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Background and purpose: Due to emerging antibiotic-resistant bacteria, finding effective alternative antimicrobials to combat the bacteria is unavoidable. Bacteriophage therapy as a biologic and highly potentiated therapeutic approach was considered recently. This study aimed to isolate lytic bacteriophage(s) against multi-drug resistant clinical isolates of Klebsiella pneumonia and evaluate the morphological and biological characteristics as candidates for phage therapy. Materials and methods: Obtained clinical isolates from Shahid Madani Hospital in Tabriz, were confirmed as K. pneumoniae, and an antibiotic sensitivity test was performed using the standard disk diffusion method. Then, the isolation of lytic bacteriophages against 16 selected antibiotic-resistant bacterial isolates was carried out. Briefly, samples for phage isolating were gained from sewage water and centrifuged. The supernatant was filtered using a 0.45 µm Filter, mixed with fresh tripton soy broth medium and magnesium sulfate and incubated at room temperature. Then, the solution was mixed with chloroform and evaluation of plaque formation was performed using the agar bilayer technique. Genomes of the bacteriophages were subjected to molecular analysis using Hind III and EcoRV enzymes. Host range determination of the isolated bacteriophages was evaluated using spot test and agar bilayer techniques. Three bacteriophages with a broader host range than others were selected for more characterization. One step growth curve of the phages was drawn and their latent period and burst size were investigated. Then, the stability and survivability of the isolated lytic bacteriophages to different temperatures (-20, 37, 50℃), different rates of pH value (3,4,5,7,9,11,12), and also UV radiation were evaluated. Finally, morphological characteristics of bacteriophage with the broadest host range (PKpMa2/20) were investigated using electron microscopy. Results: 36 clinical bacterial isolates were confirmed as K. pneumoniae based on standard biochemical tests. In the antibiotic susceptibility assay, it was revealed that the highest and lowest rates of antibiotic resistance belonged to cefazoline (80%) and tobramycin (26%) respectively. Isolation of bacteriophages against all 16 selected isolates was successfully performed. After the extraction of the genomes of the phages, a high variety of genome sequences was detected according to molecular analysis. In host range determination, PKpMa2/20 had lytic activity against 11 of 15 isolates .In a study of the one-step growth curve of the phages, with a broader host range than other isolated bacteriophages, latent periods were shown 10, 10, and 15 min and their burst size were 350, 210, and 180 pfu/ml respectively. All of the phages showed appropriate stability in temperatures of -20 and 37 ℃. In the temperature of 50℃, the survivability of PKpMa2/20 was higher than two other phages. Also, PKpMa2/20 was stable in the pH range of 3-12; in the pH value of 3, two other phages stability was ceased but PKpMa2/20 showed appropriate lytic activity. In electron microscopy, PKpMa2/20 had a short tail length of 10 nm and a head with a diameter of about 60 nm that possibly belonged to the family Podoviridae. Conclusion: Based on appropriate lytic effects, latent period, and burst size of the three isolated bacteriophages in the current study, especially PKpMa2/20, and also, their high stability to different temperatures, pH, and UV radiation, they could be considered as candidates for phage therapy to control resistant strains of K. pneumoniae infections but for the therapeutic use of theses phages, more molecular analysis is needed. [ABSTRACT FROM AUTHOR]

Abstract Background Escherichia coli is a common fecal coliform, facultative aerobic, gram-negative bacterium. Pathogenic strains of such microbes have evolved to cause diarrhea, urinary tract infections, and septicemias. The emergence of antibiotic resistance urged the identification of an alternative strategy. The use of lytic bacteriophages against the control of pathogenic E. coli in clinics and different environmental setups (waste and drink water management) has become an alternative therapy to antibiotic therapy. Thus, this study aimed to isolate and characterize lytic bacteriophage from various sources in Addis Ababa, tested them against antimicrobial-resistant diarrheagenic E. coli strains and evaluated their therapeutic potential under in vitro conditions. Methods A total of 14 samples were processed against six different diarrheagenic E. coli strains. The conventional culture and plaque analysis agar overlay method was used to recover lytic bacteriophage isolates. The phage isolates were characterized to determine their lytic effect, growth characteristics, host range activity, and stability under different temperature and pH conditions. Phage isolates were identified by scanning electron microscope (SEM), and molecular techniques (PCR). Results In total, 17 phages were recovered from 84 tested plates. Of the 17 phage isolates, 11 (65%) were Myoviridae-like phages, and 6 (35%) phage isolates were Podoviridae and Siphoviridae by morphology and PCR identification. Based on the host range test, growth characteristics, and stability test 7 potent phages were selected. These phages demonstrated better growth characteristics, including short latent periods, highest burst sizes, and wider host ranges, as well as thermal stability and the ability to survive in a wide range of pH levels. Conclusions The promising effect of the phages isolated in this study against AMR pathogenic E. coli has raised the possibility of their use in the future treatment of E. coli infections.

Background: Escherichia coli is a common fecal coliform, facultative aerobic, gram-negative bacterium. Pathogenic strains of such microbes have evolved to cause diarrhea, urinary tract infections, and septicemias. The emergence of antibiotic resistance urged the identification of an alternative strategy. The use of lytic bacteriophages against the control of pathogenic E. coli in clinics and different environmental setups (waste and drink water management) has become an alternative therapy to antibiotic therapy. Thus, this study aimed to isolate and characterize lytic bacteriophage from various sources in Addis Ababa, tested them against antimicrobial-resistant diarrheagenic E. coli strains and evaluated their therapeutic potential under in vitro conditions. Methods: A total of 14 samples were processed against six different diarrheagenic E. coli strains. The conventional culture and plaque analysis agar overlay method was used to recover lytic bacteriophage isolates. The phage isolates were characterized to determine their lytic effect, growth characteristics, host range activity, and stability under different temperature and pH conditions. Phage isolates were identified by scanning electron microscope (SEM), and molecular techniques (PCR). Results: In total, 17 phages were recovered from 84 tested plates. Of the 17 phage isolates, 11 (65%) were Myoviridae-like phages, and 6 (35%) phage isolates were Podoviridae and Siphoviridae by morphology and PCR identification. Based on the host range test, growth characteristics, and stability test 7 potent phages were selected. These phages demonstrated better growth characteristics, including short latent periods, highest burst sizes, and wider host ranges, as well as thermal stability and the ability to survive in a wide range of pH levels. Conclusions: The promising effect of the phages isolated in this study against AMR pathogenic E. coli has raised the possibility of their use in the future treatment of E. coli infections. [ABSTRACT FROM AUTHOR]

