Isolation of lytic bacteriophages and their relationships with the adherence genes of Staphylococcus saprophyticus.

OBJECTIVE: This study aimed to introduce a lytic bacteriophage against Staphylococcus saprophyticus from wastewater in Gorgan, northern Iran. RESULTS: The vB_SsapS-46 phage was isolated from urban wastewater and formed round and clear plaques on bacterial culture. It was visualized by electron microscopy and had a large head (approximately 106 nm) and a long tail (approximately 150 nm), indicating that it belongs to the Siphoviridae family. The host range of vB_SsapS-46 was determined using a spot test on 35 S. saprophyticus clinical isolates, and it was able to lyse 12 of the 35 clinical isolates (34%). Finally, the relationship between phage sensitivity and adherence genes was assessed, revealing no significant correlation between phage sensitivity and the frequency of adherence genes. The vB_SsapS-46 phage can be used alone or in a mixture in future studies to control urinary tract infections caused by this bacterium, especially in the elimination of drug-resistant pathogens.

A novel flavobacterial phage abundant during green tide, representing a new viral family, Zblingviridae.

Flavobacteriia are the dominant and active bacteria during algal blooms and play an important role in polysaccharide degradation. However, little is known about phages infecting Flavobacteriia, especially during green tide. In this study, a novel virus, vB_TgeS_JQ, infecting Flavobacteriia was isolated from the surface water of the Golden Beach of Qingdao, China. Transmission electron microscopy demonstrated that vB_TgeS_JQ had the morphology of siphovirus. The experiments showed that it was stable from -20°C to 45°C and pH 5 to pH 8, with latent and burst periods both lasting for 20 min. Genomic analysis showed that the phage vB_TgeS_JQ contained a 40,712-bp dsDNA genome with a GC content of 30.70%, encoding 74 open-reading frames. Four putative auxiliary metabolic genes were identified, encoding electron transfer-flavoprotein dehydrogenase, calcineurin-like phosphoesterase, phosphoribosyl-ATP pyrophosphohydrolase, and TOPRIM nucleotidyl hydrolase. The abundance of phage vB_TgeS_JQ was higher during Ulva prolifera (U. prolifera) blooms compared with other marine environments. The phylogenetic and comparative genomic analyses revealed that vB_TgeS_JQ exhibited significant differences from all other phage isolates in the databases and therefore was classified as an undiscovered viral family, named Zblingviridae. In summary, this study expands the knowledge about the genomic, phylogenetic diversity and distribution of flavobacterial phages (flavophages), especially their roles during U. prolifera blooms. IMPORTANCE: The phage vB_TgeS_JQ was the first flavobacterial phage isolated during green tide, representing a new family in Caudoviricetes and named Zblingviridae. The abundance of phage vB_TgeS_JQ was higher during the Ulva prolifera blooms. This study provides insights into the genomic, phylogenetic diversity, and distribution of flavophages, especially their roles during U. prolifera blooms.

Viruses participate in the organomineralization of travertines.

Travertines, which precipitate from high temperature water saturated with calcium carbonate, are generally considered to be dominated by physico-chemical and microbial precipitates. Here, as an additional influence on organomineral formation, metagenomic data and microscopic analyses clearly demonstrate that highly diverse viral, bacterial and archaeal communities occur in the biofilms associated with several modern classic travertine sites in Europe and Asia, along with virus-like particles. Metagenomic analysis reveals that bacteriophages (bacterial viruses) containing icosahedral capsids and belonging to the Siphoviridae, Myoviridae and Podoviridae families are the most abundant of all viral strains, although the bacteriophage distribution does vary across the sampling sites. Icosahedral shapes of capsids are also the most frequently observed under the microscope, occurring as non-mineralized through to mineralized viruses and virus-like particles. Viruses are initially mineralized by Ca-Si amorphous precipitates with subordinate Mg and Al contents; these then alter to nanospheroids composed of Ca carbonate with minor silicate 80-300 nm in diameter. Understanding the roles of bacteriophages in modern carbonate-saturated settings and related organomineralization processes is critical for their broader inclusion in the geological record and ecosystem models.

