Characterization of a Unique Bordetella bronchiseptica vB_BbrP_BB8 Bacteriophage and Its Application as an Antibacterial Agent.

Bordetella bronchiseptica, an emerging zoonotic pathogen, infects a broad range of mammalian hosts. B. bronchiseptica-associated atrophic rhinitis incurs substantial losses to the pig breeding industry. The true burden of human disease caused by B. bronchiseptica is unknown, but it has been postulated that some hypervirulent B. bronchiseptica isolates may be responsible for undiagnosed respiratory infections in humans. B. bronchiseptica was shown to acquire antibiotic resistance genes from other bacterial genera, especially Escherichia coli. Here, we present a new B. bronchiseptica lytic bacteriophage-vB_BbrP_BB8-of the Podoviridae family, which offers a safe alternative to antibiotic treatment of B. bronchiseptica infections. We explored the phage at the level of genome, physiology, morphology, and infection kinetics. Its therapeutic potential was investigated in biofilms and in an in vivo Galleria mellonella model, both of which mimic the natural environment of infection. The BB8 is a unique phage with a genome structure resembling that of T7-like phages. Its latent period is 75 ± 5 min and its burst size is 88 ± 10 phages. The BB8 infection causes complete lysis of B. bronchiseptica cultures irrespective of the MOI used. The phage efficiently removes bacterial biofilm and prevents the lethality induced by B. bronchiseptica in G. mellonella honeycomb moth larvae.

Genome characterization of the novel lytic Vibrio parahaemolyticus phage vB_VpP_BA6.

A lytic bacteriophage, designated Vibrio phage vB_VpP_BA6, was isolated from sewage collected in Guangzhou, China. The double-stranded DNA genome of phage BA6 is composed of 50,520 bp with a G+C content of 41.77%. It possesses 64 open reading frames relating to phage structure, packaging, host lysis, DNA metabolism, and additional functions. Three tRNAs genes (encoding Pro, Ile and Trp) were detected. Comparison of its genomic features and phylogenetic analysis revealed that phage BA6 is a novel member of the family Podoviridae. This phage may represent a potential therapeutic agent against multidrug-resistant Vibrio parahaemolyticus.

Engineered K1F bacteriophages kill intracellular Escherichia coli K1 in human epithelial cells.

Bacterial infections can be treated with bacteriophages that show great specificity towards their bacterial host and can be genetically modified for different applications. However, whether and how bacteriophages can kill intracellular bacteria in human cells remains elusive. Here, using CRISPR/Cas selection, we have engineered a fluorescent bacteriophage specific for E. coli K1, a nosocomial pathogen responsible for urinary tract infections, neonatal meningitis and sepsis. By confocal and live microscopy, we show that engineered bacteriophages K1F-GFP and E. coli EV36-RFP bacteria displaying the K1 capsule, enter human cells via phagocytosis. Importantly, we show that bacteriophage K1F-GFP efficiently kills intracellular E. coli EV36-RFP in T24 human urinary bladder epithelial cells. Finally, we provide evidence that bacteria and bacteriophages are degraded by LC3-associated phagocytosis and xenophagy.

Genomic Features and Lytic Activity of the Bacteriophage PPPL-1 Effective against Pseudomonas syringae pv. actinidiae, a Cause of Bacterial Canker in Kiwifruit.

Bacterial canker in kiwifruit is caused by Pseudomonas syringae pv. actinidiae (Psa). In this study, the bacteriophage PPPL-1 effective against Psa was characterized. Belonging to the Podoviridae family, PPPL-1 was effective against most Psa strains as well as most Pseudomonas syringae pathovars. PPPL-1 carries a 41,149-bp genome with 49 protein coding sequences and is homologous to the previously reported phiPSA2 bacteriophage. The lytic activity of PPPL-1 was stable up to 40°C, within a range of pH 3-11 and under 365 nm UV light. These results indicate that the bacteriophage PPPL-1 might be useful to control Psa in the kiwifruit field.

Novel lytic bacteriophages of Klebsiella oxytoca ABG-IAUF-1 as the potential agents for mastitis phage therapy.