Phages that infect pathogenic bacteria present a valuable resource for treating antibiotic-resistant infections. We isolated and developed a collection of 19 Enterococcus phages, including myoviruses, siphoviruses, and a podovirus, that can infect both Enterococcus faecalis and Enterococcus faecium. Several of the Myoviridae phages that we found in southern California wastewater were from the Brockvirinae subfamily (formerly Spounavirinae) and had a broad host range across both E. faecium and E. faecalis. By searching the NCBI Sequence Read Archive, we showed that these phages are prevalent globally in human and animal microbiomes. Enterococcus is a regular member of healthy human gut microbial communities; however, it is also an opportunistic pathogen responsible for an increasing number of antibiotic-resistant infections. We tested the ability of each phage to clear Enterococcus host cultures and delay the emergence of phage-resistant Enterococcus. We found that some phages were ineffective at clearing Enterococcus cultures individually but were effective when combined into cocktails. Quantitative PCR was used to track phage abundance in cocultures and revealed dynamics ranging from one dominant phage to an even distribution of phage growth. Genomic characterization showed that mutations in Enterococcus exopolysaccharide synthesis genes were consistently found in the presence of phage infection. This work will help to inform cocktail design for Enterococcus, which is an important target for phage therapy applications. IMPORTANCE Due to the rise in antibiotic resistance, Enterococcus infections are a major health crisis that requires the development of alternative therapies. Phage therapy offers an alternative to antibiotics and has shown promise in both in vitro and early clinical studies. Here, we established a collection of 19 Enterococcus phages and tested whether combining phages into cocktails could delay growth and the emergence of resistant mutants in comparison with individual phages. We showed that cocktails of two or three phages often prevented the growth of phage-resistant mutants, and we identified which phages were replicating the most in each cocktail. When resistant mutants emerged to single phages, they showed consistent accumulation of mutations in exopolysaccharide synthesis genes. These data serve to demonstrate that a cocktail approach can inform efforts to improve efficacy against Enterococcus isolates and reduce the emergence of resistance.

Background: Acinetobacter nosocomialis (A. nosocomialis) is a glucose non-fermentative, gram-negative bacillus that belongs to the Acinetobacter calcoaceticus-baumannii complex. In recent years, studies have found an increased clinical prevalence of A. nosocomialis. However, given the increasing trend of antibiotic resistance, developing new antibacterial agents is vital. Currently, research regarding bacteriophage therapy against A. nosocomialis is only limited. Methods: Two A. nosocomialis bacteriophages, TCUAN1 and TCUAN2, were isolated from sewage. Experiments such as transmission electron microscopy (TEM), host-range analysis, and sequencing were performed to determine their biological and genomic characteristics. TCUAN2 were further subjected to in vivo experiments and their derived-endolysin were cloned and tested against their bacteria host. Results: Transmission electron microscopy revealed that TCUAN1 and TCUAN2 belong to Myoviridae and Podoviridae, respectively. Both phages show a broad host spectrum and rapid adsorption efficiency. Further biological analysis showed that TCUAN2 possesses a shorter latent period and larger burst size compared to TCUAN1. Because TCUAN2 showed a better antibacterial activity, it was injected into A. nosocomialis-infected mice which resulted in a significant decrease in bacterial load levels in the blood and increased the mice's survival. Finally, genomic analysis revealed that the complete nucleotide sequence of TCUAN1 is 49, 691 bps (containing 75 open reading frames) with a G + C content of 39.3%; whereas the complete nucleotide sequence of TCUAN2 is 41, 815 bps (containing 68 open reading frames) with a G + C content of 39.1%. The endolysin gene cloned and purified from TCUAN2 also showed antibacterial activity when used with a chelator EDTA.

In the present study, two new Bacillus subtilis phages, BSTP4 and BSTP6, were isolated and studied further. Morphologically, BSTP4 and BSTP6 are podoviruses with complete genome of 19,145 (39.9% G + C content) and 19,367 bp (39.8% G + C content), respectively, which became among the smallest Bacillus phages. Three most prominent structural proteins were separated and identified as pre-neck appendage, major head, and head fiber proteins using LC–MS/MS. Both phages encode putative terminal proteins (TP) and contain short inverted terminal repeats (ITRs) which may be important for their replication. In addition, non-coding RNA (pRNA) and parS sites were identified which may be required for DNA packaging and their maintenance inside the host, respectively. Furthermore, the phage genome sequences show significant similarity to B. subtilis group species genome sequences. Finally, phylogenomic and phylogenetic analyses suggest that BSTP4 and BSTP6 may form a new species in the genus Salasvirus, subfamily Picovirinae of family Salasmaviridae. Considering the small numbers of ICTV-accepted B. subtilis phages and the importance of the host in the food industry and biotechnology, the current study helps to improve our understanding of the diversity of B. subtilis phages and shed light on the phage–host relationships. [ABSTRACT FROM AUTHOR]

Abstract Background Staphylococcus aureus (S. aureus) is one of the major causes of bovine mastitis with significant economic losses around the worldwide. The emergence of multidrug-resistant (MDR), methicillin-resistant (MRSA) and biofilm-producing strains of S. aureus challenges the treatment strategies based on the antibiotic application. Today, alternative or combinational treatment options such as bacteriophage application has received much attention. The goal of the present study was to focus on isolation and evaluation of the efficacy of bacteriophages with specific lytic activity against S. aureus strains with low cure rates (MDR, MRSA and biofilm-producing strains). Results In the present study, two phages belonging to the Podoviridae family with specific lytic activity against S. aureus were isolated from the sewage of dairy farms and designated as Staphylococcus phage M8 and Staphylococcus phage B4. Latent period and burst size for Staphylococcus phage M8 (70 min, 72 PFU/cell) and Staphylococcus phage B4 (30 min, 447 PFU/cell) were also defined. Our results revealed the susceptibility of MDR (4/20; 20%), MRSA (4/13; 30.8%) and biofilm-producing (1/10; 10%) strains to Staphylococcus phage M8. Moreover, one biofilm-producing strain (1/10; 10%) was susceptible to Staphylococcus phage B4. Furthermore, both phages kept their lytic activity in milk. They reduced the S. aureus population by about 3 logs in cultured milk after 8 h of incubation. Conclusion In conclusion, it seems that both phages had the potential to serve as biological control agents alone or in combination with other agents such as antibiotics against infections induced by S. aureus. However, further studies are needed to investigate the efficacy of these phages in vivo.

This study aimed to conduct a detailed study on murine model testing of bacteriophage against Acinetobacter baumannii. These bacteriophages were tested not only for their efficacy in healing wound of murine models infected with multidrug resistant A. baumannii but were also studied for any derangement in hematological parameters as well as liver and kidney function. The study also included any histological changes observed in hepatic and renal tissues of the bacteriophage treated murine animals. This experimental study was conducted at Institute of Basic Medical Sciences, Khyber Medical University, Peshawar and Institute of Pure and Applied Biology, Department of Microbiology and Molecular Genetics, Bahauddin Zakariya University, Multan. A. baumannii isolates were obtained from the Microbiology Department, Armed Forces Institute of Pathology, Rawalpindi. Antimicrobial susceptibility was done by using standard procedures and as per Clinical Laboratory Standards Institute guidelines. Bacteriophages were isolated from sewage water samples collected from different hospitals in Multan. These bacteriophages were characterized and finally used for treating the murine model and efficacy of phage as a therapeutic option was determined by using superficial rat wound model. In this study, the isolated lytic bacteriophage was effective in relatively faster wound healing of the infected animals. Moreover, there were no significant hematological or renal and hepatic profile changes in treated animals. Histology of renal and hepatic tissues was also normal as compared to control animals. Our study concluded that the isolated phage could serve as an attractive therapeutic candidate to combat the deadly multidrug resistant A. baumannii. [ABSTRACT FROM AUTHOR]

