Icosahedral Head Research Articles

Biological and Genomic Characteristics of MaMV-DH01, a Novel Freshwater Myoviridae Cyanophage Strain.

The genomic traits of cyanophages and their potential for metabolic reprogramming of the host cell remain unknown due to the limited number of studies on cyanophage isolates. In the present study, a lytic Microcystis cyanophage, MaMV-DH01, was isolated and identified. MaMV-DH01 has an icosahedral head approximately 100 nm in diameter and a tail 260 nm in length. Its burst size is large, with approximately 145 phage particles/infected cell; it has a latent period of 2 days, and it shows high stability under pH and temperature stresses. Multiple infection (multiplicity of infection [MOI] 0.0001 to 100) results showed that when the MOI was 0.0001, MaMV-DH01 needed a longer time to lyse host cells. Cyanophage MaMV-DH01 has a double-stranded DNA genome of 182,372 bp, with a GC content of 45.35% and 210 predicted open reading frames (ORFs). These ORFs are related to DNA metabolism, structural proteins, lysis, host-derived metabolic genes, and DNA packaging. Phylogenetic trees based on the whole genome and two conserved genes (TerL and capsid) indicate that MaMV-DH01 is clustered with Ma-LMM01 and MaMV-DC, which are independent of other cyanophages. Collinearity analysis showed that the complete genome of MaMV-DH01 was longer than those of Ma-LMM01 and MaMV-DC, with lengths of 20,263 bp and 13,139 bp, respectively. We verified the authenticity of these excess DNA fragments and found that they are involved to various degrees in the MaMV-DH01 transcription process. Map overlays of environmental virus macrogenomic reads onto the MaMV-DH01 genome revealed that viral sequences similar to that of MaMV-DH01 are widespread in the environment. IMPORTANCE A novel freshwater Myoviridae cyanophage strain, MaMV-DH01, was isolated; this strain infects Microcystis aeruginosa FACHB-524, and the biological and genomic characteristics of MaMV-DH01 provide new insights for understanding the mechanism by which cyanophages infect cyanobacterial blooms.

Isolation and genomic analysis of a novel bacteriophage IME278 infecting Enterobacter hormaechei and its biocontrol potential on pork.

Enterobacter hormaechei is an opportunistic pathogen and is found in a large variety of food including animal-derived food. In recent years, bacteria present a severe clinical challenge due to their increasing resistance to antibiotics. Bacteriophages have gained attention as a new antibacterial strategy. In this study, we isolated a novel E. hormaechei bacteriophage IME278 from hospital sewage in Beijing, China. Bacteriophage IME278 had a double-stranded linear DNA genome with 40,164 bp and 51.99% GC content. Whole-genome alignments showed IME278 shared 87% homology with other phages in the National Center for Biotechnology Information (NCBI) database. And phylogenetic analysis demonstrated that IME278 was highly similar to bacteriophages belonging to the genus Kayfunavirus, family Autographiviridae, indicating IME278 can be classified as a new member of the Autographiviridae family. Transmission electron microscopy revealed that IME278 had an icosahedral head 51.72nm in diameter and a tail 151.28nm in length. Bacteriophage IME278 was able to survive under high temperature (50°C-70°C) and its activity decreased significantly above 70°C and almost completely inactivated at 80°C. Bacteriophage IME278 could survive in a wide pH range (4.0-11.0) and it was stable in chloroform (up to 5%). The phage was sensitive to UV irradiation. Bacteriophage IME278 had a latent period of 40min and reached a plateau stage at 150min and its cleavage was approximately 8.21×108/3.66×108=2.24. The biocontrol potential of bacteriophage IME278 was evaluated in a model that artificially contaminated pork with E. hormaechei 529 and the result revealed that IME278 could effectively control bacterial contamination on pork. The in-depth analysis of the biological characteristics, whole genome sequencing, and bioinformatics of IME278 has laid the foundation for the biocontrol application and the treatment of bacteria using bacteriophages.

Isolation and genomic analysis of a type IV pili-independent Thermus thermophilus phage, φMN1 from a Japanese hot spring.

