Paenibacillus larvae is a Firmicute bacterium that causes American Foulbrood, a lethal disease in honeybees and is a major source of global agricultural losses. Although P. larvae phages were isolated prior to 2013, no full genome sequences of P. larvae bacteriophages were published or analyzed. This report includes an in-depth analysis of the structure, genomes, and relatedness of P. larvae myoviruses Abouo, Davis, Emery, Jimmer1, Jimmer2, and siphovirus phiIBB_Pl23 to each other and to other known phages.

Temperate phages are bacterial viruses that as part of their life cycle reside in the bacterial genome as prophages. They are found in many species including most clinical strains of the human pathogens, Staphylococcus aureus and Salmonella enterica serovar Typhimurium. Previously, temperate phages were considered as only bacterial predators, but mounting evidence point to both antagonistic and mutualistic interactions with for example some temperate phages contributing to virulence by encoding virulence factors. Here we show that generalized transduction, one type of bacterial DNA transfer by phages, can create conditions where not only the recipient host but also the transducing phage benefit. With antibiotic resistance as a model trait we used individual-based models and experimental approaches to show that antibiotic susceptible cells become resistant to both antibiotics and phage by i) integrating the generalized transducing temperate phages and ii) acquiring transducing phage particles carrying antibiotic resistance genes obtained from resistant cells in the environment. This is not observed for non-generalized transducing temperate phages, which are unable to package bacterial DNA, nor for generalized transducing virulent phages that do not form lysogens. Once established, the lysogenic host and the prophage benefit from the existence of transducing particles that can shuffle bacterial genes between lysogens and for example disseminate resistance to antibiotics, a trait not encoded by the phage. This facilitates bacterial survival and leads to phage population growth. We propose that generalized transduction can function as a mutualistic trait where temperate phages cooperate with their hosts to survive in rapidly-changing environments. This implies that generalized transduction is not just an error in DNA packaging but is selected for by phages to ensure their survival.

Bacteriophages (phages, bacteria-specific viruses) have biotechnological and therapeutic potential. To apply phages as pure or heterogeneous mixtures, it is essential to have a robust mechanism for transport and storage, with different phages having very different stability profiles across storage conditions. For many biologics, cryopreservation is employed for long-term storage and cryoprotectants are essential to mitigate cold-induced damage. Here, we report that poly(ethylene glycol) can be used to protect phages from cold damage, functioning at just 10 mg·mL-1 (∼1 wt %) and outperforms glycerol in many cases, which is a currently used cryoprotectant. Protection is afforded at both -20 and -80 °C, the two most common temperatures for frozen storage in laboratory settings. Crucially, the concentration of the polymer required leads to frozen solutions at -20 °C, unlike 50% glycerol (which results in liquid solutions). Post-thaw recoveries close to 100% plaque-forming units were achieved even after 2 weeks of storage with this method and kill assays against their bacterial host confirmed the lytic function of the phages. Initial experiments with other hydrophilic polymers also showed cryoprotection, but at this stage, the exact mechanism of this protection cannot be concluded but does show that water-soluble polymers offer an alternative tool for phage storage. Ice recrystallization inhibiting polymers (poly(vinyl alcohol)) were found to provide no additional protection, in contrast to their ability to protect proteins and microorganisms which are damaged by recrystallization. PEG's low cost, solubility, well-established low toxicity/immunogenicity, and that it is fit for human consumption at the concentrations used make it ideal to help translate new approaches for phage therapy.

