In vitro and in vivo evaluation of the biofilm-degrading Pseudomonas phage Motto, as a candidate for phage therapy.

Infections caused by Pseudomonas aeruginosa are becoming increasingly difficult to treat due to the emergence of strains that have acquired multidrug resistance. Therefore, phage therapy has gained attention as an alternative to the treatment of pseudomonal infections. Phages are not only bactericidal but occasionally show activity against biofilm as well. In this study, we describe the Pseudomonas phage Motto, a T1-like phage that can clear P. aeruginosa infections in an animal model and also exhibits biofilm-degrading properties. The phage has a substantial anti-biofilm activity against strong biofilm-producing isolates (n = 10), with at least a twofold reduction within 24 h. To demonstrate the safety of using phage Motto, cytotoxicity studies were conducted with human cell lines (HEK 293 and RAW 264.7 macrophages). Using a previously established in vivo model, we demonstrated the efficacy of Motto in Caenorhabditis elegans, with a 90% survival rate when treated with the phage at a multiplicity of infection of 10.

Bacteriophage Taxonomy: A Continually Evolving Discipline.

While taxonomy is an often underappreciated branch of science, it serves very important roles. Bacteriophage taxonomy has evolved from a discipline based mainly on morphology, characterized by the work of David Bradley and Hans-Wolfgang Ackermann, to the sequence-based approach that is taken today. The Bacterial Viruses Subcommittee of the International Committee on Taxonomy of Viruses (ICTV) takes a holistic approach to classifying prokaryote viruses by measuring overall DNA and protein similarity and phylogeny before making decisions about the taxonomic position of a new virus. The huge number of complete genomes being deposited with the National Center for Biotechnology Information (NCBI) and other public databases has resulted in a reassessment of the taxonomy of many viruses, and the future will see the introduction of new viral families and higher orders.

Genomic Analysis of Two Novel Bacteriophages Infecting Acinetobacter beijerinckii and halotolerans Species.

Bacteriophages are the most diverse genetic entities on Earth. In this study, two novel bacteriophages, nACB1 (Podoviridae morphotype) and nACB2 (Myoviridae morphotype), which infect Acinetobacter beijerinckii and Acinetobacter halotolerans, respectively, were isolated from sewage samples. The genome sequences of nACB1 and nACB2 revealed that their genome sizes were 80,310 bp and 136,560 bp, respectively. Comparative analysis showed that both genomes are novel members of the Schitoviridae and the Ackermannviridae families, sharing ≤ 40% overall nucleotide identities with any other phages. Interestingly, among other genetic features, nACB1 encoded a very large RNA polymerase, while nACB2 displayed three putative depolymerases (two capsular depolymerases and one capsular esterase) encoded in tandem. This is the first report of phages infecting A. halotolerans and beijerinckii human pathogenic species. The findings regarding these two phages will allow us to further explore phage-Acinetobacter interactions and the genetic evolution for this group of phages.

Four principles to establish a universal virus taxonomy.

A universal taxonomy of viruses is essential for a comprehensive view of the virus world and for communicating the complicated evolutionary relationships among viruses. However, there are major differences in the conceptualisation and approaches to virus classification and nomenclature among virologists, clinicians, agronomists, and other interested parties. Here, we provide recommendations to guide the construction of a coherent and comprehensive virus taxonomy, based on expert scientific consensus. Firstly, assignments of viruses should be congruent with the best attainable reconstruction of their evolutionary histories, i.e., taxa should be monophyletic. This fundamental principle for classification of viruses is currently included in the International Committee on Taxonomy of Viruses (ICTV) code only for the rank of species. Secondly, phenotypic and ecological properties of viruses may inform, but not override, evolutionary relatedness in the placement of ranks. Thirdly, alternative classifications that consider phenotypic attributes, such as being vector-borne (e.g., "arboviruses"), infecting a certain type of host (e.g., "mycoviruses," "bacteriophages") or displaying specific pathogenicity (e.g., "human immunodeficiency viruses"), may serve important clinical and regulatory purposes but often create polyphyletic categories that do not reflect evolutionary relationships. Nevertheless, such classifications ought to be maintained if they serve the needs of specific communities or play a practical clinical or regulatory role. However, they should not be considered or called taxonomies. Finally, while an evolution-based framework enables viruses discovered by metagenomics to be incorporated into the ICTV taxonomy, there are essential requirements for quality control of the sequence data used for these assignments. Combined, these four principles will enable future development and expansion of virus taxonomy as the true evolutionary diversity of viruses becomes apparent.

