Informing the Exposure Landscape: The Fate of Microplastics in a Large Pelagic In-Lake Mesocosm Experiment.

Understanding microplastic exposure and effects is critical to understanding risk. Here, we used large, in-lake closed-bottom mesocosms to investigate exposure and effects on pelagic freshwater ecosystems. This article provides details about the experimental design and results on the transport of microplastics and exposure to pelagic organisms. Our experiment included three polymers of microplastics (PE, PS, and PET) ranging in density and size. Nominal concentrations ranged from 0 to 29,240 microplastics per liter on a log scale. Mesocosms enclosed natural microbial, phytoplankton, and zooplankton communities and yellow perch (Perca flavescens). We quantified and characterized microplastics in the water column and in components of the food web (biofilm on the walls, zooplankton, and fish). The microplastics in the water stratified vertically according to size and density. After 10 weeks, about 1% of the microplastics added were in the water column, 0.4% attached to biofilm on the walls, 0.01% within zooplankton, and 0.0001% in fish. Visual observations suggest the remaining >98% were in a surface slick and on the bottom. Our study suggests organisms that feed at the surface and in the benthos are likely most at risk, and demonstrates the value of measuring exposure and transport to inform experimental designs and achieve target concentrations in different matrices within toxicity tests.

Environment-specific virocell metabolic reprogramming.

Viruses impact microbial systems through killing hosts, horizontal gene transfer, and altering cellular metabolism, consequently impacting nutrient cycles. A virus-infected cell, a "virocell," is distinct from its uninfected sister cell as the virus commandeers cellular machinery to produce viruses rather than replicate cells. Problematically, virocell responses to the nutrient-limited conditions that abound in nature are poorly understood. Here we used a systems biology approach to investigate virocell metabolic reprogramming under nutrient limitation. Using transcriptomics, proteomics, lipidomics, and endo- and exo-metabolomics, we assessed how low phosphate (low-P) conditions impacted virocells of a marine Pseudoalteromonas host when independently infected by two unrelated phages (HP1 and HS2). With the combined stresses of infection and nutrient limitation, a set of nested responses were observed. First, low-P imposed common cellular responses on all cells (virocells and uninfected cells), including activating the canonical P-stress response, and decreasing transcription, translation, and extracellular organic matter consumption. Second, low-P imposed infection-specific responses (for both virocells), including enhancing nitrogen assimilation and fatty acid degradation, and decreasing extracellular lipid relative abundance. Third, low-P suggested virocell-specific strategies. Specifically, HS2-virocells regulated gene expression by increasing transcription and ribosomal protein production, whereas HP1-virocells accumulated host proteins, decreased extracellular peptide relative abundance, and invested in broader energy and resource acquisition. These results suggest that although environmental conditions shape metabolism in common ways regardless of infection, virocell-specific strategies exist to support viral replication during nutrient limitation, and a framework now exists for identifying metabolic strategies of nutrient-limited virocells in nature.

Benchmarking informatics approaches for virus discovery: caution is needed when combining in silico identification methods.

