Disease-specific alterations in the enteric virome in inflammatory bowel disease.

Decreases in the diversity of enteric bacterial populations are observed in patients with Crohn's disease (CD) and ulcerative colitis (UC). Less is known about the virome in these diseases. We show that the enteric virome is abnormal in CD and UC patients. In-depth analysis of preparations enriched for free virions in the intestine revealed that CD and UC were associated with a significant expansion of Caudovirales bacteriophages. The viromes of CD and UC patients were disease and cohort specific. Importantly, it did not appear that expansion and diversification of the enteric virome was secondary to changes in bacterial populations. These data support a model in which changes in the virome may contribute to intestinal inflammation and bacterial dysbiosis. We conclude that the virome is a candidate for contributing to, or being a biomarker for, human inflammatory bowel disease and speculate that the enteric virome may play a role in other diseases.

DNA polymerase swapping in Caudoviricetes bacteriophages.

BACKGROUND: Viruses with double-stranded (ds) DNA genomes in the realm Duplodnaviria share a conserved structural gene module but show a broad range of variation in their repertoires of DNA replication proteins. Some of the duplodnaviruses encode (nearly) complete replication systems whereas others lack (almost) all genes required for replication, relying on the host replication machinery. DNA polymerases (DNAPs) comprise the centerpiece of the DNA replication apparatus. The replicative DNAPs are classified into 4 unrelated or distantly related families (A-D), with the protein structures and sequences within each family being, generally, highly conserved. More than half of the duplodnaviruses encode a DNAP of family A, B or C. We showed previously that multiple pairs of closely related viruses in the order Crassvirales encode DNAPs of different families. METHODS: Groups of phages in which DNAP swapping likely occurred were identified as subtrees of a defined depth in a comprehensive evolutionary tree of tailed bacteriophages that included phages with DNAPs of different families. The DNAP swaps were validated by constrained tree analysis that was performed on phylogenetic tree of large terminase subunits, and the phage genomes encoding swapped DNAPs were aligned using Mauve. The structures of the discovered unusual DNAPs were predicted using AlphaFold2. RESULTS: We identified four additional groups of tailed phages in the class Caudoviricetes in which the DNAPs apparently were swapped on multiple occasions, with replacements occurring both between families A and B, or A and C, or between distinct subfamilies within the same family. The DNAP swapping always occurs "in situ", without changes in the organization of the surrounding genes. In several cases, the DNAP gene is the only region of substantial divergence between closely related phage genomes, whereas in others, the swap apparently involved neighboring genes encoding other proteins involved in phage genome replication. In addition, we identified two previously undetected, highly divergent groups of family A DNAPs that are encoded in some phage genomes along with the main DNAP implicated in genome replication. CONCLUSIONS: Replacement of the DNAP gene by one encoding a DNAP of a different family occurred on many independent occasions during the evolution of different families of tailed phages, in some cases, resulting in very closely related phages encoding unrelated DNAPs. DNAP swapping was likely driven by selection for avoidance of host antiphage mechanisms targeting the phage DNAP that remain to be identified, and/or by selection against replicon incompatibility.

Genomic characterization of the novel bacteriophage IME183, infecting Klebsiella pneumoniae of capsular type K2.

Klebsiella pneumoniae causes a wide range of serious and life-threatening infections. Klebsiella phage IME183, isolated from hospital sewage, exhibited lytic activity against K. pneumoniae of capsular type K2. Transmission electron microscopy revealed that phage IME183 has a head with a diameter of 50 nm and a short tail. Its genome is 41,384 bp in length with a GC content of 52.92%. It is predicted to contain 50 open reading frames (ORFs). The results of evolutionary analysis suggest that phage IME183 should be considered a member of a new species in the genus Przondovirus.

Genomic analysis of K47-type Klebsiella pneumoniae phage IME305, a newly isolated member of the genus Teetrevirus.

