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Vibrio alginolyticus is a common foodborne pathogen existing both in contaminated seafood and the environment and can cause serious mortality in aquaculture facilities. Bacteriophages can be used as an alternative bio-control agent to eliminate and reduce pathogens. In this study, a novel lytic phage, designated vB_ValP_VA-RY-3 (referred to as S1R3Y), was isolated from sewage collected in Dalian, China. The linear double-stranded DNA genome of phage S1R3Y is 40.271 kb, which has a mol% G + C content of 43.98, containing 51 ORFs with a T7-like genomic organization. It shared the closest relationship with phage vB_CsaP_Ss1, but the homology coverage is just 6%. S1R3Y lacks tRNA and no known virulence or lysogenic genes were found. S1R3Y had a burst size of 147 PFU/cell and is stable under different temperatures (4-56 °C) and pH (5.0-7.0). A comparison of its genomic features and phylogenetic analysis revealed that phage S1R3Y is a novel member of the order Caudovirales, family Podoviridae. Our results suggest that phage S1R3Y may represent a potential therapeutic agent against Vibrio alginolyticus.

Stenotrophomonas maltophilia (S. maltophilia) is an important Gram-negative opportunistic pathogen that is widely distributed in nature. S. maltophilia is highly drug-resistant because of its intrinsic properties and acquired drug resistance involving multiple molecular mechanisms, which creates a critical situation for infection therapy. Hence, there is an urgent need for alternative antimicrobial strategies to combat S. maltophilia. Herein, a novel S. maltophilia bacteriophage (phage) in family Podoviridae, named BUCT598, was isolated from hospital sewage and characterized to evaluate its potential as an antibacterial agent. The one-step growth curve showed that its latent period and burst size were approximately 30 min and 165 PFU/cell, respectively. Furthermore, phage BUCT598 survived within an extremely broad pH range (1-11), indicating its outstanding tolerance to both extremely acidic and extremely alkaline conditions. The whole-genome sequence of phage BUCT598 showed that it was a linear double-stranded DNA genome of 43,581 bp and 60% GC content. We identified 55 putative gene products involved in DNA replication, packaging, structure, and cell lysis. Whole-genome sequence comparisons among closely related phages indicated that phage BUCT598 had the highest sequence similarity with S. maltophilia phage BUCT609, with 52% query coverage and 76.40% identity, suggesting that it is a novel phage. Our findings indicate the great potential of phage BUCT598 as an alternative antimicrobial agent to eliminate S. maltophilia, and provide additional evidence that will help to understand how phages adapt and evolve under extreme environmental conditions, thereby opening up more extensive biotechnology applications of phages.

Stenotrophomonas maltophilia is a common conditional pathogen, and it is naturally resistant to most commonly used clinical antibiotics. The bacteriophage is considered to be a potential antibiotic alternative for treating multi-drug-resistant bacteria. In this study, a bacteriophage BUCT555 was isolated from hospital sewage for lysing the clinical multi-drug resistant Stenotrophomonas maltophilia. Electron microscopy studies revealed this phage belongs to the Podoviridae family. The double-stranded DNA genome of bacteriophage BUCT555 is composed of 39,440 bp with a GC content of 61.43%. The genome contains 57 open reading frames, 14 of which had assigned functions, while no virulence related genes, antibiotic resistance genes or tRNA were identified. The burst size of BUCT555 was 204 pfu per infected cell. Structure proteins of bacteriophage BUCT555 generated by SDS-PAGE and HPLC-MS revealed that it contains seven proteins with molecular weight ranging from 19 to 89 kDa. BLASTn analysis showed that phage BUCT555 has 2% homology with other phages in NCBI database, suggesting BUCT555 is a new phage genus of Podoviridae that infects Stenotrophomonas maltophilia. Characterization of the bacteriophage BUCT555 enriches our knowledge about the diversity of Stenotrophomonas maltophilia bacteriophages.

