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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.

Peroxynitrite is a reactive intermediate formed in vivo through uncatalysed reaction of superoxide and nitric oxide radicals. Despite significant interest in detecting peroxynitrite in vivo and understanding its production, little attention has been given to the evolutionary origins of peroxynitrite signalling. Herein we focus on two enzymes that are key to the biosynthesis of superoxide and nitric oxide, NADPH oxidase 5 (NOX5) and endothelial nitric oxide synthase (eNOS), respectively. Multiple sequence alignments of both enzymes including homologues from all domains of life, coupled with a phylogenetic analysis of NOX5, suggest eNOS and NOX5 are present in animals as the result of horizontal gene transfer from ancestral cyanobacteria to ancestral eukaryotes. Therefore, biochemical studies from other laboratories on a NOX5 homologue in Cylindrospermum stagnale and an eNOS homologue in Synechococcus sp. PCC 7335 are likely to be of relevance to human NOX5 and eNOS and to the production of superoxide, nitric oxide and peroxynitrite in humans.

RuBisCO (ribulose 1,5-bisphosphate carboxylase/oxygenase) is one the most abundant enzymes on Earth. Virtually all food webs depend on its activity to supply fixed carbon. In aerobic environments, RuBisCO struggles to distinguish efficiently between CO2 and O2. To compensate, organisms have evolved convergent solutions to concentrate CO2 around the active site. The genetic engineering of such inorganic carbon concentrating mechanisms (CCMs) into plants could help facilitate future global food security for humankind. In bacteria, the carboxysome represents one such CCM component, of which two independent forms exist: α and β. Cyanobacteria are important players in the planet’s carbon cycle and the vast majority of the phylum possess a β-carboxysome, including most cyanobacteria used as laboratory models. The exceptions are the exclusively marine Prochlorococcus and Synechococcus that numerically dominate open ocean systems. However, the reason why marine systems favor an α-form is currently unknown. Here, we report the genomes of 58 cyanobacteria, closely related to marine Synechococcus that were isolated from freshwater lakes across the globe. We find all these isolates possess α-carboxysomes accompanied by a form 1A RuBisCO. Moreover, we demonstrate α-cyanobacteria dominate freshwater lakes worldwide. Hence, the paradigm of a separation in carboxysome type across the salinity divide does not hold true, and instead the α-form dominates all aquatic systems. We thus question the relevance of β-cyanobacteria as models for aquatic systems at large and pose a hypothesis for the reason for the success of the α-form in nature.

Bacteriophages are as ubiquitous as their bacterial hosts and often more abundant. Understanding how bacteriophages control their bacterial host populations requires a number of different approaches. Bacteriophages can control bacterial populations through lysis, drive evolution of bacterial immunity systems through infection, provide a conduit for horizontal gene transfer and alter host metabolism by carriage of auxiliary metabolic genes. Understanding and quantifying how bacteriophages drive these processes, requires both technological developments to take measurements in situ, and laboratory-based studies to understand mechanisms. Technological advances have allowed quantification of the number of infected cells in situ, revealing far-lower levels than expected. Understanding how observations in laboratory conditions relate to what occurs in the environment, and experimental confirmation of the predicted function of phage genes from observations in environmental omics data, remains challenging.

Extensively drug-resistant Acinetobacter baumannii (XDR-AB) is a major threat to public health worldwide. A retrospective study for 27 XDR-AB isolates from four tertiary hospitals in Thailand was c...

The bacteriophage vB_AhM_PVN02 (PVN02), infecting Aeromonas hydrophila, was isolated from a striped catfish pond water sample in Can Tho City, Vietnam. The phage had high lytic activity with a latent period and burst size of approximately 20 min and 105 plaque-forming units per cell, respectively. Observation of the phage by transmission electron microscopy indicated that PVN02 belongs to the family Myoviridae. The genome of PVN02 is a double-stranded linear DNA with a length in 51,668 bp and a content of 52% GC. Among the 64 genes, 16 were predicted to encode proteins with predicted functions. No virulence or antibiotic resistance genes were found in the genome, suggesting it would be a useful biocontrol agent. Classification of the phage based on sequence comparisons, phylogenetic analysis, and gene-sharing networks was carried out, and it was found to be the first representative of a new species within a previously undefined genus in the family Myoviridae. This study confirmed that PVN02 is a novel lytic phage that could potentially be used as an agent to control Aeromonas hydrophila in striped catfish in the Mekong Delta, Vietnam.

