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CrAssphage is a recently discovered human gut-associated bacteriophage. To validate the potential use of crAssphage for detecting human fecal contamination on environmental surfaces and hands, we tested stool samples (n = 60), hand samples (n = 30), and environmental swab samples (n = 201) from 17 norovirus outbreaks for crAssphage by real-time PCR. In addition, we tested stool samples from healthy persons (n = 173), respiratory samples (n = 113), and animal fecal specimens (n = 68) and further sequenced positive samples. Overall, we detected crAssphage in 71.4% of outbreak stool samples, 48%-68.5% of stool samples from healthy persons, 56.2% of environmental swabs, and 60% of hand rinse samples, but not in human respiratory samples or animal fecal samples. CrAssphage sequences could be grouped into 2 major genetic clusters. Our data suggest that crAssphage could be used to detect human fecal contamination on environmental surfaces and hands.

Viruses are part of the microbiome and have essential roles in immunology, evolution, biogeochemical cycles, health, and disease progression. Viruses influence a wide variety of systems and processes, and the continued discovery of novel viruses is anticipated to reveal new mechanisms influencing the biology of diverse environments. While the identity and roles of viruses continue to be discovered and understood through viral metagenomics, most of the sequences in virome datasets cannot be attributed to known viruses or may be only distantly related to species already described in public sequence databases, at best. Such viruses are known as the viral dark matter. Ongoing discoveries from the viral dark matter have provided insights into novel viruses from a variety of environments, as well as their potential in immunological processes, virus evolution, health, disease, therapeutics, and surveillance. Increased understanding of the viral dark matter will continue with a combination of cultivation, microscopy, sequencing, and bioinformatic efforts, which are discussed in the present review.

Wastewater-based epidemiology (WBE) emerged during the coronavirus disease 2019 (COVID-19) pandemic as a scalable and broadly applicable method for community-level monitoring of infectious disease burden. The lack of high-resolution fecal shedding data for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) limits our ability to link WBE measurements to disease burden. In this study, we present longitudinal, quantitative fecal shedding data for SARS-CoV-2 RNA, as well as for the commonly used fecal indicators pepper mild mottle virus (PMMoV) RNA and crAss-like phage (crAssphage) DNA. The shedding trajectories from 48 SARS-CoV-2-infected individuals suggest a highly individualized, dynamic course of SARS-CoV-2 RNA fecal shedding. Of the individuals that provided at least three stool samples spanning more than 14 days, 77% had one or more samples that tested positive for SARS-CoV-2 RNA. We detected PMMoV RNA in at least one sample from all individuals and in 96% (352/367) of samples overall. CrAssphage DNA was detected in at least one sample from 80% (38/48) of individuals and was detected in 48% (179/371) of all samples. The geometric mean concentrations of PMMoV and crAssphage in stool across all individuals were 8.7 × 10 4 and 1.4 × 10 4 gene copies/milligram-dry weight, respectively, and crAssphage shedding was more consistent for individuals than PMMoV shedding. These results provide us with a missing link needed to connect laboratory WBE results with mechanistic models, and this will aid in more accurate estimates of COVID-19 burden in sewersheds. Additionally, the PMMoV and crAssphage data are critical for evaluating their utility as fecal strength normalizing measures and for source-tracking applications. This research represents a critical step in the advancement of wastewater monitoring for public health. To date, mechanistic materials balance modeling of wastewater-based epidemiology has relied on SARS-CoV-2 fecal shedding estimates from small-scale clinical reports or meta-analyses of research using a wide range of analytical methodologies. Additionally, previous SARS-CoV-2 fecal shedding data have not contained sufficient methodological information for building accurate materials balance models. Like SARS-CoV-2, fecal shedding of PMMoV and crAssphage has been understudied to date. The data presented here provide externally valid and longitudinal fecal shedding data for SARS-CoV-2, PMMoV, and crAssphage which can be directly applied to WBE models and ultimately increase the utility of WBE.

Background The crAss-like phages are ubiquitous and highly abundant members of the human gut virome that infect commensal bacteria of the order Bacteroidales. Although incapable of lysogeny, these viruses demonstrate long-term persistence in the human gut microbiome, dominating the virome in some individuals. Results Here we show that rapid phase variation of alternate capsular polysaccharides in Bacteroides intestinalis cultures plays an important role in a dynamic equilibrium between phage sensitivity and resistance, allowing phage and bacteria to multiply in parallel. The data also suggests the role of a concomitant phage persistence mechanism associated with delayed lysis of infected cells, similar to carrier state infection. From an ecological and evolutionary standpoint, this type of phage-host interaction is consistent with the Piggyback-the-Winner model, which suggests a preference towards lysogenic or other “benign” forms of phage infection when the host is stably present at high abundance. Conclusion Long-term persistence of bacteriophage and host could result from mutually beneficial mechanisms driving bacterial strain-level diversity and phage survival in complex environments.