A Thermus thermophilus lytic phage was isolated from a Japanese hot spring using a type IV pili-deficient strain as an indicator host, and designated as φMN1. Electron microscopic (EM) examination revealed that φMN1 had an icosahedral head and a contractile tail, suggesting that φMN1 belonged to Myoviridae. An EM analysis focused on φMN1 adsorption to the Thermus host cell showed that the receptor molecules for the phage were uniformly distributed on the outer surface of the cells. The circular double-stranded DNA of φMN1 was 76,659 base pairs in length, and the guanine and cytosine content was 61.8%. It was predicted to contain 99 open reading frames, and its putative distal tail fiber protein, which is essential for non-piliated host cell surface receptor recognition, was dissimilar in terms of sequence and length with its counterpart in the type IV pili-dependent φYS40. A phage proteomic tree revealed that φMN1 and φYS40 are in the same cluster, but many genes had low sequence similarities and some seemed to be derived from both mesophilic and thermophilic organisms. The gene organization suggested that φMN1 evolved from a non-Thermus phage through large-scale recombination events of the genes determining the host specificity, followed by gradual evolution by recombination of both the thermophilic and mesophilic DNAs assimilated by the host Thermus cells. This newly isolated phage will provide evolutionary insights into thermophilic phages.

Bacillus subtilis phage phi18: genomic analysis and receptor identification.

Bacillus subtilis strains play a pivotal role in the fermentation industry. B. subtilis phages can cause severe damage by infecting bacterial cells used in industrial fermentation processes. In this work, we isolated and characterized a Bacillus subtilis-infecting phage, termed phi18. Transmission electron microscopy revealed that phage phi18 particles have typical myovirus morphology, with an icosahedral head connected to a contractile tail. Genomic analysis revealed that the phage genome is a linear double-stranded DNA molecule of 147,298 bp with terminal redundancy of 14,434 bp, and 226 protein coding genes and four tRNA genes were predicted in the genome. Phage-resistant mutants were selected from a mariner transposon-insertion library of B. subtilis 168 in which two bacterial genes, tagE and pgcA, which are required for the glycosylation of wall teichoic acid (WTA), were found to be disrupted, suggesting that WTA is the receptor for phage phi18. Comparative genomic analysis showed that phage phi18 is a new member of the genus Okubovirus of the family Herelleviridae. Finally, general characteristics of the phage-resistant mutants, including biofilm formation, growth, and sporulation, were examined. The results showed that the phage-resistant mutants grew as rapidly as the parental strain B. subtilis 168 at 42 °C, suggesting that these phage-resistant mutants may be used as starters in fermentation processes.

ISOLATION AND CHARACTERIZATION OF BACTERIOPHAGE WITH LYTIC ACTIVITY AGAINST CARBAPENEM RESISTANCE STRAIN OF KLEBSIELLA PNEUMONIA.

Aim: Klebsiella pneumonia has emerged as an increasingly important cause of community-acquired nosocomial infections and many of these strains are highly virulent and exhibit a strong propensity to spread. Infections cause by K. pneumonia produces carbapen¬emase (KPC) enzyme and can be difficult to treat since only a few antibiotics are effective against them. Bacteriophage targeting this strain can be an alternative treatment. Characterisation of bacteriophage is utmost important in assisting the application of bacteriophage in phage therapy. Materials and methods: In the present study, the lytic bacteriophage, k3w7, isolated by the host Klebsiella pneumoniae kP2 was characterised using transmission electron microscope (TEM), plaque assay, and restriction digestive enzyme to investigate mor¬phology, host spectrum, bacteriophage life cycle and stability accordingly. Results and conclusions: As shown by TEM, k3w7 was observed to have the characteristic of icosahedral heads 100 nm and contractile sheaths 120 nm suggesting it belongs to the family of myoviridae.The Investigation has done on the phage growth cycle showed a short latent period of 20 min and a burst size of approximately 220 plaque forming units per infected cell. Stability test showed the phage was stable over a wide range of pH and temperatures. According to restriction analysis, k3w7 had 50 -kb double-stranded DNA genome as well as the heterogeneous nature of genetic material. These findings suggest that K3W7 has a potential use in therapy against infections caused by K. pneumonia produces carbapenemase.