Aims This study describes the effect of phage therapy on hatching of longfin yellowtail (Seriola rivoliana) eggs challenged with Photobacterium damselae subsp. damselae. Methods and results A lytic phage (vB_Pd_PDCC-1) against P. damselae subsp. damselae was isolated and characterized. The use of phage vB_Pd_PDCC-1 increased the hatching rate of eggs, and reduced presumptive Vibrio species to non-detectable numbers, even in non-disinfected eggs. High-throughput 16S rRNA gene sequencing analysis revealed that phage vB_Pd_PDCC-1 caused significant changes in the composition and structure of the associated microbiota, allowing that members (e.g. those belonging to the family Vibrionaceae) of the class Gammaproteobacteria to be displaced by members of the class Alphaproteobacteria. Conclusions To the best of our knowledge, this represents the first study evaluating phage therapy to control potential negative effects of P. damselae subsp. damselae during hatching of longfin yellowtail eggs. Significance and impact of the study The Seriola genus includes several important commercial fish species due to its rapid growth and easy adaptability to confinement conditions. However, bacterial infections (especially those caused by Vibrio and Photobacterium species) are among the main limiting factors for the intensification of marine fish aquaculture, particularly during early development stages. Therefore, the use of phages, which are natural killers of bacteria, represents a promising strategy to reduce the mortality of farmed organisms caused by pathogenic bacteria.

Infections caused by multidrug resistant Salmonella strains are problematic in swine and are entering human food chains. Bacteriophages (phages) could be used to complement or replace antibiotics to reduce infection within swine. Here, we extensively characterised six broad host range lytic Salmonella phages, with the aim of developing a phage cocktail to prevent or treat infection. Intriguingly, the phages tested differed by one to five single nucleotide polymorphisms. However, there were clear phenotypic differences between them, especially in their heat and pH sensitivity. In vitro killing assays were conducted to determine the efficacy of phages alone and when combined, and three cocktails reduced bacterial numbers by ~2 × 103 CFU/mL within two hours. These cocktails were tested in larvae challenge studies, and prophylactic treatment with phage cocktail SPFM10-SPFM14 was the most efficient. Phage treatment improved larvae survival to 90% after 72 h versus 3% in the infected untreated group. In 65% of the phage-treated larvae, Salmonella counts were below the detection limit, whereas it was isolated from 100% of the infected, untreated larvae group. This study demonstrates that phages effectively reduce Salmonella colonisation in larvae, which supports their ability to similarly protect swine.

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.

Complete genomes of xenobiotic-degrading microorganisms provide valuable resources for researchers to understand molecular mechanisms involved in bioremediation. Despite the well-known ability of Sphingomonas paucimobilis to degrade persistent xenobiotic compounds, a complete genome sequencing is lacking for this organism. In line with this, we report the first complete genome sequence of Sphingomonas paucimobilis (strain AIMST S2), an organophosphate and hydrocarbon-degrading bacterium isolated from oil-polluted soil at Kedah, Malaysia. The genome was derived from a hybrid assembly of short and long reads generated by Illumina HiSeq and MinION, respectively. The assembly resulted in a single contig of 4,005,505 bases which consisted of 3,612 CDS and 56 tRNAs. An array of genes involved in xenobiotic degradation and plant-growth promoters were identified, suggesting its’ potential role as an effective microorganism in bioremediation and agriculture. Having reported the first complete genome of the species, this study will serve as a stepping stone for comparative genome analysis of Sphingomonas strains and other xenobiotic-degrading microorganisms as well as gene expression studies in organophosphate biodegradation., Measurement(s)genome • sequence_assemblyTechnology Type(s)DNA sequencing • genome assemblySample Characteristic - OrganismSphingomonas paucimobilisSample Characteristic - Environmentoil contaminated soilSample Characteristic - LocationMalaysia Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.9971711

