Bacteriophage therapy holds promise in addressing the antibiotic-resistance crisis, globally and in Germany. Here, we provide an overview of the current situation (2023) of applied phage therapy and supporting research in Germany. The authors, an interdisciplinary group working on patient-focused bacteriophage research, addressed phage production, phage banks, susceptibility testing, clinical application, ongoing translational research, the regulatory situation, and the network structure in Germany. They identified critical shortcomings including the lack of clinical trials, a paucity of appropriate regulation and a shortage of phages for clinical use. Phage therapy is currently being applied to a limited number of patients as individual treatment trials. There is presently only one site in Germany for large-scale good-manufacturing-practice (GMP) phage production, and one clinic carrying out permission-free production of medicinal products. Several phage banks exist, but due to varying institutional policies, exchange among them is limited. The number of phage research projects has remarkably increased in recent years, some of which are part of structured networks. There is a demand for the expansion of production capacities with defined quality standards, a structured registry of all treated patients and clear therapeutic guidelines. Furthermore, the medical field is still poorly informed about phage therapy. The current status of non-approval, however, may also be regarded as advantageous, as insufficiently restricted use of phage therapy without adequate scientific evidence for effectiveness and safety must be prevented. In close coordination with the regulatory authorities, it seems sensible to first allow some centers to treat patients following the Belgian model. There is an urgent need for targeted networking and funding, particularly of translational research, to help advance the clinical application of phages. The decreasing sensitivity of clinically relevant bacteria to antibiotics, as stated in the WHO surveillance reports of recent years, also affects Germany ,2,3. TheStaphylococcus aureus (MRSA), ~4000 with vancomycin-resistant enterococci , ~8000 with MDR Escherichia coli, ~2000 with MDR Klebsiella pneumoniae and ~4000 with MDR Pseudomonas aeruginosa). Of these, ~1500 cases (0.3%) are caused by isolates that are pandrug-resistant to almost all antibiotic classes [The proportion of patients acquiring infections in hospitals is ~3.6%, corresponding to an estimated 400,000–600,000 nosocomial infections in Germany per year [ classes .Nosocomial and community-acquired infections by MDR pathogens, according to projections, amount to ~54,500 people in Germany annually. A 2018 study on the disease burden due to MDR pathogens published by the European Centre for Disease Prevention and Control (ECDC) found the number of infection-associated deaths for Germany to be ~2400 people per year   and 2018The regulatory situation or legal framework for phage products in Germany;The extent of the existing clinical application of phages;Existing activities to produce phages for this purpose;The status of phage banks;The technical status of sensitivity testing (“phagogram”).The current situation of phage therapy in Germany is presented primarily from the perspective of clinicians, cooperating research institutions, and regulatory authorities , with whom effective exchange on the topic has been in place since 2017. Thus, an interdisciplinary network of physicians, pharmacists, microbiologists, bioengineers, bioinformaticians and regulators—all with a patient-oriented focus—were involved in describing the current situation in Germany. The goal of taking translational aspects into account in the present analysis seemed achievable within this group of authors, following scientific exchange at national and international meetings over the last five years, as well as joint research activities. An attempt was made to include only one author from each working group. The following aspects are addressed in particular:For an insight into further developments, ongoing research projects, the activities for a one-health approach, as well as the existing Germany-wide network structure and activity will be presented.To avoid a limited overview of the phage landscape in Germany, literature covering at least the years 2020–2022 was reviewed for the academic and industrial working groups that, according to the authors, have contributed to translational phage research. In addition, the authors of this article provided their opinion on the most urgent changes needed in the German-phage landscape, the most important research projects, and the biggest hurdles for the implementation of phage therapy. Multiple answers were possible.Within the European Union, phages for therapeutic use in humans are defined as biological medicinal products, in accordance with Directive 2001/83/EC. This implies the need for a marketing authorization (MA) for market access of phage-therapy medicinal products (PTMP). Article 3 of Directive 2001/83/EC (and paragraph (§) 21(2b) of the German Medicinal Products Act, ‘AMG’) define exemptions from the need for an MA, including prescription medicinal products prepared in a pharmacy for a specific patient . According to §55(8) AMG, these products must be manufactured in compliance with the recognized pharmaceutical rules . A Ph. Eur. general chapter on PTMPs is currently being drafted that will provide harmonized quality standards. According to §13 AMG, a manufacturing authorization is required for PTMPs produced on a commercial/professional basis and intended to be marketed. An exemption exists (§13(2) AMG) which includes manufacturing in pharmacies  or by physicians . PTMP manufacturing must also follow the recognized pharmaceutical rules, and is subject to notification of competent supervisory authorities (§67(2) AMG). PTMPs therefore can be made available to patients using either way, MA  or formula magistralis.While MA would primarily pertain to standardized ready-to-use PTMPs, magistral phage products could provide a more personalized approach, adapted individually. The existing regulations are often seen as an obstacle for phage therapy. Nevertheless, as indicated above, varying routes for patient access to PTMPs are currently available. In any case, the development and modification of regulatory requirements will be based on the expansion of knowledge. Once the efficacy of PTMPs is demonstrated, the regulatory framework can be amended, if required, to address the particularities of these products. However, they can be used in cases of hardship. Thus, within the framework of an official compassionate-use program, the use of PTMPs that are currently included in clinical trials or in the approval process of an MA application can be authorized by the national authorities, and thus would be available for a defined group of patients .This officially authorized compassionate-use program is distinguished from an “individual treatment trial”, which does not require notification. The latter is the application of PTMPs in an individual case, decided on by the treating physician, under his or her responsibility, within the framework of therapeutic freedom and with the patient’s consent. The individual treatment is usually applied after all standard treatment options have been exhausted or are not available, i.e., in the case of an “unmet medical need”, according to Article 37  of the Declaration of Helsinki of the World Medical Association (WMA), and if the treating physician suspects a benefit of phage use for the patient, based on scientific findings. The focus of the individual treatment trial is not on obtaining research results, but on healing the individual patient. However, the regulatory authorities explicitly emphasize that precise documentation, observation of the patient and transparent communication of the individual case, as well as scientific justification, must be in place, which also corresponds to the requirement of Article 37, Declaration of Helsinki. No therapeutic phage products are currently clinically approved in Germany; an overview of ongoing and planned clinical trials is included in ® against Shigella infections was the first phage preparation marketed by Behringwerke, near Marburg/Lahn, in July 1939 [® against Salmonella. Many German soldiers were subsequently treated with phages. Positive therapy results were reported, but also—after the initial euphoria—relevant failures. It has been speculated that many of these failures occurred because clinicians were not aware of the narrow host specificity of the phages and important application parameters, and, probably, temperate phages were also used. In addition, process-related impurities, and insufficient stability of the phage solutions impaired product quality. However, scientific proof for effectiveness as well as for the manifold speculative reasons for failure is lacking. Ultimately, these failures greatly reduced interest in phage therapy after the market launch of antibiotics (penicillin and sulfonamides) in the clinical field [At the beginning of the Second World War, phage therapy experienced a significant upswing in Germany. Dysentery-Polyfaginuly 1939 , followeal field .