Background: Staphylococcus aureus (S. aureus) is one of the major causes of bovine mastitis with significant economic losses around the worldwide. The emergence of multidrug-resistant (MDR), methicillin-resistant (MRSA) and biofilm-producing strains of S. aureus challenges the treatment strategies based on the antibiotic application. Today, alternative or combinational treatment options such as bacteriophage application has received much attention. The goal of the present study was to focus on isolation and evaluation of the efficacy of bacteriophages with specific lytic activity against S. aureus strains with low cure rates (MDR, MRSA and biofilm-producing strains). Results: In the present study, two phages belonging to the Podoviridae family with specific lytic activity against S. aureus were isolated from the sewage of dairy farms and designated as Staphylococcus phage M8 and Staphylococcus phage B4. Latent period and burst size for Staphylococcus phage M8 (70 min, 72 PFU/cell) and Staphylococcus phage B4 (30 min, 447 PFU/cell) were also defined. Our results revealed the susceptibility of MDR (4/20; 20%), MRSA (4/13; 30.8%) and biofilm-producing (1/10; 10%) strains to Staphylococcus phage M8. Moreover, one biofilm-producing strain (1/10; 10%) was susceptible to Staphylococcus phage B4. Furthermore, both phages kept their lytic activity in milk. They reduced the S. aureus population by about 3 logs in cultured milk after 8 h of incubation. Conclusion: In conclusion, it seems that both phages had the potential to serve as biological control agents alone or in combination with other agents such as antibiotics against infections induced by S. aureus. However, further studies are needed to investigate the efficacy of these phages in vivo. [ABSTRACT FROM AUTHOR]

Stenotrophomonas maltophilia (S. maltophilia) is widely distributed in nature and frequently causes nosocomial infections. In this work, the biological characteristics and genome of a new S. maltophilia phage BUCT609 isolated from hospital sewage with S. maltophilia strain No. 3015 as host was analyzed and its therapeutic effect in vivo was explored. It was observed by TEM that phage BUCT609 belongs to the Podoviridae with a 10 nm tail structure and a capsid with a diameter of about 50 nm. It has a short latent period (about 10 min) and its burst size is 382 PFU /cell when multiplicity of infection (MOI) is 0.01. Furthermore, it has a high survival rate in the environment with a pH range from 3 to 10 and temperature range from 4°C to 55°C. The complete genome of phage BUCT609 is linear double-stranded DNA of 43,145 bp in length, and the GC content is 58%. The genome sequence of phage BUCT609 shares

Objective: The increase of multidrug resistance in bacteria has increased the efforts in search of alternative methods. The aim of the present study was to isolate and characterize the lytic phages and assess their lytic activity against a number of gram-negative bacteria. Materials and Methods: The phages and their respective hosts were isolated from wastewater collected from the municipal sewer system of Trabzon, Turkey. The lytic activities of phage were determined using the agar spot test. The identification and antibiotic susceptibility of host bacteria were determined using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and Phoenix 100, respectively. The phages were characterized morphologically using transmission electron microscopy. One of the phages, Enteroc21, which has a broad-host-range, was further characterized by genome restriction endonuclease analysis and burst size. Results: Two phages infected strains of four different species, nine phages were able to infect 2-4 strains belonging to one or two species, and three phages showed lytic activity against only the hosts from which they were isolated. All phages belonged to the Siphoviridae, Myoviridae, and Podoviridae family based on transmission electron microscopy morphology. The Enteroc21 had more than 100 kb genome size and a burst size of 180 per infected cell. Most of the host strains were resistant to ampicillin, amoxicillin-clavulanic acid, and in particular, Achromobacter xylosoxidans TRAX 13 was multidrug-resistant showing resistance to cefepime, aztreonam, gentamicin, netilmicin, and ciprofloxacin. Conclusion: This study showed that the isolated phages have the potential to be used in phage therapy against various bacterial infections, including multidrug-resistant bacteria. [ABSTRACT FROM AUTHOR]

In this study, two lytic bacteriophages designated as vB_CjP and vB_CcM were isolated and evaluated for their ability to combat multidrug-resistant bacteria Campylobacter jejuni and Campylobacter coli, respectively. A morphological analysis of these phages by transmission electron microscopy revealed that the vB-CjP bacteriophage had a mean head dimension of 66.6 ± 2.1 nm and a short non-contractile tail and belongs to the Podoviridae family, whereas vB_CcM had a mean head dimension of 80 ± 3.2 nm, a contractile tail, and a length calculated to be 60 ± 2.5 nm and belongs to the Myoviridae family. The results of the host range assay showed that vB_CjP could infect 5 of 10 C. jejuni isolates, whereas vB_CcM could infect 4 of 10 C. coli isolates. Both phages were thermostable and did not lose their infectivity and ability to lyse their host following exposure to 60 °C for 10 min; furthermore, phage particles were relatively stable within a pH range of 6–8. A one-step growth curve indicated that the phages produced estimated burst sizes of 110 and 120 PFU per infected cell with latent periods of 10 and 15 min, for vB-CjP and vB-CcM, respectively. The lytic activity of these phages against planktonic Campylobacter showed that these phages were able to control the growth of Campylobacter in vitro. These results suggest that these phages have a high potential for phage applications and can reduce significantly the counts of Campylobacter spp. The lytic activity of vB-CjP and vB-CcM phages at different (MOIs) against multidrug resistance Campylobacter strains was evaluated. The bacterial growth was slightly delayed by both phages, and the highest efficiency of both phages was observed when MOI = 1 was applied. [ABSTRACT FROM AUTHOR]

Nineteen bacteriophages against five main capsular types of multidrug-resistant Acinetobacter baumannii were isolated from tertiary care hospital sewage. Eight representative phages from each capsular type were characterized and tested for their biological properties. The biological features revealed that phages T1245, T444, and T515 had a large burst size of more than 420 pfu/mL, together with a short latent period lasting less than 6 min, and were readily adsorbed to a bacterial host within 10 min. Moreover, these phages demonstrated host specificity and stability over a broad range of temperatures (−20 to 60 °C) and pH (5.0–9.0). A whole-genome analysis of six lytic and two temperate phages revealed high genomic similarity with double-stranded DNA between 40 and 50 kb and G + C content of 38–39%. The protein compositions disclosed the absence of toxin-coding genes. The phylogenic results, together with morphological micrographs, confirmed that three selected phages (T1245, T444, and T515) belong to the Podoviridae family within the order Caudovirales. The biological data and bioinformatics analysis indicated that these novel A. baumannii phages possess important enzymes, including depolymerase and endolysin, which could be further developed as promising alternative antibacterial agents to control A. baumannii infections. [ABSTRACT FROM AUTHOR]

In this study, we have presented the genomic characterisation of UFJF_PfDIW6, a novel lytic Pseudomonas fluorescens-phage with potential for biocontrol in the dairy industry. This phage showed a short linear double-stranded DNA genome (~42 kb) with a GC content of 58.3% and more than 50% of the genes encoding proteins with unknown functions. Nevertheless, UFJF_PfDIW6's genome was organised into five functional modules: DNA packaging, structural proteins, DNA metabolism, lysogenic, and host lysis. Comparative genome analysis revealed that the UFJF_PfDIW6's genome is distinct from other viral genomes available at NCBI databases, displaying maximum coverages of 5% among all alignments. Curiously, this phage showed higher sequence coverages (38–49%) when aligned with uncharacterised prophages integrated into Pseudomonas genomes. Phages compared in this study share conserved locally collinear blocks comprising genes of the modules' DNA packing and structural proteins but were primarily differentiated by the composition of the DNA metabolism and lysogeny modules. Strategies for taxonomy assignment showed that UFJF_PfDIW6 was clustered into an unclassified genus in the Podoviridae clade. Therefore, our findings indicate that this phage could represent a novel genus belonging to the Podoviridae family. [ABSTRACT FROM AUTHOR]

