A global metagenomic map of urban microbiomes and antimicrobial resistance.

We present a global atlas of 4,728 metagenomic samples from mass-transit systems in 60 cities over 3 years, representing the first systematic, worldwide catalog of the urban microbial ecosystem. This atlas provides an annotated, geospatial profile of microbial strains, functional characteristics, antimicrobial resistance (AMR) markers, and genetic elements, including 10,928 viruses, 1,302 bacteria, 2 archaea, and 838,532 CRISPR arrays not found in reference databases. We identified 4,246 known species of urban microorganisms and a consistent set of 31 species found in 97% of samples that were distinct from human commensal organisms. Profiles of AMR genes varied widely in type and density across cities. Cities showed distinct microbial taxonomic signatures that were driven by climate and geographic differences. These results constitute a high-resolution global metagenomic atlas that enables discovery of organisms and genes, highlights potential public health and forensic applications, and provides a culture-independent view of AMR burden in cities.

Emergence and Spread of Basal Lineages of Yersinia pestis during the Neolithic Decline.

Between 5,000 and 6,000 years ago, many Neolithic societies declined throughout western Eurasia due to a combination of factors that are still largely debated. Here, we report the discovery and genome reconstruction of Yersinia pestis, the etiological agent of plague, in Neolithic farmers in Sweden, pre-dating and basal to all modern and ancient known strains of this pathogen. We investigated the history of this strain by combining phylogenetic and molecular clock analyses of the bacterial genome, detailed archaeological information, and genomic analyses from infected individuals and hundreds of ancient human samples across Eurasia. These analyses revealed that multiple and independent lineages of Y. pestis branched and expanded across Eurasia during the Neolithic decline, spreading most likely through early trade networks rather than massive human migrations. Our results are consistent with the existence of a prehistoric plague pandemic that likely contributed to the decay of Neolithic populations in Europe.

Decoding the DNA and RNA viromes of a tropical urban lagoon.

Human and livestock sewage is one of the major causes of excess nutrients, leading to the eutrophication of aquatic ecosystems and potentially to the emergence or spread of pathogenic viruses. This study aimed to investigate the composition and diversity of aquatic viromes in a highly anthropized lagoon, to identify the presence of pathogenic taxa and to explore their use as possible viral indicators of faecal contamination. For this, water and sediment samples were collected in the Ebrié Lagoon (Ivory Coast) at seven stations with contrasting levels of eutrophication. The DNA viromes of the planktonic and the benthic compartments were highly divergent, but were not influenced by the level of eutrophication. Conversely, the RNA viromes in the water column were comparable to those found in sediment, but showed significant differences between the stations. We detected the presence of viral DNA and RNA sequences we had assigned as indicators of faecal contamination (smacovirus, pecovirus and pepper mild mottle virus) as well as human pathogens (human cyclovirus, coxsackie B virus and picobirnavirus), which were all enriched in the most eutrophicated sites. These findings suggest that the examination of viromes represents a promising tool for assessing the state of human-induced contamination of aquatic ecosystems.

Fishing for the Microbiome of Tropical Tuna.

Although tunas represent a significant part of the global fish economy and a major nutritional resource worldwide, their microbiome still remains poorly documented. Here, we conducted an analysis of the taxonomic composition of the bacterial communities inhabiting the gut, skin, and liver of two most consumed tropical tuna species (skipjack and yellowfin), from individuals caught in the Atlantic and Indian oceans. We hypothesized that each organ harbors a specific microbial assemblage whose composition might vary according to different biotic (sex, species) and/or abiotic (environmental) factors. Our results revealed that the composition of the tuna microbiome was totally independent of fish sex, regardless of the species and ocean considered. Instead, the main determinants of observed diversity were (i) tuna species for the gut and (ii) sampling site for the skin mucus layer and (iii) a combination of both parameters for the liver. Interestingly, 4.5% of all amplicon sequence variants (ASV) were shared by the three organs, highlighting the presence of a core-microbiota whose most abundant representatives belonged to the genera Mycoplasma, Cutibacterium, and Photobacterium. Our study also revealed the presence of a unique and diversified bacterial assemblage within the tuna liver, comprising a substantial proportion of potential histamine-producing bacteria, well known for their pathogenicity and their contribution to fish poisoning cases. These results indicate that this organ is an unexplored microbial niche whose role in the health of both the host and consumers remains to be elucidated.