®, was available from 1959 to 1965 . This preparation was, however, ineffective against Shigella flexneri type 4A, which was responsible for an epidemic in 1962. Within a very short time, production capacities of 11,000 L per week were implemented for the prophylaxis of the population /mL). At that time, phages in the GDR were not subject to the “Ordinance on the Regulation and Supervision of the Traffic in Medicinal Products”, but to the “Ordinance on the Traffic in Vaccines, Sera and Bacteriophages”. Phage prophylaxis began in Berlin on 26 April 1962. Until June 14, 1962, a total of 174,906 people received the phage preparation [In the German Democratic Republic (GDR), a ready-to-use phage preparation, Intestolysin7–108 PFU/mL; 30 min before eating; after taking an acid-binding drug). In addition, the phage lysate was introduced intraoperatively into the infected area (exposure time 30 min). From August 1979 to May 1981, 54 patients with implant-associated infections were treated , over a period of at least 5 years (unpublished data). For the treatment of periprosthetic infections, the phage lysate was administered to the patient twice daily for 14 days before surgery and for 14 days afterwards  began to be applied topically to chronic limb wounds, post-traumatic infections and burn wounds at the Hanover Medical School (B. Wippermann), as part of individual treatment trials (unpublished data). Most recently, this treatment series was continued in Hildesheim. Since 2015, 33 patients with chronic periprosthetic infections (on average 11 previous operations) have been treated topically with phages by another working group (R. Ascherl) in the Chemnitz and Tirschenreuth hospitals and, with a follow-up period of 14–42 months at that time, a complete cure of infection without severe side effects (mostly fever and chills on the 2nd–4th day) was achieved in 25 cases. .PhagoFlow research project (see below), it will be possible to treat patients with P. aeruginosa and S. aureus infections on a larger scale with magistral phage preparations, from spring 2023 onwards (www.phagoflow.de). Additionally, at the German Heart Institute Berlin, Germany, six patients were treated as a last-resort measure between December 2018 and October 2021, in collaboration with Charité—Universitätsmedizin Berlin, Germany, Center for Musculoskeletal Surgery, with phages acquired from the Belgian LabMCT and Eliava Institute of Bacteriophage, Microbiology and Virology, IBMV . Following skin closure, a 1:1 mixture of PYO-bacteriophage with 106 PFU/mL and staphylococcal phage Sb-1 with 107 PFU/mL  was applied locally—and in one case also orally and intravenously [S. aureus) remained infection-free until the end of the observation period (≥30 months) [P. aeruginosa infection, followed by Staphylococcus haemolyticus, received a magistral phage preparation applied both locally and intravenously, and remained infection-free for four months before succumbing to LVAD pump thrombosis. Another patient, with an S. aureus infection, was administered PYO-bacteriophage plus Sb-1 locally and, at a nine-month follow-up, showed no signs of a local infection [Berlin: At the Military Academic Hospital Berlin, three patients have been treated with phages from Georgia since 2016, partly in collaboration with the laboratory for molecular and cellular technology (LabMCT) at Queen Astrid Military Hospital in Brussels (Belgium) and the Charité—Universitätsmedizin Berlin, Germany, Center for Musculoskeletal Surgery. As part of the venously ,14. Out nfection .S. aureus (n = 13), P. aeruginosa (n = 12), E. coli (n = 4), E. faecium (n = 3), K. pneumoniae (n = 3), Burkholderia multivorans (n = 2), E. faecalis (n = 1), Stenotrophomonas maltophilia (n = 1), Serratia marcescens (n = 1), and Proteus spp. (n = 1).Hannover: Nowadays, phage therapy is again based at the Department of Cardiac, Thoracic, Transplantation and Vascular Surgery of the Hannover Medical School. The department’s portfolio includes 33 cases of personalized phage therapy since 2015 in critically ill patients, of which 31 were successful. The high clinical success rate (>90%) is due to a combination of modern principles of permission-free preparation using mostly self-isolated phages (see below), an interdisciplinary approach to administration of phages and an optimal, concomitant conventional treatment, including antibiotic therapy [® bacteriophage cocktail produced by the Eliava Institute was recommended, which is active against various strains of Shigella, Salmonella, E. coli, Proteus, S. aureus, P. aeruginosa, and Enterococcus. To improve local release kinetics, the phage cocktail was mixed with a hydrogel, as previously described [PhagoDAIR trial, a randomized, non-comparative, double-blinded phase I/II clinical study in patients with S. aureus knee or hip periprosthetic-joint infections with indication for debridement, antibiotic and implant retention (DAIR), combined with antibiotics .Regensburg: At the Department of Trauma Surgery University Hospital Regensburg, phage therapy has been performed since October 2022. The first case was an infected non-union of the proximal femur with different MDR gram-negative bacteria. Phage testing was performed by LabMCT at Queen Astrid Military Hospital and treatment with Intestiescribed . No locaRostock: Phage therapy is also performed at the Clinic for General, Visceral, Thoracic, Vascular and Transplant Surgery, Rostock University Medical Centre. In the field of vascular surgery, it is mainly patch- or bypass-associated infections in the groin area, followed by acute life-threatening aortic-prosthesis infections. In cardiac surgery, the most frequent indication is drive-line infection after LVAD insertion. In orthopedic-surgery patients, periprosthetic-joint infections in the hip area dominate, increasingly also after implantations of endo-exo systems in transfemoral amputations. Unfortunately, despite a high demand for treatment, there are fundamental problems with individualized phage therapy. Therefore, it has only been possible to treat patients with composite phage cocktails , which have so far been accompanied by therapeutic success of only ~30%. The focus on individualized phage therapy with on-site production planned for 2023 is expected to significantly improve success rates. Current and planned research projects are focusing on testing the thermostability of phages in bone cement, topical phage application to infected endo-exo systems at the stoma, and the planning of the study “Bacteriophage-related treatment of chronically infected Drive-Line/LVAD Systems: a randomised controlled multicentre trial” (via a German Research Foundation (DFG) application), in collaboration with the Heart Center, Leipzig, Germany.The Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM), Braunschweig, is developing a platform-like manufacturing process for natural phages as APIs using host bacteria under GMP-conforming conditions. For this purpose, the starting materials, master cell banks (MCB) and master phage stocks (MPS), are first produced according to GMP. Phage drug production begins with cultivation (up to 10 L) of the production strain from the MCB, infection with phages (MPS) and subsequent cell lysis. The phage-containing lysate is first separated by depth filtration (to reduce cell fragments), diluted in a buffer suitable for chromatography (diafiltration) and then purified chromatographically to remove endotoxins and host-cell proteins. This is followed by a second diafiltration step with a physiological buffer and 0.2 µM filtration, to reduce bioburden. Finally, the phage agent is filled into the primary packaging. The challenge in developing the manufacturing process is to find suitable conditions that allow for high titers and yields and the reduction of endotoxins and host-cell proteins. After filling, the phages must meet international quality requirements and are tested for identity (sequencing), potency (plaque assay) and purity , for release. The justification of the batch-release specifications is carried out in close exchange with the competent authorities and process-immanent aspects. The challenge here is that phages represent a completely new class of active substances, for which official quality standards remain to be defined. The first manufacturing authorization for phage production in Germany was granted in August 2022. Three phages produced this way are set to be supplied to the Military Academic Hospital Berlin in Q1 2023.https://invitris.com, accessed on 12 January 2023), a spin-off company of the Technical University of Munich. For in vitro or cell-free protein synthesis (CFPS) the cellular-expression machinery is isolated from the cell, most commonly E. coli, and combined with metabolites, precursor molecules and a DNA template specific for the intended phage. CFPS has been used to produce E. coli phages such as T7, phiX174, and MS2 with titers of up to 1012–1013 PFU/mL [E.