The human gut is a complex environment that contains a multitude of microorganisms that are collectively termed the microbiome. Multiple factors have a role to play in driving the composition of human gut bacterial communities either toward homeostasis or the instability that is associated with many disease states. One of the most important forces are likely to be bacteriophages, bacteria-infecting viruses that constitute by far the largest portion of the human gut virome. Despite this, bacteriophages (phages) are the one of the least studied residents of the gut. This is largely due to the challenges associated with studying these difficult to culture entities. Modern high throughput sequencing technologies have played an important role in improving our understanding of the human gut phageome but much of the generated sequencing data remains uncharacterised. Overcoming this requires database-independent bioinformatic pipelines and even those phages that are successfully characterized only provide limited insight into their associated biological properties, and thus most viral sequences have been characterized as "viral dark matter." Fundamental to understanding the role of phages in shaping the human gut microbiome, and in turn perhaps influencing human health, is how they interact with their bacterial hosts. An essential aspect is the isolation of novel phage-bacteria host pairs by direct isolation through various screening methods, which can transform in silico phages into a biological reality. However, this is also beset with multiple challenges including culturing difficulties and the use of traditional methods, such as plaquing, which may bias which phage-host pairs that can be successfully isolated. Phage-bacteria interactions may be influenced by many aspects of complex human gut biology which can be difficult to reproduce under laboratory conditions. Here we discuss some of the main findings associated with the human gut phageome to date including composition, our understanding of phage-host interactions, particularly the observed persistence of virulent phages and their hosts, as well as factors that may influence these highly intricate relationships. We also discuss current methodologies and bottlenecks hindering progression in this field and identify potential steps that may be useful in overcoming these hurdles.

Increased intestinal permeability and translocation of gut bacteria trigger various polyaetiological diseases associated with chronic inflammation and underlie a variety of poorly treatable pathologies. Previous studies have established a primary role of the microbiota composition and intestinal permeability in such pathologies. Using a rat model, we examined the effects of exposure to a bacteriophage cocktail on intestinal permeability and relative abundance of taxonomic units in the gut bacterial community. There was an increase in markers of impaired gut permeability, such as the lactulose/mannitol ratio, plasma endotoxin concentrations, and serum levels of inflammation-related cytokines, following the bacteriophage challenge. We observed significant differences in the alpha diversity of faecal bacterial species and found that richness and diversity index values increased following the bacteriophage challenge. There was a reduction in the abundance of Blautia, Catenibacterium, Lactobacillus, and Faecalibacterium species and an increase in Butyrivibrio, Oscillospira and Ruminococcus after bacteriophage administration. These findings provide novel insights into the role of bacteriophages as potentially pathogenic for mammals and their possible implication in the development of diseases associated with increased intestinal permeability.

Members of the genus Acinetobacter are ubiquitous in the environment and the multiple-drug resistant species A. baumannii is of significant clinical concern. This clinical relevance is currently driving research on bacterial viruses infecting A. baumannii, in an effort to implement phage therapy and phage-derived antimicrobials. Initially, a total of 42 Acinetobacter phage genome sequences were available in the international nucleotide sequence databases, corresponding to a total of 2.87 Mbp of sequence information and representing all three families of the order Caudovirales and a single member of the Leviviridae. A comparative bioinformatics analysis of 37 Acinetobacter phages revealed that they form six discrete clusters and two singletons based on genomic organisation and nucleotide sequence identity. The assignment of these phages to clusters was further supported by proteomic relationships established using OrthoMCL. The 4067 proteins encoded by the 37 phage genomes formed 737 groups and 974 orphans. Notably, over half of the proteins encoded by the Acinetobacter phages are of unknown function. The comparative analysis and clustering presented enables an updated taxonomic framing of these clades.

Pectobacterium atrosepticum is an economically important phytopathogen that is responsible for potato blackleg and soft rot, and for which current control strategies are limited. In this study, stem samples of potato crops exhibiting blackleg were taken from three farms in Co. Cork, Ireland, and they were found to be infected with P. atrosepticum. Three closely related bacteriophages (phages) that are specific to this phytopathogen were isolated and characterized, namely vB_PatP_CB1, vB_PatP_CB3, and vB_PatP_CB4 (abbreviated as CB1, CB3, and CB4). Both CB1 and CB3 were determined to infect 12 strains and CB4 10 strains of the 19 strains of P. atrosepticum tested. Morphology, latent periods, burst sizes, and their stability at various temperatures and pHs were also examined. Genome sequencing of the three phages revealed that they shared a minimum nucleotide identity of 93% with each other. Their genomes exhibited an Enquartavirinae genome organization, possessing several conserved proteins that were associated with phages of this group, like the type species Escherichia virus N4. Tandem electrospray ionization-mass spectrometry (ESI-MS/MS) allowed for the identification of ten structural proteins that form the virion of CB1, six that are conserved in phage N4. Biocontrol experiments demonstrated that the phages suppress soft rot formation upon co-inoculation with P. atrosepticum on whole tubers. The results of this study indicate that CB1 related phages could be good candidates for phage-based control.