Background Coastal environments are dynamic and rapidly changing. Living organisms in coastal environments are known to synthesise large quantities of organic osmolytes, which they use to cope with osmotic stresses. The organic osmolyte glycine betaine (GBT) is ubiquitously found in marine biota from prokaryotic Bacteria and Archaea to coastal plants, marine protozoa, and mammals. In intertidal coastal sediment, GBT represents an important precursor of natural methane emissions and as much as 90% of total methane production in these ecosystems can be originated from methanogenesis from GBT and its intermediate trimethylamine through microbial metabolism. Results We set out to uncover the microorganisms responsible for methanogenesis from GBT using stable isotope labelling and metagenomics. This led to the recovery of a near-complete genome (2.3 Mbp) of a novel clostridial bacterium involved in anaerobic GBT degradation. Phylogenetic analyses of 16S rRNA gene, functional marker genes, and comparative genomics analyses all support the establishment of a novel family Candidatus ‘Betainaceae’ fam. nov. in Clostridiales and its role in GBT metabolism. Conclusions Our comparative genomes and metagenomics analyses suggest that this bacterium is widely distributed in coastal salt marshes, marine sediments, and deep subsurface sediments, suggesting a key role of anaerobic GBT metabolism by this clostridial bacterium in these ecosystems. Electronic supplementary material The online version of this article (10.1186/s40168-019-0732-4) contains supplementary material, which is available to authorized users.

Volatile organic compounds (VOCs) are potential biomarkers for diagnosing colorectal cancer (CRC). We characterized urinary VOCs from CRC patients, their spouses/cohabitors (spouses) and first-degree relatives (relatives) to determine any differences. Correlation with stool-derived microbiomes was also undertaken.Urine from 56 CRC patients, 45 spouses and 37 relatives was assayed using liquid chromatography, field asymmetric ion mobility spectrometry (FAIMS), mass spectrometer technology. Analysis was performed using five-fold cross-validation and a random forest classifier. Faecal microbiome 16S rRNA was sequenced using Illumina MiSeq protocols and analysed using UPARSE and QIIME pipelines. VOC and microbiome profiles were also compared before and after cancer treatment.Urinary VOC profiles of CRC patients were indistinguishable from either spouses or relatives. When spouses and relatives were grouped together to form a larger non-cancer control group (n = 82), their VOC profiles became distinguishable from those of CRC patients (n = 56) with 69% sensitivity and specificity, area under the curve 0.72 (P 0.001). Microbiome analysis identified 1300 operational taxonomic units across all groups. The analysis of similarity R value was 0.067 (P 0.001), with significantly different bacterial abundances in 82 operational taxonomic units (6.2%) by Kruskal-Wallis testing. CRC patients' VOC or stool microbiome profiles were unchanged after treatment.Although CRC patients' urinary VOC profiles cannot be differentiated from those of spouses or relatives they can be differentiated from a larger non-cancer control group. Comparison of the groups' microbiomes confirmed differences in bacterial species abundance. The current FAIMS-based assay can detect a unique, but modest, signal in CRC patients' urinary VOCs, which remains unaltered after treatment.

Vibrio campbellii is widely distributed in the marine environment and is an important pathogen of aquatic organisms such as shrimp, fish, and mollusks. The emergence of multi-drug resistance among these bacteria resulted in a worldwide public health problem, which requires alternative treatment approaches such as phage therapy. In the present study, we isolated a phage vB_Vc_SrVc2 from white shrimp hepatopancreas with symptoms of AHPND. Phage vB_Vc_SrVc2 is a member of the genus Maculvirus and the family Autographiviridae, with high lytic ability against Vibrio isolates. Phage has a high resistance to a broad range of temperatures, salinity, UV radiation and chloroform. The genome size was 43,157 bp, with a GC content of 49.2% that encodes 49 putative ORFs, no tRNAs, showed three single nucleotide polymorphisms, two small deletions and one nucleotide insertion compared to SrVc9, showing slightly different infectivity profiles.. No lysogeny related genes were detected in vB_Vc_SrVc2 genome. Overall phage vB_Vc_SrVc2 has a good potential for therapeutic use in the aquaculture industry against V. campbellii infections.