Mastitis is an inflammation of the mammary gland that occurs when pathogenic microorganisms enter the udder. Even though tremendous advancements in veterinary diagnosis and therapeutics, mastitis is still the most frequent and costly disease of dairy herds overall the world. The purpose of this research was to isolate and identify the lytic phages as a potential method for biological control of bovine mastitis. In this study Klebsiella oxytoca was isolated from contaminated milk samples of Isfahan dairy herds, Isfahan, Iran and characterized as K. oxytoca ABG-IAUF-1 and its 16s-rRNA sequence was deposited in GenBank under the accession numbers of MF175803.1. Then, the four novel specific lytic bacteriophages of K. oxytoca ABG-IAUF-1 from Isfahan public wastewater were isolated and identified. The results of transmission electron microscopy indicated that theses isolated phages were related to Myoviridae and Podoviridae families of bacteriophages. Also the analysis of the growth curve of K. oxytoca ABG-IAUF-1 before and after treatment with lytic phage showed the 97% success rate of the phages in preventing of bacterial growth. This is the first report indicating the use of bacteriophages as the potential agents for eliminating the pathogenic bacteria responsible for bovine mastitis in Iran. The applications of these lytic phages could be an asset for biocontrolling of pathogenic agents in medical and veterinary biotechnology.

Isolation and characterization of a novel, T7-like phage against Aeromonas veronii.

A virulent Aeromonas veronii biovar sobria and the corresponding novel, lytic bacteriophage (VTCCBPA5) were isolated from village pond water. The phage was found to belong to family Podoviridae. PCR analysis of major capsid protein gene confirmed its classification to T7-like genus. The protein profiling by SDS-PAGE indicated the major structural protein to be ~ 45 kDa. The phage (VTCCBPA5) is host specific and is stable over a range of pH (6-10) and temperatures (4-45 °C). On the basis of restriction endonuclease analysis combined with prediction mapping, it was observed to vary significantly from previously reported podophages of Aeromonas sp., viz. phiAS7 and Ahp1. The phylogenetic analysis on the basis of PCR-amplified segment of DNA polymerase gene of phage revealed it being an outgroup from podophages of Klebsiella sp. and Pseudomonas sp. though a small internal fragment (359 bp) showed the highest identity (77%) with Vibrio sp. phages. Thus, this is the first report of a novel Podoviridae phage against A. veronii. It expands the assemblage of podophages against Aeromonas sp. and BPA5 could be potentially useful in biocontrol of environmentally acquired Aeromonas veronii infections.

Complete genome sequence of a novel, virulent Ahjdlikevirus bacteriophage that infects Enterococcus faecium.

A novel virulent bacteriophage named vB_EfaP_IME199 that specifically infects Enterococcus faecium was isolated and characterized. Its optimal multiplicity of infection was 0.01, and it had a 30 minute outbreak period. High-throughput sequencing revealed that the phage has a dsDNA genome of 18,838 bp with 22 open reading frames. The genome has very low homology to all other bacteriophage sequences in the GenBank database. Run-off sequencing experiments confirmed that vB_EfaP_IME199 has short inverted terminal repeats. Phylogenetic analysis indicated that vB_EfaP_IME199 can be taxonomically classified as a new member of the genus Ahjdlikevirus of family Podoviridae.

Isolation and characterization of wetland VSW-3, a novel lytic cold-active bacteriophage of Pseudomonas fluorescens.

Wetlands are often called the "kidneys of the Earth" and contribute substantially to environmental improvement. Pseudomonas fluorescens is a major contaminant of milk products and causes the spoilage of refrigerated foods and fresh poultry. In this study, we isolated and characterized a lytic cold-active bacteriophage named VSW-3 together with P. fluorescens SW-3 cells from the Napahai wetland in China. Electron microscopy showed that VSW-3 had an icosahedral head (56 nm) and a tapering tail (20 nm × 12 nm) and a genome size of approximate 40 kb. On the basis of the top-scoring hits in the BLASTP analysis, VSW-3 showed a high degree of module similarity to the Pseudomonas phages Andromeda and Bf7. The latent and burst periods were 45 and 20 min, respectively, with an average burst size of 90 phage particles per infected cell. The pH and thermal stability of VSW-3 were also explored. The optimal pH was found to be 7.0 and the activity decreased rapidly when the temperature exceeded 60 °C. VSW-3 is a cold-active bacteriophage, hence, it is important to research its ability to prevent product contamination caused by P. fluorescens and to characterize its relationship with its host P. fluorescens in the future.