Bacterial biofilm provides bacteria with resistance and protection against conventional antimicrobial agents and the host immune system. Bacteriophages are known to move across the biofilm to make it permeable to antimicrobials. Mineral hydroxyapatite (HA) can improve the lytic activity of bacteriophages, and, together with eicosanoic acid (C20:0), can destroy the biofilm structure. Here, we demonstrate the efficacy of the combined use of phage, HA and C20:0 against Xanthomonas campestris pv campestris (Xcc) biofilm. We used nuclear magnetic resonance (NMR)-based metabolomics to investigate the molecular determinants related to the lytic action, aiming at identifying the metabolic pathways dysregulated by phage treatment. Furthermore, we identified specific markers (amino acids, lactate and galactomannan) which are involved in the control of biofilm stability. Our data show that Xccφ1, alone or in combination with HA and C20:0, interferes with the metabolic pathways involved in biofilm formation. The approach described here might be extended to other biofilm-producing bacteria.

Bacteriophages are an important repository of genetic diversity. As one of the major constituents of terrestrial biomass, they exert profound effects on the earth's ecology and microbial evolution by mediating horizontal gene transfer between bacteria and controlling their growth. Only limited genomic sequence data are currently available for phages but even this reveals an overwhelming diversity in their gene sequences and genomes. The contribution of the T4-like phages to this overall phage diversity is difficult to assess, since only a few examples of complete genome sequence exist for these phages. Our analysis of five T4-like genomes represents half of the known T4-like genomes in GenBank.

Bacteriophages are the most abundant biological entity on the planet, having pivotal roles in bacterial ecology, animal and plant health, and in the biogeochemical cycles. Although, in principle, phages are simple entities that replicate at the expense of their bacterial hosts, due the importance of bacteria in all aspects of nature, they have the potential to influence and modify diverse processes, either in subtle or profound ways. Traditionally, the main application of bacteriophages is phage therapy, which is their utilization to combat and help to clear bacterial infections, from enteric diseases, to skin infections, chronic infections, sepsis, etc. Nevertheless, phages can also be potentially used for several other tasks, including food preservation, disinfection of surfaces, treatment of several dysbioses, and modulation of microbiomes. Phages may also be used as tools for the treatment of non-bacterial infections and pest control in agriculture; moreover, they can be used to decrease bacterial virulence and antibiotic resistance and even to combat global warming. In this review manuscript we discuss these possible applications and promote their implementation.

The antibiotic-resistant bacterium Paenibacillus larvae is the causative agent of American foulbrood (AFB), currently the most destructive bacterial disease in honeybees. Phages that infect P. larvae were isolated as early as the 1950s, but it is only in recent years that P. larvae phage genomes have been sequenced and annotated. In this study we analyze the genomes of all 48 currently sequenced P. larvae phage genomes and classify them into four clusters and a singleton. The majority of P. larvae phage genomes are in the 38⁻45 kbp range and use the cohesive ends (cos) DNA-packaging strategy, while a minority have genomes in the 50⁻55 kbp range that use the direct terminal repeat (DTR) DNA-packaging strategy. The DTR phages form a distinct cluster, while the cos phages form three clusters and a singleton. Putative functions were identified for about half of all phage proteins. Structural and assembly proteins are located at the front of the genome and tend to be conserved within clusters, whereas regulatory and replication proteins are located in the middle and rear of the genome and are not conserved, even within clusters. All P. larvae phage genomes contain a conserved N-acetylmuramoyl-l-alanine amidase that serves as an endolysin.