A novel phage vB_VpP_DE17, which infects Vibrio parahaemolyticus, was isolated from the sewer of the Huangsha aquatic market in Guangzhou. Transmission electron microscopy indicated that DE17 had an icosahedral head (47 ± 2 nm diameter) and a short, non-contractile tail (17 ± 2 nm). The genome of DE17 was a double-stranded linear DNA with a length of 43,397 bp and GC content of 49.23%. In total, 49 putative open reading frames (ORFs) were predicted and could be divided into six modules: DNA metabolism, lysis, packaging, structure, additional function, and hypothetical proteins. Taxonomic analysis revealed that the phage belonging to the genus of Maculvirus, Autographivirinae subfamily, Podoviridae family. DE17 had a short latent period of 5 min with burst size of 80 pfu/cell. Its optimum temperature and pH ranges were 4 °C–50 °C and 5–10, respectively; it was completely inactivated after 20 min of ultraviolet irradiation. No transfer RNA (tRNA), virulence associated, or antibiotic resistance genes were identified. Bacterial challenge test revealed that DE17 had a certain inhibitory effect on V. parahaemolyticus within 6 h. Characterization, genomic analysis and in vitro antibacterial assays of DE17 will further enhance our understanding of phage biology and diversity.

Hypermucoviscous (HV) strains of capsular types K1, K2 and K57 are the most virulent representatives of the Klebsiella pneumoniae species. Eight novel bacteriophages lytic for HV K. pneumoniae were isolated and characterized. Three bacteriophages, KpV41, KpV475, and KpV71 were found to have a lytic activity against mainly K. pneumoniae of capsular type K1. Two phages, KpV74, and KpV763 were lytic for K2 capsular type K. pneumoniae , and the phage KpV767 was specific to K57-type K. pneumoniae only. Two more phages, KpV766, and KpV48 had no capsular specificity. The phage genomes consist of a linear double-stranded DNA of 40,395–44,623 bp including direct terminal repeats of 180–246 bp. The G + C contents are 52.3–54.2 % that is slightly lower than that of genomes of K. pneumoniae strains being used for phage propagation. According to the genome structures, sequence similarity and phylogenetic data, the phages are classified within the genus Kp32virus and Kp34virus of subfamily Autographivirinae, family Podoviridae . In the phage genomes, genes encoding proteins with putative motifs of polysaccharide depolymerase were identified. Depolymerase genes of phages KpV71 and KpV74 lytic for hypermucoviscous K. pneumoniae of K1 and K2 capsular type, respectively, were cloned and expressed in Escherichia coli , and the recombinant gene products were purified. The specificity and polysaccharide-degrading activity of the recombinant depolymerases were demonstrated.

Cyanophages are primary regulators of cyanobacterial harmful algal blooms (CyanoHABs), and they control host cyanobacterial dynamics, frequency, and diversity in the aquatic environment. This study deals with growth characteristics of three lytic cyanophages, Myoviridae AGM-1, Myoviridae NGM-1, and Podoviridae NDP-1, newly isolated from the Nakdong River in South Korea. These isolates are capable of infecting Amazoninema brasiliense, Nododsilinea nodulosa, and Nostoc sp. The results showed that abiotic parameters such as water temperature and pH balance significantly affect the growth of a cyanophage and the interaction with its host in the aquatic environment. The optimal growth conditions of the newly isolated cyanophages are less than 37 °C and pH 9, whereas optimal conditions are 25-30 °C and pH 7 for the cyanobacteria used as hosts. However, each cyanophage was found to have significantly different growth characteristics in phage titer, latent period, and burst size, depending on the characteristics of the species. Among the three cyanophages, Podoviridae NDP-1 showed the highest burst size and infection activity. The lower the designed multiplicity of infection (MOI) ratio (0.01 to 10), the longer it takes to lyse the host cells. The minimum MOI value for sustainable biocontrol of CyanoHABs is proposed as MOI=1. These results can be used as basic information in further studies, such as pyophage control of CyanoHABs and enrichment of cyanophages with high activity.