We isolated a K47-type Klebsiella pneumoniae phage from untreated hospital sewage: vB_KpnP_IME305 (GenBank no. OK149215). Next-generation sequencing (NGS) demonstrated that IME305 has a double-stranded DNA genome of 38,641 bp with 50.9% GC content. According to BLASTn comparisons, the IME305 genome sequence shares similarity with that of Klebsiella phage 6998 (97.37% identity and 95% coverage). IME305 contains 45 open reading frames (ORFs) and no rRNA, tRNA, or virulence-related gene sequences. Bioinformatic analysis showed that IME305 belongs to the phage subfamily Studiervirinae and genus Teetrevirus.

Biological characteristics and genomic analysis of a novel Escherichia phage Kayfunavirus CY1.

As the problem of bacterial resistance becomes serious day by day, bacteriophage as a potential antibiotic substitute attracts more and more researchers' interest. In this study, Escherichia phage Kayfunavirus CY1 was isolated from sewage samples of swine farms and identified by biological characteristics and genomic analysis. One-step growth curve showed that the latent period of phage CY1 was about 10 min, the outbreak period was about 40 min and the burst size was 35 PFU/cell. Analysis of the electron microscopy and whole-genome sequence showed that the phage should be classified as a member of the Autographiviridae family, Studiervirinae subfamily. Genomic analysis of phage CY1 (GenBank accession no. OM937123) revealed a genome size of 39,173 bp with an average GC content of 50.51% and 46 coding domain sequences (CDSs). Eight CDSs encoding proteins involved in the replication and regulation of phage DNA, 2 CDSs encoded lysis proteins, 14 CDSs encoded packing and morphogenesis proteins. Genomic and proteomic analysis identified no sequence that encoded for virulence factor, integration-related proteins or antibiotic resistance genes. In summary, morphological and genomics suggest that phage CY1 is more likely a novel Escherichia phage.

Biological and molecular characterization of fEg-Eco19, a lytic bacteriophage active against an antibiotic-resistant clinical Escherichia coli isolate.

Characterization of bacteriophages facilitates better understanding of their biology, host specificity, genomic diversity, and adaptation to their bacterial hosts. This, in turn, is important for the exploitation of phages for therapeutic purposes, as the use of uncharacterized phages may lead to treatment failure. The present study describes the isolation and characterization of a bacteriophage effective against the important clinical pathogen Escherichia coli, which shows increasing accumulation of antibiotic resistance. Phage fEg-Eco19, which is specific for a clinical E. coli strain, was isolated from an Egyptian sewage sample. Phage fEg-Eco19 formed clear, sharp-edged, round plaques. Electron microscopy showed that the isolated phage is tailed and therefore belongs to the order Caudovirales, and morphologically, it resembles siphoviruses. The diameter of the icosahedral head of fEg-Eco19 is 68 ± 2 nm, and the non-contractile tail length and diameter are 118 ± 0.2 and 13 ± 0.6 nm, respectively. The host range of the phage was found to be narrow, as it infected only two out of 137 clinical E. coli strains tested. The phage genome is 45,805 bp in length with a GC content of 50.3% and contains 76 predicted genes. Comparison of predicted and experimental restriction digestion patterns allowed rough mapping of the physical ends of the phage genome, which was confirmed using the PhageTerm tool. Annotation of the predicted genes revealed gene products belonging to several functional groups, including regulatory proteins, DNA packaging and phage structural proteins, host lysis proteins, and proteins involved in DNA/RNA metabolism and replication.

Isolation and characterization of a novel Escherichia coli phage Kayfunavirus ZH4.

Escherichia coli, a gram-negative bacterium, was generally considered conditional pathogenic bacteria and the proportion of bacteria resistant to commonly used specified antibacterial drugs exceeded 50%. Phage therapeutic application has been revitalized since antibiotic resistance in bacteria was increasing. Compared with antibiotics, phage is the virus specific to bacterial hosts. However, further understanding of phage-host interactions is required. In this study, a novel phage specific to a E. coli strain, named as phage Kayfunavirus ZH4, was isolated and characterized. Transmission electron microscopy showed that phage ZH4 belongs to the family Autographiviridae. The whole-genome analysis showed that the length of phage ZH4 genome was 39,496 bp with 49 coding domain sequence (CDS) and no tRNA was detected. Comparative genome and phylogenetic analysis demonstrated that phage ZH4 was highly similar to phages belonging to the genus Kayfunavirus. Moreover, the highest average nucleotide identity (ANI) values of phage ZH4 with all the known phages was 0.86, suggesting that ZH4 was a relatively novel phage. Temperature and pH stability tests showed that phage ZH4 was stable from 4° to 50 °C and pH range from 3 to 11. Host range of phage ZH4 showed that there were only 2 out of 17 strains lysed by phage ZH4. Taken together, phage ZH4 was considered as a novel phage with the potential for applications in the food and pharmaceutical industries.