Paenibacillus larvae is a Gram-positive, spore-forming bacterium that is the causative agent of American foulbrood (AFB), the most devastating bacterial disease of the honeybee. P. larvae is antibiotic resistant, complicating treatment efforts. Bacteriophages that target P. larvae are rapidly emerging as a promising treatment. The first P. larvae phages were isolated in the 1950s, but as P. larvae was not antibiotic resistant at the time, interest in them remained scant. Interest in P. larvae phages has grown rapidly since the first P. larvae phage genome was sequenced in 2013. Since then, the number of sequenced P. larvae phage genomes has reached 48 and is set to grow further. All sequenced P. larvae phages encode a conserved N-acetylmuramoyl-l-alanine amidase that is responsible for cleaving the peptidoglycan cell wall of P. larvae. All P. larvae phages also encode either an integrase, excisionase or Cro/CI, indicating that they are temperate. In the last few years, several studies have been published on using P. larvae phages and the P. larvae phage amidase as treatments for AFB. Studies were conducted on infected larvae in vitro and also on hives in the field. The phages have a prophylactic effect, preventing infection, and also a curative effect, helping resolve infection. P. larvae phages have a narrow range, lysing only P. larvae, and are unable to lyse even related Paenibacillus species. P. larvae phages thus appear to be safe to use and effective as treatment for AFB, and interest in them in the coming years will continue to grow.

The genus Paraburkholderia encompasses mostly environmental isolates with diverse predicted lifestyles. Genome analyses have shown that bacteriophages form a considerable portion of some Paraburkholderia genomes. Here, we analyzed the evolutionary history of prophages across all Paraburkholderia spp. Specifically, we investigated to what extent the presence of prophages and their distribution affect the diversity/diversification of Paraburkholderia spp., as well as to what extent phages coevolved with their respective hosts. Particular attention was given to the presence of CRISPR-Cas arrays as a reflection of past interactions with phages. We thus analyzed 36 genomes of Paraburkholderia spp., including those of 11 new strains, next to those of three Burkholderia species. Most genomes were found to contain at least one full prophage sequence. The highest number was found in Paraburkholderia sp. strain MF2-27; the nine prophages found amount to up to 4% of its genome. Among all prophages, potential moron genes (e.g., DNA adenine methylase) were found that might be advantageous for host cell fitness. Co-phylogenetic analyses indicated the existence of complex evolutionary scenarios between the different Paraburkholderia hosts and their prophages, including short-term co-speciation, duplication, host-switching and phage loss events. Analysis of the CRISPR-Cas systems showed a record of diverse, potentially recent, phage infections. We conclude that, overall, different phages have interacted in diverse ways with their Paraburkholderia hosts over evolutionary time.

Bacteriophages (phages) are ubiquitous viruses that control the growth and diversity of bacteria. Although they have no tropism to mammalian cells, accumulated evidence suggests that phages are not neutral to the mammalian macro-host and can promote immunomodulatory and anti-tumorigenic activities. Here we demonstrate that Ff phages that do not display any proteins or peptides could inhibit the growth of subcutaneous glioblastoma tumors in mice and that this activity is mediated in part by lipopolysaccharide molecules attached to their virion. Using the intranasal route, a non-invasive approach to deliver therapeutics directly to the CNS, we further show that phages rapidly accumulate in the brains of mice and could attenuate progression of orthotopic glioblastoma. Taken together, this study provides new insight into phages non-bacterial activities and demonstrates the feasibility of delivering Ff phages intranasally to treat brain malignancies.

Xp10 and OP1 are phages of Xanthomonas oryzae pv. oryzae (Xoo), the causative agent of bacterial leaf blight in rice plants, which were isolated in 1967 in Taiwan and in 1954 in Japan, respectively. We recently isolated the Xoo phage Xop411.

Phage therapy has attracted much attention for the treatment of antibiotic-resistant bacteria in recent years. However, it is common for bacteria to obtain resistance capability in short time after interaction with a lytic phage, as observed in phage therapy and co-culture of host and phage in a lab. In order to understand the mechanisms behind resistance, Staphylococcus aureus AB91118 and its lytic phage LQ7 were studied as a model system. A mutant strain named R1-3-1 resistant to the ancestral phage LQ7 was isolated, and then phages experimentally evolved from LQ7 were able to kill R1-3-1. Genomes of the two bacterial strains and the three phages (LQ7, ELQ7P-10, and ELQ7P-20) were analyzed based on deep sequencing data of NGS. Analyses showed that a few mutations could be identified in R1-3-1 and the evolved phages. Instead, in all the genomes of the bacteria and the phages, there exists genetic polymorphism of minor alleles, which distributes in many functional genes. Specifically, in the AB91118-LQ7 system it was found that the unique polymorphism sites in R1-3-1 associated to metabolic pathways could be inhibited by chloramphenicol (CHL). The resistant mutant R1-3-1 could become sensitive to the phage LQ7 in the presence of CHL. Combined use of CHL and the evolved phage from 20 cycles (ELQ7P-20) could produce the least resistance when killing the bacteria AB91118. The genetic polymorphism of minor alleles would be a new mechanism to drive the co-evolution between a phage and its host, which may enable the phage and the host get ready and fast response to the selective pressure from one to the other.