-coli phages, including those against clinically relevant pathogens such as Yersinia pestis and even gram-positive bacteria such as Bacillus subtilis [K. pneumoniae patient isolate, from isolation to in vitro production. Utilizing the advantages of in vitro bacteriophage production, a personalized phage-therapy pipeline can be implemented, and could be applied for on-site production [Klebsiella phages, most of which initiate infection through the highly diverse capsular polysaccharide of K. pneumoniae.An alternative way to produce phages, the expression in an in vitro system, is currently being developed by INVITRIS  AMG  that uses host bacteria is currently only in use at the Hanover Medical School  according to the quality and safety requirements for sustainable-phage-therapy products. The production process uses only strictly lytic phages, which do not contain known genes encoding integrases, bacterial virulence and antibiotic-resistance factors. Despite the absence of a conventional GMP process in the production consumables, nutrient media and reagents designed for either GMP production or for clinical use are employed. For example, nutrient media free of animal components are used for cultivation. The host bacterial strains are tested for absence of inducible prophages. Phage amplification is performed on a small-scale, using dense nutrient media, which has shown stable high-phage titers and does not require optimization of the production process. After amplification, the phage lysate undergoes a multistep purification process to remove unlysed cells, nutrient-medium components, and pyrogens, followed by control of sterility, phage titer, identity, and endotoxin content. Other clinics in Germany  are currently also aiming for this type of production.E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa and Enterobacter spp.) are dominate, and are used in externally funded projects such as P. aeruginosa in Phage4Cure, some prioritized species in PhagoFlow, E. coli in IDEAL-EC and E. faecium in EVREA-Phage (there are 30–150 phages per ESKAPE species). Other large phage panels exist for Achromobacter xylosoxidans and Stenotrophomonas maltophilia; phages in lower numbers were isolated on rarer patient isolates of, e.g., Bordetella bronchiseptica, Burkholderia cepacia or Mycobacterium abscessus. The focus of the DSMZ phage bank is defined by medical need. DSMZ can search for new suitable phages at any time, at the request of clinicians, and they can then be supplied according to material-transfer agreements. However, direct application in humans is excluded, for warranty reasons. For genomic-identity confirmation, the DSMZ was granted GMP certification according to §64 (3f), German Medicinal Products Act, in 2022.In the early 2000s, a working group specializing in clinically relevant bacteria was founded at the Leibniz Institute DSMZ and, furthermore at the DZIF (German Center for Infection Research) strain repository. Both provide an essential basis for isolating, characterizing, and investigating phages at the DSMZ and for using them in the institute’s so-far exclusive applicative-phage-research projects. The bank contains >1000 phages for more than 150 bacterial species; however, phages for the ESKAPE bacteria  [K. pneumoniae (>380 isolates with a large variety of capsule types (>80)) and their specific phages (>505 plaque-purified stocks) in Europe. The group is using its phage collection for studies to extend the host range [K. pneumoniae infections [Another large phage bank is located at the Bundeswehr Institute of Microbiology (IMB), Munich, with a focus on the isolation and characterization of  groups) . The IMBst range , and engfections .Salmonella (>120 phages), K. pneumoniae (>80), Yersinia (>50), Brucella (>40), P. aeruginosa (>30), Morganella (>15), Campylobacter (>15), Burkholderia (>15) and Vibrio (>10). However, due to the different clonality of the bacteria in the human and livestock/food sector, most of the phages are much more effective on isolates of the respective compartments than on human isolates (esp. Klebsiella).Phage research at the German Federal Institute for Risk Assessment (BfR) is based on fundamental investigation of the occurrence of phages (temperate/lytic), their genetic diversity and their potential for application, especially in the veterinary and food sectors. Therefore, the culture collections of foodborne pathogens as well as bacteria recovered from livestock during annual monitoring of zoonoses are used for phage testing. Phages against foodborne pathogens at the BfR mainly originated from samples of the food-production chain , but have also been recovered from environmental sources  and/or municipal/clinical wastewater. The current collection includes phages against Conventionally, phage–host-susceptibility testing is based on the double-agar-overlay plaque assay , which ihttps://phage4cure.de, accessed on 12 January 2023, funded by the German Federal Ministry of Education and Research (BMBF)) is a first-in-human (FIH) study to investigate safety, tolerability, and preliminary efficacy of a phage cocktail in healthy volunteers and patients with chronic P. aeruginosa lung infection. The phages investigated in the clinical trial were selected with the help of the phage biobank of the Leibniz Institute DSMZ GmbH. Three phages from the order of Caudovirales were selected for the study: two myoviruses (JG005 and JG024), and one podovirus (Bhz17). JG005 and JG024 had been known previously, whereas Bhz17 was newly isolated. The phage selection aimed at providing a broad antibacterial spectrum and limiting the emergence of phage-resistant bacterial variants. Three individual IMP formulations of the respective phages were initially prepared, according to European GMP standards and will subsequently be combined in a cocktail prior to use. The cocktail will be administered as an aerosol through a CE-certified nebulizer. Efficacy and safety were previously tested, in animal models. The Phage4Cure study consists of two parts: part 1 is a classical single-ascending-dose (SAD) design in healthy volunteers, while part 2 is a multiple-dose design conducted in patients with chronic P. aeruginosa colonization of the lung, with demonstrated susceptibility to this particular phage combination. All documentation for phases 1a  and 1b  has been completed, allowing for phase 1a to start in Q2 2023. In the longer term, a scalable procedure for phage therapy will be developed, which, after initial establishment, can also be applied to other phage entities in its modified and adapted form.“Phage4Cure” . The project investigates whether, with today’s biopharmaceutical possibilities, it is possible to adapt phage preparations in the hospital pharmacy to the individual patient and prepare them in time for therapeutic use—thus, the practicability of magistral production of phage products is investigated . The project focuses on wounds on arms and legs infected by multidrug-resistant pathogens. In the first project phase, phages were isolated (DSMZ GmbH), characterized and preserved with all methodology following the OECD Best Practice Guidelines for BRCs. Subsequently, they will be produced in a biotechnological process (Fraunhofer ITEM) in such a way that they can be made available in a purified form to the hospital pharmacy, as API components. The second phase of the project is aimed at treating patients. First, pathogens from a patient’s wound material are typified for phage sensitivity and then a tailor-made phage preparation is produced. Treatment, initially with phages against multidrug-resistant P. aeruginosa, will begin in February 2023, and against S. aureus from August 2023.