Bacterial viruses (or bacteriophages) have developed formidable ways to deliver their genetic information inside bacteria, overcoming the complexity of the bacterial-cell envelope. In short-tailed phages of the Podoviridae superfamily, genome ejection is mediated by a set of mysterious internal virion proteins, also called ejection or pilot proteins, which are required for infectivity. The ejection proteins are challenging to study due to their plastic structures and transient assembly and have remained less characterized than classical components such as the phage coat protein or terminase subunit. However, a spate of recent cryo-EM structures has elucidated key features underscoring these proteins' assembly and conformational gymnastics that accompany their expulsion from the virion head through the portal protein channel into the host. In this review, we will use a phage-T7-centric approach to critically review the status of the literature on ejection proteins, decipher the conformational changes of T7 ejection proteins in the pre- and post-ejection conformation, and predict the conservation of these proteins in other Podoviridae. The challenge is to relate the structure of the ejection proteins to the mechanisms of genome ejection, which are exceedingly complex and use the host's machinery. [ABSTRACT FROM AUTHOR]

Potato common scab caused by Streptomyces scabies is one of the most economically important diseases infecting potato. It reduces the quality of potato tubers, which subsequently decreases the tuber prices and causes significant economic losses for potato growers. Biological control using bacteriophages is a promising strategy for controlling this disease. In this study, a novel bacteriophage with high lytic efficacy against S. scabies was isolated from a potato field at El-Minya, Egypt, and was designated SscP1EGY. The phage has an icosahedral head of 55 nm and a short tail of 7.5 nm, typical of a podovirus. Its infection cycle was 90 min, including 50 min of latent time and 40 min of rise period with a burst size of approximately 200 PFU per infected cell. The genome of SscP1EGY consists 51,751 nucleotides with 76 predicted genes. SscP1EGY infected and completely lysed seven tested S. scabies strains but showed no lytic activity against three beneficial Streptomyces species, other beneficial bacterial species, and non-target plant pathogenic bacteria. In greenhouse experiments, treatment of S. scabies-inoculated potato tubers with phage SscP1EGY resulted in reductions of (1) the severity of scab, (2) the number of lesions, and (3) the percentage of lesion surface, as compared to the inoculated tubers without phage treatment. Also, scab lesions appeared superficial in phage-treated tubers but pitted in non-phage-treated tubers. Our results suggest that SscP1EGY has a potential as a biological control agent for S. scabies. Based on our knowledge, SscP1EGY is the first sequenced S. scabies-infecting phage in Egypt. [ABSTRACT FROM AUTHOR]

Bacteriophages (phages) are ubiquitous entities present in every conceivable habitat as a result of their bacterial parasitism. Their prevalence and impact in the ecology of bacterial communities and their ability to control pathogens make their characterization essential, particularly of new phages, improving knowledge and potential application. The isolation and characterization of a new lytic phage against Sphaerotilus natans strain DSM 6575, named vB_SnaP-R1 (SnaR1), is here described. Besides being the first sequenced genome of a Sphaerotilus natans infecting phage, 99% of its 41507 bp genome lacks homology with any other sequenced phage, revealing its uniqueness and previous lack of knowledge. Moreover, SnaR1 is the first Podoviridae phage described infecting this bacterium. Sphaerotilus natans is an important filamentous bacterium due to its deleterious effect on wastewater treatment plants (WWTP) and thus, phages may play a role as novel biotechnological tools against filamentous overgrowth in WWTP. The lytic spectrum of SnaR1 was restricted to its host strain, infecting only one out of three S. natans strains and infection assays revealed its ability to reduce bacterial loads. Results suggest SnaR1 as the prototype of a new phage genus and demonstrates its potential as a non-chemical alternative to reduce S. natans DSM 6575 cells. [ABSTRACT FROM AUTHOR]

Nineteen bacteriophages against five main capsular types of multidrug-resistant Acinetobacter baumannii were isolated from tertiary care hospital sewage. Eight representative phages from each capsular type were characterized and tested for their biological properties. The biological features revealed that phages T1245, T444, and T515 had a large burst size of more than 420 pfu/mL, together with a short latent period lasting less than 6 min, and were readily adsorbed to a bacterial host within 10 min. Moreover, these phages demonstrated host specificity and stability over a broad range of temperatures (−20 to 60 °C) and pH (5.0–9.0). A whole-genome analysis of six lytic and two temperate phages revealed high genomic similarity with double-stranded DNA between 40 and 50 kb and G + C content of 38–39%. The protein compositions disclosed the absence of toxin-coding genes. The phylogenic results, together with morphological micrographs, confirmed that three selected phages (T1245, T444, and T515) belong to the Podoviridae family within the order Caudovirales. The biological data and bioinformatics analysis indicated that these novel A. baumannii phages possess important enzymes, including depolymerase and endolysin, which could be further developed as promising alternative antibacterial agents to control A. baumannii infections.

Vibrio parahaemolyticus is a foodborne pathogen that is frequently isolated from a variety of seafood. To control this pathogenic Vibrio spp., the implementation of bacteriophages in aquaculture and food industries have shown a promising alternative to antibiotics. In this study, six bacteriophages isolated from the seafood samples demonstrated a narrow host range specificity that infecting only the V. parahaemolyticus strains. Morphological analysis revealed that bacteriophages Vp33, Vp22, Vp21, and Vp02 belong to the Podoviridae family, while bacteriophages Vp08 and Vp11 were categorized into the Siphoviridae family. All bacteriophages were composed of DNA genome and showed distinctive restriction fragment length polymorphism. The optimal MOI for bacteriophage propagation was determined to be 0.001 to 1. One-step growth curve revealed that the latent period ranged from 10 to 20 min, and the burst size of bacteriophage was approximately 17 to 51 PFU/cell. The influence of temperature and pH levels on the stability of bacteriophages showed that all bacteriophages were optimally stable over a wide range of temperatures and pH levels. In vitro lytic activity of all bacteriophages demonstrated to have a significant effect against V. parahaemolyticus. Besides, the application of a bacteriophage cocktail instead of a single bacteriophage suspension was observed to have a better efficiency to control the growth of V. parahaemolyticus. Results from this study provided a basic understanding of the physiological and biological properties of the isolated bacteriophages before it can be readily used as a biocontrol agent against the growth of V. parahaemolyticus.

