The long and short of it: benchmarking viromics using Illumina, Nanopore and PacBio sequencing technologies.

Viral metagenomics has fuelled a rapid change in our understanding of global viral diversity and ecology. Long-read sequencing and hybrid assembly approaches that combine long- and short-read technologies are now being widely implemented in bacterial genomics and metagenomics. However, the use of long-read sequencing to investigate viral communities is still in its infancy. While Nanopore and PacBio technologies have been applied to viral metagenomics, it is not known to what extent different technologies will impact the reconstruction of the viral community. Thus, we constructed a mock bacteriophage community of previously sequenced phage genomes and sequenced them using Illumina, Nanopore and PacBio sequencing technologies and tested a number of different assembly approaches. When using a single sequencing technology, Illumina assemblies were the best at recovering phage genomes. Nanopore- and PacBio-only assemblies performed poorly in comparison to Illumina in both genome recovery and error rates, which both varied with the assembler used. The best Nanopore assembly had errors that manifested as SNPs and INDELs at frequencies 41 and 157 % higher than found in Illumina only assemblies, respectively. While the best PacBio assemblies had SNPs at frequencies 12 and 78 % higher than found in Illumina-only assemblies, respectively. Despite high-read coverage, long-read-only assemblies recovered a maximum of one complete genome from any assembly, unless reads were down-sampled prior to assembly. Overall the best approach was assembly by a combination of Illumina and Nanopore reads, which reduced error rates to levels comparable with short-read-only assemblies. When using a single technology, Illumina only was the best approach. The differences in genome recovery and error rates between technology and assembler had downstream impacts on gene prediction, viral prediction, and subsequent estimates of diversity within a sample. These findings will provide a starting point for others in the choice of reads and assembly algorithms for the analysis of viromes.

Isolation of new Klebsiella pneumoniae phage PSKP16.

Background and Objectives: Klebsiella pneumoniae is a clinically relevant opportunistic pathogen belonging to the Enterobacteriaceae family. It is in the top three bacteria associated with antimicrobial resistance deaths globally, and one of the most dangerous bacteria causing nosocomial infections. Phage therapy offers a potential option for the treatment of drug-resistant bacterial infections. Materials and Methods: Phage PSKP16 was isolated against K. pneumoniae, capsular type K2 (isolated from a wound infection). PSKP16 is a new lytic phage with a Siphovirus-like morphology. Results: PSKP16 is a linear double stranded DNA phage with a GC content of 50% and genome size of 46,712 bp, for which we predicted 67 ORFs. PSKP16 belongs to the genus Webervirus and shows high evolutionary proximity to Klebsiella phages JY917, Sushi, and B1. Conclusion: Phage isolation is fast, cheap and efficient, but it requires time and characterization (which adds expense) to ensure that the isolated phages do not pose a health risk, which is essential to safely use phage therapy to treat life-threatening bacterial infections.

Staying below the Radar: Unraveling a New Family of Ubiquitous "Cryptic" Non-Tailed Temperate Vibriophages and Implications for Their Bacterial Hosts.

Bacteriophages are the most abundant biological entities in the oceans and play key roles in bacterial activity, diversity and evolution. While extensive research has been conducted on the role of tailed viruses (Class: Caudoviricetes), very little is known about the distribution and functions of the non-tailed viruses (Class: Tectiliviricetes). The recent discovery of the lytic Autolykiviridae family demonstrated the potential importance of this structural lineage, emphasizing the need for further exploration of the role of this group of marine viruses. Here, we report the novel family of temperate phages under the class of Tectiliviricetes, which we propose to name "Asemoviridae" with phage NO16 as a main representative. These phages are widely distributed across geographical regions and isolation sources and found inside the genomes of at least 30 species of Vibrio, in addition to the original V. anguillarum isolation host. Genomic analysis identified dif-like sites, suggesting that NO16 prophages recombine with the bacterial genome based on the XerCD site-specific recombination mechanism. The interactions between the NO16 phage and its V. anguillarum host were linked to cell density and phage-host ratio. High cell density and low phage predation levels were shown to favor the temperate over the lytic lifestyle for NO16 viruses, and their spontaneous induction rate was highly variable between different V. anguillarum lysogenic strains. NO16 prophages coexist with the V. anguillarum host in a mutualistic interaction by rendering fitness properties to the host, such as increased virulence and biofilm formation through lysogenic conversion, likely contributing to their global distribution.

A new family of globally distributed lytic roseophages with unusual deoxythymidine to deoxyuridine substitution.