Genomic analysis and biological characterization of a novel Schitoviridae phage infecting Vibrio alginolyticus

Vibrio alginolyticus is a Gram-negative bacterium commonly associated with mackerel poisoning. A bacteriophage that specifically targets and lyses this bacterium could be employed as a biocontrol agent for treating the bacterial infection or improving the shelf-life of mackerel products. However, only a few well-characterized V. alginolyticus phages have been reported in the literature. In this study, a novel lytic phage, named ΦImVa-1, specifically infecting V. alginolyticus strain ATCC 17749, was isolated from Indian mackerel. The phage has a short latent period of 15min and a burst size of approximately 66 particles per infected bacterium. ΦImVa-1 remained stable for 2h at a wide temperature (27-75°C) and within a pH range of 5 to 10. Transmission electron microscopy revealed that ΦImVa-1 has an icosahedral head of approximately 60nm in diameter with a short tail, resembling those in the Schitoviridae family. High throughput sequencing and bioinformatics analysis elucidated that ΦImVa-1 has a linear dsDNA genome of 77,479 base pairs (bp), with a G + C content of ~ 38.72% and 110 predicted gene coding regions (106 open reading frames and four tRNAs). The genome contains an extremely large virion-associated RNA polymerase gene and two smaller non-virion-associated RNA polymerase genes, which are hallmarks of schitoviruses. No antibiotic genes were found in the ΦImVa-1 genome. This is the first paper describing the biological properties, morphology, and the complete genome of a V. alginolyticus-infecting schitovirus. When raw mackerel fish flesh slices were treated with ΦImVa-1, the pathogen loads reduced significantly, demonstrating the potential of the phage as a biocontrol agent for V. alginolyticus strain ATCC 17749 in the food. KEY POINTS: • A novel schitovirus infecting Vibrio alginolyticus ATCC 17749 was isolated from Indian mackerel. • The complete genome of the phage was determined, analyzed, and compared with other phages. • The phage is heat stable making it a potential biocontrol agent in extreme environments.

In Vitro and Pre-Clinical Evaluation of Locally Isolated Phages, vB_Pae_SMP1 and vB_Pae_SMP5, Formulated as Hydrogels against Carbapenem-Resistant Pseudomonas aeruginosa.

The inadequate therapeutic opportunities associated with carbapenem-resistant Pseudomonas aeruginosa (CRPA) clinical isolates impose a search for innovative strategies. Therefore, our study aimed to characterize and evaluate two locally isolated phages formulated in a hydrogel, both in vitro and in vivo, against CRPA clinical isolates. The two phages were characterized by genomic, microscopic, phenotypic characterization, genomic analysis, in vitro and in vivo analysis in a Pseudomonas aeruginosa-infected skin thermal injury rat model. The two siphoviruses belong to class Caudovirectes and were named vB_Pae_SMP1 and vB_Pae_SMP5. Each phage had an icosahedral head of 60 ± 5 nm and a flexible, non-contractile tail of 170 ± 5 nm long, while vB_Pae_SMP5 had an additional base plate containing a 35 nm fiber observed at the end of the tail. The hydrogel was prepared by mixing 5% w/v carboxymethylcellulose (CMC) into the CRPA propagated phage lysate containing phage titer 108 PFU/mL, pH of 7.7, and a spreadability coefficient of 25. The groups were treated with either Phage vB_Pae_SMP1, vB_Pae_SMP5, or a two-phage cocktail hydrogel cellular subepidermal granulation tissues with abundant records of fibroblastic activity and mixed inflammatory cell infiltrates and showed 17.2%, 25.8%, and 22.2% records of dermal mature collagen fibers, respectively. In conclusion, phage vB_Pae_SMP1 or vB_Pae_SMP5, or the two-phage cocktails formulated as hydrogels, were able to manage the infection of CRPA in burn wounds, and promoted healing at the injury site, as evidenced by the histopathological examination, as well as a decrease in animal mortality rate. Therefore, these phage formulae can be considered promising for clinical investigation in humans for the management of CRPA-associated skin infections.

