Bacteriophages: Emerging Applications in Medicine, Food, and Biotechnology

Abstract

PHAGEVol. 1, No. 2 Meeting ReportsFree AccessBacteriophages: Emerging Applications in Medicine, Food, and BiotechnologyHasan A. Sohail, Aidan Coffey, Krystyna Debrowska, Irmtraud M. Meyer, Mathias Middelboe, Muhammad Sohail, and Martha R.J. ClokieHasan A. SohailSchool of Life Sciences, University of Warwick, Coventry, United Kingdom.Search for more papers by this author, Aidan CoffeyUniversity College, Cork, and the APC Microbiome Institute, Cork, Ireland.Search for more papers by this author, Krystyna DebrowskaInstitute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland.Search for more papers by this author, Irmtraud M. MeyerFreie Universität Berlin, Institute of Chemistry and Biochemistry, Berlin, Germany.Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine, Berlin, Germany.Search for more papers by this author, Mathias MiddelboeDepartment of Biology, Marine Biological Section, University of Copenhagen, Helsingør, Denmark.Search for more papers by this author, Muhammad SohailAddress correspondence to: Muhammad Sohail, St. Hilda's College, Cowley Place, University of Oxford, Oxford, United Kingdom E-mail Address: [email protected]. Hilda's College, Cowley Place, University of Oxford, Oxford, United Kingdom.Search for more papers by this author, and Martha R.J. ClokieDepartment of Genetics and Genome Biology, University of Leicester, United Kingdom.Search for more papers by this authorPublished Online:16 Jun 2020https://doi.org/10.1089/phage.2020.29004.hasAboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail IntroductionThe repertoire of bacteriophage applications has expanded considerably in the past decade. The potential of bacteriophages to be exploited as antimicrobial agents has received particular attention in view of the rapid global emergence of multiantibiotic resistance in bacterial pathogens.Phages 2019, the ninth annual gathering at Oxford (United Kingdom), brought together phage researchers from academia and industry from across the globe. At this meeting, chaired by Prof. Martha Clokie (University of Leicester, United Kingdom), bacteriophage research was discussed from several perspectives, including basic research, their application in therapy, food, and biotechnology, as well as their commercialization. Hereunder is an extract of 2 packed days of highly stimulating presentations from this meeting.Bacteriophage Immunology and Molecular GeneticsThis set of talks largely focused on immunological and molecular aspects of phages within the context of interacting with bacteria within humans and other animals. Factors affecting their delivery to the site of bacterial infection were discussed. The conference opened with a presentation from Krystyna Dąbrowska (Hirszfeld Institute of Immunology and Experimental Therapy, Poland) on phage pharmacokinetics, where Krystyna had both exhaustively reviewed the literature and conducted new experiments. Available data suggest that smaller phages are easier to deliver than larger phages, and that the best method of phage delivery in terms of efficacy was by injection. Although oral delivery is also possible, it is not helpful to get phages to treat systemic infections, due to quick clearance from the gut. Passive and active immunity are key factors that shape the ability of phages to enter an animal or human body by various administration routes and that impact the ability of the body to clear them. Krystyna presented data that suggest that once phages successfully enter the body, they reach most organs, including the central nervous system, and are cleared by the active immune system. Innate immunity is not always considered in terms of the human immune response to phages, but data suggest that it removes phages from the body, even when no specific immune response to bacteriophages has yet developed. In terms of phage movement within the human body, when delivered systemically, phages are not detected in other tissue in large quantities, and their clearance in urine is insignificant.Antiviral immunity is a major factor that can influence the efficacy of phages and can prevent clearance of a bacterial infection. Paul Bollyky (Stanford University, CA) presented a study from his laboratory on the biology of the filamentous bacteriophage Pf that is encoded within the genomes of Pseudomonas aeruginosa. The phage has been shown to be involved in the suppression of immunity against bacterial infection and consequently the promotion of wound infections. Their study was carried out for 2 years in 113 patients and showed that Pf phages are associated with wound progression. Phages are internalized by mammalian cells and trigger antiviral responses that suppress bacterial clearance, making them a potential novel therapeutic target for vaccines against P. aeruginosa.In continuation of the excellent work on phage delivery being carried out in Dr. Dąbrowska's laboratory, Joanna Majewska (Hirszfeld Institute of Immunology and Experimental Therapy, Poland) from her group then presented data on the bioavailability of phages in the gastrointestinal (GI) tract when they are delivered orally. They also carried out molecular characteristics of the model Escherichia coli phage T4 and two therapeutic Staphylococcus phages A3R and 676Z to understand the molecular factors facilitating phage survival in the GI tract environment. Her data showed that phage-specific IgA antibodies appeared to play a major role in phage bioavailability in the gut, as their increased levels correlated with a decrease in phage titers in feces for all investigated phages. She also showed that phage structural proteins such as the T4 phage encoded gp24 differed in their individual immunogenicity, but these proteins may also aid in phage bioavailability by shaping its survival in the GI tract environment.The following two presentations in this session focused on molecular genetics of phage. Panos Kalatzis (University of Copenhagen, Denmark) presented his study where he characterized a novel