Extraordinary diversity of viruses in deep-sea sediments as revealed by metagenomics without prior virion separation.

Our current knowledge of the virosphere in deep-sea sediments remains rudimentary. Here we investigated viral diversity at both gene and genomic levels in deep-sea sediments of Southwest Indian Ocean. Analysis of 19 676 106 non-redundant genes from the metagenomic DNA sequences revealed a large number of unclassified viral groups in these samples. A total of 1106 high-confidence viral contigs were obtained after two runs of assemblies, and 217 of these contigs with sizes up to ~120 kb were shown to represent complete viral genomes. These contigs are clustered with no known viral genomes, and over 2/3 of the ORFs on the viral contigs encode no known functions. Furthermore, most of the complete viral contigs show limited similarity to known viral genomes in genome organization. Most of the classified viral contigs are derived from dsDNA viruses belonging to the order Caudovirales, including primarily members of the families Myoviridae, Podoviridae and Siphoviridae. Most of these viruses infect Proteobacteria and, less frequently, Planctomycetes, Firmicutes, Chloroflexi, etc. Auxiliary metabolic genes (AMGs), present in abundance on the viral contigs, appear to function in modulating the host ability to sense environmental gradients and community changes, and to uptake and metabolize nutrients.

Analysis of Spounaviruses as a Case Study for the Overdue Reclassification of Tailed Phages.

Tailed bacteriophages are the most abundant and diverse viruses in the world, with genome sizes ranging from 10 kbp to over 500 kbp. Yet, due to historical reasons, all this diversity is confined to a single virus order-Caudovirales, composed of just four families: Myoviridae, Siphoviridae, Podoviridae, and the newly created Ackermannviridae family. In recent years, this morphology-based classification scheme has started to crumble under the constant flood of phage sequences, revealing that tailed phages are even more genetically diverse than once thought. This prompted us, the Bacterial and Archaeal Viruses Subcommittee of the International Committee on Taxonomy of Viruses (ICTV), to consider overall reorganization of phage taxonomy. In this study, we used a wide range of complementary methods-including comparative genomics, core genome analysis, and marker gene phylogenetics-to show that the group of Bacillus phage SPO1-related viruses previously classified into the Spounavirinae subfamily, is clearly distinct from other members of the family Myoviridae and its diversity deserves the rank of an autonomous family. Thus, we removed this group from the Myoviridae family and created the family Herelleviridae-a new taxon of the same rank. In the process of the taxon evaluation, we explored the feasibility of different demarcation criteria and critically evaluated the usefulness of our methods for phage classification. The convergence of results, drawing a consistent and comprehensive picture of a new family with associated subfamilies, regardless of method, demonstrates that the tools applied here are particularly useful in phage taxonomy. We are convinced that creation of this novel family is a crucial milestone toward much-needed reclassification in the Caudovirales order.

Characterization of four virulent Klebsiella pneumoniae bacteriophages, and evaluation of their potential use in complex phage preparation.