Vibrio parahaemolyticus is a foodborne pathogen that is frequently isolated from a variety of seafood. To control this pathogenic Vibrio spp., the implementation of bacteriophages in aquaculture and food industries have shown a promising alternative to antibiotics. In this study, six bacteriophages isolated from the seafood samples demonstrated a narrow host range specificity that infecting only the V. parahaemolyticus strains. Morphological analysis revealed that bacteriophages Vp33, Vp22, Vp21, and Vp02 belong to the Podoviridae family, while bacteriophages Vp08 and Vp11 were categorized into the Siphoviridae family. All bacteriophages were composed of DNA genome and showed distinctive restriction fragment length polymorphism. The optimal MOI for bacteriophage propagation was determined to be 0.001 to 1. One-step growth curve revealed that the latent period ranged from 10 to 20 min, and the burst size of bacteriophage was approximately 17 to 51 PFU/cell. The influence of temperature and pH levels on the stability of bacteriophages showed that all bacteriophages were optimally stable over a wide range of temperatures and pH levels. In vitro lytic activity of all bacteriophages demonstrated to have a significant effect against V. parahaemolyticus. Besides, the application of a bacteriophage cocktail instead of a single bacteriophage suspension was observed to have a better efficiency to control the growth of V. parahaemolyticus. Results from this study provided a basic understanding of the physiological and biological properties of the isolated bacteriophages before it can be readily used as a biocontrol agent against the growth of V. parahaemolyticus. [ABSTRACT FROM AUTHOR]

In this study, we have presented the genomic characterisation of UFJF_PfDIW6, a novel lytic Pseudomonas fluorescens-phage with potential for biocontrol in the dairy industry. This phage showed a short linear double-stranded DNA genome (~42 kb) with a GC content of 58.3% and more than 50% of the genes encoding proteins with unknown functions. Nevertheless, UFJF_PfDIW6’s genome was organised into five functional modules: DNA packaging, structural proteins, DNA metabolism, lysogenic, and host lysis. Comparative genome analysis revealed that the UFJF_PfDIW6’s genome is distinct from other viral genomes available at NCBI databases, displaying maximum coverages of 5% among all alignments. Curiously, this phage showed higher sequence coverages (38–49%) when aligned with uncharacterised prophages integrated into Pseudomonas genomes. Phages compared in this study share conserved locally collinear blocks comprising genes of the modules’ DNA packing and structural proteins but were primarily differentiated by the composition of the DNA metabolism and lysogeny modules. Strategies for taxonomy assignment showed that UFJF_PfDIW6 was clustered into an unclassified genus in the Podoviridae clade. Therefore, our findings indicate that this phage could represent a novel genus belonging to the Podoviridae family.

Bacterial viruses (or bacteriophages) have developed formidable ways to deliver their genetic information inside bacteria, overcoming the complexity of the bacterial-cell envelope. In short-tailed phages of the Podoviridae superfamily, genome ejection is mediated by a set of mysterious internal virion proteins, also called ejection or pilot proteins, which are required for infectivity. The ejection proteins are challenging to study due to their plastic structures and transient assembly and have remained less characterized than classical components such as the phage coat protein or terminase subunit. However, a spate of recent cryo-EM structures has elucidated key features underscoring these proteins’ assembly and conformational gymnastics that accompany their expulsion from the virion head through the portal protein channel into the host. In this review, we will use a phage-T7-centric approach to critically review the status of the literature on ejection proteins, decipher the conformational changes of T7 ejection proteins in the pre- and post-ejection conformation, and predict the conservation of these proteins in other Podoviridae. The challenge is to relate the structure of the ejection proteins to the mechanisms of genome ejection, which are exceedingly complex and use the host’s machinery.

Multiple pathogenic types or serotypes restrict treatment for colibacillosis. In addition, rising antibiotic resistance has heightened public awareness to prevent and control pathogenic Escherichia coli. The bacteriophage is a viable technique to treat colibacillosis as an alternative to antibiotics. In this study, PH444, a relatively broad-spectrum obligate lytic phage, was screened from 48 Shiga toxin-producing Escherichia coli (STEC) phages isolated from farm manure samples and sewage samples in order to conduct genome-wide analysis, biological characterization, and a bacterial challenge experiment in milk. The results demonstrated that PH444 was a T7-like phage with a double-stranded DNA of 115,111 bp that belongs to the Kuravirus and was stable at temperatures between 4 and 50 °C and a pH range of 3 to 11. After adding PH444, the bacterial load in milk could be reduced from 3 × 10 3 PFU/ mL to zero within 1 h. In consideration of the biological properties of phage PH444, it was, therefore, demonstrated that PH444 has the potential to be used in phage biocontrol., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The revolution in cryo-electron microscopy has resulted in unprecedented power to resolve large macromolecular complexes including viruses. Many methods exist to explain density corresponding to proteins and thus entire protein capsids have been solved at the all-atom level. However methods for nucleic acids lag behind, and no all-atom viral double-stranded DNA genomes have been published at all. We here present a method which exploits the spiral winding patterns of DNA in icosahedral capsids. The method quickly generates shells of DNA wound in user-specified, idealized spherical or cylindrical spirals. For transition regions, the method allows guided semiflexible fitting. For the kuravirus SU10, our method explains most of the density in a semiautomated fashion. The results suggest rules for DNA turns in the end caps under which two discrete parameters determine the capsid inner diameter. We suggest that other kuraviruses viruses may follow the same winding scheme, producing a discrete rather than continuous spectrum of capsid inner diameters. Our software may be used to explain the published density maps of other double-stranded DNA viruses and uncover their genome packaging principles., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Coat proteins of tailed, dsDNA phages and in herpesviruses include a conserved core similar to the bacteriophage HK97 subunit. This core is often embellished with other domains such as the telokin Ig-like domain of phage P22. Eighty-six P22-like phages and prophages with sequenced genomes share a similar set of virion assembly genes and, based on comparisons of twelve viral assembly proteins (structural and assembly/packaging chaperones), these phages are classified into three groups (P22-like, Sf6-like, and CUS-3-like). We used cryo-electron microscopy and 3D image reconstruction to determine the structures of Sf6 procapsids and virions (~7Å resolution), and the structure of the entire, asymmetric Sf6 virion (16-Å resolution). The Sf6 coat protein is similar to that of P22 yet it has differences in the telokin domain and in its overall quaternary organization. Thermal stability and agarose gel experiments show that Sf6 virions are slightly less stable than those of P22. Finally, bacterial host outer membrane proteins A and C were identified in lipid vesicles that co-purify with Sf6 particles, but are not components of the capsid.

Phage 3A_8767 is a newly isolates phage from river water sample against Salmonella typhi 8767 (MTCC). The genome of the phage is linear, double stranded and 38,821 bp long in size. A total 49 functional ORF (open reading frame) were annotated and no tRNA was predicted. Phage 3A_8767 has icosahedral shaped head with stubby tail which comes under family Podoviridae, and genera T7 like virus.

Bacteriophages have potential for use as biological control agents (biocontrols) of pathogenic bacteria, but their low stability is limiting for their utilization as biocontrols. Understanding of the conditions conducive to storage of phages in which infectivity is maintained over long periods will be useful for their application as biocontrols. We employed a nanomechanical approach to study how external environmental factors affect surface properties and infectivity of the podovirus C22 phage, a candidate for biocontrol of Ralstonia solanacearum, the agent of bacterial wilt in crops. We performed atomic force microscopy (AFM)-based nano-indentation on the C22 phage in buffers with varying pH and ionic strength. The infectivity data from plaque assay in the same conditions revealed that an intermediate range of stiffness was associated with phage titer that remained consistently high, even after prolonged storage up to 182 days. The data are consistent with the model that C22 phage must adopt a metastable state for maximal infectivity, and external factors that alter the stiffness of the phage capsid lead to perturbation of this infective state. [ABSTRACT FROM AUTHOR]

Five bacteriophages were isolated from kimchi with the hosts of two Weissella cibaria and acid-producing bacteria, and characterized for understanding Weissella phage. By transmission electron microscope, фWC51 and фWC52 belonged to the Myoviridae and фWC005, фWC130, and фWC248 belonged to the Podoviridae. One-step growth curves showed that latent periods of bacteriophages ranged from 70 to 90 min and burst sizes ranged from 7 to 195 particles/cell. фWC51 and фWC005 were thermally resistant than the others and those D values were 71 and 34 s at 70 °C. pH stability test at pH 2.0–4.0 showed фWC005 was most stable and the others were also stable at low pH of 3.0. Interestingly, the phages showed two types of morphology and had a high tolerance under acidic condition, therefore, which might cause mortality to the Weissella after middle stage of kimchi fermentation. [ABSTRACT FROM AUTHOR]