nontailed virus, NO16, that infects a major pathogen of cultured fish and shellfish, Vibrio anguillarum. This phage has a narrow host range, restricted to its host of isolation known as A023. Complete Genome Sequence of NO16 showed that only 5 out of 23 proteins have a predicted function. Although the phage morphology resembles the already known corticovirus PM2, its genome is novel and significantly different, indicating that it represents a new genus. The sequence analysis also identified integration sites, attB and attP, used for site-specific integration into the host genome. This lysogenic conversion is carried out using the two host-encoded tyrosine recombinases, XerC and XerD. After this, Stephen Stockdale (APC Microbiome, Ireland) presented his study on ssRNA phage genomes. He pointed out that only 12 representative ssRNA phage genome sequences are available in the NCBI Genome database (June 2019). They identified 15,611 nonredundant ssRNA phage sequences, including 1015 complete or near-complete genomes, which enabled them to complete a phylogenetic assessment of both novel and known ssRNA phage genomes. He concluded that these viruses have been significantly overlooked within microbiome studies.Computational Biology, Bioinformatics, and EvolutionThe conference saw a number of exciting contributions using computational biology and bioinformatics to study bacteriophage. Aidan Brown (University of Edinburgh, United Kingdom) presented a mathematical model to investigate the spread of bacteriophages through bacterial populations. Assuming the setting of a two-dimensional system in solution (one space dimension, one time dimension), he presented a system of three coupled differential equations to explain the key features of the combined systems such as the diffusive motion of the bacterial population, the bacteriophages and their interplay. Mathematically, this model is currently set up for a population of E. coli with parameters quantifying how they divide and swim, as well as phage lysis and a burst size. The current model is already capable of deriving the critical phage concentration above which the bacterial spread is stopped. In the future, the Brown group plans to extend the model to more spatial dimensions and include additional features.Piotr Tynecki (Bialystok University of Technology, Poland) showed how machine learning methods from natural language processing can be employed to capture bacteriophage life cycle recognition to distinguish between phages undergoing lytic and lysogenic life cycles. Using training sets of genomic DNA fragments of 6-mers from known temperate and virulent phages, he employed support vector machines to classify entire phage genomes based on input vectors of >200 features. In the future, they want to use approaches such as advanced deep learning to study and predict aspects of phage–host relationships.Rob Lavigne (KU Leuven, Belgium) presented a machine learning strategy to elucidate phage–bacteria interactions, developed with local collaborators Cedric Lood and Prof. Vera Van Noort. Over time, his team has compiled a wealth of omics data using experimental genomic, proteomic, and metabolomic research to understand the molecular interactions that occur between phages and their associated bacteria. By combining mutagenesis and resistance development, complementation libraries, and prophage induction, they aim to identify key determinants of host–virus interactions, from both the bacterial host and the virus. To do this, they generated a custom database comprising genomics data and phage infectivity applied machine learning methods to identify key genomic features that determine the outcome of host–virus interactions. Out of the 270 models tested, those based on support vector machines currently provide the best classifier (accuracy 0.80 and precision 0.68). Further analysis using a random forest model (accuracy 0.80 and precision 0.70) allowed a more in-depth parameter interpretation, revealing metabolism, cell wall, and CRISPR-associated gene products that influence phage infectivity, currently under investigation. The ultimate goal is to turn this insight into predictive models of phage infection and principled rules to help with the formulation of potent phage cocktails to treat specific infections. This study highlights the importance of having good experimental data sets to extract meaningful biological insight using state-of-the-art machine learning methods.Irmtraud Meyer (Max Delbrück Center for Molecular Medicine, Germany) presented a new computational method to identify novel biological classes of trans-RNA–RNA interactions. She hypothesized that these interactions both significantly contribute to regulating individual cells, but that they also feature prominently as key players in the context of infections, such as, for example, phage–host interactions. She explained how the fully probabilistic machine learning method could be used to help interpret transcriptome-wide studies, thereby enabling the exploration of the yet unknown universe of trans-RNA–RNA interactions, for example, within the topic of phage infections or, more generally, pathogen–host interactions.Alfonso Jaramillo (University of Warwick, United Kingdom) spoke about the study carried out in his group using bacteriophage for de novo evolution. They have developed a directed evolution systems based on transducing particles of filamentous (M13), temperate (P2), and lytic (T7) phages. This approach could be extended to other phage systems to evolve proteins, nucleic acids, and phage tropism determinants, allowing for in vivo evolution in other hosts.Katarzyna Kosznik-Kwaśnicka (Institute of Biochemistry and Biophysics, Poland) presented data wherein she compared the evolution of antibiotic and phage resistance in Salmonella enterica serotypes Enteritidis and Typhimurium under laboratory conditions. Study in their laboratory has been designed to ask the pertinent question: will constant exposure to phages result in the development of phage tolerance/resistance in bacteria and will