Three Novel Bacteriophages, J5a, F16Ba, and z1a, Specific for Bacillus anthracis, Define a New Clade of Historical Wbeta Phage Relatives.

Bacillus anthracis is a potent biowarfare agent, able to be highly lethal. The bacteria dwell in the soil of certain regions, as natural flora. Bacteriophages or their lytic enzymes, endolysins, may be an alternative for antibiotics and other antibacterials to fight this pathogen in infections and to minimize environmental contamination with anthrax endospores. Upon screening environmental samples from various regions in Poland, we isolated three new siphophages, J5a, F16Ba, and z1a, specific for B. anthracis. They represent new species related to historical anthrax phages Gamma, Cherry, and Fah, and to phage Wbeta of Wbetavirus genus. We show that the new phages and their closest relatives, phages Tavor_SA, Negev_SA, and Carmel_SA, form a separate clade of the Wbetavirus genus, designated as J5a clade. The most distinctive feature of J5a clade phages is their cell lysis module. While in the historical phages it encodes a canonical endolysin and a class III holin, in J5a clade phages it encodes an endolysin with a signal peptide and two putative holins. We present the basic characteristic of the isolated phages. Their comparative genomic analysis indicates that they encode two receptor-binding proteins, of which one may bind a sugar moiety of B. anthracis cell surface.

A Novel Wide-Range Freshwater Cyanophage MinS1 Infecting the Harmful Cyanobacterium Microcystis aeruginosa.

Microcystis aeruginosa, as one of the major players in algal bloom, produces microcystins, which are strongly hepatotoxic, endangering human health and damaging the ecological environment. Biological control of the overgrowth of Microcystis with cyanophage has been proposed to be a promising solution for algal bloom. In this study, a novel strain of Microcystis cyanophage, MinS1, was isolated. MinS1 contains an icosahedral head approximately 54 nm in diameter and a 260 nm-long non-contractile tail. The phage genome consists of a linear, double-stranded 49,966 bp DNA molecule, which shares very low homology with known phages in the NCBI database (only 1% of the genome showed weak homology with known phages when analyzed by megablast). The phage contains 75 ORFs, of which 23 ORFs were predicted to code for proteins of known function, 39 ORFs were predicted to code for proteins of unknown function, and 13 ORFs showed no similarity to any protein sequences. Transmission electron microscopy and phylogenetic analysis showed that MinS1 belongs to the family Siphoviridae. Various experiments confirmed that the phage could infect several different orders of cyanobacteria, including Chroococcales, Nostocales, Oscillatoriales, Hormogonales, and Synechococcales, indicating that it has a very broad host range. In addition, MinS1 has no known antibiotic tolerance genes, virulence genes, and tRNAs, and it is tolerant to temperature, pH, UV, and salinity, suggesting that MinS1 has good potential for application as a biological control agent against cyanobacterial blooms. This study expands the diversity and knowledge of cyanophages, and it provides useful information for the development of novel prevention and control measures against cyanobacterial blooms.

BtuB-Dependent Infection of the T5-like Yersinia Phage ϕR2-01.

Yersinia enterocolitica is a food-borne Gram-negative pathogen responsible for several gastrointestinal disorders. Host-specific lytic bacteriophages have been increasingly used recently as an alternative or complementary treatment to combat bacterial infections, especially when antibiotics fail. Here, we describe the proteogenomic characterization and host receptor identification of the siphovirus vB_YenS_ϕR2-01 (in short, ϕR2-01) that infects strains of several Yersinia enterocolitica serotypes. The ϕR2-01 genome contains 154 predicted genes, 117 of which encode products that are homologous to those of Escherichia bacteriophage T5. The ϕR2-01 and T5 genomes are largely syntenic, with the major differences residing in areas encoding hypothetical ϕR2-01 proteins. Label-free mass-spectrometry-based proteomics confirmed the expression of 90 of the ϕR2-01 genes, with 88 of these being either phage particle structural or phage-particle-associated proteins. In vitro transposon-based host mutagenesis and ϕR2-01 adsorption experiments identified the outer membrane vitamin B12 receptor BtuB as the host receptor. This study provides a proteogenomic characterization of a T5-type bacteriophage and identifies specific Y. enterocolitica strains sensitive to infection with possible future applications of ϕR2-01 as a food biocontrol or phage therapy agent.

