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.

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.

Whole genome sequence analysis of Aeromonas-infecting bacteriophage AHPMCC7, a new species of genus Ahphunavirus and its application in Litopenaeus vannamei culture.

Aeromonas hydrophila, a Gram-negative pathogenic bacterium, is responsible for huge economic losses in aquaculture. In this study, we evaluated the efficacy of bacteriophage AHPMCC7 which was isolated by using A. hydrophila MTCC 1739 as a host. This bacteriophage exhibited 10 min latent period and burst size was 275. In liquid culture, bacteriophage AHPMCC7 could completely lyse A. hydrophila MTCC 1739 after 2 h. AHPMCC7 genome was 42,277 bp long with 58.9% G + C content. The genome consisted of 48 CDSs and no tRNA. The comparative genomic analyses clearly implied that AHPMCC7 might represent a novel species of the genus Aphunavirus under Autographiviridae family. Bacteriophage AHPMCC7 could survive at broad pH (3-10), temperature (4-37 °C), and salinity (0-40 ppt). In aquarium trial, AHPMCC7 could control A. hydrophila MTCC 1739 without affecting the survivability of Litopenaeus vannamei. Clearly, the bacteriophage AHPMCC7 might be used in shrimp aquaculture as a biocontrol agent.

Structure and proposed DNA delivery mechanism of a marine roseophage.

Tailed bacteriophages (order, Caudovirales) account for the majority of all phages. However, the long flexible tail of siphophages hinders comprehensive investigation of the mechanism of viral gene delivery. Here, we report the atomic capsid and in-situ structures of the tail machine of the marine siphophage, vB_DshS-R4C (R4C), which infects Roseobacter. The R4C virion, comprising 12 distinct structural protein components, has a unique five-fold vertex of the icosahedral capsid that allows genome delivery. The specific position and interaction pattern of the tail tube proteins determine the atypical long rigid tail of R4C, and further provide negative charge distribution within the tail tube. A ratchet mechanism assists in DNA transmission, which is initiated by an absorption device that structurally resembles the phage-like particle, RcGTA. Overall, these results provide in-depth knowledge into the intact structure and underlining DNA delivery mechanism for the ecologically important siphophages.

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.

StenM_174: A Novel Podophage That Infects a Wide Range of Stenotrophomonas spp. and Suggests a New Subfamily in the Family Autographiviridae.

Stenotrophomonas maltophilia was discovered as a soil bacterium associated with the rhizosphere. Later, S. maltophilia was found to be a multidrug-resistant hospital-associated pathogen. Lytic bacteriophages are prospective antimicrobials; therefore, there is a need for the isolation and characterization of new Stenotrophomonas phages. The phage StenM_174 was isolated from litter at a poultry farm using a clinical strain of S. maltophilia as the host. StenM_174 reproduced in a wide range of clinical and environmental strains of Stenotrophomonas, mainly S. maltophilia, and it had a podovirus morphotype. The length of the genomic sequence of StenM_174 was 42,956 bp, and it contained 52 putative genes. All genes were unidirectional, and 31 of them encoded proteins with predicted functions, while the remaining 21 were identified as hypothetical ones. Two tail spike proteins of StenM_174 were predicted using AlphaFold2 structural modeling. A comparative analysis of the genome shows that the Stenotrophomonas phage StenM_174, along with the phages Ponderosa, Pepon, Ptah, and TS-10, can be members of the new putative genus Ponderosavirus in the Autographiviridae family. In addition, the analyzed data suggest a new subfamily within this family.

Isolation, Characterization, and Genome Analysis of a Novel Bacteriophage, Escherichia Phage vB_EcoM-4HA13, Representing a New Phage Genus in the Novel Phage Family Chaseviridae.