it also impact antibiotic resistance. They observed that the development of resistance to streptomycin also caused a concomittant development of tolerance to individual phages and to those within their phage cocktail. However, in general the development of resistance to multiple antibiotics, upon exposure of bacteria to phage, was not detected, with the only exception being the development of resistance to streptomycin. This shows that phages in general could be used to extend the life of antibiotics.Phage–Host InteractionsTalks in this part of the conference addressed multiple aspects of phage interactions with their bacterial hosts, with a particular focus on molecular mechanisms and pathways. Mathias Middelboe (University of Copenhagen, Denmark) discussed lysis–lysogeny regulation in H20-like vibriophages that infect fish pathogenic bacteria. H20-like vibriophages have a global distribution and are seen both as prophages and free phages. Interestingly, in this group, phenotypic properties, including phage host range, can be partly regulated by the host since they are dependent on the strains used for proliferation. Moreover, induction of prophages was controlled by the quorum sensing state of the host. The presence of H20 prophages, in turn, stimulates biofilm formation and aggregate production in the low-cell density state of bacteria, but not in high densities. All these observations emphasize a dynamic regulation of the lysis–lysogeny switch, which is orchestrated by host quorum-sensing signaling.Sean Meaden (University of Exeter, United Kingdom) discussed the loss of CRISPR-mediated herd immunity from bacterial populations, addressing long-term effectiveness of CRISPR-mediated immunity—an aspect of bacterial immunity that is relatively unknown when it comes to controlling phage interactions. Experimental evolution study with deep sequencing revealed that bacterial populations initially generate high population level of CRISPR spacer diversity, causing rapid phage extinction. This diversity, however, rapidly declined after phage extinction, probably due to the molecular costs of maintaining CRISPR-Cas immunity; the population was invaded by sensitive bacteria and receptor mutants. The implications from these observations could help with determining optimal timings and modes of the delivery of phages when they are used as phage therapy treatments.Paul Fogg (University of York, United Kingdom) presented new aspects of his study that investigated the regulation of gene transfer agents (GTAs), that is, elements that have the potential to carry out high-frequency horizontal gene transfer. GTAs appear to be "selfless"—they package and disseminate the entire genome of their bacterial host. Interestingly, GTA production seems to be linked to host regulatory pathways.In two subsequent talks, Abram Aertsen and Sanne Wolput from KU Leuven focused on the approaches they have developed to study phage–host interactions. Using genetics and cell biology, they illustrated how the temperate Salmonella phage P22 can commit to a phage carrier state after infection. This state is obtained when, during the process of lysogenization, the incoming phage chromosome becomes localized in one of the poles of the host cell without immediate integration as a prophage. In turn, this carrier state allows the emergence of a phage-free subpopulation in which cytoplasmically inherited superinfection exclusion factors are able to spur complex infection and transmission dynamics that could help P22 to farm its host. For reasons currently unknown, P22 also expresses its ORFan pid gene during the carrier state, which leads to derepression of the dgo operon of the host. This operon is involved in d-galactonate metabolism and is possibly related to intracellular survival and virulence of Salmonella Typhimurium.Further impact of ecological aspects on phage development cycles was discussed by Karolina Ciemińska (University of Gdańsk, Poland), who focused on growth conditions of the host and host serotype in Salmonella. This study revealed temperature and serotype dependency in three environmental phages and, importantly, showed that these phages differed in their optimum strain in terms of antibacterial activity, suggesting that the dependencies are strain specific.The session was concluded by Gabriel Kaufmann (Tel Aviv University, Israel) who has spent many years analyzing multiple anticodon nucleases in P. aeruginosa strains. These anticodon nucleases that hint at encounters with phage are able to activate these host defenses. This involves a conserved bacterial protein (RloC). New observations indicate a host–parasite arms race where the target specificity of RloC and the nature of the viral anticodons are mutually adapted.Commercialization of Phage ProductsPhilip Webber (Dehns Patent Attorneys, United Kingdom) explained how to patent phage products and methods of using them. Although phage-based products, such as phage cocktails, and uses, such as treating bacterial infections, can be patented, it is important that the product or use has not previously been publically disclosed (e.g., by a research publication, oral presentation, or a poster at a conference) before your patent application has been filed. Also, changing a single phage in a phage cocktail will make it a new product, which can be potentially patented in its own right. Dr. Webber also explained the process of the patenting inventions, including writing and filing a priority application, filing an international (PCT) patent application, the examination procedure, and getting patents granted in various countries.Methods and TechnologiesA number of talks presented new diagnostic methods and approaches to tackling antimicrobial resistance. Rodrigo Ibarra-Chávez (University of Glasgow, United Kingdom) presented an approach using synthetic phage-inducible chromosomal islands (PICIs) as an approach to fight bacterial infections. They are satellite viruses that manipulate the life cycle of phages to hijack their machinery and spread into other host cells. They can