Background The gut phageome comprises a complex phage community of thousands of individual strains, with a few highly abundant bacteriophages. CrAss-like phages, which infect bacteria of the order Bacteroidales, are the most abundant bacteriophage family in the human gut and make an important contribution to an individual’s core virome. Based on metagenomic data, crAss-like phages form a family, with four sub-families and ten candidate genera. To date, only three representatives isolated in pure culture have been reported: ΦcrAss001 and two closely related phages DAC15 and DAC17; all are members of the less abundant candidate genus VI. The persistence at high levels of both crAss-like phage and their Bacteroidales hosts in the human gut has not been explained mechanistically, and this phage-host relationship can only be properly studied with isolated phage-host pairs from as many genera as possible. Results Faeces from a healthy donor with high levels of crAss-like phage was used to initiate a faecal fermentation in a chemostat, with selected antibiotics chosen to inhibit rapidly growing bacteria and selectively enrich for Gram-negative Bacteroidales. This had the objective of promoting the simultaneous expansion of crAss-like phages on their native hosts. The levels of seven different crAss-like phages expanded during the fermentation, indicating that their hosts were also present in the fermenter. The enriched supernatant was then tested against individual Bacteroidales strains isolated from the same faecal sample. This resulted in the isolation of a previously uncharacterised crAss-like phage of candidate genus IV of the proposed Alphacrassvirinae sub-family, ΦcrAss002, that infects the gut commensal Bacteroides xylanisolvens . ΦcrAss002 does not form plaques or spots on lawns of sensitive cells, nor does it lyse liquid cultures, even at high titres. In keeping with the co-abundance of phage and host in the human gut, ΦcrAss002 and Bacteroides xylanisolvens can also co-exist at high levels when co-cultured in laboratory media. Conclusions We report the isolation and characterisation of ΦcrAss002, the first representative of the proposed Alphacrassvirinae sub-family of crAss-like phages. ΦcrAss002 cannot form plaques or spots on bacterial lawns but can co-exist with its host, Bacteroides xylanisolvens , at very high levels in liquid culture without impacting on bacterial numbers. 9UEWUPaqFhz6FLHJmZ8En6 Video abstract

Background Viruses are the most abundant biological entities on Earth, known to be crucial components of microbial ecosystems. However, there is little information on the viral community within agricultural waste. There are currently ~ 2.7 million dairy cattle in the UK producing 7–8% of their own bodyweight in manure daily, and 28 million tonnes annually. To avoid pollution of UK freshwaters, manure must be stored and spread in accordance with guidelines set by DEFRA. Manures are used as fertiliser, and widely spread over crop fields, yet little is known about their microbial composition. We analysed the virome of agricultural slurry over a 5-month period using short and long-read sequencing. Results Hybrid sequencing uncovered more high-quality viral genomes than long or short-reads alone; yielding 7682 vOTUs, 174 of which were complete viral genomes. The slurry virome was highly diverse and dominated by lytic bacteriophage, the majority of which represent novel genera (~ 98%). Despite constant influx and efflux of slurry, the composition and diversity of the slurry virome was extremely stable over time, with 55% of vOTUs detected in all samples over a 5-month period. Functional annotation revealed a diverse and abundant range of auxiliary metabolic genes and novel features present in the community, including the agriculturally relevant virulence factor VapE, which was widely distributed across different phage genera that were predicted to infect several hosts. Furthermore, we identified an abundance of phage-encoded diversity-generating retroelements, which were previously thought to be rare on lytic viral genomes. Additionally, we identified a group of crAssphages, including lineages that were previously thought only to be found in the human gut. Conclusions The cattle slurry virome is complex, diverse and dominated by novel genera, many of which are not recovered using long or short-reads alone. Phages were found to encode a wide range of AMGs that are not constrained to particular groups or predicted hosts, including virulence determinants and putative ARGs. The application of agricultural slurry to land may therefore be a driver of bacterial virulence and antimicrobial resistance in the environment. C9xR_Dnuto-w_HvVTTDj7q Video abstract