Complete genome analysis of the novel Shewanella phage vB_Sb_QDWS.

We present here the results of the analysis of the complete genome sequence of a potentially temperate phage, vB_Sb_QDWS, which was isolated from wastewater samples collected in Qingdao, China. The genome of phage vB_Sb_QDWS is composed of a double-stranded DNA that is 47,902 bp in length with a G + C content of 63.16%. It is predicted to contain 69 putative protein-encoding genes. Microscopic and genomic analysis showed that vB_Sb_QDWS is a novel phage of the class Siphoviridae.

Characterization of the first Pseudomonas grimontii bacteriophage, PMBT3.

The complete genome sequence of the virulent bacteriophage PMBT3, isolated on the proteolytic Pseudomonas grimontii strain MBTL2-21, showed no significant similarity to other known phage genome sequences, making this phage the first reported to infect a strain of P. grimontii. Electron microscopy revealed PMBT3 to be a member of the family Siphoviridae, with notably long and flexible whiskers. The linear, double-stranded genome of 87,196 bp has a mol% G+C content of 60.4 and contains 116 predicted protein-encoding genes. A putative tellurite resistance (terB) gene, originally reported to occur in the genome of a bacterium, was detected in the genome of phage PMBT3.

A novel temperate phage, vB_PstS-pAN, induced from the naphthalene-degrading bacterium Pseudomonas stutzeri AN10.

A novel temperate phage named vB_PstS-pAN was induced by mitomycin C treatment from the naphthalene-degrading bacterium Pseudomonas stutzeri AN10. The phage particles have icosahedral heads and long non-contractile tails, and vB_PstS-pAN can therefore be morphologically classified as a member of the family Siphoviridae. The whole genome of vB_PstS-pAN is 39,466 bp in length, with an 11-nt 3' overhang cohesive end. There are 53 genes in the vB_PstS-pAN genome, including genes responsible for phage integration, replication, morphogenesis, and bacterial lysis. The vB_PstS-pAN genome has low similarity to other phage genomes in the GenBank database, suggesting that vB_PstS-pAN is a novel member of the family Siphoviridae.

Biological characteristics and genomic analysis of a Stenotrophomonas maltophilia phage vB_SmaS_BUCT548.

Stenotrophomonas maltophilia (hereinafter referred to as S. maltophilia) has developed into an important opportunistic pathogenic bacterium, which is prevalent in nosocomial and community infections, and has adverse effects on patients with a compromised immune system. Phage vB_SmaS_BUCT548 was isolated from sewage of Beijing 307 Hospital with S. maltophilia (strain No.824) as a host. Phage morphology was observed by transmission electron microscopy and its biological and genomic characteristics were determined. The electron microscope shows that the bacteriophage belonged to the Siphoviridae and MOI is 0.001. One-step growth curve shows that the incubation period is 30 min and the burst size is 134 PFU/Cell. The host range is relatively wide and it can lysis 11of 13 S. maltophilia strains. Next-Generation Sequencing (NGS) results show that the genome sequence is a dsDNA with 62354 bp length, and the GC content is 56.3% (GenBank: MN937349). One hundred and two online reading frames (ORFs) are obtained after RAST online annotation and the BlastN nucleic acid comparison shows that the phage had low homology with other phages in NCBI database. This study reports a novel S. maltophilia phage named vB_SmaS_BUCT548, which has a short incubation period, strong lytic ability, and a wide host range. The main characteristic of this bacteriophage is the novelty of the genomic sequence and the analysis of the other characteristics provides basic data for further exploring the interaction mechanism between the phage and the host.

Characterization of bacteriophage VVP001 and its application for the inhibition of Vibrio vulnificus causing seafood-borne diseases.