Marine bacterial viruses (bacteriophages) are abundant biological entities that are vital for shaping microbial diversity, impacting marine ecosystem function, and driving host evolution.1-3 The marine roseobacter clade (MRC) is a ubiquitous group of heterotrophic bacteria4,5 that are important in the elemental cycling of various nitrogen, sulfur, carbon, and phosphorus compounds.6-10 Bacteriophages infecting MRC (roseophages) have thus attracted much attention and more than 30 roseophages have been isolated,11-13 the majority of which belong to the N4-like group (Podoviridae family) or the Chi-like group (Siphoviridae family), although ssDNA-containing roseophages are also known.14 In our attempts to isolate lytic roseophages, we obtained two new phages (DSS3_VP1 and DSS3_PM1) infecting the model MRC strain Ruegeria pomeroyi DSS-3. Here, we show that not only do these phages have unusual substitution of deoxythymidine with deoxyuridine (dU) in their DNA, but they are also phylogenetically distinct from any currently known double-stranded DNA bacteriophages, supporting the establishment of a novel family ("Naomiviridae"). These dU-containing phages possess DNA that is resistant to the commonly used library preparation method for metagenome sequencing, which may have caused significant underestimation of their presence in the environment. Nevertheless, our analysis of Tara Ocean metagenome datasets suggests that these unusual bacteriophages are of global importance and more diverse than other well-known bacteriophages, e.g., the Podoviridae in the oceans, pointing to an overlooked role for these novel phages in the environment.

Isolation and Characterization of Klebsiella Phages for Phage Therapy.

Introduction: Klebsiella is a clinically important pathogen causing a variety of antimicrobial resistant infections in both community and nosocomial settings, particularly pneumonia, urinary tract infection, and sepsis. Bacteriophage (phage) therapy is being considered a primary option for the treatment of drug-resistant infections of these types. Methods: We report the successful isolation and characterization of 30 novel, genetically diverse Klebsiella phages. Results: The isolated phages span six different phage families and nine genera, representing both lysogenic and lytic lifestyles. Individual Klebsiella phage isolates infected up to 11 of the 18 Klebsiella capsule types tested, and all 18 capsule-types were infected by at least one of the phages. Conclusions: Of the Klebsiella-infecting phages presented in this study, the lytic phages are most suitable for phage therapy, based on their broad host range, high virulence, short lysis period and given that they encode no known toxin or antimicrobial resistance genes. Phage isolates belonging to the Sugarlandvirus and Slopekvirus genera were deemed most suitable for phage therapy based on our characterization. Importantly, when applied alone, none of the characterized phages were able to suppress the growth of Klebsiella for more than 12 h, likely due to the inherent ease of Klebsiella to generate spontaneous phage-resistant mutants. This indicates that for successful phage therapy, a cocktail of multiple phages would be necessary to treat Klebsiella infections.

From Trees to Clouds: PhageClouds for Fast Comparison of ∼640,000 Phage Genomic Sequences and Host-Centric Visualization Using Genomic Network Graphs.

Background: Fast and computationally efficient strategies are required to explore genomic relationships within an increasingly large and diverse phage sequence space. Here, we present PhageClouds, a novel approach using a graph database of phage genomic sequences and their intergenomic distances to explore the phage genomic sequence space. Methods: A total of 640,000 phage genomic sequences were retrieved from a variety of databases and public virome assemblies. Intergenomic distances were calculated with dashing, an alignment-free method suitable for handling massive data sets. These data were used to build a Neo4j® graph database. Results: PhageClouds supported the search of related phages among all complete phage genomes from GenBank for a single query phage in just 10 s. Moreover, PhageClouds expanded the number of closely related phage sequences detected for both finished and draft phage genomes, in comparison with searches exclusively targeting phage entries from GenBank. Conclusions: PhageClouds is a novel resource that will facilitate the analysis of phage genomic sequences and the characterization of assembled phage genomes.

A new family of "megaphages" abundant in the marine environment.

Megaphages, bacteriophages harbouring extremely large genomes, have recently been found to be ubiquitous, being described from a variety of microbiomes ranging from the animal gut to soil and freshwater systems. However, no complete marine megaphage has been identified to date. Here, using both short and long read sequencing, we assembled >900 high-quality draft viral genomes from water in the English Channel. One of these genomes included a novel megaphage, Mar_Mega_1 at >650 Kb, making it one of the largest phage genomes assembled to date. Utilising phylogenetic and network approaches, we found this phage represents a new family of megaphages. Genomic analysis showed Mar_Mega_1 shares relatively few homologues with its closest relatives, but, as with other megaphages Mar_Mega_1 contained a variety of auxiliary metabolic genes responsible for carbon metabolism and nucleotide biosynthesis, including a NADP-dependent isocitrate dehydrogenase [Idh] and nicotinamide-nucleotide amidohydrolase [PncC], which have not previously been identified in megaphages. Mar_Mega_1 was abundant in a marine virome sample and related phages are widely prevalent in the oceans.

Genome Sequence and Characterization of Coliphage vB_Eco_SLUR29.