Background/aimsThe gut virome is mainly composed of bacteriophages and influences gut homeostasis and pathogenic conditions. In this study, we analyzed the gut prokaryotic virome in Japanese patients with Crohn’s disease (CD).Materials/methodsWe collected 19 fecal samples from CD patients and 16 samples from healthy controls. The gut bacteriome was analyzed by 16S rRNA gene sequencing and the virome was profiled by shotgun metagenomic sequencing.ResultsDespite no differences in richness and evenness, there was a significant difference in the overall structure of the gut virome between CD patients and controls (P = 0.013). CrAssphage and Staphylococcus virus, belonging to the order Caudovirales, were dominant in the gut virome of controls and CD patients. The abundance of crAssphage was significantly greater in CD patients than controls (P = 0.021). Lactococcus, Enterococcus and Lactobacillus phages were present only in CD patients, while Xanthomonas and Escherichia phages were unique to the controls. In the gut bacteriome of CD patients, richness and evenness were significantly lower, and a significant difference in the overall structure was observed between groups (P = 0.014). The gut bacteriome of CD patients was characterized by a decrease of the genera Faecalibacterium, Roseburia, and Ruminococcus and an increase of the family Enterobacteriaceae. There were more significant correlations between viruses and bacteria in CD patients than controls.ConclusionsThe gut virome of CD patients was distinct from that of healthy controls in a Japanese population. An altered gut virome may be one of the factors associated with the bacterial dysbiosis of CD.

An increased abundance of antibiotic resistance genes (ARGs) in aquatic environments has been linked to environmental pollution. Mining polluted sites with high concentration of metals could favor the in situ coselection of ARGs, whereas wastewater discharges release fecal antibiotic resistant bacteria in the environment. To study the effect of human fecal contamination and mining pollution, water and sediment samples affected by mining activities and sewage discharges were collected from three lakes in Bolivia, the pristine Andean lake Pata Khota, the Milluni Chico lake directly impacted by acid mine drainage, and the Uru-Uru lake located close to Oruro city and highly polluted by mining activities and human wastewater discharges. Physicochemical parameters, including metal composition, were analyzed in water and sediment samples. ARGs were screened for and verified by quantitative polymerase chain reaction (PCR) together with the mobile element class 1 integron (intl1), as well as crAssphage, a marker of human fecal pollution. The gene intl1 was positively correlated with sul1, sul2, tetA, and blaOXA-2. CrAssphage was only detected in the Uru-Uru lake, and its tributaries and significantly higher abundance of ARGs were found in these sites. Multivariate analysis showed that crAssphage abundance, electrical conductivity, and pH were positively correlated with higher levels of intl1 and ARGs. Taken together, our results suggest that fecal pollution is the major driver of higher levels of ARGs and intl1 in environments contaminated by wastewater and mining activities.

Objectives crAssphage is a newly found phage described as the most abundant virus in the human gut microbiome. The majority of the crAssphage proteins are unknown in sequences databases, and its pathogenicity and epidemiology in humans are yet unclear. Hence, being one of the most abundant phages in the human gut microbiome more investigation at the genomic level is necessary to improve our understanding, especially in the Latin American population. Data description In this article, we provide the whole genome of a crAssphage isolated from the human gut microbiome of the Mexican population, which was named Mexican-crAssphage. The genome consists of 96,283 bp, G+C content of 29.24% and 87 coding sequences. Notably, we did not find any transfer RNA genes in the genome sequence. We also sequenced viral-like enriched particles from 28 fecal samples, and we detected the presence of the Mexican-crAssphage genome in 8 samples (28.5%). To our knowledge, our data is the first whole genome report of the crAssphage isolated from the Latin American Population and provides valuable information for the experimental characterization of the most abundant human gut bacteriophage. The whole genome shotgun project of the Mexican-crAssphage is available at DDBJ/ENA/GenBank under the GenBank MK069403.

crAssphage is a newly discovered gut bacteriophage. However, its pathogenicity and molecular epidemiology in humans are as yet unclear. In this study, we investigated the association between crAssphage and diarrhoea, as well as the molecular epidemiology of crAssphage in Chinese patients from our hospital. Our results indicated that there were no significant differences in the crAssphage-positive ratio and viral loads in faecal supernatants between adults with diarrhoea and healthy adults. Of infants and children with diarrhoea, 2·8% were found to be crAssphage-positive, including two infants aged <1 month. Markedly, of all confirmed crAssphage-positive strains, 100% had the ORF00039 deletion and 77·8% had low identity of ORF00018 compared to crAssphage (GenBank accession no. NC_024711, designated genotype 1). Thus, crAssphage was not associated with diarrhoea and most strains of crAssphage in Chinese patients (designated genotype 2) were characterized by the ORF00039 deletion and low identity of ORF00018.