Vibrio vulnificus is a major food-borne pathogen that causes septicemia and cellulitis with a mortality rate of >50%. However, there are no efficient natural food preservatives or biocontrol agents to control V. vulnificus in seafood. In this study, we isolated and characterized a novel bacteriophage VVP001. Host range and transmission electron microscopy morphology observations revealed that VVP001 belongs to the family Siphoviridae and specifically infects V. vulnificus. Phage stability tests showed that VVP001 is stable at a broad temperature range of -20 °C to 65 °C and a pH range from 3 to 11, which are conditions for food applications (processing, distribution, and storage). In vitro challenge assays revealed that VVP001 inhibited V. vulnificus MO6-24/O (a clinical isolate) growth up to a 3.87 log reduction. In addition, complete genome analysis revealed that the 76 kb VVP001 contains 102 open reading frames with 49.64% G + C content and no gene encoding toxins or other virulence factors, which is essential for food applications. Application of VVP001 to fresh abalone samples contaminated with V. vulnificus demonstrated its ability to inhibit V. vulnificus growth, and an in vivo mouse survival test showed that VVP001 protects mice against high mortality (survival rate >70% at a multiplicity of infection of 1000 for up to 7 days). Therefore, the bacteriophage VVP001 can be used as a good natural food preservative and biocontrol agent for food applications.

A bacteriophage infecting Mesorhizobium species has a prolate capsid and shows similarities to a family of Caulobacter crescentus phages.

Mesorhizobium phage vB_MloS_Cp1R7A-A1 was isolated from soil planted with chickpea in Saskatchewan. It is dissimilar in sequence and morphology to previously described rhizobiophages. It is a B3 morphotype virus with a distinct prolate capsid and belongs to the tailed phage family Siphoviridae. Its genome has a GC content of 60.3% and 238 predicted genes. Putative functions were predicted for 57 genes, which include 27 tRNA genes with anticodons corresponding to 18 amino acids. This represents the highest number of tRNA genes reported yet in a rhizobiophage. The gene arrangement shows a partially modular organization. Most of the structural genes are found in one module, whereas tRNA genes are in another. Genes for replication, recombination, and nucleotide metabolism form the third module. The arrangement of the replication module resembles the replication module of Enterobacteria phage T5, raising the possibility that it uses a recombination-based replication mechanism, but there is also a suggestion that a T7-like replication mechanism could be used. Phage termini appear to be long direct repeats of just over 12 kb in length. Phylogenetic analysis revealed that Cp1R7A-A1 is more closely related to PhiCbK-like Caulobacter phages and other B3 morphotype phages than to other rhizobiophages sequenced thus far.

Bacillus-infecting bacteriophage Izhevsk harbors thermostable endolysin with broad range specificity.

Several bacterial species belonging to the Bacillus cereus group are known to be causative agents of food poisoning and severe human diseases. Bacteriophages and their lytic enzymes called endolysins have been widely shown to provide for a supplemental or primary means of treating bacterial infections. In this work we present a new broad-host-range phage Izhevsk, which infects the members of the Bacillus cereus group. Transmission electron microscopy, genome sequencing and comparative analyses revealed that Izhevsk is a temperate phage with Siphoviridae morphology and belongs to the same genus as the previously described but taxonomically unclassified bacteriophages Tsamsa and Diildio. The Ply57 endolysin of Izhevsk phage has broad-spectrum activity against B. cereus sensu lato. The thermolability of Ply57 is higher than that of the PlyG of Wß phage. This work contributes to our current understanding of phage biodiversity and may be useful for further development of efficient antimicrobials aimed at diagnosing and treating infectious diseases and food contaminations caused by the Bacillus cereus group of bacteria.

Architecture of the flexible tail tube of bacteriophage SPP1.

Bacteriophage SPP1 is a double-stranded DNA virus of the Siphoviridae family that infects the bacterium Bacillus subtilis. This family of phages features a long, flexible, non-contractile tail that has been difficult to characterize structurally. Here, we present the atomic structure of the tail tube of phage SPP1. Our hybrid structure is based on the integration of structural restraints from solid-state nuclear magnetic resonance (NMR) and a density map from cryo-EM. We show that the tail tube protein gp17.1 organizes into hexameric rings that are stacked by flexible linker domains and, thus, form a hollow flexible tube with a negatively charged lumen suitable for the transport of DNA. Additionally, we assess the dynamics of the system by combining relaxation measurements with variances in density maps.

