradictory results. Here, we examine potential confounders in COVID-19 respiratory microbiome studies by analyzing the upper ( n  = 58) and lower ( n  = 35) respiratory tract microbiome in well-phenotyped COVID-19 patients and controls combining microbiome sequencing, viral load determination, and immunoprofiling. We find that time in the intensive care unit and type of oxygen support, as well as associated treatments such as antibiotic usage, explain the most variation within the upper respiratory tract microbiome, while SARS-CoV-2 viral load has a reduced impact. Specifically, mechanical ventilation is linked to altered community structure and significant shifts in oral taxa previously associated with COVID-19. Single-cell transcriptomics of the lower respiratory tract of COVID-19 patients identifies specific oral bacteria in physical association with proinflammatory immune cells, which show higher levels of inflammatory markers. Overall, our findings suggest confounders are driving contradictory results in current COVID-19 microbiome studies and careful attention needs to be paid to ICU stay and type of oxygen support, as bacteria favored in these conditions may contribute to the inflammatory phenotypes observed in severe COVID-19 patients. Here, the authors profile the respiratory microbiome of COVID-19 patients and link clinical practices, such as mechanical ventilation, with vast changes in the microbiota. In the lungs, oral bacteria are found physically associated with proinflammatory immune cells, thus possibly contributing to exacerbated immune responses in severe disease

Marine viruses impact global biogeochemical cycles via their influence on host community structure and function, yet our understanding of viral ecology is constrained by limitations in host culturing and a lack of reference genomes and ‘universal’ gene markers to facilitate community surveys. Short-read viral metagenomic studies have provided clues to viral function and first estimates of global viral gene abundance and distribution, but their assemblies are confounded by populations with high levels of strain evenness and nucleotide diversity (microdiversity), limiting assembly of some of the most abundant viruses on Earth. Such features also challenge assembly across genomic islands containing niche-defining genes that drive ecological speciation. These populations and features may be successfully captured by single-virus genomics and fosmid-based approaches, at least in abundant taxa, but at considerable cost and technical expertise. Here we established a low-cost, low-input, high throughput alternative sequencing and informatics workflow to improve viral metagenomic assemblies using short-read and long-read technology. The ‘VirION’ (Viral, long-read metagenomics via MinION sequencing) approach was first validated using mock communities where it was found to be as relatively quantitative as short-read methods and provided significant improvements in recovery of viral genomes. We then then applied VirION to the first metagenome from a natural viral community from the Western English Channel. In comparison to a short-read only approach, VirION: (i) increased number and completeness of assembled viral genomes; (ii) captured abundant, highly microdiverse virus populations, and (iii) captured more and longer genomic islands. Together, these findings suggest that VirION provides a high throughput and cost-effective alternative to fosmid and single-virus genomic approaches to more comprehensively explore viral communities in nature.

Background The role of commensal viruses in humans is poorly understood, and the impact of the virome on lung health and smoking-related disease is particularly understudied. Methods Genetic material from acellular bronchoalveolar lavage fluid was sequenced to identify and quantify viral members of the lower respiratory tract which were compared against concurrent bronchoalveolar lavage bacterial, metabolite, cytokine and cellular profiles, and clinical data. Twenty smoker and 10 nonsmoker participants with no significant comorbidities were studied. Results Viruses that infect bacteria (phages) represented the vast majority of viruses in the lung. Though bacterial communities were statistically indistinguishable across smokers and nonsmokers as observed in previous studies, lung viromes and metabolic profiles were significantly different between groups. Statistical analyses revealed that changes in viral communities correlate most with changes in levels of arachidonic acid and IL-8, both potentially relevant for chronic obstructive pulmonary disease (COPD) pathogenesis based on prior studies. Conclusions Our assessment of human lung DNA viral communities reveals that commensal viruses are present in the lower respiratory tract and differ between smokers and nonsmokers. The associations between viral populations and local immune and metabolic tone suggest a significant role for virome-host interaction in smoking related lung disease.