Genes essential for the morphogenesis of the Shiga toxin 2-transducing phage from Escherichia coli O157:H7.

Shiga toxin 2 (Stx2), one of the most important virulence factors of enterohaemorrhagic Escherichia coli (EHEC), is encoded by phages. These phages (Stx2 phages) are often called lambda-like. However, most Stx2 phages are short-tailed, thus belonging to the family Podoviridae, and the functions of many genes, especially those in the late region, are unknown. In this study, we performed a systematic genetic and morphological analysis of genes with unknown functions in Sp5, the Stx2 phage from EHEC O157:H7 strain Sakai. We identified nine essential genes, which, together with the terminase genes, determine Sp5 morphogenesis. Four of these genes most likely encoded portal, major capsid, scaffolding and tail fiber proteins. Although exact roles/functions of the other five genes are unknown, one was involved in head formation and four were required for tail formation. One of the four tail genes encoded an unusually large protein of 2,793 amino-acid residues. Two genes that are likely required to maintain the lysogenic state were also identified. Because the late regions of Stx2 phages from various origins are highly conserved, the present study provides an important basis for better understanding the biology of this unique and medically important group of bacteriophages.

Identification of structural and morphogenesis genes of Pseudoalteromonas phage φRIO-1 and placement within the evolutionary history of Podoviridae.

The virion proteins of Pseudoalteromonas phage φRIO-1 were identified and quantitated by mass spectrometry and gel densitometry. Bioinformatic methods customized to deal with extreme divergence defined a φRIO-1 tail structure homology group of phages, which was further related to T7 tail and internal virion proteins (IVPs). Similarly, homologs of tubular tail components and internal virion proteins were identified in essentially all completely sequenced podoviruses other than those in the subfamily Picovirinae. The podoviruses were subdivided into several tail structure homology groups, in addition to the RIO-1 and T7 groups. Molecular phylogeny indicated that these groups all arose about the same ancient time as the φRIO-1/T7 split. Hence, the T7-like infection mechanism involving the IVPs was an ancestral property of most podoviruses. The IVPs were found to variably host both tail lysozyme domains and domains destined for the cytoplasm, including the N4 virion RNA polymerase embedded within an IVP-D homolog.

Complete genome sequence of novel T7-like virus vB_KpnP_KpV289 with lytic activity against Klebsiella pneumoniae.

A novel bacteriophage, vB_KpnP_KpV289, lytic for hypermucoviscous strains of Klebsiella pneumoniae, was attributed to the family Podoviridae, subfamily Autographivirinae, genus T7likevirus based on transmission electron microscopy and genome analysis. The complete genome of the bacteriophage vB_KpnP_KpV289 consists of a linear double-stranded DNA of 41,054 bp including 179-bp direct-repeat sequences at the ends and 51 open reading frames (ORFs). The G+C content is 52.56 %. The phage was shown to lyse 15 out of 140 (10.7 %) K. pneumoniae strains belonged to the capsular types K-1, K-2, and K-57 and strains without a determined capsular type, including a hypermucoviscous strain of the novel sequence type ST-1554.

A suggested new bacteriophage genus, "Kp34likevirus", within the Autographivirinae subfamily of Podoviridae.

Klebsiella pneumoniae phages vB_KpnP_SU503 (SU503) and vB_KpnP_SU552A (SU552A) are virulent viruses belonging to the Autographivirinae subfamily of Podoviridae that infect and kill multi-resistant K. pneumoniae isolates. Phages SU503 and SU552A show high pairwise nucleotide identity to Klebsiella phages KP34 (NC_013649), F19 (NC_023567) and NTUH-K2044-K1-1 (NC_025418). Bioinformatic analysis of these phage genomes show high conservation of gene arrangement and gene content, conserved catalytically active residues of their RNA polymerase, a common and specific lysis cassette, and form a joint cluster in phylogenetic analysis of their conserved genes. Also, we have performed biological characterization of the burst size, latent period, host specificity (together with KP34 and NTUH-K2044-K1-1), morphology, and structural genes as well as sensitivity testing to various conditions. Based on the analyses of these phages, the creation of a new phage genus is suggested within the Autographivirinae, called "Kp34likevirus" after their type phage, KP34. This genus should encompass the recently genome sequenced Klebsiella phages KP34, SU503, SU552A, F19 and NTUH-K2044-K1-1.