Assessment of diversity of archaeal communities in Algerian chott.

Halophilic archaea are the dominant type of microorganisms in hypersaline environments. The diversity of halophilic archaea in Zehrez-Chergui (Saharian chott) was analyzed and compared by both analysis of a library of PCR amplified 16S rRNA genes and by cultivation approach. This work, represents the first of its type in Algeria. A total cell count was estimated at 3.8 × 103 CFU/g. The morphological, biochemical, and physiological characterizations of 45 distinct strains, suggests that all of them might be members of the class Halobacteria. Among stains, 23 were characterized phylogenetically and are related to 6 genera of halophilic archaea.The dominance of the genus Halopiger, has not been reported yet in other hypersaline environments. The 100 clones obtained by the molecular approach, were sequenced, and analyzed. The ribosomal library of 61 OTUs showed that the archaeal diversity included uncultured haloarcheon, Halomicrobium, Natronomonas, Halomicroarcula, Halapricum, Haloarcula, Halosimplex, Haloterrigena, Halolamina, Halorubellus, Halorussus and Halonotius. The results of rarefaction analysis indicated that the analysis of an increasing number of clones would have revealed additional diversity. Surprisingly, no halophilic archaea were not shared between the two approaches. Combining both types of methods was considered the best approach to acquire better information on the characteristics of soil halophilic archaea.

Metagenomics and the Human Virome in Asymptomatic Individuals.

High-throughput sequencing technologies have revolutionized how we think about viruses. Investigators can now go beyond pathogenic viruses and have access to the thousands of viruses that inhabit our bodies without causing clinical symptoms. By studying their interactions with each other, with other microbes, and with host genetics and immune systems, we can learn how they affect health and disease. This article reviews current knowledge of the composition and diversity of the human virome in physiologically healthy individuals. It focuses on recent results from metagenomics studies and discusses the contribution of bacteriophages and eukaryotic viruses to human health.

Metagenomic Analysis of Microdissected Valvular Tissue for Etiological Diagnosis of Blood Culture-Negative Endocarditis.

BACKGROUND: Etiological diagnosis is a key to therapeutic adaptation and improved prognosis, particularly for infections such as endocarditis. In blood culture-negative endocarditis (BCNE), 22% of cases remain undiagnosed despite an updated comprehensive syndromic approach. This prompted us to develop a new diagnostic approach. METHODS: Eleven valves from 10 BCNE patients were analyzed using a method that combines human RNA bait-depletion with phi29 DNA polymerase-based multiple displacement amplification and shotgun DNA sequencing. An additional case in which a microbe was serendipitously visualized by immunofluorescence was analyzed using the same method, but after laser capture microdissection. RESULTS: Background DNA prevented any diagnosis in cases analyzed without microdissection because the majority of sequences were contaminants. Moraxella sequences were dramatically enriched in the stained microdissected region of the additional case. A consensus genome sequence of 2.4 Mbp covering more than 94% of the Moraxella osloensis KSH reference genome was reconstructed with 234X average coverage. Several antibiotic-resistance genes were observed. Etiological diagnosis was confirmed using Western blot and specific polymerase chain reaction with sequencing on a different valve sample. CONCLUSIONS: Microdissection could be a key to the metagenomic diagnosis of infectious diseases when a microbe is visualized but remains unidentified despite an updated optimal approach. Moraxella osloensis should be tested in blood culture-negative endocarditis.

Detection of novel RNA viruses from free-living gorillas, Republic of the Congo: genetic diversity of picobirnaviruses.