BackgroundIn order to optimize phage therapy, we need to understand how bacteria evolve against phage attack. One of the main problems of the phage therapy is the appearance of bacterial resistance variants. The use of genomics to track antimicrobial resistance is increasingly developed and used in clinical laboratories. For that reason, it is important to consider, in an emerging future with phage therapy, to detect and avoid phage resistant strains, that can be overcomed by the analysis of metadata provided by WGS. Here, we identified genes associated with phage resistance in 18 Acinetobacter baumannii clinical strain belonging to the ST-2 clonal complex during a decade (Ab2000 vs 2010): 9 from 2000 and 9 from 2010.ResultsThe presence of genes putatively associated to phage resistance were detected. Genes detected were associated with an abortive infection system, restriction-modification system, genes predicted to be associated with defence systems but with unknown function and CRISPR-Cas system. Between 118 and 171 genes were found in the 18 clinical strains. On average, 26% of these genes were detected inside genomic islands (GIs) in the 2000 strains and 32% in 2010 strains. Furthermore, 38 potential CRISPR arrays in 17 of 18 of the strains were found, as well as 705 proteins associated with CRISPR-Cas systems.ConclusionsA moderately higher presence of these genes in the strains of the 2010 in comparison to those of the 2000 were found, especially those related to the R-M system and CRISPR-Cas system. The presence of these genes in GIs in a higher rate in the strains of the 2010 compared to those of the 2000 was also detected. WGS and bioinformatics could be powerful tools to avoid drawbacks when a personalized therapy is applied. In this study, it allows us to take care of the phage resistance in A. baumannii clinical strains to prevent a failure in a possible phage therapy.

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 bacteriophages. Genomic analysis showed Mar_Mega_1 shares relatively few homologues with its closest relatives, but, as with other mega-phages Mar_Mega_1 contained a variety of auxiliary metabolic genes responsible for carbon metabolism and nucleotide biosynthesis, including isocitrate dehydrogenase [NADP] and nicotinamide-nucleotide amidohydrolase [PncC] which have not previously been identified in megaphages. The results of this study indicate that phages containing extremely large genomes can be found in abundance in the marine environment and augment host metabolism by mechanisms not previously described.

Phage therapy recently passed a key milestone with success of the first regulated clinical trial using systemic administration. In this single-arm non-comparative safety study, phages were administered intravenously to patients with invasive Staphylococcus aureus infections with no adverse reactions reported. Here, we examined features of 78 lytic S. aureus phages, most of which were propagated using a S. carnosus host modified to be broadly susceptible to staphylococcal phage infection. Use of this host eliminates the threat of contamination with staphylococcal prophage — the main vector of S. aureus horizontal gene transfer. We determined the host range of these phages against an international collection of 185 S. aureus isolates with 56 different multilocus sequence types that included multiple representatives of all epidemic MRSA and MSSA clonal complexes. Forty of our 78 phages were able to infect > 90% of study isolates, 15 were able to infect > 95%, and two could infect all 184 clinical isolates, but not a phage-resistant mutant generated in a previous study. We selected the 10 phages with the widest host range for in vitro characterization by planktonic culture time-kill analysis against four isolates:- modified S. carnosus strain TM300H, methicillin-sensitive isolates D329 and 15981, and MRSA isolate 252. Six of these 10 phages were able to rapidly kill, reducing cell numbers of at least three isolates. The four best-performing phages, in this assay, were further shown to be highly effective in reducing 48 h biofilms on polystyrene formed by eight ST22 and eight ST36 MRSA isolates. Genomes of 22 of the widest host-range phages showed they belonged to the Twortvirinae subfamily of the order Caudovirales in three main groups corresponding to Silviavirus, and two distinct groups of Kayvirus. These genomes assembled as single-linear dsDNAs with an average length of 140 kb and a GC content of c. 30%. Phages that could infect > 96% of S. aureus isolates were found in all three groups, and these have great potential as therapeutic candidates if, in future studies, they can be formulated to maximize their efficacy and eliminate emergence of phage resistance by using appropriate combinations.