Structure and mechanism of DNA delivery of a gene transfer agent.

Alphaproteobacteria, which are the most abundant microorganisms of temperate oceans, produce phage-like particles called gene transfer agents (GTAs) that mediate lateral gene exchange. However, the mechanism by which GTAs deliver DNA into cells is unknown. Here we present the structure of the GTA of Rhodobacter capsulatus (RcGTA) and describe the conformational changes required for its DNA ejection. The structure of RcGTA resembles that of a tailed phage, but it has an oblate head shortened in the direction of the tail axis, which limits its packaging capacity to less than 4,500 base pairs of linear double-stranded DNA. The tail channel of RcGTA contains a trimer of proteins that possess features of both tape measure proteins of long-tailed phages from the family Siphoviridae and tail needle proteins of short-tailed phages from the family Podoviridae. The opening of a constriction within the RcGTA baseplate enables the ejection of DNA into bacterial periplasm.

Complete genome sequence of Phobos: a novel bacteriophage with unusual genomic features that infects Pseudomonas syringae.

The GenBank database contains over 2580 complete genome sequences from bacteriophages. However, limited reports are available concerning phages can that lyse members of Pseudomonas syringae, although this is a widespread bacterial species that can infect almost 200 plant species. In the present study, we isolated and characterized a new Siphoviridae phage, named "Pseudomonas phage vB_PsyS_Phobos" (for brevity, referred to here as Phobos). To our knowledge, this is one of the first genome sequences reported for a phage with lytic activity against P. syringae pv. syringae. The genome of Phobos is dsDNA of 56,734 bp with a GC content of 63.3%, containing 65 ORFs. Genome analysis revealed that Phobos is a novel lytic phage with unique genomic features and low similarity to other phages, suggesting that Phobos represents a new phage genus. Genome sequencing did not reveal sequences with significant similarity to known virulence factors, antibiotic resistance genes, potential immunoreactive allergens, or lysogeny-related proteins, suggesting suggests that phage Phobos is strictly lytic. Therefore, Phobos may be suitable for formulation as a biocontrol agent against P. syringae pv. syringae.

Novel Genus of Phages Infecting Streptococcus thermophilus: Genomic and Morphological Characterization.

Streptococcus thermophilus is a lactic acid bacterium commonly used for the manufacture of yogurt and specialty cheeses. Virulent phages represent a major risk for milk fermentation processes worldwide, as they can inactivate the added starter bacterial cells, leading to low-quality fermented dairy products. To date, four genetically distinct groups of phages infecting S. thermophilus have been described. Here, we describe a fifth group. Phages P738 and D4446 are virulent siphophages that infect a few industrial strains of S. thermophilus The genomes of phages P738 and D4446 were sequenced and found to contain 34,037 and 33,656 bp as well as 48 and 46 open reading frames, respectively. Comparative genomic analyses revealed that the two phages are closely related to each other but display very limited similarities to other S. thermophilus phages. In fact, these two novel S. thermophilus phages share similarities with streptococcal phages of nondairy origin, suggesting that they emerged recently in the dairy environment.IMPORTANCE Despite decades of research and adapted antiphage strategies such as CRISPR-Cas systems, virulent phages are still a persistent risk for the milk fermentation industry worldwide, as they can cause manufacturing failures and alter product quality. Phages P738 and D4446 are novel virulent phages that infect the food-grade Gram-positive bacterial species Streptococcus thermophilus These two related viruses represent a fifth group of S. thermophilus phages, as they are significantly distinct from other known S. thermophilus phages. Both phages share similarities with phages infecting nondairy streptococci, suggesting their recent emergence and probable coexistence in dairy environments. These findings highlight the necessity of phage surveillance programs as the phage population evolves in response to the application of antiphage strategies.