Characterization of a lytic phage KFS-EC3 infecting multiple foodborne pathogens

The purpose of this study was to characterize lytic and genomic properties of phage KFS-EC3 used as a biocontrol agent. The morphology of KFS-EC was observed by transmission electron microscopy (TEM). The polyvalent bactericidal activity of KFS-EC3 was determined against various host bacteria by enumerating bacterial number after phage treatment at multiplicities of infection (MOIs) of 0.001, 0.1, and 1.0 for 4 h. Finally, whole-genome sequencing was performed and annotated to identify its lytic property, safety, and novelty. TEM observation revealed that KFS-EC had morphology of myophage with an icosahedral head and contractile tail. In addition, KFS-EC3 could reduce E. coli, The Salmonella, Shigella sonnei, and their bacterial cocktail by ∼3 log CFU/mL, even at the lowest MOI of 0.001. The genomic analyses revealed that KFS-EC3 consisted of 167,440 bp and 151 functional genes, without any genes related to antibiotic resistance, virulence, allergenicity, and lysogenicity. Among the genes involved in host cell lysis, endolysin, holin, and a receptor binding protein were identified. Furthermore, KFS-EC3 was classified into a new phage of the Tequatrovirus genus in the Myoviridae family. In conclusion, lytic and safe phage KFS-EC3 with a broad host range can be used as a biocontrol agent to control foodborne pathogens.

Identification and Characterization of Erwinia Phage IT22: A New Bacteriophage-Based Biocontrol against Erwinia amylovora.

Erwinia amylovora is a quarantine phytopathogenic bacterium that is the causal agent of fire blight, a destructive disease responsible for killing millions of fruit-bearing plants worldwide, including apple, pear, quince, and raspberry. Efficient and sustainable control strategies for this serious bacterial disease are still lacking, and traditional methods are limited to the use of antibiotics and some basic agricultural practices. This study aimed to contribute to the development of a sustainable control strategy through the identification, characterization, and application of bacteriophages (phages) able to control fire blight on pears. Phages isolated from wastewater collected in the Apulia region (southern Italy) were characterized and evaluated as antibacterial agents to treat experimental fire blight caused by E. amylovora. Transmission electron microscopy (TEM) conducted on purified phages (named EP-IT22 for Erwinia phage IT22) showed particles with icosahedral heads of ca. 90 ± 5 nm in length and long contractile tails of 100 ± 10 nm, typical of the Myoviridae family. Whole genome sequencing (WGS), assembly, and analysis of the phage DNA generated a single contig of 174.346 bp representing a complete circular genome composed of 310 open reading frames (ORFs). EP-IT22 was found to be 98.48% identical to the Straboviridae Erwinia phage Cronus (EPC) (GenBank Acc. n° NC_055743) at the nucleotide level. EP-IT22 was found to be resistant to high temperatures (up to 60 °C) and pH values between 4 and 11, and was able to accomplish a complete lytic cycle within one hour. Furthermore, the viability-qPCR and turbidity assays showed that EP-IT22 (MOI = 1) lysed 94% of E. amylovora cells in 20 h. The antibacterial activity of EP-IT22 in planta was evaluated in E. amylovora-inoculated pear plants that remained asymptomatic 40 days post inoculation, similarly to those treated with streptomycin sulphate. This is the first description of the morphological, biological, and molecular features of EP-IT22, highlighting its promising potential for biocontrol of E. amylovora against fire blight disease.

Biological characteristics and genomic analysis of a novel Vibrio parahaemolyticus phage phiTY18 isolated from the coastal water of Xiamen China.