Shiga toxin-producing Escherichia coli (STEC) is one of the leading causes of foodborne illnesses in North America and can lead to severe symptoms, with increased fatality risk for young children. While E. coli O157:H7 remains the dominant STEC serotype associated with foodborne outbreaks, there has been an increasing number of non-O157 STEC outbreaks in recent years. For the food industry, lytic bacteriophages offer an organic, self-limiting alternative to pathogen reduction-one that could replace or reduce the use of chemical and physical food processing methods. From EHEC-enriched sewage, we isolated a novel bacteriophage, vB_EcoM-4HA13 (4HA13). Phenotypic characterizations revealed 4HA13 to possess a myoviral morphotype, with a high specificity to non-motile O111 serotype, and a long latent period (90 min). Through genomic analyses, this 52,401-bp dsDNA phage was found to contain 81 CDS, but no detectable presence of antibiotic resistance, integrase, or virulence genes. A BLASTn search for each of the identified 81 CDS yielded homologues with low levels of similarity. Comparison of RNA polymerase and terminase large subunit amino acid sequences led to the proposal and acceptance of a new bacteriophage family, Chaseviridae, with 4HA13 representing a new species and genus. The discovery of this phage has broadened our current knowledge of bacteriophage diversity.

The small genome, virulent, non-contractile tailed bacteriophages that infect Enterobacteriales hosts.

Tailed bacteriophages are abundant and extremely diverse. Understanding this diversity is a challenge, and here we examine a small slice of that diversity in some detail. We contrast and compare the small genome, virulent, non-contractile tailed phages that infect the bacterial order Enterobacteriales. These phages, with genomes in the 35-60 kbp range, have very similar virions that are often difficult to distinguish by negative stain electron microscopy. There are currently 651 genome sequences of such phages in the public database. We show that these can be robustly parsed into fifteen well-defined clusters that have very different nucleotide sequences. We examine the similarities and differences among these clusters, as well as genetic exchange among clusters and the relationships between host species and phage clusters.

Isolation and genomic characterization of a novel Autographiviridae bacteriophage IME184 with lytic activity against Klebsiella pneumoniae.

Klebsiella pneumoniae, a multidrug resistant bacterium that causes nosocomial infections including septicemia, pneumonia etc. Bacteriophages are potential antimicrobial agents for the treatment of antibiotic resistant bacteria. In this study, a novel bacteriophage IME184, was isolated from hospital sewage against clinical multi-drug resistant Klebsiella pneumoniae. Transmission electron microscopy and genomic characterization exhibited this phage belongs to the Molineuxvirinae genus, Autographiviridae family. Phage IME184 possessed a double-stranded DNA genome composed of 44,598 bp with a GC content of 50.3%. The phage genome encodes 57 open reading frames, out of 26 are hypothetical proteins while 31 had assigned putative functions. No tRNA, virulence related or antibiotic resistance genes were found in phage genome. Comparative genomic analysis showed that phage IME184 has 94% similarity with genomic sequence of Klebsiella phage K1-ULIP33 (MK380014.1). Multiplicity of infection, one step growth curve and host range of phage were also measured. According to findings, Phage IME184 is a promising biological agent that infects Klebsiella pneumoniae and can be used in future phage therapies.

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.

Characterization of Three Novel Virulent Aeromonas Phages Provides Insights into the Diversity of the Autographiviridae Family.

In this study, we isolated and characterized three novel virulent Autographiviridae bacteriophages, vB_AspA_Bolek, vB_AspA_Lolek, and vB_AspA_Tola, which infect different Aeromonas strains. These three host-pathogen pairs were derived from the same sampling location-the arsenic-containing microbial mats of the Zloty Stok gold mine. Functional analysis showed they are psychrotolerant (4-25 °C), albeit with a much wider temperature range of propagation for the hosts (≤37 °C). Comparative genomic analyses revealed a high nucleotide and amino acid sequence similarity of vB_AspA_Bolek and vB_AspA_Lolek, with significant differences exclusively in the C-terminal region of their tail fibers, which might explain their host range discrimination. The protein-based phage network, together with a phylogenetic analysis of the marker proteins, allowed us to assign vB_AspA_Bolek and vB_AspA_Lolek to the Beijerinckvirinae and vB_AspA_Tola to the Colwellvirinae subfamilies, but as three novel species, due to their low nucleotide sequence coverage and identity with other known phage genomes. Global comparative analysis showed that the studied phages are also markedly different from most of the 24 Aeromonas autographiviruses known so far. Finally, this study provides in-depth insight into the diversity of the Autographiviridae phages and reveals genomic similarities between selected groups of this family as well as between autographiviruses and their relatives of other Caudoviricetes families.

