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.

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.

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.

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.

Structural investigations of a Podoviridae streptococcus phage C1, implications for the mechanism of viral entry.

The Podoviridae phage C1 was one of the earliest isolated bacteriophages and the first virus documented to be active against streptococci. The icosahedral and asymmetric reconstructions of the virus were calculated using cryo-electron microscopy. The capsid protein has an HK97 fold arranged into a T = 4 icosahedral lattice. The C1 tail is terminated with a ϕ29-like knob, surrounded by a skirt of twelve long appendages with novel morphology. Several C1 structural proteins have been identified, including a candidate for an appendage. The crystal structure of the knob has an N-terminal domain with a fold observed previously in tube forming proteins of Siphoviridae and Myoviridae phages. The structure of C1 suggests the mechanisms by which the virus digests the cell wall and ejects its genome. Although there is little sequence similarity to other phages, conservation of the structural proteins demonstrates a common origin of the head and tail, but more recent evolution of the appendages.

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.

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.

The evolution and distribution of phage ST160 within Salmonella enterica serotype Typhimurium.

Salmonellosis is an internationally important disease of mammals and birds. Unique epidemics in New Zealand in the recent past include two Salmonella serovars: Salmonella enterica subsp. enterica serovar Typhimurium definitive type (DT) 160 (S. Typhimurium DT160) and S. Brandenburg. Although not a major threat internationally, in New Zealand S. Typhimurium DT160 has been the most common serovar isolated from humans, and continues to cause significant losses in wildlife. We have identified DNA differences between the first New Zealand isolate of S. Typhimurium DT160 and the genome-sequenced strain, S. Typhimurium LT2. All the differences could be accounted for in one cryptic phage ST64B, and one novel P22-like phage, ST160. The majority of the ST160 genome is almost identical to phage SE1 but has two regions not found in SE1 which are identical to the P22-like phage ST64T, suggesting that ST160 evolved from SE1 via two recombination events with ST64T. All of the New Zealand isolates of DT160 were identical indicating the clonal spread of this particular Salmonella. Some overseas isolates of S. Typhimurium DT160 differed from the New Zealand strain and contained SE1 phage rather than ST160. ST160 was also identified in New Zealand isolates of S. Typhimurium DT74 and S. Typhimurium RDNC-April06 and in S. Typhimurium DT160 isolates from the USA. The emergence of S. Typhimurium DT160 as a significant pathogen in New Zealand is postulated to have occurred due to the sensitivity of the Salmonella strains to the ST160 phage when S. Typhimurium DT160 first arrived.

In vitro management of hospital Pseudomonas aeruginosa biofilm using indigenous T7-like lytic phage.

Pseudomonas aeruginosa, a human pathogen capable of forming biofilm and contaminating medical settings, is responsible for 65% mortality in the hospitals all over the world. This study was undertaken to isolate lytic phages against biofilm forming Ps. aeruginosa hospital isolates and to use them for in vitro management of biofilms in the microtiter plate. Multidrug resistant strains of Ps. aeruginosa were isolated from the hospital environment in and around Pimpri-Chinchwad, Maharashtra by standard microbiological methods. Lytic phages against these strains were isolated from the Pavana river water by double agar layer plaque assay method. A wide host range phage bacterial virus Ps. aeruginosa phage (BVPaP-3) was selected. Electron microscopy revealed that BVPaP-3 phage is a T7-like phage and is a relative of phage species gh-1. A phage at MOI-0.001 could prevent biofilm formation by Ps. aeruginosa hospital strain-6(HS6) on the pegs within 24 h. It could also disperse pre-formed biofilms of all hospital isolates (HS1-HS6) on the pegs within 24 h. Dispersion of biofilm was studied by monitoring log percent reduction in cfu and log percent increase in pfu of respective bacterium and phage on the peg as well as in the well. Scanning electron microscopy confirmed that phage BVPaP-3 indeed causes biofilm reduction and bacterial cell killing. Laboratory studies prove that BVPaP-3 is a highly efficient phage in preventing and dispersing biofilms of Ps. aeruginosa. Phage BVPaP-3 can be used as biological disinfectant to control biofilm problem in medical devices.

Characterization of a T7-like lytic bacteriophage of Klebsiella pneumoniae B5055: a potential therapeutic agent.

Characterization of bacteriophages to be used prophylactically or therapeutically is mandatory, as use of uncharacterized bacteriophages is considered as one of the major reasons of failure of phage therapy in preantibiotic era. In the present study, one lytic bacteriophage, KPO1K2, specific for Klebsiella pneumoniae B5055, with broad host range was selected for characterization. As shown by TEM, morphologically KPO1K2 possessed icosahedral head with pentagonal nature with apex to apex head diameter of about 39 nm. Presence of short noncontractile tail (10 nm) suggested its inclusion into family Podoviridae with a designation of T7-like lytic bacteriophage. The phage growth cycle with a latent period of 15 min and a burst size of approximately 140 plaque forming units per infected cell as well as a genome of 42 kbps and structural protein pattern of this bacteriophage further confirmed its T7-like characteristics. Phage was stable over a wide pH range of 4-11 and demonstrated maximum activity at 37 degrees C. After injection into mice, at 6 h, a high phage titer was seen in blood as well as in kidney and urinary bladder, though titers in kidney and urinary bladder were higher as compared to blood. Phage got cleared completely in 36 h from blood while from kidneys and urinary bladder its clearance was delayed. We propose the use of this characterized phage, KPO1K2, as a prophylactic/therapeutic agent especially for the treatment of catheter associated UTI caused by Klebsiella pneumoniae.

Characterization of a T7-like lytic bacteriophage (phiSG-JL2) of Salmonella enterica serovar gallinarum biovar gallinarum.

PhiSG-JL2 is a newly discovered lytic bacteriophage infecting Salmonella enterica serovar Gallinarum biovar Gallinarum but is nonlytic to a rough vaccine strain of serovar Gallinarum biovar Gallinarum (SG-9R), S. enterica serovar Enteritidis, S. enterica serovar Typhimurium, and S. enterica serovar Gallinarum biovar Pullorum. The phiSG-JL2 genome is 38,815 bp in length (GC content, 50.9%; 230-bp-long direct terminal repeats), and 55 putative genes may be transcribed from the same strand. Functions were assigned to 30 genes based on high amino acid similarity to known proteins. Most of the expected proteins except tail fiber (31.9%) and the overall organization of the genomes were similar to those of yersiniophage phiYeO3-12. phiSG-JL2 could be classified as a new T7-like virus and represents the first serovar Gallinarum biovar Gallinarum phage genome to be sequenced. On the basis of intraspecific ratios of nonsynonymous to synonymous nucleotide changes (Pi[a]/Pi[s]), gene 2 encoding the host RNA polymerase inhibitor displayed Darwinian positive selection. Pretreatment of chickens with phiSG-JL2 before intratracheal challenge with wild-type serovar Gallinarum biovar Gallinarum protected most birds from fowl typhoid. Therefore, phiSG-JL2 may be useful for the differentiation of serovar Gallinarum biovar Gallinarum from other Salmonella serotypes, prophylactic application in fowl typhoid control, and understanding of the vertical evolution of T7-like viruses.

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.

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.

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.

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.

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.