Most of the emerging infectious diseases reported so far originated in wildlife. Therefore, virological surveillance of animals and particularly great apes is of great interest to establish the repertory of viruses associated with healthy hosts. This will further help to identify the emergence of new viruses and predict the possibility of interspecies transmission. In this study, we performed shotgun viral metagenomics on stool samples collected from seventeen free-living wild gorillas from the Republic of the Congo. The analysis revealed the presence of novel RNA viruses (picobirnaviruses, partitivirus, and Picornavirales (posa-like and dicistrovirus-like viruses)). Among these, picobirnavirus-related sequences were abundantly covered in the stools. Based on genetic variations both in capsid and RdRp proteins of picobirnaviruses, at least 96 variants were identified and most of them were novel. Among the 96, 22 variants had a nearly complete genome or segment. A comprehensive sequence analysis identified a potential new genogroup/genetic cluster and the presence of a short linear amino acid motif (ExxRxNxxxE) in a hypothetical protein. The sequence analysis of posa-like virus and dicistrovirus showed that these two viruses were novel members in the respective viral families. In conclusion, the identification of novel RNA viruses and their genetic diversity increases our knowledge about viruses that are associated with stools of wild gorillas and contributes to the initiatives in the search for potential emerging zoonotic viruses.

Human Polyomavirus-6 Infecting Lymph Nodes of a Patient With an Angiolymphoid Hyperplasia With Eosinophilia or Kimura Disease.

Human polyomavirus 6 (HPyV6) is most often detected at the skin surface of healthy individuals. Here, we demonstrate for the first time that HPyV6 also infects internal tissues. We provide direct evidence of HPyV6 infecting a lymph node of a patient with an angiolymphoid hyperplasia with eosinophilia or Kimura disease.

Zoonotic viruses: how to monitor their emergence?

Zoonoses are responsible of more than two thirds of human viral infections. In addition, with increasing contacts between humans and the wildlife and the domestic fauna, the emergence and reemergence of zoonotic viruses is accelerating. The development and democratization of high-throughput sequencing tools and their application in metagenomics allow inventorying the viral communities of various reservoirs and vectors in order to detect the emergence of viruses before their transmission to humans. The prediction of future emerging zoonotic viruses is very difficult, if not impossible. However, the characterization of viral communities present in the different actors of zoonotic transmission cycle is a first step to evaluate potential risks of transmission to humans. In this article, we report a brief summary of the concepts of emergence and zoonoses before reviewing the current tools available to monitor their emergence.

Absence of giant blood Marseille-like virus DNA detection by polymerase chain reaction in plasma from healthy US blood donors and serum from multiply transfused patients from Cameroon.

BACKGROUND: A new Marseilleviridae virus family member, giant blood Marseille-like (GBM) virus, was recently reported in persons from France in the serum of an infant with adenitis, in the blood of 4% of healthy blood donors, and in 9% of multiply transfused thalassemia patients. These results suggested the presence of a nucleocytoplasmic large DNA virus potentially transmissible by blood product transfusion. STUDY DESIGN AND METHODS: To investigate this possibility we tested the plasma from 113 US blood donors and 74 multiply transfused Cameroon patients for GBM viral DNA using highly sensitive polymerase chain reaction (PCR) assays. RESULTS: GBM DNA was not detected by nested PCR in any of these 187 human specimens. CONCLUSIONS: Further testing is required to confirm the occurrence of human GBM virus infections.

Provirophages and transpovirons as the diverse mobilome of giant viruses.

A distinct class of infectious agents, the virophages that infect giant viruses of the Mimiviridae family, has been recently described. Here we report the simultaneous discovery of a giant virus of Acanthamoeba polyphaga (Lentille virus) that contains an integrated genome of a virophage (Sputnik 2), and a member of a previously unknown class of mobile genetic elements, the transpovirons. The transpovirons are linear DNA elements of ~7 kb that encompass six to eight protein-coding genes, two of which are homologous to virophage genes. Fluorescence in situ hybridization showed that the free form of the transpoviron replicates within the giant virus factory and accumulates in high copy numbers inside giant virus particles, Sputnik 2 particles, and amoeba cytoplasm. Analysis of deep-sequencing data showed that the virophage and the transpoviron can integrate in nearly any place in the chromosome of the giant virus host and that, although less frequently, the transpoviron can also be linked to the virophage chromosome. In addition, integrated fragments of transpoviron DNA were detected in several giant virus and Sputnik genomes. Analysis of 19 Mimivirus strains revealed three distinct transpovirons associated with three subgroups of Mimiviruses. The virophage, the transpoviron, and the previously identified self-splicing introns and inteins constitute the complex, interconnected mobilome of the giant viruses and are likely to substantially contribute to interviral gene transfer.