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 the First Temperate Virus Infecting Psychrobacillus from Marine Sediments.

Viruses are far more abundant than cellular microorganisms in the marine ecosystem. However, very few viruses have so far been isolated from marine sediments, especially hydrothermal vent sediments, hindering the understanding of the biology and ecological functions of these tiny organisms. Here, we report the isolation and characterization of a temperate bacteriophage, named PVJ1, which infects Psychrobacillus from a hydrothermal vent field in Okinawa Trough. PVJ1 belongs to the Myoviridae family of the order Caudovirales. The tailed phage possesses a 53,187 bp linear dsDNA genome, with 84 ORFs encoding structural proteins, genome replication, host lysis, etc. in a modular pattern. The phage genome is integrated into the host chromosome near the 3'-end of deoD, a gene encoding purine nucleoside phosphorylase (PNP). The phage integration does not appear to disrupt the function of PNP. The phage DNA is packaged by the headful mechanism. Release of PVJ1 from the host cell was drastically enhanced by treatment with mitomycin C. Phages encoding an MCP sharing significant similarity (≥70% identical amino acids) with that of PVJ1 are widespread in diverse environments, including marine and freshwater sediments, soils, artificial ecosystems, and animal intestines, and primarily infect Firmicutes. These results are valuable to the understanding of the lifestyle and host interactions of bacterial viruses at the bottom of the ocean.

Phages from Genus Bruynoghevirus and Phage Therapy: Pseudomonas Phage Delta Case.

The applicability and safety of bacteriophage Delta as a potential anti-Pseudomonas aeruginosa agent belonging to genus Bruynoghevirus (family Podoviridae) was characterised. Phage Delta belongs to the species Pseudomonas virus PaP3, which has been described as a temperate, with cos sites at the end of the genome. The phage Delta possesses a genome of 45,970 bp that encodes tRNA for proline (Pro), aspartic acid (Asp) and asparagine (Asn) and does not encode any known protein involved in lysogeny formation or persistence. Analysis showed that phage Delta has 182 bp direct terminal repeats at the end of genome and lysogeny was confirmed, neither upon infection at low nor at high multiplicity of infection (MOI). The turbid plaques that appear on certain host lawns can result from bacteriophage insensitive mutants that occur with higher frequency (10-4). In silico analysis showed that the genome of Delta phage does not encode any known bacterial toxin or virulence factor, determinants of antibiotic resistance and known human allergens. Based on the broad host range and high lytic activity against planktonic and biofilm cells, phage Delta represents a promising candidate for phage therapy.

Novel Viruses That Lyse Plant and Human Strains of Kosakonia cowanii.

Kosakonia cowanii (syn. Enterobacter cowanii) is a highly competitive bacterium that lives with plant, insect, fish, bird, and human organisms. It is pathogenic on some plants and an opportunistic pathogen of human. Nine novel viruses that lyse plant pathogenic strains and/or human strains of K. cowanii were isolated, sequenced, and characterized. Kc166A is a novel kayfunavirus, Kc261 is a novel bonnellvirus, and Kc318 is a new cronosvirus (all Autographiviridae). Kc237 is a new sortsnevirus, but Kc166B and Kc283 are members of new genera within Podoviridae. Kc304 is a new winklervirus, and Kc263 and Kc305 are new myoviruses. The viruses differ in host specificity, plaque phenotype, and lysis kinetics. Some of them should be suitable also as pathogen control agents.