Mobile Loops and Electrostatic Interactions Maintain the Flexible Tail Tube of Bacteriophage Lambda.

The long flexible tail tube of bacteriophage lambda connects its capsid to the tail tip. On infection, a DNA ejection signal is passed from the tip, along the tube to the capsid that triggers passage of the DNA down the tube and into the host bacterium. The tail tube is built from repeating units of the major tail protein, gpV, which has two distinctive domains. Its N-terminal domain has the same fold as proteins that form the rigid inner tubes of contractile tail phages, such as T4, and its C-terminal domain adopt an Ig-like fold of unknown function. We determined structures of the lambda tail tube in free tails and in virions before and after DNA ejection using cryoelectron microscopy. Modeling of the density maps reveals how electrostatic interactions and a mobile loop participate in assembly and also impart flexibility to the tube while maintaining its integrity. We also demonstrate how a common protein fold produces rigid tubes in some phages but flexible tubes in others.

New Genus Fibralongavirus in Siphoviridae Phages of Staphylococcus pseudintermedius.

Bacteriophages of the significant veterinary pathogen Staphylococcus pseudintermedius are rarely described morphologically and genomically in detail, and mostly include phages of the Siphoviridae family. There is currently no taxonomical classification for phages of this bacterial species. Here we describe a new phage designated vB_SpsS_QT1, which is related to phage 2638A originally described as a Staphylococcus aureus phage. Propagating strain S. aureus 2854 of the latter was reclassified by rpoB gene sequencing as S. pseudintermedius 2854 in this work. Both phages have a narrow but different host range determined on 54 strains. Morphologically, both of them belong to the family Siphoviridae, share the B1 morphotype, and differ from other staphylococcal phage genera by a single long fibre at the terminus of the tail. The complete genome of phage vB_SpsS_QT1 was sequenced with the IonTorrent platform and expertly annotated. Its linear genome with cohesive ends is 43,029 bp long and encodes 60 predicted genes with the typical modular structure of staphylococcal siphophages. A global alignment found the genomes of vB_SpsS_QT1 and 2638A to share 84% nucleotide identity, but they have no significant similarity of nucleotide sequences with other phage genomes available in public databases. Based on the morphological, phylogenetic, and genomic analyses, a novel genus Fibralongavirus in the family Siphoviridae is described with phage species vB_SpsS_QT1 and 2638A.

A newly isolated roseophage represents a distinct member of Siphoviridae family.

BACKGROUND: Members of the Roseobacter lineage are a major group of marine heterotrophic bacteria because of their wide distribution, versatile lifestyles and important biogeochemical roles. Bacteriophages, the most abundant biological entities in the ocean, play important roles in shaping their hosts' population structures and mediating genetic exchange between hosts. However, our knowledge of roseophages (bacteriophages that infect Roseobacter) is far behind that of their host counterparts, partly reflecting the need to isolate and analyze the phages associated with this ecologically important bacterial clade. METHODS: vB_DshS-R4C (R4C), a novel virulent roseophage that infects Dinoroseobacter shibae DFL12T, was isolated with the double-layer agar method. The phage morphology was visualized with transmission electron microscopy. We characterized R4C in-depth with a genomic analysis and investigated the distribution of the R4C genome in different environments with a metagenomic recruitment analysis. RESULTS: The double-stranded DNA genome of R4C consists of 36,291 bp with a high GC content of 66.75%. It has 49 genes with low DNA and protein homologies to those of other known phages. Morphological and phylogenetic analyses suggested that R4C is a novel member of the family Siphoviridae and is most closely related to phages in the genus Cronusvirus. However, unlike the Cronusvirus phages, R4C encodes an integrase, implying its ability to establish a lysogenic life cycle. A terminal analysis shows that, like that of λ phage, the R4C genome utilize the 'cohesive ends' DNA-packaging mechanism. Significantly, homologues of the R4C genes are more prevalent in coastal areas than in the open ocean. CONCLUSIONS: Information about this newly discovered phage extends our understanding of bacteriophage diversity, evolution, and their roles in different environments.