International audience; Evolution of virulence traits from adaptation to environmental niches other than the host is probably a common feature of marine microbial pathogens, whose knowledge might be crucial to understand their emergence and pathogenetic potential. Here we report genome sequence analysis of a novel marine bacterial species, Vibrio bathopelagicus sp. nov., isolated from warm bathypelagic waters (3309m depth) of the Mediterranean Sea. Interestingly, V. bathopelagicus sp. nov. is closely related to coastal Vibrio strains pathogenic to marine bivalves. V. bathopelagicus sp. nov. genome encodes genes involved in environmental adaptations to the deep-sea but also in virulence, such as the R5.7 element, MARTX toxin cluster, Type VI secretion system and zinc-metalloprotease, previously associated with Vibrio infection in farmed oysters. The results of functional in vitro assays on immunocytes (hemocytes) of the Mediterranean mussel Mytilus galloprovincialis and the Pacific oyster Crassostrea gigas, and of the early larval development assay in Mytilus support strong toxicity of V. bathopelagicus sp. nov. towards bivalves. V. bathopelagicus sp. nov., isolated from a remote Mediterranean bathypelagic site, is an example of a planktonic marine bacterium with genotypic and phenotypic traits associated with animal pathogenicity, that might have played an evolutionary role in the origin of coastal marine pathogens.

BackgroundWith advances in sequencing technology and decreasing costs, the number of bacteriophage genomes that have been sequenced has increased markedly in the last decade.Materials and MethodsWe developed an automated retrieval and analysis system for bacteriophage genomes, INPHARED (https://github.com/RyanCook94/inphared), that provides data in a consistent format.ResultsAs of January 2021, 14,244 complete phage genomes have been sequenced. The data set is dominated by phages that infect a small number of bacterial genera, with 75% of phages isolated only on 30 bacterial genera. There is further bias with significantly more lytic phage genomes than temperate within the database, resulting in ~54% of temperate phage genomes originating from just three host genera. Within phage genomes, putative antibiotic resistance genes were found in higher frequencies in temperate phages than lytic phages.ConclusionWe provide a mechanism to reproducibly extract complete phage genomes and highlight some of the biases within this data, that underpins our current understanding of phage genomes.

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.

There is currently a renaissance in research on bacteriophages as alternatives to antibiotics. Phage specificity to their bacterial host, in addition to a plethora of other advantages, makes them ideal candidates for a broad range of applications, including bacterial detection, drug delivery, and phage therapy in particular. One issue obstructing phage efficiency in phage therapy settings is their poor localization to the site of infection in the human body. Here, we engineered phage T7 with lung tissue targeting homing peptides. We then used in vitro studies to demonstrate that the engineered T7 phages had a more significant association with the lung epithelium cells than wild-type T7. In addition, we showed that, in general, there was a trend of increased association of engineered phages with the lung epithelium cells but not mouse fibroblast cells, allowing for targeted tissue specificity. These results indicate that appending phages with homing peptides would potentially allow for greater phage concentrations and greater efficacy at the infection site.

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.

We characterized the complete genome sequence of the lytic Salmonella enterica bacteriophage PRF-SP1, isolated from Penang National Park, a conserved rainforest in northern Malaysia. The novel phage species from the Autographiviridae family has a 39,966-bp double-stranded DNA (dsDNA) genome containing 49 protein-encoding genes and shares 90.96% similarity with Escherichia phage DY1.

Prophages are phages that are integrated into bacterial genomes and which are key to understanding many aspects of bacterial biology. Their extreme diversity means they are challenging to detect using sequence similarity, yet this remains the paradigm and thus many phages remain unidentified. We present a novel, fast and generalizing machine learning method based on feature space to facilitate novel prophage discovery. To validate the approach, we reanalyzed publicly available marine viromes and single-cell genomes using our feature-based approaches and found consistently more phages than were detected using current state-of-the-art tools while being notably faster. This demonstrates that our approach significantly enhances bacteriophage discovery and thus provides a new starting point for exploring new biologies.