Isolation and characterization of T7-like lytic bacteriophages infecting multidrug resistant Pseudomonas aeruginosa isolated from Egypt.

In this study, two lytic phages designated as ϕPSZ1 and ϕPSZ2 infecting multidrug resistant Pseudomonas aeruginosa were isolated from sewage samples collected in Zagazig, Egypt. Morphological analysis by transmission electron microscopy revealed that both phages belong to the podoviridae family and resembles typical T7-like phages. ϕPSZ1 has a head of about 60 ± 5 nm in diameter with a short tail of 19 ± 2 nm in length, while ϕPSZ2 has a head of about 57 ± 5 nm in diameter with a short tail of 14 ± 2 nm in length. Both phages were shown to be able to infect 13 different P. aeruginosa strains and has no effect on other tested bacteria. In spite of morphological similarity, these phages showed diverged genomic sequences revealed by restriction enzyme digestion analysis. One-step growth curves of bacteriophages revealed eclipse and latent periods of 12 min for ϕPSZ1 and 15 min for ϕPSZ2, respectively, with burst sizes of about 100 per infected cell. Phage treatment prevented the growth of P. aeruginosa for up to 18 h with multiplicity of infection ratios of 1. These results suggest that both phages have a high potential for phage application to control P. aeruginosa.

Characterization and genome sequence of Dev2, a new T7-like bacteriophage infecting Cronobacter turicensis.

Cronobacter spp. are opportunistic pathogenic bacteria that are responsible for severe infections in neonates. Powdered infant formula was confirmed to be the source in some cases. Bacteriophages offer a safe means for eliminating this pathogen. In the present study, we investigated the growth parameters and genome organization of a new bacteriophage, Dev2, isolated from sewage. The Dev2 phage contains DNA with a length of 39 kb and belongs to the T7 branch of the subfamily Autographivirinae, with the highest degree of identity to the phage K1F. The host specificity of Dev2 is limited to C. turicensis strains of the CT O:1 serotype. With a lower efficiency, this phage also infects some Salmonella and E. coli strains. The Dev2 phage can inactivate sensitive Cronobacter strains in reconstituted milk formula. The results obtained in this study are an important prerequisite for application of Dev2 in food control.

Isolation and partial characterization of bacteriophages infecting Pseudomonas syringae pv. actinidiae, causal agent of kiwifruit bacterial canker.

The phytopathogen Pseudomonas syringae pv. actinidiae (Psa) is the causal agent of bacterial canker of kiwifruit. In the last years, it has caused severe economic losses to Actinidia spp. cultivations, mainly in Italy and New Zealand. Conventional strategies adopted did not provide adequate control of infection. Phage therapy may be a realistic and safe answer to the urgent need for novel antibacterial agents aiming to control this bacterial pathogen. In this study, we described the isolation and characterization of two bacteriophages able to specifically infect Psa. φPSA1, a member of the Siphoviridae family, is a temperate phage with a narrow host range, a long latency, and a burst size of 178; φPSA2 is a lytic phage of Podoviridae family with a broader host range, a short latency, a burst size of 92 and a higher bactericidal activity as determined by the TOD value. The genomic sequence of φPSA1 has a length of 51,090 bp and a low sequence homology with the other siphophages, whereas φPSA2 has a length of 40 472 bp with a 98% homology with Pseudomonas putida bacteriophage gh-1. Of the two phages examined, φPSA2 may be considered as a candidate for phage therapy of kiwifruit disease, while φPSA1 seems specific toward the recent outbreak's isolates and could be useful for Psa typing.

Characterization and genomic analysis of a plaque purified strain of cyanophage PP.