Vibrio parahaemolyticus is a common pathogen usually controlled by antibiotics in mariculture. Notably, traditional antibiotic therapy is becoming less effective because of the emergence of bacterial resistance, hence new strategies need to be found to overcome this challenge. Bacteriophages, a class of viruses that lyse bacteria, can help us control drug-resistant bacteria. In this study, a novel Vibrio parahaemolyticus phage phiTY18 isolated from the coastal water of Xiamen was explored. Transmission electron microscopy showed that phiTY18 had an icosahedral head of 130.0 ± 1.2 nm diameter and a contractile tail of length of 66.7 ± 0.6 nm. The phage titer could reach 7.2×1010 PFU/mL at the optimal MOI (0.01). The phage phiTY18 had a degree of tolerance to heat and acid and base. At the temperature of 50°C (pH7.0, 1h) the survival phages reached 1.28×106 PFU/mL, and at pH 5-9 (30°C, 1h), the survival phages was greater than 6.37×107 PFU/mL Analysis of the phage one-step growth curve revealed that it had a latent period of 10min, a rise period of 10min, and an average burst size of the phage was 48 PFU/cell. Genome sequencing and analysis drew that phage phiTY18 had double-stranded DNA (191,500 bp) with 34.90% G+C content and contained 117 open reading frames (ORFs) and 24 tRNAs. Phylogenetic tree based on major capsid protein (MCP) revealed that phage phiTY18 (MW451250) was highly related to two Vibrio phages phiKT1024 (OM249648) and Va1 (MK387337). The NCBI alignment results showed that the nucleotide sequence identity was 97% and 93%, respectively. In addition, proteomic tree analysis indicated that phage phiTY18, phiKT1024, and Va1 were belong to the same virus sub-cluster within Myoviridae. This study provides a theoretical basis for understanding the genomic characteristics and the interaction between Vibrio parahaemolyticus phages and their host.

Escherichia coli phage phi2013: genomic analysis and receptor identification.

Escherichia coli is an important foodborne pathogen that can cause severe human disease. Here, we report the isolation and characterization of the lytic virus phi2013, which is specific for Escherichia coli laboratory strains. Transmission electron microscopy showed that phage phi2013 has an icosahedral head and a long, fragile, noncontractile tail, exhibiting the typical form of a siphovirus. Evidence revealed that the phi2013 genome is a linear double-stranded DNA molecule of 49,833 bp with 79 predicted genes without any known antibiotic resistance genes, virulence factor genes, or integrase genes. Moreover, the conserved outer membrane protein FhuA, which is present in members of several genera of the family Enterobacteriaceae, was identified as the receptor of phage phi2013. To evaluate the potential of phage phi2013 as a biocontrol agent for controlling E. coli contamination, it was tested in several foods, including sterilized milk, ready-to-eat beef, and crisphead lettuce. The data showed that phage phi2013 can efficiently inhibit E. coli growth in the tested foods at 4°C and 25°C. We therefore conclude that phage phi2013 or cocktails containing phi2013 may be used as an antimicrobial agent in extending the shelf-life of food products by effectively controlling the growth of E. coli.

Anti-Salmonella polyvinyl alcohol coating containing a virulent phage PBSE191 and its application on chicken eggshell

The use of antibiotics in the food industry is avoided due to the increase of antibiotic-resistant bacteria. Therefore, the bacteriophage is emerging as an alternative agent. Here, we characterized the Salmonella Enteritidis phage PBSE191 and applied it to a polyvinyl alcohol (PVA) film. Transmission electron microscopic analysis revealed that it belonged to the Caudoviricetes class, with an icosahedral head and flexible tails. The phage showed rapid and strong lytic activity within 1 h. It was active against a broad range of Salmonella isolates, including six serotypes. In 25 min, 99 % of the initial population was adsorbed to the bacterial cell surface. The phage was also applied to 10 % (w/v) PVA films and coatings, which were then characterized in terms of phage stability and antibacterial performance, both in vitro and in foods. The phage remained stable in the 10 % (w/v) PVA solution containing 20 % (w/w, based on PVA weight) sorbitol (PVAS20), indicating that the phage was stable under dry conditions and strongly released in the polymer. Furthermore, significant bacterial cell reduction (2.0 × 105 CFU/film within 2 h) was observed in the phage-containing PVAS20 films. In addition, the PBSE191-containing PVAS20 coating on the chicken eggshell surface showed significant anti-Salmonella efficiency (about 2 log CFU reduction) within 24 h. Overall, the PBSE191 phage possesses a high potential as a biocontrol agent for use as an additive, or as an active antibacterial packaging to improve food safety against Salmonella contamination.