Green approach to phytopathogen: Characterization of lytic bacteriophages of Pseudomonas sp., an etiology of the bacterial blight of pomegranate.

Two morphologically different bacteriophages were isolated from the river and soil samples from various locations of Maharashtra, India against the phytopathogen Pseudomonas sp. that was recently reported to cause a new bacterial blight of pomegranate. Both the phages belonged to the order Caudovirales representing the families Siphoviridae (vB_Psp.S_PRɸL2) and Myoviridae (vB_Psp.M_SSɸL8). The multiplicity of infection ranged from 0.01 to 0.1, phage adsorption rate from 39% to 66%, latent period from 10 to 20 min with a burst size of 24-85 phage particles per infected host cell. The genome size of phages PRɸL2 and SSɸL8 was approximately 25.403 kb and 29.877 kb respectively. Restriction digestion pattern of phage genomic DNA was carried out for phage PRɸL2, Eco RI resulted in two bands and Hind III resulted in three bands while for phage SSɸL8, both Eco RI and Hind III each resulted in three bands. SDS-PAGE protein profile showed six bands for PRɸL2 and nine bands for SSɸL8 of different proteins. Phages showed high pH stability over a range of 4-9, temperature stability over a range of 4-50 °C and UV radiation showed a reduction up to 89.36% for PRɸL2 and 96% for SSɸL8. In short, the present research work discusses for the first time in-detailed characterization of phages of a phytopathogen Pseudomonas sp. from Maharashtra, India, which can be further efficiently used for biological control of the causative agent of a new bacterial blight disease of pomegranate.

Characterization of SE-P3, P16, P37, and P47 bacteriophages infecting Salmonella Enteritidis.

The aim of this study was to identify and characterize the SE-P3, P16, P37, and P47 phages infecting Salmonella Enteritidis. Transmission electron microscopy analysis showed that the SE phages belonged to the Myoviridae or Siphoviridae family and had plaque sizes between 0.622 ± 0.027 and 1.630 ± 0.036 mm in diameter. sefC, pefA, spvC, sopE, and gipA virulent gene regions were absent in their genome and their calculated genome sizes were between 35.9 and 37.8 kbp. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the protein profiles of each phage were different. The SE phages had a short latent period (10-20 min), large burst size (76-356 PFU/cell), and a short burst time (25-35 min). The multiplicity of infection values and mutant frequency of the phages were 0.01-0.0001 and 10-7 , respectively. They were very infective against their host bacteria when applied at 20°C, 30°C, or 37°C and adsorbed to their host cells by 96.20-97.65% in the first 5 min of incubation, and also Ca2+ ions did not have a significant effect on their adsorption. The SE phages were resistant to wide pH ranges and high temperatures. These results indicate that the SE phages are good candidates as therapeutic and biocontrol agents against foodborne pathogenic S. Enteritidis.

Bacillus Phage vB_BtS_B83 Previously Designated as a Plasmid May Represent a New Siphoviridae Genus.

The Bacillus cereus group of bacteria includes, inter alia, the species known to be associated with human diseases and food poisoning. Here, we describe the Bacillus phage vB_BtS_B83 (abbreviated as B83) infecting the species of this group. Transmission electron microscopy (TEM) micrographs indicate that B83 belongs to the Siphoviridae family. B83 is a temperate phage using an arbitrium system for the regulation of the lysis-lysogeny switch, and is probably capable of forming a circular plasmid prophage. Comparative analysis shows that it has been previously sequenced, but was mistaken for a plasmid. B83 shares common genome organization and >46% of proteins with other the Bacillus phage, BMBtp14. Phylograms constructed using large terminase subunits and a pan-genome presence-absence matrix show that these phages form a clade distinct from the closest viruses. Based on the above, we propose the creation of a new genus named Bembunaquatrovirus that includes B83 and BMBtp14.