Using different Sinorhizobium meliloti strains as hosts, we isolated eight new virulent phages from the rhizosphere of the coastal legume Medicago marina. Half of the isolated phages showed a very narrow host range while the other half exhibited a wider host range within the strains tested. Electron microscopy studies showed that phages M_ort18, M_sf1.2, and M_sf3.33 belonged to the Myoviridae family with feature long, contractile tails and icosaedral head. Phages I_sf3.21 and I_sf3.10T appeared to have filamentous shape and produced turbid plaques, which is a characteristic of phages from the Inoviridae family. Phage P_ort11 is a member of the Podoviridae , with an icosahedral head and a short tail and was selected for further characterization and genome sequencing. P_ort11 contained linear, double-stranded DNA with a length of 75239 bp and 103 putative open reading frames. BLASTP analysis revealed strong similarities to Escherichia phage N4 and other N4-like phages. This is the first report of filamentous and N4-like phages that infect S. meliloti. [ABSTRACT FROM AUTHOR]

Edwardsiella tarda phage (ETP-1) was isolated from marine fish farm water to characterize its effect against pathogenic multidrug-resistant E. tarda. According to transmission electron microscopy results, ETP-1 is classified as a member of the family Podoviridae. ETP-1 showed MOI dependent E. tarda growth inhibition, a latent period of 60 min, and burst size of 100 PFU per infected cells. In host range tests, five out of eight E. tarda strains were sensitive to ETP-1 which had efficiency of plating index in the range 1–1.28. ETP-1 was stable over a broad range of pH and temperature. The size of the ETP-1 genome was predicted to be approximately 40 kb. Zebrafish exposed to ETP-1 showed no adverse gene responses to the inflammatory mediator cytokines, il1-β , tnf-α , il-6 , and il-10 , the chemokine, cxcl-8a , and reactive oxygen species, sod-1. When zebrafish were bath exposed to ETP-1 for 12 days and simultaneously challenged with E. tarda (1.08 × 105 CFU fish−1), the survival rate was higher in phage exposed fish (68%) compared to that of the control (18%) until 4 days post challenge. Our results suggest that ETP-1 can be used as a potential bio-therapeutic candidate to control multi-drug resistant E. tarda infection in aquaculture. [ABSTRACT FROM AUTHOR]

Enterotoxigenic Escherichia coli (ETEC) strains are an important cause of bacterial diarrheal illness in humans and animals. Infections arising from ETEC could potentially be treated through the use of bacteriophage (phage) therapy, as phages encode for enzymes capable of bacterial cell lysis. vB_EcoP_SU7 was isolated from the Käppala wastewater treatment plant in Stockholm, Sweden, and propagated on an ETEC strain exhibiting the O:139 serovar. Transmission electron microscopy confirmed that vB_EcoP_SU7 belongs to the Podoviridae family and has the rare C3 morphotype of an elongated head. Bioinformatic analyses showed that the genome was 76,626 base pairs long and contained 35 genes with predicted functions. A total of 81 open reading frames encoding proteins with hypothetical function and two encoding proteins of no significant similarity were also found. A putative tRNA gene, which may aid in vB_EcoP_SU7’s translation, was also identified. Phylogenetic analyses showed that compared to other Podoviridae, vB_EcoP_SU7 is a rare Kuravirus and is closely related to E. coli phages with the uncommon C3 morphotype, such as ECBP2, EK010, vB_EcoP_EcoN5, and vB_EcoP_SU10. Phage vB_EcoP_SU7 has a narrow host range, infecting 11 out of the 137 E. coli strains tested, a latency period of 30 min, a burst size of 12 PFU/cell, and an adsorption rate of 8.78 × 10−9 mL/min five minutes post infection. With a limited host range and poor infection kinetics, it is unlikely that SU7 can be a standalone phage used for therapeutic purposes; rather, it must be used in combination with other phages for broad-spectrum therapeutic success.

Phage therapy is now reconsidered with interest in the treatment of bacterial infections. A major piece of information for this application is the definition of the molecular targets exploited by phages to infect bacteria. Here, the genetic basis of resistance to the lytic phage φBO1E by its susceptible host Klebsiella pneumoniae KKBO-1 has been investigated. KKBO-1 phage-resistant mutants were obtained by infection at high multiplicity. One mutant, designated BO-FR-1, was selected for subsequent experiments, including virulence assessment in a Galleria mellonella infection model and characterization by whole-genome sequencing. Infection with BO-FR-1 was associated with a significantly lower mortality when compared to that of the parental strain. The BO-FR-1 genome differed from KKBO-1 by a single nonsense mutation into the wbaP gene, which encodes a glycosyltransferase involved in the first step of the biosynthesis of the capsular polysaccharide (CPS). Phage susceptibility was restored when BO-FR-1 was complemented with the constitutive wbaP gene. Our results demonstrated that φBO1E infects KKBO-1 targeting the bacterial CPS. Interestingly, BO-FR-1 was less virulent than the parental strain, suggesting that in the context of the interplay among phage, bacterial pathogen and host, the emergence of phage resistance may be beneficial for the host.

Background: Bacteriophage SU10, from the family Podoviridae, infects a wide range of E. coli strains. The phage has 77kbp dsDNA genome, prolate capsid with the length of 135 nm and the diameter of 42 nm. Contrary to what was observed in most Podoviridae phages, bacteriophage SU10 has 27 nm long tail. Furthermore, the baseplate of SU10 changes conformation upon attachment to the host. Methods: We used cryo-electron microscopy and localized single particle reconstruction techniques to determine the structure of capsid, portal, tail and base plate of SU10. Furthermore, we also characterized their conformational changes associated with cell-wall binding and genome ejection. Results: Prolate capsid is formed by 11 pentons and 110 hexons of major capsid protein. Major capsid protein has HK97 fold. The dodecameric portal complex has prolonged crown-barrel, similar to that of phage P22. Three types of fibers, all present in six copies, decorate the phage particle. Collar fibers are connected to the neck protein. Long tail fibers connected to the upper tail protein are similar to that of phage T4 and short tail fibers are positioned on bottom part of the base plate. Conclusion: Our studies of structural changes of tail and base plate extend the current knowledge of the mechanism of host recognition and genome delivery of bacteriophage SU10.

Yersinia enterocolitica is a major agent of foodborne diseases worldwide. Prophage plays an important role in the genetic evolution of the bacterial genome. Little is known about the genetic information about prophages in the genome of Y. enterocolitica, and no pathogenic Y. enterocolitica prophages have been described. In this study, we induced and described the genomes of six prophages from pathogenic Y. enterocolitica for the first time. Phylogenetic analysis based on whole genome sequencing revealed that these novel Yersinia phages are genetically distinct from the previously reported phages, showing considerable genetic diversity. Interestingly, the prophages induced from O:3 and O:9 Y. enterocolitica showed different genomic sequences and morphology but highly conserved among the same serotype strains, which classified into two diverse clusters. The three long-tailed Myoviridae prophages induced from serotype O:3 Y. enterocolitica were highly conserved, shared ≥99.99% identity and forming genotypic cluster A; the three Podoviridae prophages induced from the serotype O:9 strains formed cluster B, also shared more than 99.90% identity with one another. Cluster A was most closely related to O:5 non-pathogenic Y. enterocolitica prophage PY54 (61.72% identity). The genetic polymorphism of these two kinds of prophages and highly conserved among the same serotype strains, suggested a possible shared evolutionary past for these phages: originated from distinct ancestors, and entered pathogenic Y. enterocolitica as extrachromosomal genetic components during evolution when facing selective pressure. These results are critically important for further understanding of phage roles in host physiology and the pathology of disease.