An insight into the therapeutic potential of a novel lytic Pseudomonas phage isolated from the river Ganga

Bacteriophages are effective natural antimicrobial agents against drug-resistant pathogens. Therefore, identification and detailed characterization of bacteriophages become essential to explore their therapeutic potential. This study aims to isolate and characterize a lytic bacteriophage against drug-resistant Pseudomonas aeruginosa. The Pseudomonas phage AIIMS-Pa-A1, isolated from the river Ganga water against drug-resistant P. aeruginosa, showed a clear lytic zone on spot assay. The phage revealed an icosahedral head (58.20 nm diameter) and a small tail (6.83 nm) under a transmission electron microscope. The growth kinetics showed an adsorption constant of 1.5 × 10-9 phage particles cell-1 ml-1 min-1 and a latent period of approximately 15 min with the burst size of 27 phages per infected cell. The whole-genome sequencing depicted a GC-rich genome of 40.97 kb having a lysis cassette of holin, endolysin and Rz protein, with features of the family Autographiviridae. The comparative genome analysis, Ortho-average nucleotide identity value, and phylogenetic analysis indicated the novelty of the phage AIIMS-Pa-A1. The study concludes that the Pseudomonas phage AIIMS-Pa-A1 is a novel member of the Autographiviridae family, truly lytic in nature for drug-resistant P. aeruginosa. The Pseudomonas phage AIIMS-Pa-A1 is having promising potential for future therapeutic intervention to treat drug-resistant P. aeruginosa infections.

Isolation, characterization, and application of a novel polyvalent lytic phage STWB21 against typhoidal and nontyphoidal Salmonella spp.

Salmonella is one of the common causal agents of bacterial gastroenteritis-related morbidity and mortality among children below 5 years and the elderly populations. Salmonellosis in humans is caused mainly by consuming contaminated food originating from animals. The genus Salmonella has several serovars, and many of them are recently reported to be resistant to multiple drugs. Therefore, isolation of lytic Salmonella bacteriophages in search of bactericidal activity has received importance. In this study, a Salmonella phage STWB21 was isolated from a lake water sample and found to be a novel lytic phage with promising potential against the host bacteria Salmonella typhi. However, some polyvalence was observed in their broad host range. In addition to S. typhi, the phage STWB21 was able to infect S. paratyphi, S. typhimurium, S. enteritidis, and a few other bacterial species such as Sh. flexneri 2a, Sh. flexneri 3a, and ETEC. The newly isolated phage STWB21 belongs to the Siphoviridae family with an icosahedral head and a long flexible non-contractile tail. Phage STWB21 is relatively stable under a wide range of pH (4–11) and temperatures (4°C–50°C) for different Salmonella serovars. The latent period and burst size of phage STWB21 against S. typhi were 25 min and 161 plaque-forming units per cell. Since Salmonella is a foodborne pathogen, the phage STWB21 was applied to treat a 24 h biofilm formed in onion and milk under laboratory conditions. A significant reduction was observed in the bacterial population of S. typhi biofilm in both cases. Phage STWB21 contained a dsDNA of 112,834 bp in length, and the GC content was 40.37%. Also, genomic analysis confirmed the presence of lytic genes and the absence of any lysogeny or toxin genes. Overall, the present study reveals phage STWB21 has a promising ability to be used as a biocontrol agent of Salmonella spp. and proposes its application in food industries.