Understanding the ecological impacts of viruses on natural and engineered ecosystems relies on the accurate identification of viral sequences from community sequencing data. To maximize viral recovery from metagenomes, researchers frequently combine viral identification tools. However, the effectiveness of this strategy is unknown. Here, we benchmarked combinations of six widely used informatics tools for viral identification and analysis (VirSorter, VirSorter2, VIBRANT, DeepVirFinder, CheckV, and Kaiju), called "rulesets." Rulesets were tested against mock metagenomes composed of taxonomically diverse sequence types and diverse aquatic metagenomes to assess the effects of the degree of viral enrichment and habitat on tool performance. We found that six rulesets achieved equivalent accuracy [Matthews Correlation Coefficient (MCC) = 0.77, Padj ≥ 0.05]. Each contained VirSorter2, and five used our "tuning removal" rule designed to remove non-viral contamination. While DeepVirFinder, VIBRANT, and VirSorter were each found once in these high-accuracy rulesets, they were not found in combination with each other: combining tools does not lead to optimal performance. Our validation suggests that the MCC plateau at 0.77 is partly caused by inaccurate labeling within reference sequence databases. In aquatic metagenomes, our highest MCC ruleset identified more viral sequences in virus-enriched (44%-46%) than in cellular metagenomes (7%-19%). While improved algorithms may lead to more accurate viral identification tools, this should be done in tandem with careful curation of sequence databases. We recommend using the VirSorter2 ruleset and our empirically derived tuning removal rule. Our analysis provides insight into methods for in silico viral identification and will enable more robust viral identification from metagenomic data sets. IMPORTANCE: The identification of viruses from environmental metagenomes using informatics tools has offered critical insights in microbial ecology. However, it remains difficult for researchers to know which tools optimize viral recovery for their specific study. In an attempt to recover more viruses, studies are increasingly combining the outputs from multiple tools without validating this approach. After benchmarking combinations of six viral identification tools against mock metagenomes and environmental samples, we found that these tools should only be combined cautiously. Two to four tool combinations maximized viral recovery and minimized non-viral contamination compared with either the single-tool or the five- to six-tool ones. By providing a rigorous overview of the behavior of in silico viral identification strategies and a pipeline to replicate our process, our findings guide the use of existing viral identification tools and offer a blueprint for feature engineering of new tools that will lead to higher-confidence viral discovery in microbiome studies.

Genomic insights into the coupling of a Chlorella-like microeukaryote and sulfur bacteria in the chemocline of permanently stratified Lake Cadagno.

Meromictic Lake Cadagno is a permanently stratified system with a persistent microbial bloom within the oxic-anoxic boundary called the chemocline. The association between oxygenic and anoxygenic photosynthesis within the chemocline has been known for at least two decades. Although anoxygenic purple and green sulfur bacteria have been well studied, reports on oxygenic phytoplankton have remained sparse since their discovery in the 1920s. Nearly a century later, this study presents the first near-complete genome of a photosynthetic microbial eukaryote from the chemocline of Lake Cadagno, provisionally named Chlorella-like MAG. The 18.9 Mbp nuclear genome displays a high GC content (71.5%), and the phylogenetic placement suggests that it is a novel species of the genus Chlorella of Chlorophytes. Functional annotation of the Chlorella-like metagenome-assembled genome predicted 10,732 protein-coding genes, with an approximate 0.6% proportion potentially involved in carbon, sulfur, and nitrogen (C, N, and S) metabolism. In addition to C4 photosynthesis, this study detected genes for heat shock proteins (HSPs) in the Chlorella-like algae, consistent with the other Chlorella species. Altogether, the genomic insights in this study suggest the cooperation of photosynthetic algae with phototrophic sulfur bacteria via C, N, and S metabolism, which may aid their collective persistence in the Lake Cadagno chemocline. Furthermore, this work additionally presents the chloroplast genome of Cryptomonas-like species, which was likely to be presumed as cyanobacteria in previous studies because of the presence of phycobilisomes.

MetaboDirect: an analytical pipeline for the processing of FT-ICR MS-based metabolomic data.