“PhagoFlow”  characterize phage-resistance development during in vivo preclinical treatment, (2) decipher the impact of these cocktails on the respiratory and intestinal microbiota, as well as on microbiota-dependent immune responses, (3) evaluate the efficacy of the cocktails in penetrating biofilms produced in vitro and ex vivo on explanted human-lung tissue, (4) investigate the direct interaction of phages with the immune system, and the mechanistic basis for the synergy between innate immune cells and phages during therapy, and (5) characterize by mathematical modeling the efficacy of these cocktails in vivo, and propose optimized treatment regimens [“MAPVAP” . The MAPVAP project is a collaborative research program financed by France and Germany through the 2019 dedicated call on antimicrobial resistance ) . Ventilaregimens .IDEAL-EC project focused on evaluating phages against extended-spectrum beta-lactamase-producing clinical E. coli samples. The WHO has recently classified E. coli as a pathogen of international concern. Antibiotic-resistant E. coli strains are on the rise, and can cause severe infections, especially in immunocompromised patients. During the project period, an E. coli phage cocktail was developed, which is currently being investigated in preclinical models . The results of this project will serve as the basis for subsequent preclinical and clinical studies investigating the effects of E. coli phages on the human microbiota and their applicability for therapeutic purposes.“IDEAL-EC” . In collaboration with the Leibniz Institute DSMZ and DZIF, the EVREA-Phage envisages the characterization of new phages and the composing of a phage cocktail against E. faecium for oral application, to specifically decolonize intestinal VRE E. faecium in immunocompromised patients. Considering potential concomitant colonization by different E. faecium strains in individual patients involving not only VRE but also VSE (vancomycin-sensitive) or VRE+ strains being additionally resistant to linezolid, the phages selected for the cocktail will be tested against panels of all mentioned E. faecium variants, to ensure the broadest-possible host coverage. Large phage and strain panels were compiled to perform the full biological-phage characterization. Bacterial strains are clinical isolates from German university hospitals of the DZIF network and from the Robert Koch Institute, and all phages were newly isolated by the Leibniz Institute DSMZ. EVREA-Phage envisages the demonstration of phage efficiency by in vitro and in vivo gut models at the University Hospital Bonn and the Helmholtz Centre for Infection Research, Braunschweig, to confirm phage effects, according to regulatory requirements and the BfArM scientific advice. All derived data of EVREA-Phage will flow into the preparation of an IMPD  required for later authorization of a clinical trial, applying highly purified approved phage preparations.“EVREA-Phage” . The DZIF-funded, translational preclinical project www.sprind.org). The project is currently identifying top groups of applied research in Europe to form clusters of excellence and to focus efforts, for example, on innovative and rapid approaches to match patient isolates to phages, on improving the yield of phage-production processes and on exploiting synergies between phages and antibiotics. In addition, with a focus on ESKAPE pathogens, requirements for large-scale production of phage cocktails for bacteria with broad-spectrum phages  or small-scale on-site production for individualized phage therapy for pathogens with narrow-spectrum phages  are being evaluated.“Phage2030”  aims to identify feasible research efforts and manufacturing pathways, the coordination and targeted support of which would represent a “leap forward” in making phage therapy available to the German public by 2030. The validation study required for this is funded by the Federal Agency for Disruptive Innovation, SPRIN-D . Another project with close cooperation between the Leibniz Institute DSMZ and the DZIF will focus on the pre-clinical evaluation of phage–antibiotic synergy in multidrug-resistant https://github.com/deng-lab/viroprofiler/, accessed on 12 January 2023”) [PHARMS”  aims to discover novel antibacterials against A. baumannii, Helicobacter pylori, and Haemophilus influenza, by studying phage–host interactions using culture-independent and multi-omics approaches. “COVPHA”  aims to identify the co-infecting MDR bacteria in COVID-19 patients using metagenomics and to isolate effective phages against them. In addition, multiple phage endolysins have been identified. The efficacy of the phages and endolysins will be tested in vivo and ultimately applied to patients, under compassionate use and in an early clinical trial. In addition, the Munich Phage Center led by L.D., which is part of the “Center for Integrated Infection Prevention (ZIP)” at the Technical University of Munich , aims to develop innovative phage-based therapeutics against MDR pathogens in humans and animals. To this end, a high-throughput phage-culturomics pipeline to automatically isolate and characterize phages as well as a well-characterized phage bank against highly-critical bacteria, including ESKAPE pathogens, are currently being built.Helmholtz Centre Munich and Technical University of Munich (head L.D.) Conducting multiple projects funded by the European Research Council Innovative Training Networks , DFG priority program ‘Novel Concepts in Phage Biology’ (duration 2021–2024) and sequencing program ‘Asthma-Phage’(2023–2025) that aim to explore the cross-talk between virome, microbiome and the human host in dysbiosis-associated digestive and respiratory diseases  and to eradicate causative bacteria using synthesized-phage communities (phageome therapy). To this end, multiple tools have been developed, including Replidec to predict phage replication cycles  and Viroy 2023”) ,30. The Klebsiella phages and their bacterial hosts are characterized, optimized and prepared for therapeutic applications with a one-health perspective . The aim is to expand the host range of phages, optimize cell-free production, support phage therapy in clinical trials and individual healing-trial protocols with personalized phage preparations, and improve therapeutic approaches of clinical partners [The ‘Therapeutic Phage Group’ of the Bundeswehr Institute of Microbiology, Munich (head J.B.): In collaboration with the European Commission, Salmonella enterica, Campylobacter spp.), but so far, no general permission for the use of phage products exists in Germany. As the clonality and the target bacteria in the veterinary/food sector are broadly overlapping, products for animal use  may also be suitable along the food chain. So far, various research projects have confirmed the beneficial effect of different applications, such as bioremediation, biocontrol and bio preservation between animal housing (pre-harvest) to the food product (post-harvest) [Salmonella, Campylobacter, Yersinia, Klebsiella, Vibrio) have been developed to gain access to a broad spectrum of phages suitable for potential treatment application. In addition, questions addressing the safety of phages are increasingly being studied to close the knowledge gaps for the final risk assessment.