Characterization and Application of a Lytic Phage D10 against Multidrug-Resistant Salmonella.

Salmonella is a widely distributed foodborne pathogen that is a serious threat to human health. The accelerated development of drug resistance and the increased demand for natural foods invoke new biocontrol agents to limit contamination by multidrug-resistant (MDR) Salmonella strains. In this study, a lytic Salmonella phage named D10 was characterized at the biological and genomic levels. D10 possesses a short latent period (10 min) and a large burst size (163 PFU/cell), as well as adequate stability under a range of pH conditions and moderate thermal tolerance. D10 effectively lysed different MDR Salmonella serovars and repressed their dynamic growth in the medium. Genomic analysis disclosed that D10 is a new member of the Siphoviridae family and lacks the genes implicated in lysogeny, pathogenicity, or antibiotic resistance. A three-ingredient phage cocktail was then developed by mixing D10 with previously identified myovirus D1-2 and podovirus Pu20. The cocktail significantly reduced the count of MDR strains in liquid eggs, regardless of the temperature applied (4 and 25 °C). These results suggest that phage D10 is a promising tool to prevent food contamination by MDR Salmonella.

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.

Characterization of a triad of genes in cyanophage S-2L sufficient to replace adenine by 2-aminoadenine in bacterial DNA.

Cyanophage S-2L is known to profoundly alter the biophysical properties of its DNA by replacing all adenines (A) with 2-aminoadenines (Z), which still pair with thymines but with a triple hydrogen bond. It was recently demonstrated that a homologue of adenylosuccinate synthetase (PurZ) and a dATP triphosphohydrolase (DatZ) are two important pieces of the metabolism of 2-aminoadenine, participating in the synthesis of ZTGC-DNA. Here, we determine that S-2L PurZ can use either dATP or ATP as a source of energy, thereby also depleting the pool of nucleotides in dATP. Furthermore, we identify a conserved gene (mazZ) located between purZ and datZ genes in S-2L and related phage genomes. We show that it encodes a (d)GTP-specific diphosphohydrolase, thereby providing the substrate of PurZ in the 2-aminoadenine synthesis pathway. High-resolution crystal structures of S-2L PurZ and MazZ with their respective substrates provide a rationale for their specificities. The Z-cluster made of these three genes - datZ, mazZ and purZ - was expressed in E. coli, resulting in a successful incorporation of 2-aminoadenine in the bacterial chromosomal and plasmidic DNA. This work opens the possibility to study synthetic organisms containing ZTGC-DNA.

The Bacteriophage pEp_SNUABM_08 Is a Novel Singleton Siphovirus with High Host Specificity for Erwinia pyrifoliae.

Species belonging to the genus Erwinia are predominantly plant pathogens. A number of bacteriophages capable of infecting Erwinia have been used for the control of plant diseases such as fire blight. Public repositories provide the complete genome information for such phages, which includes genomes ranging from 30 kb to 350 kb in size. However, limited information is available regarding bacteriophages belonging to the family Siphoviridae. A novel lytic siphophage, pEp_SNUABM_08, which specifically infects Erwinia pyrifoliae, was isolated from the soil of an affected apple orchard in South Korea. A comprehensive genome analysis was performed using the Erwinia-infecting siphophage. The whole genome of pEp_SNUABM_08 comprised 62,784 bp (GC content, 57.24%) with 79 open reading frames. The genomic characteristics confirmed that pEp_SNUABM_08 is a singleton lytic bacteriophage belonging to the family Siphoviridae, and no closely related phages have been reported thus far. Our study not only characterized a unique phage, but also provides insight into the genetic diversity of Erwinia bacteriophages.