BACKGROUND: Microbiomes are now recognized as the main drivers of ecosystem function ranging from the oceans and soils to humans and bioreactors. However, a grand challenge in microbiome science is to characterize and quantify the chemical currencies of organic matter (i.e., metabolites) that microbes respond to and alter. Critical to this has been the development of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), which has drastically increased molecular characterization of complex organic matter samples, but challenges users with hundreds of millions of data points where readily available, user-friendly, and customizable software tools are lacking. RESULTS: Here, we build on years of analytical experience with diverse sample types to develop MetaboDirect, an open-source, command-line-based pipeline for the analysis (e.g., chemodiversity analysis, multivariate statistics), visualization (e.g., Van Krevelen diagrams, elemental and molecular class composition plots), and presentation of direct injection high-resolution FT-ICR MS data sets after molecular formula assignment has been performed. When compared to other available FT-ICR MS software, MetaboDirect is superior in that it requires a single line of code to launch a fully automated framework for the generation and visualization of a wide range of plots, with minimal coding experience required. Among the tools evaluated, MetaboDirect is also uniquely able to automatically generate biochemical transformation networks (ab initio) based on mass differences (mass difference network-based approach) that provide an experimental assessment of metabolite connections within a given sample or a complex metabolic system, thereby providing important information about the nature of the samples and the set of microbial reactions or pathways that gave rise to them. Finally, for more experienced users, MetaboDirect allows users to customize plots, outputs, and analyses. CONCLUSION: Application of MetaboDirect to FT-ICR MS-based metabolomic data sets from a marine phage-bacterial infection experiment and a Sphagnum leachate microbiome incubation experiment showcase the exploration capabilities of the pipeline that will enable the research community to evaluate and interpret their data in greater depth and in less time. It will further advance our knowledge of how microbial communities influence and are influenced by the chemical makeup of the surrounding system. The source code and User's guide of MetaboDirect are freely available through ( https://github.com/Coayala/MetaboDirect ) and ( https://metabodirect.readthedocs.io/en/latest/ ), respectively. Video Abstract.

Evaluating the ecological hypothesis: early life salivary microbiome assembly predicts dental caries in a longitudinal case-control study.

BACKGROUND: Early childhood caries (ECC)-dental caries (cavities) occurring in primary teeth up to age 6 years-is a prevalent childhood oral disease with a microbial etiology. Streptococcus mutans was previously considered a primary cause, but recent research promotes the ecologic hypothesis, in which a dysbiosis in the oral microbial community leads to caries. In this incident, density sampled case-control study of 189 children followed from 2 months to 5 years, we use the salivary bacteriome to (1) prospectively test the ecological hypothesis of ECC in salivary bacteriome communities and (2) identify co-occurring salivary bacterial communities predicting future ECC. RESULTS: Supervised classification of future ECC case status using salivary samples from age 12 months using bacteriome-wide data (AUC-ROC 0.78 95% CI (0.71-0.85)) predicts future ECC status before S. mutans can be detected. Dirichlet multinomial community state typing and co-occurrence network analysis identified similar robust and replicable groups of co-occurring taxa. Mean relative abundance of a Haemophilus parainfluenzae/Neisseria/Fusobacterium periodonticum group was lower in future ECC cases (0.14) than controls (0.23, P value < 0.001) in pre-incident visits, positively correlated with saliva pH (Pearson rho = 0.33, P value < 0.001) and reduced in individuals who had acquired S. mutans by the next study visit (0.13) versus those who did not (0.20, P value < 0.01). In a subset of whole genome shotgun sequenced samples (n = 30), case plaque had higher abundances of antibiotic production and resistance gene orthologs, including a major facilitator superfamily multidrug resistance transporter (MFS DHA2 family PBH value = 1.9 × 10-28), lantibiotic transport system permease protein (PBH value = 6.0 × 10-6) and bacitracin synthase I (PBH value = 5.6 × 10-6). The oxidative phosphorylation KEGG pathway was enriched in case plaque (PBH value = 1.2 × 10-8), while the ABC transporter pathway was depleted (PBH value = 3.6 × 10-3). CONCLUSIONS: Early-life bacterial interactions predisposed children to ECC, supporting a time-dependent interpretation of the ecological hypothesis. Bacterial communities which assemble before 12 months of age can promote or inhibit an ecological succession to S. mutans dominance and cariogenesis. Intragenera competitions and intergenera cooperation between oral taxa may shape the emergence of these communities, providing points for preventive interventions. Video Abstract.

Bacterial, Phytoplankton, and Viral Distributions and Their Biogeochemical Contexts in Meromictic Lake Cadagno Offer Insights into the Proterozoic Ocean Microbial Loop.