‘One-health’ phage applications are increasingly discussed worldwide, but have so far hardly found their way into a holistic approach with respect to the German food chain and veterinary/environmental use. For over a decade, German experts of the different ‘one-health’ compartments and the legislative authorities have been discussing the routine implementation of phages. Major concerns hampering a final decision include esp. (A) a lack of risk assessment for phages released into habitats, (B) the bacterial diversity associated with low reduction and phage-efficiency rates, (C) a spread of phage-resistant bacterial subpopulations associated with a loss of treatment options, and (D) potential disadvantageous effects or changes in microbial communities affecting plant, animal, and human health . Neverthharvest) ,33,34, bOctober 2017: Two symposia, both organized by the University of Hohenheim in Stuttgart, brought the potential of the German phage-researchers’ community to light. The first symposium, in 2017, with 170 participants from 20 countries, addressed the full spectrum of phage research and application. The plenary included experts from the two regulatory bodies BfArM and Paul-Ehrlich-Institut (PEI), as well as from the Federal Ministry of Education and Research (BMBF), who discussed “quo vadis, German phage research”. The regulators stated that there was no clearly defined product to prompt an immediate licensing activity, and urged interested companies to approach the authorities proactively for an early detailed exchange, to avoid costly procedures. They declared their openness to dialogue to support companies planning to develop a phage product. It was also stated that close international cooperation should compensate for the limited number of phage applications worldwide.www.sto.nato.int, accessed on 12 January 2023”) was held at the Military Academic Hospital, Berlin. A total of 67 invited participants from 17 nations exchanged views on the topic of the re-introduction of phages into today’s medical space. From individual working groups as well as from the plenary, it was emphasized that the creation of an international register and an easier exchange of already isolated phages would be particularly important to advance phage therapy.December 2019: A workshop within the framework of the NATO HFM-313 Research Task Group (RTG) “Re-introduction of phage therapy in military medicine” , developments in phage diagnostics and therapy were presented and discussed. The topics of talks included reporter-phage-based detection of bacterial pathogens, phage-receptor-binding proteins for pathogen identification, and an enzyme-linked receptor-binding-protein assay. Further topics included the genetic modification with bactericidal transgenes, non-cellular phage production, issues with GMP production of therapeutic phages in Germany, the efficacy of commercial phage preparations against MRSA, and the possibilities of overcoming the antibiotic resistance in dormant bacteria, as well as the genetic modification of phage TUN1, specific for 4 MRGN K. pneumoniae.October 2021: Phage therapy was also a topic at the 17th Medical Biodefense Conference in Munich, Germany 2021 (MBDC21). In several sessions , several externally funded projects emerged and DZIF launched campaign-like activity in the area. In addition, cooperation with the regulators has intensified considerably, as a result.DZIF TransPhage-Net) was founded and a roadmap for translational phage research was drafted.July 2022: To promote translational phage research, DZIF organized a first strategic meeting on “Bacteriophages in Science and Clinical Use”. The symposium took place in Frankfurt am Main, with 75 national and international participants, including physicians, scientists and industrial partners. Following expert talks on important phage-related research aspects, regulatory requirements and first experiences with phage treatments, all participants actively engaged in further identification of specific needs to promote phage research and clinical application in Germany. As major results of the symposium, a DZIF Translational Phage-Network , originated in Germany in 2020. The combined efforts and expertise of phage scientists, physicians, pharmacists, microbiologists, bioengineers, bioinformaticians, and regulatory affairs officers have joined together to support physicians who intend to treat patients with phages. Topics of the monthly meetings are the presentation of activities of the individual network partners, preparation of funding applications and the identification of suitable partners for clinical and scientific studies. Invited presentations from members and guests on new approaches to phage therapy and the required regulatory processes aim to inform and integrate successful approaches in Europe, Israel, and the US.DZIF has a strong interest in promoting translational phage research, i.e., the successful transfer of research results from bench to bedside. Within the DZIF translational thematic unit “Healthcare-Associated and Antibiotic-Resistant Bacterial Infections” (TTU HAARBI) several initiatives have started up to support phage research in Germany. To provide physicians with an official directive for safe phage therapy, the German Society for Infectious Diseases initiated an official A more complete overview of the current phage landscape in Germany is provided through a literature review of recent years and the personal communication of various research groups. The working groups of institutions and companies contributing to translational/clinical phage research are compiled in Each author provided one or more answers to the three questions presented below in the legends to As seen in Research topics that the participants would like to see prioritized are mainly related to optimizing phage therapy and investigating safety aspects . SpecifiThe biggest hurdles towards implementation of phage therapy in Germany, as seen by the participants, include the current lack of approved phage therapeutics, missing uniform guidelines and quality standards, the limited access to phages for clinical use and the lacking infrastructure for phage production, sensitivity testing and application in the clinics . MoreoveThe aim of this article was to present the current German-phage-therapy landscape from the perspective of clinicians, researchers working closely with them, and those responsible for regulatory aspects, i.e., a group representing the present efforts of physicians, pharmacists, microbiologists, bioengineers, bioinformaticians and regulators.Phage4Cure and PhagoFlow, mainly with phages against P. aeruginosa and S. aureus. Due to the current and previous limitations, there are probably hardly 100 patients who have been treated in Germany in the immediate past, in marked contrast to the many thousands of phage applications between the 1930s and the 1960s. Nevertheless, the treatment volume will increase from 2023 onwards, due to the imminent start of clinical phases of the ongoing research projects. It is also becoming apparent that numerous clinics will take advantage of permission-free production of medicinal products (§13 (2b) German Medicinal Products Act) in the foreseeable future, to produce phages for their patients.As phages are currently not clinically approved, phage therapy is only being applied to a limited number of patients in university hospitals and a German armed-forces hospital, which operate without exception under the umbrella of Article 37 of the Declaration of Helsinki. Either ready-to-use composite phage products are used  or phages are produced within the framework of permission-free production in the respective hospital for their own patients, under supervision of the treating physician. To date, there is only one place in Germany that can produce phages on a larger scale, according to GMP (Fraunhofer ITEM in Braunschweig). These GMP phages will initially benefit the publicly funded research projects It is striking that different phage banks exist in Germany, but, due to their different institutional backgrounds and the policies to which they are bound , they do not operate in comparable modes, and do not freely exchange all their phage holdings. There is also no full genome sequencing of the respective complete phage collections. Thus, a complete and common data collection of all bioinformatic data and the respective host areas is not yet available. However, the DSMZ envisages sequencing the genomes of all clinically relevant phages quickly.SPP2330—aims to elucidate the regulation of the phage life cycle, identify new bacterial anti-phage defense systems, and study the impact of phages on viral communities and biofilms . These research efforts will also help to further improve the efficacy of phage therapy.A major limitation of the present analysis is certainly the arbitrary selection of authors, which cannot cover all the important groups contributing to phage therapy and research in Germany. However, the group encompasses stakeholders from most—if not all—relevant German phage-research consortia, so that overall a valid portrait of the situation in Germany could be generated. In addition, there are numerous research groups engaged in translational research, for example, to