Isolation and Characterization of a Newly Discovered Phage, V-YDF132, for Lysing Vibrio harveyi

A newly discovered lytic bacteriophage, V-YDF132, which efficiently infects the pathogenic strain of Vibrio harveyi, was isolated from aquaculture water collected in Yangjiang, China. Electron microscopy studies revealed that V-YDF132 belonged to the Siphoviridae family, with an icosahedral head and a long noncontractile tail. The phage has a latent period of 25 min and a burst size of 298 pfu/infected bacterium. V-YDF132 was stable from 37 to 50 °C. It has a wide range of stability (pH 5–11) and can resist adverse external environments. In addition, in vitro the phage V-YDF132 has a strong lytic effect on the host. Genome sequencing results revealed that V-YDF132 has a DNA genome of 84,375 bp with a GC content of 46.97%. In total, 115 putative open reading frames (ORFs) were predicted in the phage V-YDF132 genome. Meanwhile, the phage genome does not contain any known bacterial virulence genes or antimicrobial resistance genes. Comparison of the genomic features of the phage V-YDF132 and phylogenetic analysis revealed that V-YDF132 is a newly discovered Vibrio phage. Multiple genome comparisons and comparative genomics showed that V-YDF132 is in the same genus as Vibrio phages vB_VpS_PG28 (MT735630.2) and VH2_2019 (MN794238.1). Overall, the results indicate that V-YDF132 is potentially applicable for biological control of vibriosis.

Biological and genomic characterization of two newly isolated Elizabethkingia anophelis bacteriophages

Elizabethkingia anophelis is an opportunistic pathogen that infects newborns and immunocompromised patients. Because the infection is associated with high mortality as a result of its intrinsic resistance to antibiotics, alternative treatment methods are needed. Our previous study successfully isolated the world's first E. anophelis phage, TCUEAP1, which showed beneficial protection to E. anophelis-infected mice. More new bacteriophages are needed in order to provide sufficient choices to combat E. anophelis infections. In the current study, two new phages infecting E. anophelis were isolated from wastewater and were designated as TCUEAP2 and TCUEAP3. Further experiments, namely, transmission electron microscopy (TEM), infection assay, host-range analysis, and sequencing were performed to determine their biological and genomic characteristics. TEM analysis revealed that both TCUEAP2 and TCUEAP3 possess an icosahedral head with a non-contractile tail, and belong to the Siphoviridae family. Further experiments revealed that TCUEAP3 has a longer latent period and higher burst size compared to TCUEAP2. Host range analysis showed that both TCUEAP2 and TCUEAP3 have a narrow host range, infecting only their respective hosts. The genomic size of phage TCUEAP2 was 42,403bps containing 61 predicted open reading frames (ORFs), whereas the genome size of TCUEAP3 was 37,073bps containing 40 predicted ORFs. Due to the distinct biological characteristics of TCUEAP2 and TCUEAP3, they may be satisfactory for clinical uses such as preparation of phage cocktails or decontamination in clinical settings.

Characterization and Genomic Analysis of Novel Vibrio parahaemolyticus Phage vB_VpaP_DE10

In the present study, a novel lytic Vibrio parahaemolyticus phage, vB_VpaP_DE10, was isolated from sewage samples collected in Guangzhou city, China. Transmission electron microscopy revealed that phage vB_VpaP_DE10 has an icosahedral head (52.4 ± 2.5 nm) and a short non-contracted tail (21.9 ± 1.0 nm). Phage vB_VpaP_DE10 lysed approximately 31% (8/26) of the antibiotic-resistant V. parahaemolyticus strains tested. A one-step growth curve showed that phage vB_VpaP_DE10 has a relatively long latency time of 25 min and a burst size of ~19 PFU per cell. The genome of phage vB_VpaP_DE10 is a 42,871-bp-long dsDNA molecule with a G + C content of 49.19% and is predicted to contain 46 open reading frames, 26 of which are predicted to be related to functions such as phage structure, packaging, host lysis, and DNA metabolism. Sequence comparisons suggested that vB_VpaP_DE10 is a member of the genus Maculvirus within the family Autographiviridae. Morphological and genomic analysis indicated that vB_VpaP_DE10 is a novel V. parahaemolyticus phage.