Wound healing is a dynamic and complex process where infection prevention is essential. Chitosan, thanks to its bactericidal activity against gram-positive and gram-negative bacteria, as well as anti-inflammatory and hemostatic properties, is an excellent candidate to design dressings for difficult-to-heal wound treatment. The great advantage of this biopolymer is its capacity to be chemically modified, which allows for the production of various functional forms, depending on the needs and subsequent use. Moreover, chitosan can be an excellent polymer matrix for bacteriophage (phage) packing as a novel alternative/supportive antibacterial therapy approach. This study is focused on the preparation and characteristics of chitosan-based material in the form of a film with the addition of Pseudomonas lytic phages (KTN4, KT28, and LUZ19), which would exhibit antibacterial activity as a potential dressing that accelerates the wound healing. We investigated the method of producing a polymer based on microcrystalline chitosan (MKCh) to serve as the matrix for phage deposition. We described some important parameters such as average molar mass, swelling capacity, surface morphology, phage release profile, and antibacterial activity tested in the Pseudomonas aeruginosa bacterial model. The chitosan polysaccharide turned out to interact with phage particles immobilizing them within a material matrix. Nevertheless, with the high hydrophilicity and swelling features of the prepared material, the external solution of bacterial culture was absorbed and phages went in direct contact with bacteria causing their lysis in the polymer matrix. KEY POINTS: • A novel chitosan-based matrix with the addition of active phages was prepared • Phage interactions with the chitosan matrix were determined as electrostatic • Phages in the matrix work through direct contact with the bacterial cells.

We have developed phiSITE, database of gene regulation in bacteriophages. To date it contains detailed information about more than 700 experimentally confirmed or predicted regulatory elements (promoters, operators, terminators and attachment sites) from 32 bacteriophages belonging to Siphoviridae, Myoviridae and Podoviridae families. The database is manually curated, the data are collected mainly form scientific papers, cross-referenced with other database resources (EMBL, UniProt, NCBI taxonomy database, NCBI Genome, ICTVdb, PubMed Central) and stored in SQL based database system. The system provides full text search for regulatory elements, graphical visualization of phage genomes and several export options. In addition, visualizations of gene regulatory networks for five phages (Bacillus phage GA-1, Enterobacteria phage lambda, Enterobacteria phage Mu, Enterobacteria phage P2 and Mycoplasma phage P1) have been defined and made available. The phiSITE is accessible at http://www.phisite.org/.

The bacteriophage population is vast, dynamic, old, and genetically diverse. The genomics of phages that infect bacterial hosts in the phylum Actinobacteria show them to not only be diverse but also pervasively mosaic, and replete with genes of unknown function. To further explore this broad group of bacteriophages, we describe here the isolation and genomic characterization of 116 phages that infect Microbacterium spp. Most of the phages are lytic, and can be grouped into twelve clusters according to their overall relatedness; seven of the phages are singletons with no close relatives. Genome sizes vary from 17.3 kbp to 97.7 kbp, and their G+C% content ranges from 51.4% to 71.4%, compared to ~67% for their Microbacterium hosts. The phages were isolated on five different Microbacterium species, but typically do not efficiently infect strains beyond the one on which they were isolated. These Microbacterium phages contain many novel features, including very large viral genes (13.5 kbp) and unusual fusions of structural proteins, including a fusion of VIP2 toxin and a MuF-like protein into a single gene. These phages and their genetic components such as integration systems, recombineering tools, and phage-mediated delivery systems, will be useful resources for advancing Microbacterium genetics.

The Bacillus cereus group is an assemblage of highly related firmicute bacteria that cause a variety of diseases in animals, including insects and humans. We announce three high-quality, complete genome sequences of bacteriophages we isolated from soil samples taken at the bases of fruit trees in Utah County, Utah. While two of the phages (Shanette and JL) are highly related myoviruses, the bacteriophage Basilisk is a siphovirus.