Lake Cadagno, a permanently stratified high-alpine lake with a persistent microbial bloom in its chemocline, has long been considered a model for the low-oxygen, high-sulfide Proterozoic ocean. Although the lake has been studied for over 25 years, the absence of concerted study of the bacteria, phytoplankton, and viruses, together with primary and secondary production, has hindered a comprehensive understanding of its microbial food web. Here, the identities, abundances, and productivity of microbes were evaluated in the context of Lake Cadagno biogeochemistry. Photosynthetic pigments together with 16S rRNA gene phylogenies suggest the prominence of eukaryotic phytoplankton chloroplasts, primarily chlorophytes. Chloroplasts closely related to those of high-alpine-adapted Ankyra judayi persisted with oxygen in the mixolimnion, where photosynthetic efficiency was high, while chloroplasts of Closteriopsis-related chlorophytes peaked in the chemocline and monimolimnion. The anoxygenic phototrophic sulfur bacterium Chromatium dominated the chemocline along with Lentimicrobium, a genus of known fermenters. Secondary production peaked in the chemocline, which suggested that anoxygenic primary producers depended on heterotrophic nutrient remineralization. The virus-to-microbe ratio peaked with phytoplankton abundances in the mixolimnion and were at a minimum where Chromatium abundance was highest, trends that suggest that viruses may play a role in the modulation of primary production. Through the combined analysis of bacterial, eukaryotic, viral, and biogeochemical spatial dynamics, we provide a comprehensive synthesis of the Lake Cadagno microbial loop. This study offers a new ecological perspective on how biological and geochemical connections may have occurred in the chemocline of the Proterozoic ocean, where eukaryotic microbial life is thought to have evolved. IMPORTANCE As a window into the past, this study offers insights into the potential role that microbial guilds may have played in the production and recycling of organic matter in ancient Proterozoic ocean chemoclines. The new observations described here suggest that chloroplasts of eukaryotic algae were persistent in the low-oxygen upper chemocline along with the purple and green sulfur bacteria known to dominate the lower half of the chemocline. This study provides the first insights into Lake Cadagno's viral ecology. High viral abundances suggested that viruses may be essential components of the chemocline, where their activity may result in the release and recycling of organic matter. The integration of diverse geochemical and biological data types provides a framework that lays the foundation to quantitatively resolve the processes performed by the discrete populations that comprise the microbial loop in this early anoxic ocean analogue.

A snapshot of the global drinking water virome: Diversity and metabolic potential vary with residual disinfectant use.

Viruses are important drivers of microbial community ecology and evolution, influencing microbial mortality, metabolism, and horizontal gene transfer. However, the effects of viruses remain largely unknown in many environments, including in drinking water systems. Drinking water metagenomic studies have offered a whole community perspective of bacterial impacts on water quality, but have not yet considered the influences of viruses. In this study, we address this gap by mining viral DNA sequences from publicly available drinking water metagenomes from distribution systems in six countries around the world. These datasets provide a snapshot of the taxonomic diversity and metabolic potential of the global drinking water virome; and provide an opportunity to investigate the effects of geography, climate, and drinking water treatment practices on viral diversity. Both environmental conditions and differences in sample processing were found to influence the viral composition. Using free chlorine as the residual disinfectant was associated with clear differences in viral taxonomic diversity and metabolic potential, with significantly fewer viral populations and less even viral community structures than observed in distribution systems without residual disinfectant. Additionally, drinking water viruses carry antibiotic resistance genes (ARGs), as well as genes to survive oxidative stress and nitrogen limitation. Through this study, we have demonstrated that viral communities are diverse across drinking water systems and vary with the use of residual disinfectant. Our findings offer directions for future research to develop a more robust understanding of how virus-bacteria interactions in drinking water distribution systems affect water quality.

Cross-Hemisphere Study Reveals Geographically Ubiquitous, Plastic-Specific Bacteria Emerging from the Rare and Unexplored Biosphere.