Background: Bacteriophages that infect Escherichia coli are relatively easily isolated, with >600 coliphage genomes sequenced to date. Despite this there is still much to be discovered about the diversity of coliphage genomes. Materials and Methods: Within this study, we isolated a coliphage from cattle slurry collected from a farm in rural England. Results: Transmission electron microscopy identified the phage as member of the Siphoviridae family. Phylogenetic analysis and comparative genomics further placed it within the subfamily Tunavirinae and forms part of a new genus. Conclusions: Characterization of the lytic properties of vB_Eco_SLUR29 reveals that it is rapidly able to lyse its host when infected at high multiplicity of infection, but not at low multiplicity of infection.

Generation of a Prophage-Free Variant of the Fast-Growing Bacterium Vibrio natriegens.

The fast-growing marine bacterium Vibrio natriegens represents an emerging strain for molecular biology and biotechnology. Genome sequencing and quantitative PCR analysis revealed that the first chromosome of V. natriegens ATCC 14048 contains two prophage regions (VNP1 and VNP2) that are both inducible by the DNA-damaging agent mitomycin C and exhibit spontaneous activation under standard cultivation conditions. Their activation was also confirmed by live cell imaging of an mCherry fusion to the major capsid proteins of VNP1 and VNP2. Transmission electron microscopy visualized the release of phage particles belonging to the Siphoviridae family into the culture supernatant. Freeing V. natriegens from its proviral load, followed by phenotypic characterization, revealed an improved robustness of the prophage-free variant toward DNA-damaging conditions, reduced cell lysis under hypo-osmotic conditions, and an increased pyruvate production compared to wild-type levels. Remarkably, the prophage-free strain outcompeted the wild type in a competitive growth experiment, emphasizing that this strain is a promising platform for future metabolic engineering approaches.IMPORTANCE The fast-growing marine bacterium Vibrio natriegens represents an emerging model host for molecular biology and biotechnology, featuring a reported doubling time of less than 10 minutes. In many bacterial species, viral DNA (prophage elements) may constitute a considerable fraction of the whole genome and may have detrimental effects on the growth and fitness of industrial strains. Genome analysis revealed the presence of two prophage regions in the V. natriegens genome that were shown to undergo spontaneous induction under standard cultivation conditions. In this study, we generated a prophage-free variant of V. natriegens Remarkably, the prophage-free strain exhibited a higher tolerance toward DNA damage and hypo-osmotic stress. Moreover, it was shown to outcompete the wild-type strain in a competitive growth experiment. In conclusion, our study presents the prophage-free variant of V. natriegens as a promising platform strain for future biotechnological applications.

Riding the wave of genomics to investigate aquatic coliphage diversity and activity.

Bacteriophages infecting Escherichia coli (coliphages) have been used as a proxy for faecal matter and water quality from a variety of environments. However, the diversity of coliphages that is present in seawater remains largely unknown, with previous studies largely focusing on morphological diversity. Here, we isolated and characterized coliphages from three coastal locations in the United Kingdom and Poland. Comparative genomics and phylogenetic analysis of phage isolates facilitated the identification of putative new species within the genera Rb69virus and T5virus and a putative new genus within the subfamily Tunavirinae. Furthermore, genomic and proteomic analysis combined with host range analysis allowed the identification of a putative tail fibre that is likely responsible for the observed differences in host range of phages vB_Eco_mar003J3 and vB_Eco_mar004NP2.

Metagenomics of the Viral Community in Three Cattle Slurry Samples.

The diversity of viruses in slurries from dairy farming remains largely uncharacterized. Here we report viral diversity found in cattle slurry from a dairy farm in the East Midlands in the United Kingdom. The same slurry tank was sampled in three consecutive years, and the viral fraction was isolated and sequenced.

Comparative Genomics of Bacteriophage of the Genus Seuratvirus.

Despite being more abundant and having smaller genomes than their bacterial host, relatively few bacteriophages have had their genomes sequenced. Here, we isolated 14 bacteriophages from cattle slurry and performed de novo genome sequencing, assembly, and annotation. The commonly used marker genes polB and terL showed these bacteriophages to be closely related to members of the genus Seuratvirus. We performed a core-gene analysis using the 14 new and four closely related genomes. A total of 58 core genes were identified, the majority of which has no known function. These genes were used to construct a core-gene phylogeny, the results of which confirmed the new isolates to be part of the genus Seuratvirus and expanded the number of species within this genus to four. All bacteriophages within the genus contained the genes queCDE encoding enzymes involved in queuosine biosynthesis. We suggest these genes are carried as a mechanism to modify DNA in order to protect these bacteriophages against host endonucleases.

Draft Genome Sequence of Bacteriophage vB_Eco_swan01.

Bacteriophage vB_Eco_swan01 was isolated from an ornamental pool using Escherichia coli MG1655 as the host. Bacteriophage vB_Eco_swan01 has limited similarity with other known phages at the nucleotide level and likely represents a new bacteriophage species within the Tunavirinae.