BACKGROUND: Nowadays, hundreds of thousands of deaths per year are caused by antibiotic resistant nosocomial infections and the prognosis for future years is much worse, as evidenced by modern research. Bacteria of the Klebsiella genus are one of the main pathogens that cause nosocomial infections. Among the many antimicrobials offered to replace or supplement traditional antibiotics, bacteriophages are promising candidates. METHODS: This article presents microbiological, physicochemical and genomic characterization of 4 virulent bacteriophages belonging to Siphoviridae, Myoviridae and Podoviridae families. Phages were studied by electron microscopy; their host range, lytic activity, adsorption rate, burst size, latent period, frequency of phage-resistant forms generation, lysis dynamics and sensitivity of phage particles to temperature and pH were identified; genomes of all 4 bacteriophages were studied by restriction digestion and complete genome sequence. RESULTS: Studied phages showed wide host range and high stability at different temperature and pH values. In contrast with single phages, a cocktail of bacteriophages lysed all studied bacterial strains, moreover, no cases of the emergence of phage-resistant bacterial colonies were detected. Genomic data proved that isolated viruses do not carry antibiotic resistance, virulence or lysogenic genes. Three out of four bacteriophages encode polysaccharide depolymerases, which are involved in the degradation of biofilms and capsules. CONCLUSIONS: The bacteriophages studied in this work are promising for further in vivo studies and might be used in phage therapy as part of a complex therapeutic and prophylactic phage preparation. The conducted studies showed that the complex preparation is more effective than individual phages. The use of the complex phage cocktail allows to extend the lytic spectrum, and significantly reduces the possibility of phage-resistant forms generation.

Predicting host taxonomic information from viral genomes: A comparison of feature representations.

The rise in metagenomics has led to an exponential growth in virus discovery. However, the majority of these new virus sequences have no assigned host. Current machine learning approaches to predicting virus host interactions have a tendency to focus on nucleotide features, ignoring other representations of genomic information. Here we investigate the predictive potential of features generated from four different 'levels' of viral genome representation: nucleotide, amino acid, amino acid properties and protein domains. This more fully exploits the biological information present in the virus genomes. Over a hundred and eighty binary datasets for infecting versus non-infecting viruses at all taxonomic ranks of both eukaryote and prokaryote hosts were compiled. The viral genomes were converted into the four different levels of genome representation and twenty feature sets were generated by extracting k-mer compositions and predicted protein domains. We trained and tested Support Vector Machine, SVM, classifiers to compare the predictive capacity of each of these feature sets for each dataset. Our results show that all levels of genome representation are consistently predictive of host taxonomy and that prediction k-mer composition improves with increasing k-mer length for all k-mer based features. Using a phylogenetically aware holdout method, we demonstrate that the predictive feature sets contain signals reflecting both the evolutionary relationship between the viruses infecting related hosts, and host-mimicry. Our results demonstrate that incorporating a range of complementary features, generated purely from virus genome sequences, leads to improved accuracy for a range of virus host prediction tasks enabling computational assignment of host taxonomic information.

Diversity of limestone bacteriophages infecting Dickeya solani isolated in the Czech Republic.

Seven novel tailed lytic viruses (Ds3CZ, Ds5CZ, Ds9CZ, Ds16CZ, Ds20CZ, Ds23CZ, Ds25CZ) infecting the bacterium Dickeya solani were isolated in the Czech Republic. Genomes of these viruses are dsDNA, 149,364 to 155,285 bp in length, and the genome arrangement is very similar to that of the type virus Dickeya virus LIMEstone 1. All but the Ds25CZ virus should be regarded as strains of a single species. Most of the sequence differences are due to the presence or absence of homing endonuclease (HE) genes, with 23 HEs found in Ds3CZ, Ds5CZ, and Ds20CZ, 22 in Ds9CZ, 19 in Ds16CZ, 18 in Ds25CZ, and 15 in Ds23CZ.

Identification of a phage-derived depolymerase specific for KL64 capsule of Klebsiella pneumoniae and its anti-biofilm effect.

The increasing prevalence of Carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a serious threat to global health. Phages and phage-derived enzymes gained increasing attention for controling CRKP infections. In this study, a lytic phage P510 infecting KL64 type K. pneumoniae was isolated and characterized. Whole genome analysis and electron microscopy analysis showed that phage P510 belonged to genus Przondovirus, family Autographiviridae, the order Caudovirales. The tail fiber protein of the phage was predicted to encode capsule depolymerase. Further analysis demonstrated that recombinant depolymerase P510dep had polysaccharide-degrading activity against KL64-types capsule of K. pneumoniae, and its lysis spectrum matched to host range of phage P510. We also demonstrated that the recombinant depolymerase was able to significantly inhibit biofilm formation. The discovery of the phage-derived depolymerase lays the foundation for controlling the spread of CRKPs.

