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

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

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

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.Peer reviewe

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, 2, 3 The marine roseobacter clade (MRC) is a ubiquitous group of heterotrophic bacteria[4],[5] that are important in the elemental cycling of various nitrogen, sulfur, carbon, and phosphorus compounds.6, 7, 8, 9, 10 Bacteriophages infecting MRC (roseophages) have thus attracted much attention and more than 30 roseophages have been isolated,11, 12, 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

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

Phages infecting marine Vibrios: prevalence, diversity and role in the dissemination of antibiotic resistance genes

Antimicrobial resistance (AMR) is a major public health issue. Recent studies indicate that antimicrobial resistance genes (ARGs) can be readily identified in both bacteriophages and bacteria found within the marine environment. Furthermore, experiments show that bacteria can obtain AMR when transduced with phage genetic material, suggesting that bacteriophages might be responsible for the transfer and spread of antimicrobial resistance in these systems. This project sought to determine the role of phages in mediating the transfer of ARGs within the marine environment, focusing on Vibrio spp. as model organisms due to their high abundance and impact caused on both public health and the seafood industry. First, a framework for the analysis of marine phages was established (Chapter 3). Genomic, p,hylogenetic, proteomic, host range and infection parameters of a range of novel marine coliphage isolates were determined and their ability to transduce ARGs was assessed. This suggested that host DNA encapsidation rate should be determined as a proxy for the transduction rate, due to the inability of stopping secondary phage infections during the transduction assay. The established framework was used for the analysis of marine Vibrio phages (Chapter 4). Experiments showed a high prevalence of Inoviridae prophages derived from the host in the obtained phage lysates, necessitating further studies to determine their exact composition. Nevertheless, the study resulted in the isolation and exhaustive characterisation of two novel Vibrio phages, including the second largest Vibrio phage isolated to date. Encapsidation of host DNA by the analysed phage was not observed, indicating this process may be below the experimental detection thresholds, which is not unexpected for generalised transducing phages. Bioinformatics analysis of all publicly available Vibrio genomes was performed to identify prophages encoding ARGs (Chapter 5) leading to the creation of the largest Vibrio prophage database to date. Analyses showed a high prevalence of the Inoviridae family of filamentous prophages in the Vibrio genomes and indicated that ~25% of the detected Vibrio prophages represent novel sequences not related to any known phages. Subsequent analysis of prophage-encoded ARGs indicated the presence of a range of ARGs, with genes encoding β-lactamases such as TEM-1, CTX-M and OXA being the most common. Selected ARGs were subsequently synthesised and their functionality was determined by heterologous expression in E. coli (Chapter 6). This not only showed that detected ARGs are often functional, but also led to the discovery of a novel cat ARG encoded on an intact Vibrio prophage conferring resistance to chloramphenicol. Finally, metagenomics and functional metagenomics analyses were performed to determine the prevalence of ARGs in marine phageomes (Chapter 7). This led to the reconstruction of a plethora of novel phage genomes, including a representative of megaphages, the largest known bacterial viruses. Surprisingly, genomics and functional genomics analyses failed to detect known or novel ARGs, respectively, suggesting that their distribution across the marine environment might not be uniform. This study represents a major step in our knowledge of lytic and temperate phages and their ability to encode functional ARGs and transfer them within the marine environment. Furthermore, it pinpoints the areas which require further investigation to fully understand the importance of generalized and specialized transduction in the dissemination of the ARGs in the environment

Western English Channel and Plymouth Sound Virome

Genome sequence and annotation of Mar_Mega1 in embl format (MARMEGA.embl.tar.gz) Alignment of terL genes from megaphages (all_TerL.faa.aln.tar.gz)All metagenome assembled viral contigs (Plym_L4_META.fasta.tar.gz) Supplementary tables of vOTU quality ( SupTables.tar.gz) </div

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

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