Infections by multidrug resistant bacteria (MDR) are becoming increasingly difficult to treat and alternative approaches like phage therapy, which is unhindered by drug resistance, are urgently needed to tackle MDR bacterial infections. During phage therapy phage cocktails targeting different receptors are likely to be more effective than monophages. In the present study, phages targeting carbapenem resistant clinical isolate of E. coli U1007 was isolated from Ganges River (U1G), Cooum River (CR) and Hospital waste water (M). Capsid architecture discerned using TEM identified the phage families as Podoviridae for U1G, Myoviridae for CR and Siphoviridae for M phage. Genome sequencing showed the phage genomes varied in size U1G (73,275 bp) CR (45,236 bp) and M (45,294 bp). All three genomes lacked genes encoding tRNA sequence, antibiotic resistant or virulent genes. A machine learning (ML) based multi-class classification model using Random Forest, Logistic Regression, and Decision Tree were employed to predict the host receptor targeted by receptor binding protein of all 3 phages and the best performing algorithm Random Forest predicted LPS O antigen, LamB or OmpC for U1G; FhuA, OmpC for CR phage; and FhuA, LamB, TonB or OmpF for the M phage. OmpC was validated as receptor for U1G by physiological experiments. In vivo intramuscular infection study in zebrafish showed that cocktail of dual phages (U1G + M) along with colsitin resulted in a significant 3.5 log decline in cell counts. Our study highlights the potential of ML tool to predict host receptor and proves the utility of phage cocktail to restrict E. coli U1007 in vivo., (© 2023. The Author(s).)

Bacteriophages or phages used as an alternative therapy for treating multi-drug resistant infections require formulation consideration. Current strategies to produce phage formulations involving organic solvents are based on empirical practices without a good understanding of phage stability during formulation development. In this study, we investigated the effect of common formulation organic solvents (ethanol, isopropyl alcohol, tetrahydrofuran (THF) and dimethyl sulfoxide (DMSO)) on the stability of Pseudomonas aeruginosa-specific myovirus (PEV1, PEV20) and podovirus (PEV31) phages using biological assay, transmission electron microscopy (TEM) and scattering near field optical microscopy (SNOM). The three phages were mixed with the solvents at different concentrations (25%, 50%, and 75% (v/v)) for 20 min. All phages were fully viable in the organic solvents at 25% (v/v) showing negligible titre changes. At the higher solvent concentration of 50% (v/v), the myoviruses PEV1 and PEV20 remained relatively stable (titre loss 0.4-1.3 log 10 ), whereas the podovirus PEV31 became less stable (titre loss 0.25-3.8 log 10 ), depending on the solvent used. Increasing the solvent level to 75% (v/v) caused increased morphological changes in TEM and decreased viability as indicated by the titre loss (0.32-7.4 log 10 ), with DMSO being the most phage-destabilising solvent. SNOM spectra showed differences in the signal intensity and peak positions in the amide I and amide II regions, revealing altered phage proteins by the solvents. In conclusion, the choice of the solvents for phage formulation depends on both the phages and solvent types. Our results showed (1) the phages are more stable in the alcohols than DMSO and THF, and (2) the myoviruses tend to be more stable than the podovirus in the solvents. Overall, a low to moderate (25-50 % v/v) level of organic solvents (except 50% THF) can be used in formulation of the phages without a substantial titre loss., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Acinetobacter nosocomialis (A. nosocomialis) is a glucose non-fermentative, gram-negative bacillus that belongs to the Acinetobacter calcoaceticus-baumannii complex. In recent years, studies have found an increased clinical prevalence of A. nosocomialis. However, given the increasing trend of antibiotic resistance, developing new antibacterial agents is vital. Currently, research regarding bacteriophage therapy against A. nosocomialis is only limited., Methods: Two A. nosocomialis bacteriophages, TCUAN1 and TCUAN2, were isolated from sewage. Experiments such as transmission electron microscopy (TEM), host-range analysis, and sequencing were performed to determine their biological and genomic characteristics. TCUAN2 were further subjected to in vivo experiments and their derived-endolysin were cloned and tested against their bacteria host., Results: Transmission electron microscopy revealed that TCUAN1 and TCUAN2 belong to Myoviridae and Podoviridae, respectively. Both phages show a broad host spectrum and rapid adsorption efficiency. Further biological analysis showed that TCUAN2 possesses a shorter latent period and larger burst size compared to TCUAN1. Because TCUAN2 showed a better antibacterial activity, it was injected into A. nosocomialis-infected mice which resulted in a significant decrease in bacterial load levels in the blood and increased the mice's survival. Finally, genomic analysis revealed that the complete nucleotide sequence of TCUAN1 is 49, 691 bps (containing 75 open reading frames) with a G + C content of 39.3%; whereas the complete nucleotide sequence of TCUAN2 is 41, 815 bps (containing 68 open reading frames) with a G + C content of 39.1%. The endolysin gene cloned and purified from TCUAN2 also showed antibacterial activity when used with a chelator EDTA., Competing Interests: Declaration of competing interest All authors declare that they have no conflict of interest., (Copyright © 2023. Published by Elsevier B.V.)

Bacteriophage (phage) taxonomy has been in flux since its inception over four decades ago. Genome sequencing has put pressure on the classification system and recent years have seen significant changes to phage taxonomy. Here, we reflect on the state of phage taxonomy and provide a roadmap for the future, including the abolition of the order Caudovirales and the families Myoviridae, Podoviridae, and Siphoviridae. Furthermore, we specify guidelines for the demarcation of species, genus, subfamily and family-level ranks of tailed phage taxonomy.

A lytic phage (CF7) specific to Aeromonas hydrophilawas isolated and characterized from the aquaculture pond. Phage isolation and host range determination were carried out by soft agar overlay assay. Phage morphology was determined by transmission electron microscopy (TEM). The multiplicity of infection, phage adsorption rate to the host cell and burst size were also determined. Phage genome was subjected to next-generation sequencing (NGS) and de novo assembly. Stability of phage preparations at various storage temperatures was studied over a period of one year. Based on TEM analysis, CF7 belonged to family Podovirdae.CF7 also had a very narrow host range with no lytic activity against other bacterial isolates. Adsorption rate and burst size were determined to be 69.7% and 93±6, respectively.NGS and de novo assembly generated phage genome of 42,439 bp with G+C content of 58.95%. Proteins involved in major functions such as phage structure formation and packaging, DNA replication and repair, DNA transcription and host cell lysis were encoded by the phage genome. Phage titers in preparations stored at 4oC declined by only one log10 unit over a period of one year. During storage at -80°C, without or with (15%, 30%, and 45%) glycerol, phage titers declined by <2 log10 units over the same period. The very narrow host range of CF7 suggests that it could be a good candidate for phage typing applications. Genes encoding for holin and endolysin could be ideal candidates for recombinant cloning and expression of potentially antimicrobial proteins. [ABSTRACT FROM AUTHOR]