facilitate the bacteria–phage matching process  or exploTherefore, although the funding situation and awareness require further improvement, important steps have been taken en route to addressing the antibiotic-resistance challenge with phage therapy. It must also be emphasized that large sums of public funding are already being used for research projects, and that the expert report on phage therapy commissioned by the German Bundestag in 2021—expected to be published in 2023—also underlines the serious interest of political decision makers. In the opinion of the authors, following the demonstration of the clinical efficacy of phage therapeutics, the current legal framework for the use and production of phages  should be adapted to the special requirements of phages, to ensure patient access in a timely manner. However, the present regulatory situation can also be viewed positively. The current relatively low level of information in the population and the legal situation mostly prevents uncritical use of ill-defined phage preparations. The broad application of phage therapy without appropriate rules and scientific exchange of treatment results would probably result in unnecessary failures, and may contribute to discrediting the therapy. The worst case would be that phage therapy would be abandoned again, before proper implementation—a situation resembling the first half of the 1940s in the US and Germany. Thus, establishing controlled, high-quality phage production and therapy is paramount.Against the background of these considerations, the German regulatory authorities support phage therapy and the respective approaches for patient access, i.e., a pragmatic approach of magistral production for selected research, and clinical facilities in Germany. Options such as the Belgian model, i.e., a legal framework that allows for individualized phage therapy using magistral formulations (see reference  for detaA nation- or EU-wide common approach to modern phage therapy, developed together with international actors, is the ideal. Indeed, institutional dependencies and personally motivated efforts to commercialize phage production and therapy may lead to demarcations and limited exchange of information. Nevertheless, the current situation shows activity supported by great enthusiasm in investigating the potential and implementing phage therapy in Germany as quickly as possible. So far, the stakeholders have been substantially supported by the regulatory authorities. At present, there is an urgent need to have more phages available for clinical testing and use, to expand production capacities, develop quality standards for production and clinical use, establish a registry with structured documentation of all treated patients, and gain more scientific evidence for the effectiveness of phage therapy. Current important scientific questions should urgently be answered through targeted networking and funding of translationally oriented research institutions, so that the clinical use of phage products in Germany can take a crucial step forward in the fight against multi-resistant bacteria.


Streptococcus suis as a model, we describe the prevalence of prophages and the anti-viral defence arsenal in the genome of the pathogen as a means to define the genetic building blocks that are available for the (synthetic) development of phage-based treatments. The data and approach described herein may provide a framework for the development of therapeutics against an array of bacterial pathogens.Bacterial infections of livestock threaten the sustainability of agriculture and public health through production losses and contamination of food products. While prophylactic and therapeutic application of antibiotics has been successful in managing such infections, the evolution and spread of antibiotic-resistant strains along the food chain and in the environment necessitates the development of alternative or adjunct preventive and/or therapeutic strategies. Additionally, the growing consumer preference for “greener” antibiotic-free food products has reinforced the need for novel and safer approaches to controlling bacterial infections. The use of bacteriophages (phages), which can target and kill bacteria, are increasingly considered as a suitable measure to reduce bacterial infections and contamination in the food industry. This review primarily elaborates on the recent veterinary applications of phages and discusses their merits and limitations. Furthermore, using  Campylobacter spp., Salmonella spp., E. coli, Clostridium spp., Listeria monocytogenes and Streptococcus suis take a significant toll on animal welfare and exert enormous financial losses on the industry [The global livestock industry is a major contributor to food security and economic development, with a value of about 1.4 trillion dollars . Howeverindustry . It has industry . In Austindustry .In the 20th century, the concept of “One Medicine” became a popular topic in public health . This coStreptococci (GAS-SLOdm) and pilus 2a backbone protein of Group B Streptococci (GBS-BP-2a) were the selected prototypes of bacterial proteins (antigens) [Vaccination with autogenous bacterins, a suspension of attenuated or killed bacteria isolated from a specific herd and administered to nonimmune animals within the herd/flock, is a standard practice in infectious disease prevention . Althougntigens) . This vaThe administration of antibiotics is the most widely implemented measure to prevent (prophylaxis), control (metaphylaxis) and treat (therapeutic) bacterial infections. While the other strategies have proven effective, the use of antimicrobials on farms—particularly as a therapeutic measure—remains a widely adopted practice . TetracyS. suis.With recent increased stringency in regulatory frameworks pertaining to antibiotic usage, new prophylactic and therapeutic strategies are required and actively being explored. These include medium chain fatty acids (MCFA) ,39, lyso30 to 1032 particles on Earth [Bacteriophages or “bacteria eaters” are viruses that infect and kill a cognate bacterial host. These bacterial viruses are ubiquitous and have an estimated abundance of 10on Earth . Their pon Earth . The genon Earth ,46.After their discovery, many studies investigated the potential of phages as an antibacterial in clinical cases, often with contradictory treatment efficacies reported. However, some of the challenges encountered in early phage therapy trials have been attributed to knowledge gaps in phage biology, procedural errors in experiments, and inadequate technology to study host–phage interactions . These cPhages target bacteria in a highly host-specific manner but do not infect eukaryotic cells and have no reported serious adverse effect on treatment subjects. These desirable properties render some theoretical advantages to phages relative to antibiotics as prohylactic or treatment candidates. Conventionally, phage application in therapy, biocontrol or decontamination began with phage isolation followed by screening against a panel of host strains. In vitro characterisation such as determining phage titres, pH, thermal stability and growth kinetics was carried out, and phage stocks were prepared and stored for future investigations or application A. Novel While the use of phages has not been approved for commercial use in most countries, they are used on a case-by-case basis in treating patients for whom currently approved therapeutics have failed (compassionate phage therapy). The world is currently moving toward “greener” crops and animal food products, with an attitudinal shift in consumer preference for antibiotic-free and sustainably farmed and or processed products . On farmSalmonella spp. remains a leading aetiological agent of human food poisoning and gastrointestinal infections in swine, poultry and other livestock. As an enteric pathogen, intestinal contents are usually affected, which predisposes carcasses to contamination during slaughter and packing. To prevent this, controlling colonisation of the pathogen prior to slaughter is practised. Thanki et al. determined the efficacy of a two-phage cocktail in reducing Salmonella enterica colonisation in piglets. Weaner meals infused with spray-dried phages were fed to experimentally infected piglets over five days. Phage treatment significantly reduced Salmonella carriage in the stomach tissue by 1 × 101 CFU/g, duodenum tissue (1.05 × 102 CFU/g), colon content (1 × 101 CFU/g) and caecum (1 × 101 CFU/g). A 