While it is now appreciated that the millions of tons of plastic pollution travelling through marine systems carry complex communities of microorganisms, it is still unknown to what extent these biofilm communities are specific to the plastic or selected by the surrounding ecosystem. To address this, we characterized and compared the microbial communities of microplastic particles, nonplastic (natural and wax) particles, and the surrounding waters from three marine ecosystems (the Baltic, Sargasso and Mediterranean seas) using high-throughput 16S rRNA gene sequencing. We found that biofilm communities on microplastic and nonplastic particles were highly similar to one another across this broad geographical range. The similar temperature and salinity profiles of the Sargasso and Mediterranean seas, compared to the Baltic Sea, were reflected in the biofilm communities. We identified plastic-specific operational taxonomic units (OTUs) that were not detected on nonplastic particles or in the surrounding waters. Twenty-six of the plastic-specific OTUs were geographically ubiquitous across all sampled locations. These geographically ubiquitous plastic-specific OTUs were mostly low-abundance members of their biofilm communities and often represented uncultured members of marine ecosystems. These results demonstrate the potential for plastics to be a reservoir of rare and understudied microbes, thus warranting further investigations into the dynamics and role of these microbes in marine ecosystems. IMPORTANCE This study represents one of the largest comparisons of biofilms from environmentally sampled plastic and nonplastic particles from aquatic environments. By including particles sampled through three separate campaigns in the Baltic, Sargasso, and Mediterranean seas, we were able to make cross-geographical comparisons and discovered common taxonomical signatures that define the plastic biofilm. For the first time, we identified plastic-specific bacteria that reoccur across marine regions. Our data reveal that plastics have selective properties that repeatedly enrich for similar bacteria regardless of location, potentially shifting aquatic microbial communities in areas with high levels of plastic pollution. Furthermore, we show that bacterial communities on plastic do not appear to be strongly influenced by polymer type, suggesting that other properties, such as the absorption and/or leaching of chemicals from the surface, are likely to be more important in the selection and enrichment of specific microorganisms.

The genetic and ecophysiological diversity of Microcystis.

Microcystis is a cyanobacterium that forms toxic blooms in freshwater ecosystems around the world. Biological variation among taxa within the genus is apparent through genetic and phenotypic differences between strains and via the spatial and temporal distribution of strains in the environment, and this fine-scale diversity exerts strong influence over bloom toxicity. Yet we do not know how varying traits of Microcystis strains govern their environmental distribution, the tradeoffs and links between these traits, or how they are encoded at the genomic level. Here we synthesize current knowledge on the importance of diversity within Microcystis and on the genes and traits that likely underpin ecological differentiation of taxa. We briefly review spatial and environmental patterns of Microcystis diversity in the field and genetic evidence for cohesive groups within Microcystis. We then compile data on strain-level diversity regarding growth responses to environmental conditions and explore evidence for variation of community interactions across Microcystis strains. Potential links and tradeoffs between traits are identified and discussed. The resulting picture, while incomplete, highlights key knowledge gaps that need to be filled to enable new models for predicting strain-level dynamics, which influence the development, toxicity and cosmopolitan nature of Microcystis blooms.

Comparison of ultrafiltration and iron chloride flocculation in the preparation of aquatic viromes from contrasting sample types.

Viral metagenomes (viromes) are a valuable untargeted tool for studying viral diversity and the central roles viruses play in host disease, ecology, and evolution. Establishing effective methods to concentrate and purify viral genomes prior to sequencing is essential for high quality viromes. Using virus spike-and-recovery experiments, we stepwise compared two common approaches for virus concentration, ultrafiltration and iron chloride flocculation, across diverse matrices: wastewater influent, wastewater secondary effluent, river water, and seawater. Viral DNA was purified by removing cellular DNA via chloroform cell lysis, filtration, and enzymatic degradation of extra-viral DNA. We found that viral genomes were concentrated 1-2 orders of magnitude more with ultrafiltration than iron chloride flocculation for all matrices and resulted in higher quality DNA suitable for amplification-free and long-read sequencing. Given its widespread use and utility as an inexpensive field method for virome sampling, we nonetheless sought to optimize iron flocculation. We found viruses were best concentrated in seawater with five-fold higher iron concentrations than the standard used, inhibition of DNase activity reduced purification effectiveness, and five-fold more iron was needed to flocculate viruses from freshwater than seawater-critical knowledge for those seeking to apply this broadly used method to freshwater virome samples. Overall, our results demonstrated that ultrafiltration and purification performed better than iron chloride flocculation and purification in the tested matrices. Given that the method performance depended on the solids content and salinity of the samples, we suggest spike-and-recovery experiments be applied when concentrating and purifying sample types that diverge from those tested here.