Abolishment of morphology-based taxa and change to binomial species names: 2022 taxonomy update of the ICTV bacterial viruses subcommittee.

This article summarises the activities of the Bacterial Viruses Subcommittee of the International Committee on Taxonomy of Viruses for the period of March 2021-March 2022. We provide an overview of the new taxa proposed in 2021, approved by the Executive Committee, and ratified by vote in 2022. Significant changes to the taxonomy of bacterial viruses were introduced: the paraphyletic morphological families Podoviridae, Siphoviridae, and Myoviridae as well as the order Caudovirales were abolished, and a binomial system of nomenclature for species was established. In addition, one order, 22 families, 30 subfamilies, 321 genera, and 862 species were newly created, promoted, or moved.

Complete Genome Sequence of Pseudomonas Phage Motto.

We describe the complete genome sequence of bacteriophage Motto, which infects clinical strains of Pseudomonas aeruginosa. Motto is a T1-like siphovirus related to members of the family Drexlerviridae and has a capsid width of ~57 nm and a tail length of ~255 nm. The 49.9-kb genome contains 84 protein-coding genes.

ICTV Virus Taxonomy Profile: Chaseviridae 2022.

Members of the family Chaseviridae are lytic bacterial viruses infecting representatives of the bacterial class Gammaproteobacteria. Chaseviruses have a global distribution. Virions of members of this family have a myovirus morphology (icosahedral head with contractile tail). Genomes are dsDNA of 52-56 kbp with G+C content ranging from 39.3-52.5 %. Chaseviruses, like members of the family Autographiviridae, encode a large single subunit RNA polymerase, but unlike those viruses their promoter sequences have not yet been identified. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Chaseviridae, which is available at ictv.global/report/chaseviridae.

Diversity of carbapenem-resistant Acinetobacter baumannii and bacteriophage-mediated spread of the Oxa23 carbapenemase.

Carbapenem-resistant Acinetobacter baumannii are prevalent in low- and middle-income countries such as Egypt, but little is known about the molecular epidemiology and mechanisms of resistance in these settings. Here, we characterize carbapenem-resistant A. baumannii from Alexandria, Egypt, and place it in a regional context. Fifty-four carbapenem-resistant isolates from Alexandria Main University Hospital (AMUH), Alexandria, Egypt, collected between 2010 and 2015 were genome sequenced using Illumina technology. Genomes were de novo assembled and annotated. Genomes for 36 isolates from the Middle East region were downloaded from GenBank. The core-gene compliment was determined using Roary, and analyses of recombination were performed in Gubbins. Multilocus sequence typing (MLST) sequence type (ST) and antibiotic-resistance genes were identified. The majority of Egyptian isolates belonged to one of three major clades, corresponding to Pasteur MLST clonal complex (CCPAS) 1, CCPAS2 and STPAS158. Strains belonging to STPAS158 have been reported almost exclusively from North Africa, the Middle East and Pakistan, and may represent a region-specific lineage. All isolates carried an oxa23 gene, six carried blaNDM-1 and one carried blaNDM-2. The oxa23 gene was located on a variety of different mobile elements, with Tn2006 predominant in CCPAS2 strains, and Tn2008 predominant in other lineages. Of particular concern, in 8 of the 13 CCPAS1 strains, the oxa23 gene was located in a temperate bacteriophage phiOXA, previously identified only once before in a CCPAS1 clone from the USA military. The carbapenem-resistant A. baumannii population in AMUH is very diverse, and indicates an endemic circulating population, including a region-specific lineage. A major mechanism for oxa23 dissemination in CCPAS1 isolates appears to be a bacteriophage, presenting new concerns about the ability of these carbapenemases to spread throughout the bacterial population.

Genomic Diversity of Bacteriophages Infecting the Genus Acinetobacter.

The number of sequenced Acinetobacter phage genomes in the International Nucleotide Sequence Database Collaboration has increased significantly in recent years, from 37 in 2017 to a total of 139 as of January 2021 with genome sizes ranging from 31 to 378 kb. Here, we explored the genetic diversity of the Acinetobacter phages using comparative genomics approaches that included assessment of nucleotide similarity, shared gene content, single gene phylogeny, and the network-based classification tool vConTACT2. Phages infecting Acinetobacter sp. are genetically diverse and can be grouped into 8 clusters (subfamilies) and 46 sub-clusters (genera), of which 8 represent genomic singletons (additional genera). We propose the creation of five new subfamilies and suggest a reorganisation of the genus Obolenskvirus. These results provide an updated view of the viruses infecting Acinetobacter species, providing insights into their diversity.