Paenibacillus larvae cause an American foulbrood disease (AFB) that is responsible for the extinction of honeybee colonies and is a honeybee bacterial disease that has to be obligatory notified worldwide. Recently, bacteriophage studies targeting Paenibacillus larvae have emerged as a promising alternative treatment method. The inability of bacteria to create resistance against bacteriophages makes this method advantageous. As a consequence, this study was conducted to describe the genome and biological characteristics of a novel phage capable of lysing Paenibacillus larvae samples isolated from honeybee larva samples in Turkey. The Paenibacillus phage SV21 (vB_PlaP_SV21) was isolated by inducing Paenibacillus larvae strain SV21 with Mitomycin-C. Whole-genome sequencing, comparative genomics, and phylogenetic analysis of vB_PlaP_SV21 were performed. Transmission electron microscopy images showed that vB_PlaP_SV21 phage was a Podovirus morphology. The vB_PlaP_SV21 phage specific for Paenibacillus larvae was determined to belong to the Podoviridae family. Host range and specificity, burst size, lytic activity, and morphological characteristics of the phage were determined. Bioinformatic analysis of the Paenibacillus phage SV21 showed 77 coding sequences in its linear 44,949 bp dsDNA genome with a GC content of 39.33%. In this study, we analysed the genomes of all of the currently sequenced P. larvae phage genomes and classified them into five clusters and a singleton. According to molecular, morphological, and bioinformatics results, it was observed that API480 (podovirus), which was reported as a singleton in previous studies and public databases, and Paenibacillus phage SV21 phage could form a new cluster together

Klebsiella pneumoniae (K. pneumoniae) infection exist widely in the farming and medical. The treatment of K. pneumoniae infection is primarily based on antibiotics, which not only leads to a large economic burden but also the development of antibiotic resistance. Bacteriophages therapy present a promising alternative. The object of this study was identifying comprehensively a lytic lethal K. pneumoniae phage vB_KpnP_Bp5, and evaluating the phage as an anti-infective agent in an experimental K. pneumoniae infection murine model. The phage Bp5 had the following characteristics: the optimal number of infections was 0.001, the latent period was 5 min, the outbreak period was 40 min, the burst size was 24 plaque-forming unit (PFU)/cell, the phage could withstand 50 °C temperature and the optimal pH value was 4.0-10.0. According to electron microscopy and whole-genome sequence analysis, the phage should be classified as a member of order Caudovirales, family Podoviridae, subfamily Autographiviridae. Meantime, phylogenetic analysis showed high conservation of gene arrangement and gene content. We demonstrated that administration of phage Bp5 significantly reduced colony formation by K. pneumoniae and alleviated damage to lung tissue. In addition, different therapy time point was closely related to body health and the degree of tissue damage. Once treated promptly, it will greatly reduce mortality and alveolar inflammatory exudation and injury.

Shigella fiexneri phage SGF2 is a novel lytic phage isolated from a sewage sample. Morphological characterization indicates that phage SGF2 is a member of the Podoviridae family, producing virions with an isometric head (82.6 ± 8 nm diameter) and a short non-contractile tail (length 52 ± 8 nm). This phage specifically infected the Shigella fiexneri. One-step growth curves indicated that the burst period of phage SGF2 is 30 min, with an approximate burst size of 38. The full-length genome was sequenced and potential virulence genes were detected. We will discuss the potential application of phage SGF2 in phage therapy.

The freshwater cyanobacterial virus (cyanophage) A-4L, a podovirus, can infect the model cyanobacterium Anabaena sp. strain PCC 7120 resulting in a high burst size and forming concentric plaques on its lawns. The complete genome sequence of A-4L was determined by the combination of high-throughput sequencing, terminal transferase-mediated polymerase chain reaction and restriction mapping. It contains 41,750 bp with 810 bp direct terminal repeats and 38 potential open reading frames. As compared with other cyanobacterial podoviruses in diverse ecosystems, the A-4L has the longest terminal repeat and shares similar genome organizations with freshwater members. Furthermore, phylogenetic analysis based on concatenated sequences of eight core proteins indicated that freshwater cyanobacterial podoviruses were clustered together and distinct from marine counterparts, suggesting a clear divergence in the cyanobacterial podovirus lineage between freshwater and marine ecosystems. Our findings uncover the unique genome structure of A-4L which contains long direct terminal repeats, and create the first model system to address knowledge gaps in understanding cyanobacterial virus-host interactions at the molecular level. (C) 2015 Elsevier B.V. All rights reserved.

The virulent bacteriophage MJG that specifically infects Aeromonas hydrophila was isolated from a water sample from a river in Harbin, China. The genome of phage MJG was a double-stranded linear DNA with 45,057 bp, possessing 50.11% GC content. No virulence or resistance genes were found in the phage genome. Morphological observation, genomic characterization, and phylogenetic analysis indicated that MJG was closely related to phages belonging to the genus Sp6virus in the Podoviridae family. This phage is a novel member within Sp6virus that could infect and lyse A. hydrophila. This study could serve as a genomic reference of A. hydrophila phages and provide a potential agent for phage therapy.