Isolation and Characterization of a Lytic Vibrio parahaemolyticus Phage vB_VpaP_GHSM17 from Sewage Samples.

Vibrio parahaemolyticus is a major foodborne pathogen and the main cause of diarrheal diseases transmitted by seafood such as fish, shrimp, and shellfish. In the current study, a novel lytic phage infecting V. parahaemolyticus, vB_VpaP_GHSM17, was isolated from the sewage of a seafood market, Huangsha, Guangzhou, and its morphology, biochemistry, and taxonomy features were identified. Morphological observation revealed that GHSM17 had an icosahedral head with a short, non-contractile tail. The double-stranded DNA genome of GHSM17 consisted of 43,228 bp with a GC content of 49.42%. In total, 45 putative ORFs were identified in the GHSM17 genome. Taxonomic analysis indicated GHSM17 belonging to genus Maculvirus, family Autographiviridae. In addition, GHSM17 was stable over a wide range of temperatures (20–60 °C) and pH (5–11) and was completely inactivated after 70 min of ultraviolet irradiation. The bacterial inhibition assay revealed that GHSM17 could inhibit the growth of V. parahaemolyticus within 8 h. The results support that phage GHSM17 may be a potential candidate in the biological control of V. parahaemolyticus contamination in aquaculture.

Genome sequence of the novel freshwater Microcystis cyanophage Mwe-Yong1112-1.

The freshwater cyanophage Mwe-Yong1112-1 was isolated using Microcystis wesenbergii as a host and found to have an icosahedral head, about 45 nm in diameter, and a flexible tail, approximately 133 nm in length and 4.5 nm in width. The complete genome of the cyanophage is 39,679 bp in length with a G+C content of 66.6%. Mwe-Yong1112-1 shared the highest pairwise average nucleotide identity (ANI) value of 67.7% (below the ≥95% boundary to define a species) and the highest nucleotide sequence similarity of 17.48% (below the >70% boundary to define a genus) with the most closely related phage. In a proteomic tree, Mwe-Yong1112-1 and three unclassified phages formed a monophyletic clade between the families Saparoviridae and Pyrstoviridae, but Mwe-Yong1112-1 occupied a separate branch from the other three phages, suggesting that it represents a new evolutionary lineage. This study enriches the available information about freshwater cyanophages.

Isolation and characterization of two homolog phages infecting Pseudomonas aeruginosa

Bacteriophages (phages) are capable of infecting specific bacteria, and therefore can be used as a biological control agent to control bacteria-induced animal, plant, and human diseases. In this study, two homolog phages (named PPAY and PPAT) that infect Pseudomonas aeruginosa PAO1 were isolated and characterized. The results of the phage plaque assay showed that PPAT plaques were transparent dots, while the PPAY plaques were translucent dots with a halo. Transmission electron microscopy results showed that PPAT (65 nm) and PPAY (60 nm) strains are similar in size and have an icosahedral head and a short tail. Therefore, these belong to the short-tailed phage family Podoviridae. One-step growth curves revealed the latent period of 20 min and burst time of 30 min for PPAT and PPAY. The burst size of PPAT (953 PFUs/infected cell) was higher than that of PPAY (457 PFUs/infected cell). Also, the adsorption rate constant of PPAT (5.97 × 10−7 ml/min) was higher than that of PPAY (1.32 × 10−7 ml/min) at 5 min. Whole-genome sequencing of phages was carried out using the Illumina HiSeq platform. The genomes of PPAT and PPAY have 54,888 and 50,154 bp, respectively. Only 17 of the 352 predicted ORFs of PPAT could be matched to homologous genes of known function. Likewise, among the 351 predicted ORFs of PPAY, only 18 ORFs could be matched to genes of established functions. Homology and evolutionary analysis indicated that PPAT and PPAY are closely related to PA11. The presence of tail fiber proteins in PPAY but not in PPAT may have contributed to the halo effect of its plaque spots. In all, PPAT and PPAY, newly discovered P. aeruginosa phages, showed growth inhibitory effects on bacteria and can be used for research and clinical purposes.