promote their own spread and can infect and integrate in bacterial genome at any stage. He presented a rapid and general method to manipulate and engineer these mobile genetic elements, which can also be applied to engineer temperate phages. In contrast with phages, PICIs can adapt modules that can be used in different applications in biotechnology and synthetic biology, for example, within the evolution of proteins and targeted antimicrobials with CRISPR-Cas systems. As there is no anti-PICI system in nature, they can be engineered to seek and destroy bacteria.Sílvio B. Santos (University of Minho, Portugal) presented his method of multiplex detection of Enterococcus and Staphylococcus, whose fast and accurate detection and identification are critical to design efficient control measures. Dr. Santos's laboratory studies the highly specific interactions between phage and its host through receptor binding proteins (RBPs) that can be used as diagnostic They and of Enterococcus phage and Staphylococcus phage with a and and designed a multiplex method that enabled detection of these two by three different and This fast and sensitive method has the potential to be extended to the detection of other time to treat an (University of United Kingdom) showed that phages can be used as efficient cell lysis for bacteria that are to such as allowing sensitive detection of levels of bacteria. Using phage as a lytic the bacterial detection method to which was significantly than other This is also successfully used to in and has the potential of being extended to other in this (University of United Kingdom) presented a phage which involved phage in Using different including and one and one they were able to phage without a loss of compared with of the phage. Their method could be and applied to a large number of in and potential applications of bacteriophages in and are also number of talks were presented in this the use of phage as (University of United Kingdom) and addressed the of Salmonella infection in with the view that infections caused by Salmonella are They Salmonella phages, all of the used as a they were able to Salmonella in as well as in infection these phages were at and could be from to and with extended Their showed that these phages Salmonella in presented his study on the use of bacteriophages to the of within the They and characterized >200 phages from and and of these phages genome of in group or the phages in in group or They also the host of all phages against strains including of and strain of and They are currently of these phages to determine their as Institute, presented a study from his laboratory on tackling the of of levels of (e.g., and in products, such as by the bacteria. They and characterized three phages against Enterococcus which are largely for the of and in of their phages was able to the level of in products by well the of making phage a for use in the in this session was by (KU Leuven, Belgium) on phage in as to using and such as streptomycin to control bacterial infections. and phages to Pseudomonas and infections. In to they are currently out phage to determine their resistance infection and their potential to be used as and Applications of of the key of in phages application is their use as antimicrobial which a part of the conference in this session the potential use of phage as antimicrobials with in vivo but also the antimicrobial potential of and Martha Clokie (University of Leicester, United Kingdom) presented a study from her laboratory that is being carried out on phages as therapeutic She the of phage that has been characterized in her laboratory, which strains. She showed how she is new of the biology such as the that phage and how studies can of when are and help interpret their function. Their phage cocktail is in cells, and it has been patented and granted in the and the United Prof. Clokie also showed that the phages can also be for example, by phage to their lytic her study shows that phages are antimicrobials and could be to treat patients with this, United Kingdom) discussed the molecular for host by phages using phage proteins host He presented data on their in or the host bacterial and their potential use as antibacterial agents and to study bacterial In his model phage, two proteins, and to the E. coli and as They used Salmonella of as their model system and showed that intracellular induction of not bacterial in induction of both and formation of Salmonella as well as by Salmonella. these proteins can potentially also be used to control and bacterial to several of using phage as as host of many phages, and host immune Aidan and (University College, Cork, Ireland) are as which can several when using phages. They have and characterized several from phages. of these from phage was characterized in including of its of this to a bacterial in of as well as it in without on the animals. it also has They are also these studies to bacteriophages and those of other to use phage as antimicrobial and (University of Minho, Portugal) are phage which play a role in different of bacterial They have and characterized several phages the all of which narrow host they have identified that and that proteins bacterial as for phage Moreover, all are active in a of environmental conditions and are highly Using a a single of of was able to of This shows the of these as novel of Prof. and the Institute for Medical presented and data phage delivery to treat Staphylococcus infections in patients not to antibiotic They used phage cocktail in with the antibiotics, to treat patients from and as and then as under of They that phage therapy was well by their and phage in were by DNA and in of there were no after a days of phage parameters of process also declined after phage In of bacterial and all showed therapy, without any in the that this is the study to a that and of phage therapy in and (University of Warwick, United Kingdom) the part of the phage session with her on the and potential for in with antibiotic to of the

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