Long-Read Metagenomics Improves the Recovery of Viral Diversity from Complex Natural Marine Samples.

The recovery of DNA from viromes is a major obstacle in the use of long-read sequencing to study their genomes. For this reason, the use of cellular metagenomes (>0.2-µm size range) emerges as an interesting complementary tool, since they contain large amounts of naturally amplified viral genomes from prelytic replication. We have applied second-generation (Illumina NextSeq; short reads) and third-generation (PacBio Sequel II; long reads) sequencing to compare the diversity and features of the viral community in a marine sample obtained from offshore waters of the western Mediterranean. We found that a major wedge of the expected marine viral diversity was directly recovered by the raw PacBio circular consensus sequencing (CCS) reads. More than 30,000 sequences were detected only in this data set, with no homologues in the long- and short-read assembly, and ca. 26,000 had no homologues in the large data set of the Global Ocean Virome 2 (GOV2), highlighting the information gap created by the assembly bias. At the level of complete viral genomes, the performance was similar in both approaches. However, the hybrid long- and short-read assembly provided the longest average length of the sequences and improved the host assignment. Although no novel major clades of viruses were found, there was an increase in the intraclade genomic diversity recovered by long reads that produced an enriched assessment of the real diversity and allowed the discovery of novel genes with biotechnological potential (e.g., endolysin genes). IMPORTANCE We explored the vast genetic diversity of environmental viruses by using a combination of cellular metagenome (as opposed to virome) sequencing using high-fidelity long-read sequences (in this case, PacBio CCS). This approach resulted in the recovery of a representative sample of the viral population, and it performed better (more phage contigs, larger average contig size) than Illumina sequencing applied to the same sample. By this approach, the many biases of assembly are avoided, as the CCS reads recovers (typically around 5 kb) complete genes and even operons, resulting in a better discovery of the viral gene diversity based on viral marker proteins. Thus, biotechnologically promising genes, such as endolysin genes, can be very efficiently searched with this approach. In addition, hybrid assembly produces more complete and longer contigs, which is particularly important for studying little-known viral groups such as the nucleocytoplasmic large DNA viruses (NCLDV).

Ecological diversification reveals routes of pathogen emergence in endemic Vibrio vulnificus populations.

Pathogen emergence is a complex phenomenon that, despite its public health relevance, remains poorly understood. Vibrio vulnificus, an emergent human pathogen, can cause a deadly septicaemia with over 50% mortality rate. To date, the ecological drivers that lead to the emergence of clinical strains and the unique genetic traits that allow these clones to colonize the human host remain mostly unknown. We recently surveyed a large estuary in eastern Florida, where outbreaks of the disease frequently occur, and found endemic populations of the bacterium. We established two sampling sites and observed strong correlations between location and pathogenic potential. One site is significantly enriched with strains that belong to one phylogenomic cluster (C1) in which the majority of clinical strains belong. Interestingly, strains isolated from this site exhibit phenotypic traits associated with clinical outcomes, whereas strains from the second site belong to a cluster that rarely causes disease in humans (C2). Analyses of C1 genomes indicate unique genetic markers in the form of clinical-associated alleles with a potential role in virulence. Finally, metagenomic and physicochemical analyses of the sampling sites indicate that this marked cluster distribution and genetic traits are strongly associated with distinct biotic and abiotic factors (e.g., salinity, nutrients, or biodiversity), revealing how ecosystems generate selective pressures that facilitate the emergence of specific strains with pathogenic potential in a population. This knowledge can be applied to assess the risk of pathogen emergence from environmental sources and integrated toward the development of novel strategies for the prevention of future outbreaks.

RaFAH: Host prediction for viruses of Bacteria and Archaea based on protein content.

Culture-independent approaches have recently shed light on the genomic diversity of viruses of prokaryotes. One fundamental question when trying to understand their ecological roles is: which host do they infect? To tackle this issue we developed a machine-learning approach named Random Forest Assignment of Hosts (RaFAH), that uses scores to 43,644 protein clusters to assign hosts to complete or fragmented genomes of viruses of Archaea and Bacteria. RaFAH displayed performance comparable with that of other methods for virus-host prediction in three different benchmarks encompassing viruses from RefSeq, single amplified genomes, and metagenomes. RaFAH was applied to assembled metagenomic datasets of uncultured viruses from eight different biomes of medical, biotechnological, and environmental relevance. Our analyses led to the identification of 537 sequences of archaeal viruses representing unknown lineages, whose genomes encode novel auxiliary metabolic genes, shedding light on how these viruses interfere with the host molecular machinery. RaFAH is available at https://sourceforge.net/projects/rafah/.

Dysbiosis in marine aquaculture revealed through microbiome analysis: reverse ecology for environmental sustainability.