Bacterial Viruses Subcommittee and Archaeal Viruses Subcommittee of the ICTV: update of taxonomy changes in 2021.

In this article, we - the Bacterial Viruses Subcommittee and the Archaeal Viruses Subcommittee of the International Committee on Taxonomy of Viruses (ICTV) - summarise the results of our activities for the period March 2020 - March 2021. We report the division of the former Bacterial and Archaeal Viruses Subcommittee in two separate Subcommittees, welcome new members, a new Subcommittee Chair and Vice Chair, and give an overview of the new taxa that were proposed in 2020, approved by the Executive Committee and ratified by vote in 2021. In particular, a new realm, three orders, 15 families, 31 subfamilies, 734 genera and 1845 species were newly created or redefined (moved/promoted).

A Roadmap for Genome-Based Phage Taxonomy.

Bacteriophage (phage) taxonomy has been in flux since its inception over four decades ago. Genome sequencing has put pressure on the classification system and recent years have seen significant changes to phage taxonomy. Here, we reflect on the state of phage taxonomy and provide a roadmap for the future, including the abolition of the order Caudovirales and the families Myoviridae, Podoviridae, and Siphoviridae. Furthermore, we specify guidelines for the demarcation of species, genus, subfamily and family-level ranks of tailed phage taxonomy.

Phage Annotation Guide: Guidelines for Assembly and High-Quality Annotation.

All sequencing projects of bacteriophages (phages) should seek to report an accurate and comprehensive annotation of their genomes. This article defines 14 questions for those new to phage genomics that should be addressed before submitting a genome sequence to the International Nucleotide Sequence Database Collaboration or writing a publication.

From Orphan Phage to a Proposed New Family-the Diversity of N4-Like Viruses.

Escherichia phage N4 was isolated in 1966 in Italy and has remained a genomic orphan for a long time. It encodes an extremely large virion-associated RNA polymerase unique for bacterial viruses that became characteristic for this group. In recent years, due to new and relatively inexpensive sequencing techniques the number of publicly available phage genome sequences expanded rapidly. This revealed new members of the N4-like phage group, from 33 members in 2015 to 115 N4-like viruses in 2020. Using new technologies and methods for classification, the Bacterial and Archaeal Viruses Subcommittee of the International Committee on Taxonomy of Viruses (ICTV) has moved the classification and taxonomy of bacterial viruses from mere morphological approaches to genomic and proteomic methods. The analysis of 115 N4-like genomes resulted in a huge reassessment of this group and the proposal of a new family "Schitoviridae", including eight subfamilies and numerous new genera.

Taxonomy of prokaryotic viruses: 2018-2019 update from the ICTV Bacterial and Archaeal Viruses Subcommittee.

This article is a summary of the activities of the ICTV's Bacterial and Archaeal Viruses Subcommittee for the years 2018 and 2019. Highlights include the creation of a new order, 10 families, 22 subfamilies, 424 genera and 964 species. Some of our concerns about the ICTV's ability to adjust to and incorporate new DNA- and protein-based taxonomic tools are discussed.

Taxonomic assignment of uncultivated prokaryotic virus genomes is enabled by gene-sharing networks.

Microbiomes from every environment contain a myriad of uncultivated archaeal and bacterial viruses, but studying these viruses is hampered by the lack of a universal, scalable taxonomic framework. We present vConTACT v.2.0, a network-based application utilizing whole genome gene-sharing profiles for virus taxonomy that integrates distance-based hierarchical clustering and confidence scores for all taxonomic predictions. We report near-identical (96%) replication of existing genus-level viral taxonomy assignments from the International Committee on Taxonomy of Viruses for National Center for Biotechnology Information virus RefSeq. Application of vConTACT v.2.0 to 1,364 previously unclassified viruses deposited in virus RefSeq as reference genomes produced automatic, high-confidence genus assignments for 820 of the 1,364. We applied vConTACT v.2.0 to analyze 15,280 Global Ocean Virome genome fragments and were able to provide taxonomic assignments for 31% of these data, which shows that our algorithm is scalable to very large metagenomic datasets. Our taxonomy tool can be automated and applied to metagenomes from any environment for virus classification.

661W Photoreceptor Cell Line as a Cell Model for Studying Retinal Ciliopathies.