Metagenomic Quantification of Genes with Internal Standards.

We demonstrate that an assembly-independent and spike-in facilitated metagenomic quantification approach can be used to screen and quantify over 2,000 genes simultaneously, while delivering absolute gene concentrations comparable to those for quantitative PCR (qPCR). DNA extracted from dairy manure slurry, digestate, and compost was spiked with genomic DNA from a marine bacterium and sequenced using the Illumina HiSeq4000. We compared gene copy concentrations, in gene copies per mass of sample, of five antimicrobial resistance genes (ARGs) generated with (i) our quantitative metagenomic approach, (ii) targeted qPCR, and (iii) a hybrid quantification approach involving metagenomics and qPCR-based 16S rRNA gene quantification. Although qPCR achieved lower quantification limits, the metagenomic method avoided biases caused by primer specificity inherent to qPCR-based methods and was able to detect orders of magnitude more genes than is possible with qPCR assays. We used the approach to simultaneously quantify ARGs in the Comprehensive Antimicrobial Resistance Database (CARD). We observed that the total abundance of tetracycline resistance genes was consistent across different stages of manure treatment on three farms, but different samples were dominated by different tetracycline resistance gene families.IMPORTANCE qPCR and metagenomics are central molecular techniques that have offered insights into biological processes for decades, from monitoring spatial and temporal gene dynamics to tracking ARGs or pathogens. Still needed is a tool that can quantify thousands of relevant genes in a sample as gene copies per sample mass or volume. We compare a quantitative metagenomic approach with traditional qPCR approaches in the quantification of ARG targets in dairy manure samples. By leveraging the benefits of nontargeted community genomics, we demonstrate high-throughput absolute gene quantification of all known ARG sequences in environmental samples.

Exopolymeric Substances Control Microbial Community Structure and Function by Contributing to both C and Fe Nutrition in Fe-Limited Southern Ocean Provinces.

Organic ligands such as exopolymeric substances (EPS) are known to form complexes with iron (Fe) and modulate phytoplankton growth. However, the effect of organic ligands on bacterial and viral communities remains largely unknown. Here, we assessed how Fe associated with organic ligands influences phytoplankton, microbial, and viral abundances and their diversity in the Southern Ocean. While the particulate organic carbon (POC) was modulated by Fe chemistry and bioavailability in the Drake Passage, the abundance and diversity of microbes and viruses were not governed by Fe bioavailability. Only following amendments with bacterial EPS did bacterial abundances increase, while phenotypic alpha diversity of bacterial and viral communities decreased. The latter was accompanied by significantly enhanced POC, pointing toward the relief of C limitation or other drivers of the microbial loop. Based on the literature and our findings, we propose a conceptual framework by which EPS may affect phytoplankton, bacteria, and viruses. Given the importance of the Southern Ocean for Earth's climate as well as the prevalence of viruses and their increasingly recognized impact on marine biogeochemistry and C cycling; the role of microbe-virus interactions on primary productivity in the Southern Ocean needs urgent attention.

Uptake and Retention of Nanoplastics in Quagga Mussels.