Background Microbes and their viruses are hidden engines driving Earth’s ecosystems from the oceans and soils to humans and bioreactors. Though gene marker approaches can now be complemented by genome-resolved studies of inter-(macrodiversity) and intra-(microdiversity) population variation, analytical tools to do so remain scattered or under-developed. Results Here, we introduce MetaPop, an open-source bioinformatic pipeline that provides a single interface to analyze and visualize microbial and viral community metagenomes at both the macro - and microdiversity levels. Macrodiversity estimates include population abundances and α- and β-diversity. Microdiversity calculations include identification of single nucleotide polymorphisms, novel codon-constrained linkage of SNPs, nucleotide diversity ( π and θ ), and selective pressures (pN/pS and Tajima’s D ) within and fixation indices ( F ST ) between populations. MetaPop will also identify genes with distinct codon usage. Following rigorous validation, we applied MetaPop to the gut viromes of autistic children that underwent fecal microbiota transfers and their neurotypical peers. The macrodiversity results confirmed our prior findings for viral populations (microbial shotgun metagenomes were not available) that diversity did not significantly differ between autistic and neurotypical children. However, by also quantifying microdiversity, MetaPop revealed lower average viral nucleotide diversity ( π ) in autistic children. Analysis of the percentage of genomes detected under positive selection was also lower among autistic children, suggesting that higher viral π in neurotypical children may be beneficial because it allows populations to better “bet hedge” in changing environments. Further, comparisons of microdiversity pre- and post-FMT in autistic children revealed that the delivery FMT method (oral versus rectal) may influence viral activity and engraftment of microdiverse viral populations, with children who received their FMT rectally having higher microdiversity post-FMT. Overall, these results show that analyses at the macro level alone can miss important biological differences. Conclusions These findings suggest that standardized population and genetic variation analyses will be invaluable for maximizing biological inference, and MetaPop provides a convenient tool package to explore the dual impact of macro - and microdiversity across microbial communities. BTPkfw67nTyd2JA5vx1spg Video abstract

Background Genetic recombination is a driving force in genome evolution. Among viruses it has a dual role. For genomes with higher fitness, it maintains genome integrity in the face of high mutation rates. Conversely, for genomes with lower fitness, it provides immediate access to sequence space that cannot be reached by mutation alone. Understanding how recombination impacts the cohesion and dissolution of individual whole genomes within viral sequence space is poorly understood across double-stranded DNA bacteriophages (a.k.a phages) due to the challenges of obtaining appropriately scaled genomic datasets. Results Here we explore the role of recombination in both maintaining and differentiating whole genomes of 142 wild double-stranded DNA marine cyanophages. Phylogenomic analysis across the 51 core genes revealed ten lineages, six of which were well represented. These phylogenomic lineages represent discrete genotypic populations based on comparisons of intra- and inter- lineage shared gene content, genome-wide average nucleotide identity, as well as detected gaps in the distribution of pairwise differences between genomes. McDonald-Kreitman selection tests identified putative niche-differentiating genes under positive selection that differed across the six well-represented genotypic populations and that may have driven initial divergence. Concurrent with patterns of recombination of discrete populations, recombination analyses of both genic and intergenic regions largely revealed decreased genetic exchange across individual genomes between relative to within populations. Conclusions These findings suggest that discrete double-stranded DNA marine cyanophage populations occur in nature and are maintained by patterns of recombination akin to those observed in bacteria, archaea and in sexual eukaryotes.

Human immunodeficiency virus (HIV) remains prevalent in sub-Saharan Africa, where it has been linked to adverse birth outcomes. Suboptimal vaginal microbiomes (VMBs) have shown similar links. This pilot study fills critical gaps in understanding how HIV interacts with the pregnant VMB in populations underrepresented in microbiome research, like the Democratic Republic of the Congo (DRC). We identified maternal systemic immune factors associated with suboptimal VMBs that have been linked to poor birth outcomes. In a global meta-analysis, we found significant taxonomic and functional differences in the VMBs between pregnant people living with and without HIV, revealing potential biomarkers that increase the risk of adverse birth outcomes. These findings provide crucial insights into VMB features that may influence pregnancy health in PLWH-ART, guiding future research and tailored interventions to support safer pregnancies in the DRC and similar populations. This study is registered with NCT03048669 .