Characterization of two newly isolated Staphylococcus aureus bacteriophages from Japan belonging to the genus Silviavirus.

Two Staphylococcus aureus bacteriophages, KSAP7 and KSAP11, were isolated from sewage and characterized. Based on morphology and DNA sequences, they were assigned to the genus Silviavirus, subfamily Twortvirinae, family Herelleviridae, whose members are hypothesized to be suitable for bacteriophage therapy. The KSAP7 and KSAP11 genomes were 137,950 and 138,307 bp in size, respectively. Although their DNA sequences were almost identical, evidence of site-specific DNA rearrangements was found in two regions. Changes in the number of PIEPEK amino acid sequence repeats encoded by orf10 and the insertion/deletion of a 541-bp sequence that includes a possible tail-related gene were identified.

New Insights on the Feature and Function of Tail Tubular Protein B and Tail Fiber Protein of the Lytic Bacteriophage φYeO3-12 Specific for Yersinia enterocolitica Serotype O:3.

For the first time, we are introducing TTPBgp12 and TFPgp17 as new members of the tail tubular proteins B (TTPB) and tail fiber proteins (TFP) family, respectively. These proteins originate from Yersinia enterocolitica phage φYeO3-12. It was originally thought that these were structural proteins. However, our results show that they also inhibit bacterial growth and biofilm formation. According to the bioinformatic analysis, TTPBgp12 is functionally and structurally similar to the TTP of Enterobacteria phage T7 and adopts a ß-structure. TFPgp17 contains an intramolecular chaperone domain at its C-terminal end. The N-terminus of TFPgp17 is similar to other representatives of the TFP family. Interestingly, the predicted 3D structure of TFPgp17 is similar to other bacterial S-layer proteins. Based on the thermal unfolding experiment, TTPBgp12 seems to be a two-domain protein that aggregates in the presence of sugars such as maltose and N-acetylglucosamine (GlcNAc). These sugars cause two unfolding events to transition into one global event. TFPgp17 is a one-domain protein. Maltose and GlcNAc decrease the aggregation temperature of TFPgp17, while the presence of N-acetylgalactosamine (GalNAc) increases the temperature of its aggregation. The thermal unfolding analysis of the concentration gradient of TTPBgp12 and TFPgp17 indicates that with decreasing concentrations, both proteins increase in stability. However, a decrease in the protein concentration also causes an increase in its aggregation, for both TTPBgp12 and TFPgp17.

Novel Caudovirales associated with Marine Group I Thaumarchaeota assembled from metagenomes.

Marine Group I (MGI) Thaumarchaeota are some of the most abundant microorganisms in the deep ocean and responsible for much of the ammonia oxidation occurring in this environment. In this work, we present 35 sequences assembled from metagenomic samples of the first uncultivated Caudovirales viruses associated with Thaumarchaeota, which we designated marthavirus. Most of the sequences were obtained from cellular metagenomes confirming that they represent an important tool to study environmental viral communities due to cells retrieved while undergoing viral lysis. Metagenomic recruitment showed that this viral population is formed by very divergent entities with high intrapopulation homogeneity. However, metatranscriptomic analyses revealed the same differential expression profile with the capsid as major transcript, indicative of viruses during the lytic cycle. The cobalamine biosynthesis gene cobS, an auxiliary metabolic gene, was also highly expressed during the infection. These analyses expand our understanding of the global diversity of archaeal viruses.

Comparative genomics groups phages of Negativicutes and classical Firmicutes despite different Gram-staining properties.