Therapeutic Effect of a Newly Isolated Lytic Bacteriophage against Multi-Drug-Resistant Cutibacterium acnes Infection in Mice.

Acne vulgaris, which is mostly associated with the colonization of Cutibacterium acnes (C. acnes), is a common skin inflammatory disease in teenagers. However, over the past few years, the disease has extended beyond childhood to chronically infect approximately 40% of adults. While antibiotics have been used for several decades to treat acne lesions, antibiotic resistance is a growing crisis; thus, finding a new therapeutic target is urgently needed. Studies have shown that phage therapy may be one alternative for treating multi-drug-resistant bacterial infections. In the present study, we successfully isolated a C. acnes phage named TCUCAP1 from the skin of healthy volunteers. Morphological analysis revealed that TCUCAP1 belongs to the family Siphoviridae with an icosahedral head and a non-contractile tail. Genome analysis found that TCUCAP1 is composed of 29,547 bp with a G+C content of 53.83% and 56 predicted open reading frames (ORFs). The ORFs were associated with phage structure, packing, host lysis, DNA metabolism, and additional functions. Phage treatments applied to mice with multi-drug-resistant (MDR) C.-acnes-induced skin inflammation resulted in a significant decrease in inflammatory lesions. In addition, our attempt to formulate the phage into hydroxyethyl cellulose (HEC) cream may provide new antibacterial preparations for human infections. Our results demonstrate that TCUCAP1 displays several features that make it an ideal candidate for the control of C. acnes infections.

Exploring Codon Adjustment Strategies towards Escherichia coli-Based Production of Viral Proteins Encoded by HTH1, a Novel Prophage of the Marine Bacterium Hypnocyclicus thermotrophus.

Marine viral sequence space is immense and presents a promising resource for the discovery of new enzymes interesting for research and biotechnology. However, bottlenecks in the functional annotation of viral genes and soluble heterologous production of proteins hinder access to downstream characterization, subsequently impeding the discovery process. While commonly utilized for the heterologous expression of prokaryotic genes, codon adjustment approaches have not been fully explored for viral genes. Herein, the sequence-based identification of a putative prophage is reported from within the genome of Hypnocyclicus thermotrophus, a Gram-negative, moderately thermophilic bacterium isolated from the Seven Sisters hydrothermal vent field. A prophage-associated gene cluster, consisting of 46 protein coding genes, was identified and given the proposed name Hypnocyclicus thermotrophus phage H1 (HTH1). HTH1 was taxonomically assigned to the viral family Siphoviridae, by lowest common ancestor analysis of its genome and phylogeny analyses based on proteins predicted as holin and DNA polymerase. The gene neighbourhood around the HTH1 lytic cassette was found most similar to viruses infecting Gram-positive bacteria. In the HTH1 lytic cassette, an N-acetylmuramoyl-L-alanine amidase (Amidase_2) with a peptidoglycan binding motif (LysM) was identified. A total of nine genes coding for enzymes putatively related to lysis, nucleic acid modification and of unknown function were subjected to heterologous expression in Escherichia coli. Codon optimization and codon harmonization approaches were applied in parallel to compare their effects on produced proteins. Comparison of protein yields and thermostability demonstrated that codon optimization yielded higher levels of soluble protein, but codon harmonization led to proteins with higher thermostability, implying a higher folding quality. Altogether, our study suggests that both codon optimization and codon harmonization are valuable approaches for successful heterologous expression of viral genes in E. coli, but codon harmonization may be preferable in obtaining recombinant viral proteins of higher folding quality.

The life cycle of SPß and related phages.