Yersinia enterocolitica is a major agent of foodborne diseases worldwide. Prophage plays an important role in the genetic evolution of the bacterial genome. Little is known about the genetic information about prophages in the genome of Y. enterocolitica , and no pathogenic Y. enterocolitica prophages have been described. In this study, we induced and described the genomes of six prophages from pathogenic Y. enterocolitica for the first time. Phylogenetic analysis based on whole genome sequencing revealed that these novel Yersinia phages are genetically distinct from the previously reported phages, showing considerable genetic diversity. Interestingly, the prophages induced from O:3 and O:9 Y. enterocolitica showed different genomic sequences and morphology but highly conserved among the same serotype strains, which classified into two diverse clusters. The three long-tailed Myoviridae prophages induced from serotype O:3 Y. enterocolitica were highly conserved, shared ≥99.99% identity and forming genotypic cluster A; the three Podoviridae prophages induced from the serotype O:9 strains formed cluster B, also shared more than 99.90% identity with one another. Cluster A was most closely related to O:5 non-pathogenic Y. enterocolitica prophage PY54 (61.72% identity). The genetic polymorphism of these two kinds of prophages and highly conserved among the same serotype strains, suggested a possible shared evolutionary past for these phages: originated from distinct ancestors, and entered pathogenic Y. enterocolitica as extrachromosomal genetic components during evolution when facing selective pressure. These results are critically important for further understanding of phage roles in host physiology and the pathology of disease. [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract It was previously demonstrated that the loss of infectivity of a myovirus PEV44 after jet nebulization was closely related to a change in bacteriophage (phage) structure. In this follow-up study, we further examined the impact of jet nebulization on tailed phages, which constitute 96% of all known phages, from three different families, Podoviridae (PEV2), Myoviridae (PEV40) and Siphoviridae (D29). Transmission electron microscopy (TEM) identified major changes in phage structures after jet nebulization, correlating with their loss of infectivity. For the podovirus PEV2 , jet nebulization had a negligible impact on its activity (0.04 log 10 pfu/mL loss) and structural change. On the other hand, the proportion of intact phages in the nebulized samples dropped from 50% to ∼27% for PEV40 and from 15% to ∼2% for D29. Phage deactivation of PEV40 measured by the TEM structural damage (0.52 log 10 pfu/mL) was lower than that obtained by plaque assay (1.02 log 10 pfu/mL), but within the range of variation (±0.5 log 10 pfu/mL). However, TEM quantification considerably underestimated the titer reduction of D29 phage, ∼2 log pfu/mL lower than that obtained in plaque assay (3.25 log 10 pfu/mL loss). In conclusion, nebulization-induced titre loss was correlated with morphological damage to phages and in particular, the tail length may be an important consideration for selection of phages in inhaled therapy using jet nebulization. [ABSTRACT FROM AUTHOR]

A lytic Ralstonia solanacearum-infecting phage designated Ralstonia phage RsoP1IDN was isolated from soil in Indonesia. The phage has a linear double-stranded DNA genome of 41,135 bp with 413-bp terminal repeats, and contains 41 annotated open reading frames. The phage is most closely related to Ralstonia phage RSB1, but different from RSB1 mainly in containing a putative HNH homing endonuclease and having a narrower host range. Our phylogenetic and genomic analyses revealed that both phages RsoP1IDN and RSB1 belong to the genus Pradovirus or a new genus, and not Phikmvvirus as previously reported for phage RSB1. RsoP1IDN is the first sequenced and characterized R. solanacearum-infecting phage isolated from Indonesia in the proposed species Ralstonia virus RsoP1IDN. [ABSTRACT FROM AUTHOR]

This project sought to investigate the domestic caprid rumen virome by developing a robust viral DNA isolation and enrichment protocol (utilizing membrane filtration, ultra-centrifugation, overnight PEG treatment and nuclease treatment) and using RSD-PCR and high throughput sequencing (HTS) techniques. 3.53% of the reads obtained were analogous to those of viruses denoting Siphoviridae, Myoviridae, Podoviridae, Mimiviridae, Microviridae, Poxviridae, Tectiviridae and Marseillevirus. Most of the sequenced reads from the rumen were similar to those of phages, which are critical in maintaining the rumen microbial populations under its carrying capacity. Though identified in the rumen, most of these viruses have been reported in other environments as well. Improvements in the viral DNA enrichment and isolation protocol are required to obtain data that are more representative of the rumen virome. The 102,130 unknown reads (92.31%) for the goat and 36,241 unknown reads (93.86%) for the sheep obtained may represent novel genomes that need further study. [ABSTRACT FROM AUTHOR]

Abstract Ballast water is a common vector for the transport of invasive species to new marine and aquatic environments. We used a metagenomics approach to examine the composition and diversity of viral communities in ballast water from ships originating in Mexico, Saudi Arabia, New York, and Panama, and in water from the port of their destination in Busan, Korea. Myoviridae was the most abundant virus family in ballast water, followed Podoviridae and Siphoviridae. We also identified viruses that infect invertebrates, amoebas, and algae in ballast water and in the Busan port water. Interestingly, there were several viruses that infect humans or other animals (Swinepox virus , Raccoonpox virus , Suid herpesvirus , and Human endogenous retrovirus) in the samples from New York and Panama. In addition, there were giant viruses in all the ballast water samples, especially, identified Megavirus chilensis in New York and Panama, and Pandoravirus salinus in Mexico and Saudi Arabia. These results provide detailed descriptions of the characteristics of the viruses present in ballast water, document significant viral diversity, and indicate the potential translocation of viruses via ballast water. Highlights • Viral diversity and composition in ballast water samples • Comparison of the ballast water and port water samples • Potential translocation of viruses via ballast water [ABSTRACT FROM AUTHOR]

Phage therapy is the use of lytic bacteriophages to cure infections caused by bacteria. The aim of this study is to isolate and to characterize the bacteriophages against Escherichia coli isolated from clinical samples. For isolation of bacteriophages, water samples were collected from the Ganges River, and phage enrichment method was followed for phage isolation. Microbiological, genomic and lyophilization experiments were carried out to characterize the bacteriophage. Galleria mellonella was used to study the potential of phages against E. coli infection. Escherichia phage myPSH1131 belonging to Podoviridae family and found to have broad host range infectivity (n = 31) to infect Enterohemorrhagic E. coli (n = 9), Enteropathogenic E. coli (n = 6), Enterotoxigenic E. coli (n = 3), Enteroaggregative E. coli (n = 3), Uropathogenic E. coli (n = 9) and one unknown E. coli. The genome size is 76,163 base pairs (97 coding regions) and their genes show high similarity to SU10 phage. Lyophilization studies showed that the use of 1M sucrose, 2% gelatin and the combination of both 0.5M sucrose plus 1% gelatin could restore phage viability up to 20 months at 4°C. For in vivo studies, it was observed that a single phage dose can reduce the E. coli infection but to achieve 100% survival rate the infected larvae should be treated with three phage doses (20 μL, 103 PFU/mL) at 6 hours interval. The characterized Escherichia phage myPSH1131 was found to have broad host range activity against E. coli pathogens and in vivo studies showed that multiple doses are required for effective treatment. [ABSTRACT FROM AUTHOR]