Viruses in a 14th-century coprolite.

Coprolites are fossilized fecal material that can reveal information about ancient intestinal and environmental microbiota. Viral metagenomics has allowed systematic characterization of viral diversity in environmental and human-associated specimens, but little is known about the viral diversity in fossil remains. Here, we analyzed the viral community of a 14th-century coprolite from a closed barrel in a Middle Ages site in Belgium using electron microscopy and metagenomics. Viruses that infect eukaryotes, bacteria, and archaea were detected, and we confirmed the presence of some of them by ad hoc suicide PCR. The coprolite DNA viral metagenome was dominated by sequences showing homologies to phages commonly found in modern stools and soil. Although their phylogenetic compositions differed, the metabolic functions of the viral communities have remained conserved across centuries. Antibiotic resistance was one of the reconstructed metabolic functions detected.

The virophage as a unique parasite of the giant mimivirus.

Viruses are obligate parasites of Eukarya, Archaea and Bacteria. Acanthamoeba polyphaga mimivirus (APMV) is the largest known virus; it grows only in amoeba and is visible under the optical microscope. Mimivirus possesses a 1,185-kilobase double-stranded linear chromosome whose coding capacity is greater than that of numerous bacteria and archaea1, 2, 3. Here we describe an icosahedral small virus, Sputnik, 50 nm in size, found associated with a new strain of APMV. Sputnik cannot multiply in Acanthamoeba castellanii but grows rapidly, after an eclipse phase, in the giant virus factory found in amoebae co-infected with APMV4. Sputnik growth is deleterious to APMV and results in the production of abortive forms and abnormal capsid assembly of the host virus. The Sputnik genome is an 18.343-kilobase circular double-stranded DNA and contains genes that are linked to viruses infecting each of the three domains of life Eukarya, Archaea and Bacteria. Of the 21 predicted protein-coding genes, eight encode proteins with detectable homologues, including three proteins apparently derived from APMV, a homologue of an archaeal virus integrase, a predicted primase-helicase, a packaging ATPase with homologues in bacteriophages and eukaryotic viruses, a distant homologue of bacterial insertion sequence transposase DNA-binding subunit, and a Zn-ribbon protein. The closest homologues of the last four of these proteins were detected in the Global Ocean Survey environmental data set5, suggesting that Sputnik represents a currently unknown family of viruses. Considering its functional analogy with bacteriophages, we classify this virus as a virophage. The virophage could be a vehicle mediating lateral gene transfer between giant viruses.

Functional metagenomic profiling of nine biomes.

Microbial activities shape the biogeochemistry of the planet and macroorganism health. Determining the metabolic processes performed by microbes is important both for understanding and for manipulating ecosystems (for example, disruption of key processes that lead to disease, conservation of environmental services, and so on). Describing microbial function is hampered by the inability to culture most microbes and by high levels of genomic plasticity. Metagenomic approaches analyse microbial communities to determine the metabolic processes that are important for growth and survival in any given environment. Here we conduct a metagenomic comparison of almost 15 million sequences from 45 distinct microbiomes and, for the first time, 42 distinct viromes and show that there are strongly discriminatory metabolic profiles across environments. Most of the functional diversity was maintained in all of the communities, but the relative occurrence of metabolisms varied, and the differences between metagenomes predicted the biogeochemical conditions of each environment. The magnitude of the microbial metabolic capabilities encoded by the viromes was extensive, suggesting that they serve as a repository for storing and sharing genes among their microbial hosts and influence global evolutionary and metabolic processes.

Biodiversity and biogeography of phages in modern stromatolites and thrombolites.