Here, a set of experiments to assess the feasibility of using an invasive and widespread freshwater mussel (Dreissena rostrformis bugensis) as a sentinel species for nanoplastic detection is reported. Under laboratory experimental conditions, mussels ingest and retain fluorescent polystyrene (PS) beads with carboxylic acid (-COOH) termination over a size range of 200-2000 nm. The number of beads the mussels ingested is quantified using fluorescence spectroscopy and the location of the beads in the mussels is imaged using fluorescence microscopy. PS beads of similar size (1000-2000 nm) to mussels' preferred food are trafficked in the ciliated food grooves of the gills. Beads of all sizes are observed in the mussels' digestive tracts, indicating that the mussels do not efficiently reject the beads as unwanted foreign material, regardless of size. Fluorescence microscopy shows all sizes of beads are concentrated in the siphons and are retained there for longer than one month postexposure. Combined atomic force microscopy-infrared spectroscopy and photothermal infrared spectroscopy are used to locate, image, and chemically identify the beads in the mussel siphons. In sum, these experiments demonstrate the potential for using mussels, specifically their siphons, to monitor environmental accumulation of aquatic nanoplastics.

Microbial and Viral Communities and Their Antibiotic Resistance Genes Throughout a Hospital Wastewater Treatment System.

Antibiotic resistance poses a serious threat to global public health, and antibiotic resistance determinants can enter natural aquatic systems through discharge of wastewater effluents. Hospital wastewater in particular is expected to contain high abundances of antibiotic resistance genes (ARGs) compared to municipal wastewater because it contains human enteric bacteria that may include antibiotic-resistant organisms originating from hospital patients, and can also have high concentrations of antibiotics and antimicrobials relative to municipal wastewater. Viruses also play an important role in wastewater treatment systems since they can influence the bacterial community composition through killing bacteria, facilitating transduction of genetic material between organisms, and modifying the chromosomal content of bacteria as prophages. However, little is known about the fate and connections between ARGs, viruses, and their associated bacteria in hospital wastewater systems. To address this knowledge gap, we characterized the composition and persistence of ARGs, dsDNA viruses, and bacteria from influent to effluent in a pilot-scale hospital wastewater treatment system in Israel using shotgun metagenomics. Results showed that ARGs, including genes conferring resistance to antibiotics of high clinical relevance, were detected in all sampling locations throughout the pilot-scale system, with only 16% overall depletion of ARGs per genome equivalent between influent and effluent. The most common classes of ARGs detected throughout the system conferred resistance to aminoglycoside, cephalosporin, macrolide, penam, and tetracycline antibiotics. A greater proportion of total ARGs were associated with plasmid-associated genes in effluent compared to in influent. No strong associations between viral sequences and ARGs were identified in viral metagenomes from the system, suggesting that phage may not be a significant vector for ARG transfer in this system. The majority of viruses in the pilot-scale system belonged to the families Myoviridae, Podoviridae, and Siphoviridae. Gammaproteobacteria was the dominant class of bacteria harboring ARGs and the most common putative viral host in all samples, followed by Bacilli and Betaproteobacteria. In the total bacterial community, the dominant class was Betaproteobacteria for each sample. Overall, we found that a variety of different types of ARGs and viruses were persistent throughout this hospital wastewater treatment system, which can be released to the environment through effluent discharge.

Phage-specific metabolic reprogramming of virocells.

Ocean viruses are abundant and infect 20-40% of surface microbes. Infected cells, termed virocells, are thus a predominant microbial state. Yet, virocells and their ecosystem impacts are understudied, thus precluding their incorporation into ecosystem models. Here we investigated how unrelated bacterial viruses (phages) reprogram one host into contrasting virocells with different potential ecosystem footprints. We independently infected the marine Pseudoalteromonas bacterium with siphovirus PSA-HS2 and podovirus PSA-HP1. Time-resolved multi-omics unveiled drastically different metabolic reprogramming and resource requirements by each virocell, which were related to phage-host genomic complementarity and viral fitness. Namely, HS2 was more complementary to the host in nucleotides and amino acids, and fitter during infection than HP1. Functionally, HS2 virocells hardly differed from uninfected cells, with minimal host metabolism impacts. HS2 virocells repressed energy-consuming metabolisms, including motility and translation. Contrastingly, HP1 virocells substantially differed from uninfected cells. They repressed host transcription, responded to infection continuously, and drastically reprogrammed resource acquisition, central carbon and energy metabolisms. Ecologically, this work suggests that one cell, infected versus uninfected, can have immensely different metabolisms that affect the ecosystem differently. Finally, we relate phage-host genome complementarity, virocell metabolic reprogramming, and viral fitness in a conceptual model to guide incorporating viruses into ecosystem models.