16 S rRNA gene sequencing analysis demonstrated no deleterious effect on the overall microbiome following phage treatment. However, a group that received a phage diet yielded higher abundance of Prevotellaceae—a Bacteroidete that has been associated with disease resilience and growth performance in pigs [Salmonella in the caecal samples (95%) and ileal samples (90%) when pigs were orally administered a microencapsulated phage cocktail post-bacterial challenge [Salmonella Typhimurium phage in diets significantly reduced bacterial shedding in pig faeces while increasing growth performance [Salmonella and other infections associated with poultry has been extensively reviewed by Mosimann et al. [ in pigs . This st in pigs . A similhallenge . Gebru eformance . Assessmn et al. . Contrasn et al. ,66,67. TEscherichia coli (APEC) is an extra-intestinal E. coli that causes local and systemic infections in avian species such as chicken, turkey and ducks. Some common colibacillosis caused by APEC include egg peritonitis, pericarditis, salpingitis, airsacculitis, cellulitis and osteomyelitis [E. coli O78. Birds that were treated with phages following challenge presented less severe clinical manifestations compared to the challenged untreated group. In addition, administration of phages offered complete protection, whereas in the untreated group, a mortality rate of 26.7% was recorded [Avian pathogenic myelitis . Collectmyelitis . APEC mamyelitis ,71. Tawamyelitis . A similrecorded . The suprecorded .Campylobacter is a zoonotic pathogen responsible for ~25% of all human diarrheal illnesses globally [C. jejuni populations in the gut of broiler chickens by 2.4 log CFU/g without disturbing the resident gut microbiome [C. jejuni infection is the reported in vitro and in vivo emergence of phage-resistant isolates (up to 13% of isolates) [Campylobacter phages were grouped into I, II and III [C. jejuni counts in broiler chickens by 3.0 log units compared to a two-phage cocktail made of only group III phages (1.0 log unit). In addition to the higher reduction, combining group II and III resulted in lower levels of phage-resistant isolates compared to using a single phage or a homogenous phage cocktail [Campylobacter phage cocktail design has been validated in vitro using several group II and III phages [C. jejuni infection in chickens has been reported by other studies with varying degrees of efficacy [globally . The baccrobiome . One majsolates) . However and III . In a 31cocktail . The effI phages . The appefficacy ,81,82.Clostridium perfringens Type A strains form part of the resident microbiota of animals; however, toxin-producing strains are implicated in a number of livestock diseases, including necrotic enteritis in poultry, necrohaemorrhagic enteritis in cattle, haemorrhagic diarrhoea in swine, and pigbel in humans. The multi-host nature of this pathogen makes it an ideal target for phage therapy. The phage cocktail “INT-401” was examined for its effectiveness in chicken necrotic enteritis (NE). As part of the study, three methods of oral administration were tested. Throughout the 42 days of observation, all phage treatments significantly (p < 0.05) reduced mortalities due to NE to 0–14.0% compared to the mortality rate among the challenged untreated birds (64%) or the antibiotic/medicated group (50%). In-water delivery of phages was the most effective delivery method, with 0% mortality due to NE [C. perfringens via oral gavage. Phage treatment via similar routes significantly improved clinical parameters and reduced gross lesions compared to the infected untreated birds. The mortality rate was 30% in the phage-treated group compared to the infected untreated group (55%) [ue to NE . In a reup (55%) .Aeromonas hydrophila infection in Nile tilapia. A. hydrophila is a zoonotic pathogen reported as the leading cause of septicaemia in freshwater fish [A. hydrophila in fish as well as stimulated the development of specific IgM against the MDR A. hydrophila [Vibrio harveyi and administered in-feed phage [Aquaculture is one of the fastest growing industries in agriculture. The FAO reported that fish accounted for 17% of all animal protein consumed globally in 2017 . Aquaculter fish . In theidrophila . The immdrophila ,91,92. Hed phage . The difed phage . It is led phage . Other fed phage . Encapsued phage ,98. Phaged phage ,100,101.p < 0.05) and reduced the incidence of diarrhoea in weaned piglets (p < 0.001) [Phages have been demonstrated to play a significant role in growth promotion and improving gut health. Upadhaya et al.  assessed< 0.001) . Studies< 0.001) ,83,104.Pseudomonas syringae, Pectobacterium spp., Ralstonia solanacearum, Dickeya solani and Xanthomonas spp. are some of the leading causes of bacterial diseases in crops. Phages that target these plant pathogens have been isolated and characterised as safer alternatives to conventional broad spectrum chemotherapeutics. Bacterial canker caused by Pseudomonas syringae is an economically important disease in farming. The efficacy of single phages in controlling canker was evaluated by spraying the leaves of two-year-old cherry trees with P. syringae. Phage treatment almost completely cleared the bacterial population (down to < 10 CFU) by the fifth week [Dickeya solani and Pectobacterium spp. account for half of all Enterobacteriaceae soft rot (SRE) in angiosperms, including food and ornamental crops. A six phage cocktail was used to control soft rot in potato tubers following a 5-day incubation. Washing tubers in a phage preparation significantly reduced (p < 0.0001) maceration in D. solani infection [Pectobacterium atrosepticum [Phages have also been evaluated as biocontrol agents in crop production. fth week . Dickeyanfection . Phage tsepticum .After harvest, both animal and plant products are susceptible to bacterial contamination and spoilage while in storage or undergoing postharvest processing. The FAO estimates show that the global economy loses $220 billion annually due to plant diseases . MoreoveEnterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species) bacteria have been characterised and assessed in vivo for their potential in veterinary medicine and the food industry, with several commercial phage preparations  based on the antigenicity of their capsular polysaccharides (CPS) [Streptococcus parasuis, 32 and 34 as Streptococcus orisratti, and 33 as Streptococcus ruminantium [S. suis. The prevalence of serotypes varies among different locations; however, most clinical cases worldwide have been associated with serotype 2 [ry tract  and, to ry tract . However piglets ,134. S. es (CPS) . DNA hominantium . For diarotype 2 .S. suis, proteins such as suilysin (Sly), DNase, muramidase-release protein (Mrp), surface antigen protein, VirA, and extracellular protein factor (Epf) contribute to the overall pathogenicity of the bacterium [In addition to the CPS, which is a critical virulence factor of acterium ,140,141.acterium . Colonisacterium .S. suis is the most prevalent bacterium implicated in systemic infections in swine [S. suis infections is not obligatory in many countries and hence data on global prevalence is limited [S. suis on Spanish, Dutch and German farms determined that the cost per pig was € 0.60–1.30. Using a stochastic model, the study reported higher morbidities and mortalities in nurseries than in any other production stage. This reflected the higher cost of metaphylactic  measures in piglets compared to that of autogenous vaccines for sows [S. suis type II infections alone were £100,000–1.3 million annually in Great Britain [in swine . Regarde limited . This ha limited . An evalfor sows . In 2005 Britain . The los Britain .S. suis is not limited to the porcine industry [S. suis infection among piglets, a report of a human infection was identified among meningitis patients in Denmark [S. suis meningitis and septicaemia among patients were recorded in the Netherlands and other European countries, suggesting that the pathogen may have crossed the species barrier long before it was first reported [S. suis genomes suggest the emergence of human-associated clade from swine isolates originating from western Europe. Using representative samples from six continents, Dong et al. traced the most common recent ancestry of human-associated clade to 1802–1855 [suis-carrying pigs in enclosed and often poorly ventilated spaces—and the export of selectively bred pigs from Europe and United States to different continents [S. suis