Phages are viruses of bacteria and are the smallest and most common biological entities in the environment. They can reproduce immediately after infection or integrate as a prophage into their host genome. SPß is a prophage of the Gram-positive model organism Bacillus subtilis 168, and it has been known for more than 50 years. It is sensitive to dsDNA damage and is induced through exposure to mitomycin C or UV radiation. When induced from the prophage, SPß requires 90 min to produce and release about 30 virions. Genomes of sequenced related strains range between 128 and 140 kb, and particle-packed dsDNA exhibits terminal redundancy. Formed particles are of the Siphoviridae morphotype. Related isolates are known to infect other B. subtilis clade members. When infecting a new host, SPß presumably follows a two-step strategy, adsorbing primarily to teichoic acid and secondarily to a yet unknown factor. Once in the host, SPß-related phages pass through complex lysis-lysogeny decisions and either enter a lytic cycle or integrate as a dormant prophage. As prophages, SPß-related phages integrate at the host chromosome's replication terminus, and frequently into the spsM or kamA gene. As a prophage, it imparts additional properties to its host via phage-encoded proteins. The most notable of these functional proteins is sublancin 168, which is used as a molecular weapon by the host and ensures prophage maintenance. In this review, we summarise the existing knowledge about the biology of the phage regarding its life cycle and discuss its potential as a research object.

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.

Complete genome sequence of the newly discovered temperate Clostridioides difficile bacteriophage phiCDKH01 of the family Siphoviridae.

A temperate siphovirus, phiCDKH01, was obtained from a clinical isolate of Clostridioides difficile. The phage genome is a 45,089-bp linear double-stranded DNA molecule with an average G+C content of 28.7%. It shows low similarity to known phage genomes, except for phiCD24-1. Genomic and phylogenetic analysis revealed that phiCDKH01 is a newly discovered phage. Sixty-six putative ORFs were predicted in the genome, 37 of which code for proteins with predicted functions. The phiCDKH01 prophage was localized in the host genome. The results of this study increase our knowledge about the genetic diversity of tailed phages.

A novel vieuvirus from multidrug-resistant Acinetobacter baumannii.

Bacteriophages are considered the most abundant biological entities on earth, and they are able to modulate the populations of their bacterial hosts. Although the potential of bacteriophages has been accepted as an alternative strategy to combat multidrug-resistant pathogenic bacteria, there still exists a considerable knowledge gap regarding their genetic diversity, which hinders their use as antimicrobial agents. In this study, we undertook a genomic and phylogenetic characterization of the phage Ab11510-phi, which was isolated from a multidrug-resistant Acinetobacter baumannii strain (Ab11510). We found that Ab11510-phi has a narrow host range and belongs to a small group of transposable phages of the genus Vieuvirus that have only been reported to infect Acinetobacter bacteria. Finally, we showed that Ab11510-phi (as well as other vieuvirus phages) has a high level of mosaicism. On a broader level, we demonstrate that comparative genomics and phylogenetic analysis are necessary tools for the proper characterization of phage diversity.

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.

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.

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.

Characterization of Novel Lytic Bacteriophages of Achromobacter marplantensis Isolated from a Pneumonia Patient.

Achromobacter spp. are becoming increasingly associated with lung infections in patients suffering from cystic fibrosis (CF). A. marplatensis, which is closely related to A. xylosoxidans, has been isolated from the lungs of CF patients and other human infections. This article describes the isolation, morphology and characterization of two lytic bacteriophages specific for an A. marplatensis strain isolated from a pneumonia patient. This host strain was the causal agent of hospital acquired pneumonia-the first clinical report of such an occurrence. Full genome sequencing revealed bacteriophage genomes ranging in size from 45901 to 46,328 bp. Transmission electron microscopy revealed that the two bacteriophages AMA1 and AMA2 belonged to the Siphoviridae family. Host range analysis showed that their host range did not extend to A. xylosoxidans. The possibility exists for future testing of such bacteriophages in the control of Achromobacter infections such as those seen in CF and other infections of the lungs. The incidence of antibiotic resistance in this genus highlights the importance of seeking adjuncts and alternatives in CF and other lung infections.