Revealing the full biosphere structure and versatile metabolic functions in the deepest ocean sediment of the Challenger Deep.

BACKGROUND: The full biosphere structure and functional exploration of the microbial communities of the Challenger Deep of the Mariana Trench, the deepest known hadal zone on Earth, lag far behind that of other marine realms. RESULTS: We adopt a deep metagenomics approach to investigate the microbiome in the sediment of Challenger Deep, Mariana Trench. We construct 178 metagenome-assembled genomes (MAGs) representing 26 phyla, 16 of which are reported from hadal sediment for the first time. Based on the MAGs, we find the microbial community functions are marked by enrichment and prevalence of mixotrophy and facultative anaerobic metabolism. The microeukaryotic community is found to be dominated by six fungal groups that are characterized for the first time in hadal sediment to possess the assimilatory and dissimilatory nitrate/sulfate reduction, and hydrogen sulfide oxidation pathways. By metaviromic analysis, we reveal novel hadal Caudovirales clades, distinctive virus-host interactions, and specialized auxiliary metabolic genes for modulating hosts' nitrogen/sulfur metabolism. The hadal microbiome is further investigated by large-scale cultivation that cataloged 1070 bacterial and 19 fungal isolates from the Challenger Deep sediment, many of which are found to be new species specialized in the hadal habitat. CONCLUSION: Our hadal MAGs and isolates increase the diversity of the Challenger Deep sediment microbial genomes and isolates present in the public. The deep metagenomics approach fills the knowledge gaps in structure and diversity of the hadal microbiome, and provides novel insight into the ecology and metabolism of eukaryotic and viral components in the deepest biosphere on earth.

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.

Ecology of inorganic sulfur auxiliary metabolism in widespread bacteriophages.

Microbial sulfur metabolism contributes to biogeochemical cycling on global scales. Sulfur metabolizing microbes are infected by phages that can encode auxiliary metabolic genes (AMGs) to alter sulfur metabolism within host cells but remain poorly characterized. Here we identified 191 phages derived from twelve environments that encoded 227 AMGs for oxidation of sulfur and thiosulfate (dsrA, dsrC/tusE, soxC, soxD and soxYZ). Evidence for retention of AMGs during niche-differentiation of diverse phage populations provided evidence that auxiliary metabolism imparts measurable fitness benefits to phages with ramifications for ecosystem biogeochemistry. Gene abundance and expression profiles of AMGs suggested significant contributions by phages to sulfur and thiosulfate oxidation in freshwater lakes and oceans, and a sensitive response to changing sulfur concentrations in hydrothermal environments. Overall, our study provides fundamental insights on the distribution, diversity, and ecology of phage auxiliary metabolism associated with sulfur and reinforces the necessity of incorporating viral contributions into biogeochemical configurations.

Dynamic changes occur in the DNA gut virome of female cynomolgus macaques during aging.

Aging is a critical factor affecting physical health and disease in mammals. Emerging evidence indicates that aging may affect the gut bacteriome in cynomolgus macaques, but little is known about whether or how the gut virome changes with age. Here, we compared the DNA gut viral composition of 16 female cynomolgus monkeys (Macaca fascicularis) at three life stages (young, adult, and old) using the shotgun metagenome sequencing method. We found that the DNA gut virome from these monkeys differed substantially among the three groups. The gut viruses were dominated by bacteriophages, the most abundant of which was the Caudovirales order (i.e., Siphoviridae, Myoviridae, and Podoviridae families). Additionally, the co-occurrence analysis revealed that the age-related bacteriophages were correlated in an extensive and complex manner with the main intestinal bacteria (i.e., Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria phyla). Furthermore, the age-related DNA gut viral functions were enriched for genetic information processing, nucleotide, and folate metabolism. Our gut virome analysis provides new insight into how aging influences the gut virome of non-human primates.

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.