Aims This study describes the physicochemical and genomic characterization of phage vB_Vc_SrVc9 and its potential for phage therapy application against a pathogenic Vibrio campbellii strain. Methods and results A lytic phage vB_Vc_SrVc9 against Vibrio campbellii was isolated from shrimp farm sediment, and characterized physicochemical and genomically. The use of vB_Vc_SrVc9 phage increased the survival in Brine shrimp Artemia franciscana and reduced presumptive Vibrio campbellii to non-detectable numbers. Genomic analysis showed a genome with a single contig of 43.15 kb, with 49 predicted genes and no tRNAs, capable of recognizing and generating complete inhibition zones of three Vibrio species. Conclusions To our knowledge vB_Vc_SrVc9 is a lytic phage that could be used against Vibrio infections, reducing vibrio presence without any apparent impact over the natural microbiota at the family level in 28 libraries tested. Significance and impact of study vB_Vc_SrVC9 is a novel phage and ecofriendly alternative for therapeutic applications and biotechnological purposes because is stable at different environmental conditions, has a potential to eliminate several strains, and has a short latent period with a good burst size. Therefore, the use of phages, which are natural killers of bacteria, represents a promising strategy to reduce the mortality of farmed organisms caused by pathogenic bacteria.

Background Viruses are the most abundant biological entities on Earth, known to be crucial components of microbial ecosystems. However, there is little information on the viral community within agricultural waste. There are currently ~ 2.7 million dairy cattle in the UK producing 7–8% of their own bodyweight in manure daily, and 28 million tonnes annually. To avoid pollution of UK freshwaters, manure must be stored and spread in accordance with guidelines set by DEFRA. Manures are used as fertiliser, and widely spread over crop fields, yet little is known about their microbial composition. We analysed the virome of agricultural slurry over a 5-month period using short and long-read sequencing. Results Hybrid sequencing uncovered more high-quality viral genomes than long or short-reads alone; yielding 7682 vOTUs, 174 of which were complete viral genomes. The slurry virome was highly diverse and dominated by lytic bacteriophage, the majority of which represent novel genera (~ 98%). Despite constant influx and efflux of slurry, the composition and diversity of the slurry virome was extremely stable over time, with 55% of vOTUs detected in all samples over a 5-month period. Functional annotation revealed a diverse and abundant range of auxiliary metabolic genes and novel features present in the community, including the agriculturally relevant virulence factor VapE, which was widely distributed across different phage genera that were predicted to infect several hosts. Furthermore, we identified an abundance of phage-encoded diversity-generating retroelements, which were previously thought to be rare on lytic viral genomes. Additionally, we identified a group of crAssphages, including lineages that were previously thought only to be found in the human gut. Conclusions The cattle slurry virome is complex, diverse and dominated by novel genera, many of which are not recovered using long or short-reads alone. Phages were found to encode a wide range of AMGs that are not constrained to particular groups or predicted hosts, including virulence determinants and putative ARGs. The application of agricultural slurry to land may therefore be a driver of bacterial virulence and antimicrobial resistance in the environment.

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.

Extensively drug-resistant

Graphical abstract, The viromes of the mammalian lower gut were shown to be heavily dominated by bacteriophages; however, only for humans were the composition and intervariability of the bacteriophage communities studied in depth. Here we present an ecogenomics survey of dsDNA bacteriophage diversity in the feces of horses (Equus caballus), comparing two groups of stabled horses, and a further group of feral horses that were isolated on an island. Our results indicate that the dsDNA viromes of the horse feces feature higher richness than in human viromes, with more even distribution of genotypes. No over-represented phage genotypes, such as CrAssphage-related viruses found in humans, were identified. Additionally, many bacteriophage genus-level clusters were found to be present in all three geographically isolated populations. The diversity of the horse intestinal bacteriophages is severely undersampled, and so consequently only a minor fraction of the phage contigs could be linked with the bacteriophage genomes. Our study indicates that bacteriophage ecological parameters in the intestinal ecosystems in horses and humans differ significantly, leading them to shape their corresponding viromes in different ways. Therefore, the diversity and structure of the intestinal virome in different animal species needs to be experimentally studied.