Genome Sequence and Characterization of Five Bacteriophages Infecting Streptomyces Coelicolor and Streptomyces Venezuelae: Alderaan, Coruscant, Dagobah, Endor1 and Endor2.

Streptomyces are well-known antibiotic producers, also characterized by a complex morphological differentiation. Streptomyces, like all bacteria, are confronted with the constant threat of phage predation, which in turn shapes bacterial evolution. However, despite significant sequencing efforts recently, relatively few phages infecting Streptomyces have been characterized compared to other genera. Here, we present the isolation and characterization of five novel Streptomyces phages. All five phages belong to the Siphoviridae family, based on their morphology as determined by transmission electron microscopy. Genome sequencing and life style predictions suggested that four of them were temperate phages, while one had a lytic lifestyle. Moreover, one of the newly sequenced phages shows very little homology to already described phages, highlighting the still largely untapped viral diversity. Altogether, this study expands the number of characterized phages of Streptomyces and sheds light on phage evolution and phage-host dynamics in Streptomyces.

Development of a broad-spectrum Salmonella phage cocktail containing Viunalike and Jerseylike viruses isolated from Thailand.

Salmonella is one of the most common agents of foodborne disease worldwide. As natural alternatives to traditional antimicrobial agents, bacteriophages (phages) are emerging as highly effective biocontrol agents against Salmonella and other foodborne bacteria. Due to the high diversity within the Salmonella genus and emergence of drug resistant strains, improved efforts are necessary to find broad range and strictly lytic Salmonella phages for use in food biocontrol. Here, we describe the isolation and characterization of two Salmonella phages: ST-W77 isolated on S. Typhimurium and SE-W109 isolated on S. Enteritidis with extraordinary Salmonella specificity. Whole genome sequencing identified ST-W77 as a Myovirus within the Viunalikevirus genus and SE-W109 as a Siphovirus within the Jerseylikevirus genus. Infectivity studies using a panel of S. Typhimurium cell wall mutants revealed both phages require the lipopolysaccharide O-antigen, with SE-W109 also recognizing the flagella, during infection of Salmonella. A combination of both phages was capable of prolonged (one-week) antibacterial activity when added to milk or chicken meat contaminated with Salmonella. Due to their broad host ranges, strictly lytic lifestyles and lack of lysogeny-related genes or virulence genes in their genomes, ST-W77 and SE-W109 are ideal phages for further development as Salmonella biocontrol agents for food production.

Isolation, characterization, and efficacy of bacteriophages isolated against Citrobacter spp. an in vivo approach in a zebrafish model (Danio rerio).

Citrobacter infections are becoming an increasingly significant health problem in aquaculture in South-Eastern countries. The objective of this study was to isolate and evaluate the potential of lytic bacteriophages against Citrobacter infections. TEM analysis revealed that the isolated phages Citrophage MRM19 and Citrophage MRM57 were identified to be Siphovirus and Podovirus family of the order Caudovirales. The phage life-cycle studies showed that Citrophage MRM19 had an adsorption time of 18 ± 1 min and a latency period of 25 ± 3 min with burst size of 110 ± 20 phages/infected cell and Citrophage MRM57 had an adsorption time of 15 ± 1 min and a latency period of 25 ± 2 min with burst size of 50 ± 5 phages/infected cell. In vitro studies indicated that the bacterial load was reduced by 5 and 7 log units within 12 h by Citrophage MRM19 and Citrophage MRM57. The in vivo efficacy of the phages was studied using zebrafish (Danio rerio) as a model organism in low-scale tanks. The study unveiled that the use of phages increased the survival up to 17%, 23%, and 26% in the case of Citrophage MRM19, Citrophage MRM57, and phage cocktail treatment, respectively. Our study indicated that bacteriophages are suitable biocontrol agents against Citrobacter spp. especially in aquaculture industry.