Viruses play an important role in the ecology and biogeochemistry of marine ecosystems. Beyond mortality and gene transfer, viruses can reprogram microbial metabolism during infection by expressing auxiliary metabolic genes (AMGs) involved in photosynthesis, central carbon metabolism, and nutrient cycling. While previous studies have focused on AMG diversity in the sunlit and dark ocean, less is known about the role of viruses in shaping metabolic networks along redox gradients associated with marine oxygen minimum zones (OMZs). Here, we analyzed relatively quantitative viral metagenomic datasets that profiled the oxygen gradient across Eastern Tropical South Pacific (ETSP) OMZ waters, assessing whether OMZ viruses might impact nitrogen (N) cycling via AMGs. Identified viral genomes encoded six N-cycle AMGs associated with denitrification, nitrification, assimilatory nitrate reduction, and nitrite transport. The majority of these AMGs (80%) were identified in T4-like Myoviridae phages, predicted to infect Cyanobacteria and Proteobacteria , or in unclassified archaeal viruses predicted to infect Thaumarchaeota . Four AMGs were exclusive to anoxic waters and had distributions that paralleled homologous microbial genes. Together, these findings suggest viruses modulate N-cycling processes within the ETSP OMZ and may contribute to nitrogen loss throughout the global oceans thus providing a baseline for their inclusion in the ecosystem and geochemical models.

The metagenome of uncultured, Pacific Ocean viruses linked to a ubiquitous cyanobacteria is characterized using viral-tagging, revealing discrete populations in viral sequence space that includes previously cultivated populations and new populations missed in isolate-based studies. Ocean cyanobacterial viruses characterized Viruses outnumber microbial cells by a factor of about ten to one in marine systems, so they are important players in ocean ecology. It has so far proved particularly challenging to link viruses to their hosts experimentally on scales relevant to naturally occurring diversity. Here Matthew Sullivan and colleagues apply their previously developed high-throughput viral tagging methodology to a metagenomic survey of surface water samples collected from Monterey Bay, California. A screen of 10 7 viruses against the marine cyanobacterium Synechococcus WH7803 reveals the presence of previously cultivated cyanophage, as well as new viral types missed by decades of isolate-based studies. The analysis provides useful benchmarks for metagenomics and genome-based viral species definitions and new insights into the structure of the viral population. Microbes and their viruses drive myriad processes across ecosystems ranging from oceans and soils to bioreactors and humans 1 , 2 , 3 , 4 . Despite this importance, microbial diversity is only now being mapped at scales relevant to nature 5 , while the viral diversity associated with any particular host remains little researched. Here we quantify host-associated viral diversity using viral-tagged metagenomics, which links viruses to specific host cells for high-throughput screening and sequencing. In a single experiment, we screened 10 7 Pacific Ocean viruses against a single strain of Synechococcus and found that naturally occurring cyanophage genome sequence space is statistically clustered into discrete populations. These population-based, host-linked viral ecological data suggest that, for this single host and seawater sample alone, there are at least 26 double-stranded DNA viral populations with estimated relative abundances ranging from 0.06 to 18.2%. These populations include previously cultivated cyanophage and new viral types missed by decades of isolate-based studies. Nucleotide identities of homologous genes mostly varied by less than 1% within populations, even in hypervariable genome regions, and by 42–71% between populations, which provides benchmarks for viral metagenomics and genome-based viral species definitions. Together these findings showcase a new approach to viral ecology that quantitatively links objectively defined environmental viral populations, and their genomes, to their hosts.