Viruses, and more particularly phages (viruses that infect bacteria), represent one of the most abundant living entities in aquatic and terrestrial environments. The biogeography of phages has only recently been investigated and so far reveals a cosmopolitan distribution of phage genetic material (or genotypes). Here we address this cosmopolitan distribution through the analysis of phage communities in modern microbialites, the living representatives of one of the most ancient life forms on Earth. On the basis of a comparative metagenomic analysis of viral communities associated with marine (Highborne Cay, Bahamas) and freshwater (Pozas Azules II and Rio Mesquites, Mexico) microbialites, we show that some phage genotypes are geographically restricted. The high percentage of unknown sequences recovered from the three metagenomes (>97%), the low percentage similarities with sequences from other environmental viral (n = 42) and microbial (n = 36) metagenomes, and the absence of viral genotypes shared among microbialites indicate that viruses are genetically unique in these environments. Identifiable sequences in the Highborne Cay metagenome were dominated by single-stranded DNA microphages that were not detected in any other samples examined, including sea water, fresh water, sediment, terrestrial, extreme, metazoan-associated and marine microbial mats. Finally, a marine signature was present in the phage community of the Pozas Azules II microbialites, even though this environment has not been in contact with the ocean for tens of millions of years. Taken together, these results prove that viruses in modern microbialites display biogeographical variability and suggest that they may be derived from an ancient community.

Mimivirus shows dramatic genome reduction after intraamoebal culture.

Most phagocytic protist viruses have large particles and genomes as well as many laterally acquired genes that may be associated with a sympatric intracellular life (a community-associated lifestyle with viruses, bacteria, and eukaryotes) and the presence of virophages. By subculturing Mimivirus 150 times in a germ-free amoebal host, we observed the emergence of a bald form of the virus that lacked surface fibers and replicated in a morphologically different type of viral factory. When studying a 0.40-µm filtered cloned particle, we found that its genome size shifted from 1.2 (M1) to 0.993 Mb (M4), mainly due to large deletions occurring at both ends of the genome. Some of the lost genes are encoding enzymes required for posttranslational modification of the structural viral proteins, such as glycosyltransferases and ankyrin repeat proteins. Proteomic analysis allowed identification of three proteins, probably required for the assembly of virus fibers. The genes for two of these were found to be deleted from the M4 virus genome. The proteins associated with fibers are highly antigenic and can be recognized by mouse and human antimimivirus antibodies. In addition, the bald strain (M4) was not able to propagate the sputnik virophage. Overall, the Mimivirus transition from a sympatric to an allopatric lifestyle was associated with a stepwise genome reduction and the production of a predominantly bald virophage resistant strain. The new axenic ecosystem allowed the allopatric Mimivirus to lose unnecessary genes that might be involved in the control of competitors.

Viral metagenomics on animals as a tool for the detection of zoonoses prior to human infection?

Many human viral infections have a zoonotic, i.e., wild or domestic animal, origin. Several zoonotic viruses are transmitted to humans directly via contact with an animal or indirectly via exposure to the urine or feces of infected animals or the bite of a bloodsucking arthropod. If a virus is able to adapt and replicate in its new human host, human-to-human transmissions may occur, possibly resulting in an epidemic, such as the A/H1N1 flu pandemic in 2009. Thus, predicting emerging zoonotic infections is an important challenge for public health officials in the coming decades. The recent development of viral metagenomics, i.e., the characterization of the complete viral diversity isolated from an organism or an environment using high-throughput sequencing technologies, is promising for the surveillance of such diseases and can be accomplished by analyzing the viromes of selected animals and arthropods that are closely in contact with humans. In this review, we summarize our current knowledge of viral diversity within such animals (in particular blood-feeding arthropods, wildlife and domestic animals) using metagenomics and present its possible future application for the surveillance of zoonotic and arboviral diseases.

Marseillevirus-like virus recovered from blood donated by asymptomatic humans.

The study of the human virome is still in its infancy, especially with regard to the viral content of the blood of people who are apparently disease free. In this study, the genome of a new giant virus that is related to the amoeba-infecting pathogen Marseillevirus was recovered from donated blood, using high-throughput sequencing. Viral antigens were identified by an immunoconversion assay. The virus was visualized with transmission electron microscopy and fluorescence in situ hybridization and was grown in human T lymphocytes. Specific antibody reactions were used to identify viral proteins in blood specimens from polymerase chain reactive-positive donors. Finally, we tested 20 blood specimens from additional donors. Three had antibodies directed against this virus, and 2 had circulating viral DNA. This study shows that giant viruses, which are missed by the use of ultrafilters, are part of the human blood virome. The putative pathogenic role of giant viruses in humans remains undefined.