In the present study two new phages (phT4A and ECA2) were characterized and their efficacy was evaluated separately and in cocktail (phT4A/ECA2) to control Escherichia coli. The isolated phages, phT4A and ECA2, belonged to the Myoviridae and Podoviridae family, respectively and both are safe (no integrase and toxin codifying genes) to be used in bacterial control. In general, the increase of multiplicity of infection (MOI) from 1 to 100 promoted a significant increase in the efficiency of phage phT4A and phage cocktail phT4A/ECA2. Both phages were effective against E. coli, but phage phT4A (reduction of 5.8 log CFU/mL after 8h treatment) was more effective than phage ECA2 phage (reduction of 4.7 log CFU/mL after 8h treatment). The use of a cocktail phT4A/ECA2 was significantly more effective (reductions of 6.2 log CFU/mL after 6h treatment) than the use single phage suspensions of phT4A and ECA2 (reductions 5.3 log CFU/mL and 4.9 log CFU/mL, respectively, after 6h treatment). The rate of emergence of phage-resistant mutants was lower for phage phT4A when compared with phage ECA2 and phage cocktail phT4A/ECA2.The results indicate that in addition to the efficacy, the potential development of phage-resistant mutants must also be considered in the design of phage cocktails.

For filoviruses, such as Ebolavirus and the closely related Marburgvirus, transcriptional regulation is poorly understood. The open reading frames (ORFs) that encode the viral proteins are separated by regulatory regions composed of the 3' nontranslated region (NTR) of the upstream gene, highly conserved transcription stop and start signals, and the 5'NTR of the downstream gene. The conserved transcription stop and start signals either overlap, or they are separated by intergenic regions (IGRs) of different lengths. To assess the contribution of the regulatory regions to transcription, we established bicistronic minireplicons in which these regions were flanked by upstream and downstream ORFs, the Ebolavirus leader and trailer regions, and by T7 RNA polymerase promoter and ribozyme sequences. We found that the individual viral regulatory regions differ in their ability to direct protein synthesis from the upstream or downstream ORFs. Deletion or modification of the NTRs, IGRs, or transcription stop and start signals affected protein expression levels to various extents; for example, 5'NTRs appear to affect efficient protein expression from the downstream ORF, whereas 3'NTRs seem to attenuate protein expression from the upstream ORF. Overall, our data suggest that the regulation of Ebolavirus protein levels is complex.

Pseudomonas aeruginosa phages belonging to the family Podoviridae are one of the well-characterized phage groups. In this study, a novel P. aeruginosa phage, KPP25, was isolated and characterized. Phage KPP25's morphology was indicative of the family Podoviridae; however, analyses of the whole genome and the virion proteins suggested that it did not belong to any of the known podophage genera. Based on these analyses, phage KPP25 appears to be a novel podophage infecting P. aeruginosa.

A DNA containing bacteriophage, Kvp1, was isolated from the water of a very polluted river, the Matanza river, near the central district of Buenos Aires City. This bacteriophage infects bacteria belonging to the Kluyvera cryocrescens species (strain 21 g) isolated from the same river. Kvp1 is a lytic bacteriophage and its propagation characteristics are: burst size 30, latent period 13 min and rise period 10 min. Morphologically, Kvp1 is a small icosahedral bacteriophage, 59.1 nm in diameter, which possesses a short wedge-shaped tail. Its buoyant density in ClCs is 1.517 g/cm3. Kvp1 DNA is linear, double stranded and approximately 40,000 bp in size. The viral particle is composed of at least nine proteins. SDS-PAGE patterns of these proteins and of those produced during the host infection, in addition to its morphological and genomic characteristics, suggested that Kvp1 is similar to the coliphage T7. Molecular cloning, sequencing and computer-assisted analysis of Kvp1 DNA fragments confirmed the relationship to the coliphage. Taking this into account, the partial sequence of the phage RNA polymerase was used to construct phylogenetic relationships between Kvp1 and other related phages. To our knowledge, Kvp1 is the first bacteriophage described which uses as host a member of the Kluyvera bacterial genus.