Minimum Information about an Uncultivated Virus Genome (MIUViG).

We present an extension of the Minimum Information about any (x) Sequence (MIxS) standard for reporting sequences of uncultivated virus genomes. Minimum Information about an Uncultivated Virus Genome (MIUViG) standards were developed within the Genomic Standards Consortium framework and include virus origin, genome quality, genome annotation, taxonomic classification, biogeographic distribution and in silico host prediction. Community-wide adoption of MIUViG standards, which complement the Minimum Information about a Single Amplified Genome (MISAG) and Metagenome-Assembled Genome (MIMAG) standards for uncultivated bacteria and archaea, will improve the reporting of uncultivated virus genomes in public databases. In turn, this should enable more robust comparative studies and a systematic exploration of the global virosphere.

Diagnostic Potential and Interactive Dynamics of the Colorectal Cancer Virome.

Human viruses (those that infect human cells) have been associated with many cancers, largely due to their mutagenic and functionally manipulative abilities. Despite this, cancer microbiome studies have focused almost exclusively on bacteria instead of viruses. We began evaluating the cancer virome by focusing on colorectal cancer, a primary cause of morbidity and mortality throughout the world and a cancer linked to altered colonic bacterial community compositions but with an unknown association with the gut virome. We used 16S rRNA gene, whole shotgun metagenomic, and purified virus metagenomic sequencing of stool to evaluate the differences in human colorectal cancer virus and bacterial community composition. Through random forest modeling, we identified differences in the healthy and colorectal cancer viromes. The cancer-associated virome consisted primarily of temperate bacteriophages that were also predicted to be bacterium-virus community network hubs. These results provide foundational evidence that bacteriophage communities are associated with colorectal cancer and potentially impact cancer progression by altering the bacterial host communities.IMPORTANCE Colorectal cancer is a leading cause of cancer-related death in the United States and worldwide. Its risk and severity have been linked to colonic bacterial community composition. Although human-specific viruses have been linked to other cancers and diseases, little is known about colorectal cancer virus communities. We addressed this knowledge gap by identifying differences in colonic virus communities in the stool of colorectal cancer patients and how they compared to bacterial community differences. The results suggested an indirect role for the virome in impacting colorectal cancer by modulating the associated bacterial community. These findings both support the idea of a biological role for viruses in colorectal cancer and provide a new understanding of basic colorectal cancer etiology.

Biogeography and environmental conditions shape bacteriophage-bacteria networks across the human microbiome.

Viruses and bacteria are critical components of the human microbiome and play important roles in health and disease. Most previous work has relied on studying bacteria and viruses independently, thereby reducing them to two separate communities. Such approaches are unable to capture how these microbial communities interact, such as through processes that maintain community robustness or allow phage-host populations to co-evolve. We implemented a network-based analytical approach to describe phage-bacteria network diversity throughout the human body. We built these community networks using a machine learning algorithm to predict which phages could infect which bacteria in a given microbiome. Our algorithm was applied to paired viral and bacterial metagenomic sequence sets from three previously published human cohorts. We organized the predicted interactions into networks that allowed us to evaluate phage-bacteria connectedness across the human body. We observed evidence that gut and skin network structures were person-specific and not conserved among cohabitating family members. High-fat diets appeared to be associated with less connected networks. Network structure differed between skin sites, with those exposed to the external environment being less connected and likely more susceptible to network degradation by microbial extinction events. This study quantified and contrasted the diversity of virome-microbiome networks across the human body and illustrated how environmental factors may influence phage-bacteria interactive dynamics. This work provides a baseline for future studies to better understand system perturbations, such as disease states, through ecological networks.