The increasing demand for products for human consumption is leading to the fast-growing expansion of numerous food sectors such as marine aquaculture (mariculture). However, excessive input of nutrients and pollutants modifies marine ecosystems. Here, we applied a metagenomic approach to investigate these perturbations in samples from marine farms of gilthead seabream cultures. Results revealed dysbiosis and functional imbalance within the net cage with a unique structure, with little interference with samples from the fish microbiota or those collected far away from the coast. Remarkably, below the cage the prokaryotic community was highly similar to the marine microbiome of photic offshore samples. We recovered 48 novel metagenome-assembled genomes. Metagenomic recruitment revealed a significant change in the microbial community which was dominated by several Proteobacteria orders (Sphingomonadales, Pseudomonadales, Caudobacterales and Rhizobiales). Genomic potential for bioremediation processes, including nitrate removal through aerobic denitrification, and degradation of aromatic compounds and other toxic products were enriched in these microbes. The detrimental side effects were the increased number of antimicrobial resistance genes and the presence of potentially emergent pathogens. Knowledge of this metabolic diversity and the microbes involved in ecological balance recovery can be used to reduce the environmental impact of these practices.

Metagenome Mining Reveals Hidden Genomic Diversity of Pelagimyophages in Aquatic Environments.

The SAR11 clade is one of the most abundant bacterioplankton groups in surface waters of most of the oceans and lakes. However, only 15 SAR11 phages have been isolated thus far, and only one of them belongs to the Myoviridae family (pelagimyophages). Here, we have analyzed 26 sequences of myophages that putatively infect the SAR11 clade. They have been retrieved by mining ca. 45 Gbp aquatic assembled cellular metagenomes and viromes. Most of the myophages were obtained from the cellular fraction (0.2 µm), indicating a bias against this type of virus in viromes. We have found the first myophages that putatively infect Candidatus Fonsibacter (freshwater SAR11) and another group putatively infecting bathypelagic SAR11 phylogroup Ic. The genomes have similar sizes and maintain overall synteny in spite of low average nucleotide identity values, revealing high similarity to marine cyanomyophages. Pelagimyophages recruited metagenomic reads widely from several locations but always much more from cellular metagenomes than from viromes, opposite to what happens with pelagipodophages. Comparing the genomes resulted in the identification of a hypervariable island that is related to host recognition. Interestingly, some genes in these islands could be related to host cell wall synthesis and coinfection avoidance. A cluster of curli-related proteins was widespread among the genomes, although its function is unclear.IMPORTANCE SAR11 clade members are among the most abundant bacteria on Earth. Their study is complicated by their great diversity and difficulties in being grown and manipulated in the laboratory. On the other hand, and due to their extraordinary abundance, metagenomic data sets provide enormous richness of information about these microbes. Given the major role played by phages in the lifestyle and evolution of prokaryotic cells, the contribution of several new bacteriophage genomes preying on this clade opens windows into the infection strategies and life cycle of its viruses. Such strategies could provide models of attack of large-genome phages preying on streamlined aquatic microbes.

Evolutionary Model of Cluster Divergence of the Emergent Marine Pathogen Vibrio vulnificus: From Genotype to Ecotype.

Vibrio vulnificus, an opportunistic pathogen, is the causative agent of a life-threatening septicemia and a rising problem for aquaculture worldwide. The genetic factors that differentiate its clinical and environmental strains remain enigmatic. Furthermore, clinical strains have emerged from every clade of V. vulnificus In this work, we investigated the underlying genomic properties and population dynamics of the V. vulnificus species from an evolutionary and ecological point of view. Genome comparisons and bioinformatic analyses of 113 V. vulnificus isolates indicate that the population of V. vulnificus is made up of four different clusters. We found evidence that recombination and gene flow between the two largest clusters (cluster 1 [C1] and C2) have drastically decreased to the point where they are diverging independently. Pangenome and phenotypic analyses showed two markedly different lifestyles for these two clusters, indicating commensal (C2) and bloomer (C1) ecotypes, with differences in carbohydrate utilization, defense systems, and chemotaxis, among other characteristics. Nonetheless, we identified frequent intra- and interspecies exchange of mobile genetic elements (e.g., antibiotic resistance plasmids, novel "chromids," or two different and concurrent type VI secretion systems) that provide high levels of genetic diversity in the population. Surprisingly, we identified strains from both clusters in the mucosa of aquaculture species, indicating that manmade niches are bringing strains from the two clusters together. We propose an evolutionary model of V. vulnificus that could be broadly applicable to other pathogenic vibrios and facultative bacterial pathogens to pursue strategies to prevent their infections and emergence.IMPORTANCEVibrio vulnificus is an emergent marine pathogen and is the cause of a deadly septicemia. However, the genetic factors that differentiate its clinical and environmental strains and its several biotypes remain mostly enigmatic. In this work, we investigated the underlying genomic properties and population dynamics of the V. vulnificus species to elucidate the traits that make these strains emerge as a human pathogen. The acquisition of different ecological determinants could have allowed the development of highly divergent clusters with different lifestyles within the same environment. However, we identified strains from both clusters in the mucosa of aquaculture species, indicating that manmade niches are bringing strains from the two clusters together, posing a potential risk of recombination and of emergence of novel variants. We propose a new evolutionary model that provides a perspective that could be broadly applicable to other pathogenic vibrios and facultative bacterial pathogens to pursue strategies to prevent their infections.