Cyanophages are ubiquitous and essential components of the aquatic environment and play an important role in the termination of algal blooms. As such, they have attracted widespread interest. PP was the first isolated cyanophage in China, which infects Plectonema boryanum and Phormidium foveolarum. In this study, this cyanophage was purified three times by a double-agar overlay plaque assay and characterized. Its genome was extracted, totally sequenced and analyzed. Electron microscopy revealed a particle with an icosahedral head connected to a short stubby tail. Bioassays showed that PP was quite virulent. The genome of PP is a 42,480 base pair (bp), linear, double-stranded DNA molecule with 222 bp terminal repeats. It has high similarity with the known Pf-WMP3 sequence. It contains 41 open reading frames (ORFs), 17 of which were annotated. Intriguingly, the genome can be divided into two completely different parts, which differ both in orientation and function.

Biocontrol of Pectobacterium carotovorum subsp. carotovorum using bacteriophage PP1.

Pectobacterium carotovorum subsp. carotovorum (formerly Erwinia carotovora subsp. carotovora) is a plant pathogen that causes soft rot and stem rot diseases in several crops, including Chinese cabbage, potato, and tomato. To control this bacterium, we isolated a bacteriophage, PP1, with lytic activity against P. carotovorum subsp. carotovorum. Transmission electron microscopy revealed that the PP1 phage belongs to the Podoviridae family of the order Caudovirales, which exhibit icosahedral heads and short non-contractile tails. PP1 phage showed high specificity for P. carotovorum subsp. carotovorum, and several bacteria belonging to different species and phyla were resistant to PP1. This phage showed rapid and strong lytic activity against its host bacteria in liquid medium and was stable over a broad range of pH values. Disease caused by P. carotovorum subsp. carotovorum was significantly reduced by PP1 treatment. Overall, PP1 bacteriophage effectively controls P. carotovorum subsp. carotovorum.

Isolation and characterization of a rare waterborne lytic phage of Salmonella enterica serovar Paratyphi B.

A lytic phage of Salmonella serovar Paratyphi B, named φSPB, was isolated from surface waters of the Pavana River in India. Phage φSPB is a member of the Podoviridae family and is morphologically similar to the 7-11 phages of the C3 morphotype of tailed phages, characterized by a very long, cigar-shaped head. The head measured approximately 153 × 57 nm, and the tail size was 12 × 7 nm. The phage was stable over a wide range of pH (4-9) and temperature (4-40 °C). The adsorption rate constant was 4.7 × 10(-10). Latent and eclipse periods were 10 and 15 min, respectively, and the burst size was 100 plaque-forming units/infected cell after 25 min at 37 °C. The phage DNA was 59 kb in size. Ten major proteins were observed on SDS-PAGE, although some of these proteins could be bacterial contaminants. This is the first report of Salmonella enterica subsp. enterica serovar Paratyphi B phage of C3 morphotype from India that has many unique features, such as high replication potential, short replication time, and stability over a wide range of pH and temperature, making it a promising biocontrol agent against the drug-resistant strains of Salmonella Paratyphi B.

[Characterization of a novel podoviridae-phage infecting Serratia marcescens isolated in China].

Serratia marcescens jn01 was employed as the host for the isolation of phages from environmental sewage. One strain of phage named SmPjn was purified by picking transparent plaque with 2mm diameter and clear edge on the double-layer agar repeatedly. Electron micrographs indicated that the phage head was icosahedral with head size and tail length of (58 +/- 2.16) x (55 +/- 0.47) nm and (7 +/- 1.25) nm, respectively. On the basis of the morphology, this phage belongs to the family Podoviridae. Host-range determination revealed that the phage was capable of infecting the other two isolates of S. marcescens, P25 and CMCC41002. The optimal multiplicity of infection was 1. A one-step growth curve of SmPjn indicated that the latent period and burst size were estimated at 50 min and 1,125 pfu/cell, respectively . Genomic DNA of SmPjn was above 27kb in size and could be digested by Hind Ill and EcoR I into 11 and 9 visible fragments after electrophoresis, respectively. A novel Podoviridae-phage infecting S. marcescens was firstly reported in China.