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 as a primary option for the treatment of drugresistant infections of these types. We report the successful isolation and characterisation of 30 novel, genetically diverse Klebsiella phages. 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. 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. Importantly, when applied alone, none of the characterised phages were able to suppress the growth of Klebsiella for more than 12 hours, with some phages only able to suppress growth for 3 hours, likely due to 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.

Bacteriophage possess a variety of auxiliary metabolic genes (AMGs) of bacterial origin. These proteins enable them to maximise infection efficiency, subverting bacterial metabolic processes for the purpose of viral genome replication and synthesis of the next generation of virion progeny. Here, we examined the enzymatic activity of a cyanophage MazG protein – a putative pyrophosphohydrolase previously implicated in regulation of the stringent response via reducing levels of the central alarmone molecule (p)ppGpp. We demonstrate however, that the purified viral MazG shows no binding or hydrolysis activity against (p)ppGpp. Instead, dGTP and dCTP appear to be the preferred substrates of this protein, consistent with a role preferentially hydrolysing deoxyribonucleotides from the high GC content host Synechococcus genome. This showcases a new example of the fine‐tuned nature of viral metabolic processes.\ud

In this study, we examined the association between antimicrobial resistance, CRISPR/Cas systems and virulence with phage susceptibility in Acinetobacter baumannii and investigated draft genomes of phage susceptible multidrug resistant A. baumannii strains from Thailand. We investigated 230 A. baumannii strains using 17 lytic A. baumannii phages and the phage susceptibility was 46.5% (107/230). Phage susceptibility was also associated with resistance to numerous antibiotics (p-value ompA gene among phage susceptible A. baumannii strains (p-value A. baumannii isolates carrying cas5 or combinations of two or three other cas genes, showed a significant increase in phage resistance. Whole-genome sequences of seven phage susceptible A. baumannii isolates revealed that six groups of antibiotic resistance genes were carried by all seven phage susceptible A. baumannii. All strains carried biofilm associated genes and two strains harbored complete prophages, acquired copper tolerance genes, and CRISPR-associated (cas) genes. In conclusion, our data exhibits an association between virulence determinants and biofilm formation among phage susceptible A. baumannii strains. These data help to understand the bacterial co-evolution with phages.

Marine picocyanobacteria of the genus Synechococcus are ubiquitous phototrophs in oceanic systems. Consistent with these organisms occupying vast tracts of the nutrient impoverished ocean, most marine Synechococcus so far studied are monoploid, i.e., contain a single chromosome copy. The exception is the oligoploid strain Synechococcus sp. WH7803, which on average possesses around 4 chromosome copies. Here, we set out to understand the role of resource availability (through nutrient deplete growth) and physical stressors (UV, exposure to low and high temperature) in regulating ploidy level in this strain. Using qPCR to assay ploidy status we demonstrate the relative stability of chromosome copy number in Synechococcus sp. WH7803. Such robustness in maintaining an oligoploid status even under nutrient and physical stress is indicative of a fundamental role, perhaps facilitating recombination of damaged DNA regions as a result of prolonged exposure to oxidative stress, or allowing added flexibility in gene expression via possessing multiple alleles.

In this study, we aimed to investigate the spatio-temporal distribution patterns of the picocyanobacterium Synechococcus, an important contributor to primary production in many freshwater lakes. Our study sites were four lakes with different trophic states within the Osterseen Lake District in Southern Germany. Flow cytometry counts and next-generation sequencing were used from April to October 2015 to analyse the occurrence of Synechococcus and heterotrophic prokaryotes in relation to physical and chemical habitat properties. Synechococcus was identified as the main representative of the autotrophic picoplankton, but cell counts varied widely. The Synechococcus taxa identified by flow cytometry were confirmed by sequencing data, but the comparison of the flow cytometry counts and sequence data revealed discrepancies for cells in the exponential phase. The eutrophic Lake Schiffhuettensee was dominated by algae and had the highest abundance of heterotrophic prokaryotes. The presence of distinct operational taxonomic units of Synechococcus varied seasonally and was lake-specific, indicating local niche adaptation. Our study sheds light on the ecology of these important primary producers in freshwater systems. Furthermore, the discrepancy observed with the direct comparison of the widely used methods of next-generation sequencing and flow cytometry should serve as a caveat for future data analysis.