Negativicutes are gram-negative bacteria characterized by two cell membranes, but they are phylogenetically a side-branch of gram-positive Firmicutes that contain only a single membrane. We asked whether viruses (phages) infecting Negativicutes were horizontally acquired from gram-negative Proteobacteria, given the shared outer cell structure of their bacterial hosts, or if Negativicute phages co-evolved vertically with their hosts and thus resemble gram-positive Firmicute prophages. We predicted and characterized 485 prophages (mostly Caudovirales) from gram-negative Firmicute genomes plus 2977 prophages from other bacterial clades, and we used virome sequence data from 183 human stool samples to support our predictions. The majority of identified Negativicute prophages were lambdoids closer related to prophages from other Firmicutes than Proteobacteria by sequence relationship and genome organization (position of the lysis module). Only a single Mu-like candidate prophage and no clear P2-like prophages were identified in Negativicutes, both common in Proteobacteria. Given this collective evidence, it is unlikely that Negativicute phages were acquired from Proteobacteria. Sequence-related prophages, which occasionally harboured antibiotic resistance genes, were identified in two distinct Negativicute orders (Veillonellales and Acidaminococcales), possibly suggesting horizontal cross-order phage infection between human gut commensals. Our results reveal ancient genomic signatures of phage and bacteria co-evolution despite horizontal phage mobilization.

Characterization and complete genomic analysis of two Salmonella phages, SenALZ1 and SenASZ3, new members of the genus Cba120virus.

Salmonella phages SenALZ1 and SenASZ3, two novel phages infecting Salmonella enterica, were isolated and analyzed. The genomes of these two phages consist of 154,811 and 157,630 base pairs (bp), with G+C contents of 44.56% and 44.74%, respectively. Fifty-nine of 199 open reading frames (ORFs) in the SenALZ1 genome, and 60 of the 204 in the SenASZ3 genome show similarity to reference sequences in the NCBI nr database that encode putative phage proteins with predicted functions. Based on the results of transmission electron microscopy (TEM) examination, complete genome sequence alignment, phylogenetic analysis, and gene annotation, we propose that these two phages are representative isolates of two new species of the genus Cba120virus, subfamily Cvivirinae, family Ackermannviridae.

Analysis of metagenomic data reveals common features of halophilic viral communities across continents.

Microbial communities from hypersaline ponds, dominated by halophilic archaea, are considered specific of such extreme conditions. The associated viral communities have accordingly been shown to display specific features, such as similar morphologies among different sites. However, little is known about the genetic diversity of these halophilic viral communities across the Earth. Here, we studied viral communities in hypersaline ponds sampled on the coast of Senegal (8-36% of salinity) using metagenomics approach, and compared them with hypersaline viromes from Australia and Spain. The specificity of hyperhalophilic viruses could first be demonstrated at a community scale, salinity being a strong discriminating factor between communities. For the major viral group detected in all samples (Caudovirales), only a limited number of halophilic Caudovirales clades were highlighted. These clades gather viruses from different continents and display consistent genetic composition, indicating that they represent related lineages with a worldwide distribution. Non-tailed hyperhalophilic viruses display a greater rate of gene transfer and recombination, with uncharacterized genes conserved across different kind of viruses and plasmids. Thus, hypersaline viral communities around the world appear to form a genetically consistent community that are likely to harbour new genes coding for enzymes specifically adapted to these environments.

Metagenomic analysis of the viral community in Namib Desert hypoliths.

Hypolithic microbial communities are specialized desert communities inhabiting the underside of translucent rocks. Here, we present the first study of the viral fraction of these communities isolated from the hyperarid Namib Desert. The taxonomic composition of the hypolithic viral communities was investigated and a functional assessment of the sequences determined. Phylotypic analysis showed that bacteriophages belonging to the order Caudovirales, in particular the family Siphoviridae, were most prevalent. Functional analysis and comparison with other metaviromes revealed a relatively high frequency of cell wall-degrading enzymes, ribonucleotide reductases (RNRs) and phage-associated genes. Phylogenetic analyses of terL and phoH marker genes indicated that many of the sequences were novel and distinct from known isolates, and the class distribution of the RNRs suggests that this is a novel environment. The composition of the viral hypolith fraction containing many Bacillus-infecting phages was not completely consistent with Namib hypolith phylotypic surveys of the bacterial hosts, in which the cyanobacterial genus Chroococcidiopsis was found to be dominant. This could be attributed to the lack of sequence information about hypolith viruses/bacteria in public databases or the possibility that hypolithic communities incorporate viruses from the surrounding soil.