The retina contains several ciliated cell types, including the retinal pigment epithelium (RPE) and photoreceptor cells. The photoreceptor cilium is one of the most highly modified sensory cilia in the human body. The outer segment of the photoreceptor is a highly elaborate primary cilium, containing stacks or folds of membrane where the photopigment molecules are located. Perhaps unsurprisingly, defects in cilia often lead to retinal phenotypes, either as part of syndromic conditions involving other organs, or in isolation in the so-called retinal ciliopathies. The study of retinal ciliopathies has been limited by a lack of retinal cell lines. RPE1 retinal pigment epithelial cell line is commonly used in such studies, but the existence of a photoreceptor cell line has largely been neglected in the retinal ciliopathy field. 661W cone photoreceptor cells, derived from mouse, have been widely used as a model for studying macular degeneration, but not described as a model for studying retinal ciliopathies such as retinitis pigmentosa. Here, we characterize the 661W cell line as a model for studying retinal ciliopathies. We fully characterize the expression profile of these cells, using whole transcriptome RNA sequencing, and provide this data on Gene Expression Omnibus for the advantage of the scientific community. We show that these cells express the majority of markers of cone cell origin. Using immunostaining and confocal microscopy, alongside scanning electron microscopy, we show that these cells grow long primary cilia, reminiscent of photoreceptor outer segments, and localize many cilium proteins to the axoneme, membrane and transition zone. We show that siRNA knockdown of cilia genes Ift88 results in loss of cilia, and that this can be assayed by high-throughput screening. We present evidence that the 661W cell line is a useful cell model for studying retinal ciliopathies.

Pectobacterium atrosepticum Phage vB_PatP_CB5: A Member of the Proposed Genus 'Phimunavirus'.

Pectobacterium atrosepticum is a phytopathogen of economic importance as it is the causative agent of potato blackleg and soft rot. Here we describe the Pectobacterium phage vB_PatP_CB5 (abbreviated as CB5), which specifically infects the bacterium. The bacteriophage is characterized in detail and TEM micrographs indicate that it belongs to the Podoviridae family. CB5 shares significant pairwise nucleotide identity (≥80%) with P. atrosepticum phages φM1, Peat1, and PP90 and also shares common genome organization. Phylograms constructed using conserved proteins and whole-genome comparison-based amino acid sequences show that these phages form a distinct clade within the Autographivirinae. They also possess conserved RNA polymerase recognition and specificity loop sequences. Their lysis cassette resembles that of KP34virus, containing in sequential order a U-spanin, a holin, and a signal⁻arrest⁻release (SAR) endolysin. However, they share low pairwise nucleotide identity with the type phage of the KP34virus genus, Klebsiella phage KP34. In addition, phage KP34 does not possess several conserved proteins associated with these P. atrosepticum phages. As such, we propose the allocation of phages CB5, Peat1, φM1, and PP90 to a separate new genus designated Phimunavirus.

The Sequence of Two Bacteriophages with Hypermodified Bases Reveals Novel Phage-Host Interactions.

Bacteriophages SP-15 and ΦW-14 are members of the Myoviridae infecting Bacillus subtilis and Delftia (formerly Pseudomonas) acidovorans, respectively. What links them is that in both cases, approximately 50% of the thymine residues are replaced by hypermodified bases. The consequence of this is that the physico-chemical properties of the DNA are radically altered (melting temperature (Tm), buoyant density and susceptibility to restriction endonucleases). Using 454 pyrosequencing technology, we sequenced the genomes of both viruses. Phage ΦW-14 possesses a 157-kb genome (56.3% GC) specifying 236 proteins, while SP-15 is larger at 222 kb (38.6 mol % G + C) and encodes 318 proteins. In both cases, the phages can be considered genomic singletons since they do not possess BLASTn homologs. While no obvious genes were identified as being responsible for the modified base in ΦW-14, SP-15 contains a cluster of genes obviously involved in carbohydrate metabolism.

Visualization of Phage Genomic Data: Comparative Genomics and Publication-Quality Diagrams.

The presentation of bacteriophage genomes as diagrams allows the location and organization of features to be communicated in a clear and effective manner. A wide range of software applications are available for the clear and accurate visualization of genomic data. Several of these applications incorporate comparative analysis tools, allowing for insertions, deletions, rearrangements and variations in syntenic regions to be visualized. In this chapter, freely available software and resources for the generation of high-quality graphical maps of bacteriophage genomes are listed and discussed.