strains, new epidemic lineages appear to have emerged in Asia, where reports of human and porcine infections remain prevalent [S. suis infections among humans with no direct contact with pigs are becoming increasingly common [S. suis infections and S. suis isolates have been recently identified in cattle, lamb, dogs and other animals [As is the case with many viral and bacterial pathogens, the threat posed by industry . Within  Denmark ,150. In reported . Recent 802–1855 . A populntinents . Followirevalent . Cases oy common ,157,158. animals ,161,162.S. suis are limited. To date, only five lytic phages  have becreening . Other sisolates ,166,167.equences ,169. HowS. suis phage landscape, we analysed 133 publicly available whole genome sequences of S. suis for the presence of prophages and anti-phage defence systems. The sequences/strains (https://padloc.otago.ac.nz/padloc/) (accessed on 22 June 2022) [Understanding host–phage interactions is key in the evaluation of phages as therapeutic agents against specific bacteria. These interactions include the preservation of phage populations through integration (prophage) in host genomes and the defence mounted by host bacteria via the expression of anti-phage systems. To shed light on the /strains  were ranne 2022) . Detectine 2022) . The prene 2022) . A phagene 2022)  and visuS. suis genomes encoded a total of 1890 anti-viral proteins representing 20 distinct anti-viral systems  of RM systems in most prokaryotic genomes [S. suis genomes could be grouped into three categories: DNA/RNA degradation, abortive infection, and systems of unknown mechanisms  per sequence (ranged 13–141 CDS), among which, an average of 49.7% could be assigned a putative function. Bacterial proteins associated with virulence, such as toxin/antitoxin, efflux pumps, zeta toxin, and virulence-associated protein E, were detected in the prophages. In addition, anti-phage defence genes such as RMs were detected in prophage sequences, indicating that some bacterial defence systems are prophage-encoded . The funp < 0.0001). The influence of host genome size on the association between prevalence of prophages and number of defence systems was controlled using a correlation matrix (p < 0.0001). Similarly, a moderately positive correlation was observed between the host genome length and the number of prophages . Up until 2.5 Mb, the number of prophages increased with increasing genome length  [S. thermophilus, in which all strains possibly encode the system, or in S. mutans, where 95% encode at least one type of CRISPR-Cas [S. suis. We categorised the defence systems on the basis of their molecular mechanism. Nucleic acid degradation systems were the most common mechanism in the S. suis defence arsenal. This is in line with the estimation for prokaryotes [Persistent lysogeny and anti-viral mechanisms are critical phage–host interactions that affect the success of phage infection, particularly in the application of phages as therapeutic agents. We investigated the prevalence of prophages in e system . Interesa (~50%) . MoreoveISPR-Cas . Recent karyotes .S. suis is higher than has been reported for other Streptococcal species such as S. pneumoniae (1.4 per genome) [S. mutans (1.5 per genome) [S. suis chromosomes. However, this observation could also be a function of the sample size. An all-against-all genomic similarity revealed little proteomic relatedness among full-length prophages suggesting a high genetic diversity. Two patterns can be described for phage groups with low gene relatedness. Firstly, along the genomes of phages of common ancestry, several gene homologs of little similarity exist. Alternatively, phage mosaicism may arise from genetic transfer between dissimilar phages such that only a small fraction of the genome would contain highly similar homologs. The latter trend is consistent with the low inter-cluster relatedness as we qualitatively show in a schematic representation of functional genome alignment   and S. m genome) . Of the lignment . Furtherosaicism . Future S. suis host genome size, the prevalence of prophages, and the abundance of defence systems exists. In Salmonella and E. coli, larger genomes were associated with more integration hotspots. In particular, there seemed to be a selection for integration sites within intergenic and regions of low gene expression rather than highly transcribed regions. In highly transcribed regions, spillovers from transcribed genes could trigger phage gene expression, which can reduce host abundance and consequently the temperate phage population following a switch to lytic cycle [S. suis and how this influences prophage prevalence among different strains. Following integration, temperate phages can confer fitness to the host. For instance, in the context of the correlation between defence systems and the number of prophages, annotations revealed the presence of phage-encoded RM systems , CRISPR-Cas systems, and in vitro synthetic genome assembly have been leveraged to custom-design phages with desired characteristics [Klebsiella sp. and Yersinia sp. in a mixed population [S. thermophilus has been used to introduce specific point mutations, large deletions, and ORF replacement, with relatively high efficiencies. This model was adapted for the construction of escape phages through the introduction of a methyltransferase gene such that the resulting phage mutants evaded the R/M system of the host. This modification significantly improved the bacteriolytic efficiency of the S. thermophilus phage 2972 [Mycobacterium abscessus in cystic fibrosis patients [S. suis using their prophages as building blocks.A potential approach to explore untapped (pro)phages against eristics . Modificpulation . Alternaage 2972 . A synthage 2972 , allowinpatients ,58. SimiK. pneumoniae infection [S. suis have demonstrated flexibility in engineering, low toxicity profiles, and broad efficacy against the pathogen and other streptococcal species, which support their potential for translational development [Moreover, co-evolution “phage training” experiments have been adopted to adapt phages to oppose host defences and widen the host range . The connfection . Howeverelopment ,193,194.Although naturally occurring phages possess a green appeal, the incorporation of genetically modified organisms (GMOs) in food may interfere with their approval by regulatory bodies. To date, the application of genetically modified phages in the treatment of infections in humans have been limited to rare cases with specific therapeutic circumstances meriting exemptions for their usage. Any additional barriers to approval will lessen commercialisation. Similarly, consumer reticence towards GMOs will decrease the likelihood of their application. Compelling research will be needed to prove the efficacy, safety and scalability of genetically engineered phages required by authorising bodies. Researchers also have a duty to engage with the public and policy groups to impress upon them the importance of phage solutions to tackle the catastrophe of antimicrobial resistance. As the prevalence of the problem continues to grow, this consumer reticence towards GMOs may become less of a challenge.S. suis, for which phage applications remain underexplored. We provide insights on the S. suis phage landscape and also paint a quantitative picture of the anti-phage arsenal of S. suis. Our findings reveal the diversity and abundance of S. suis prophages, which can be used as building blocks for synthesising safe lytic phages for application in food and medicine.The emergence of antibiotic-resistant zoonotic bacteria poses a threat that transcends the food industry, affecting the overall health of the environment and other animal populations. The efficacy and safety of phages have been demonstrated in several bacterial infections, with some commercial products already approved in several countries for use in food production. However, there are other zoonotic bacteria, including


In the field of phage applications and clinical treatment, virulent phages have been in the spotlight whereas temperate phages received, relatively speaking, less attention. The fact that temperate phages often carry virulent or drug-resistant genes is a constant concern and drawback in temperate phage applications. However, temperate phages also play a role in bacterial regulation. This review elucidates the biological properties of temperate phages based on their life cycle and introduces the latest work on temperate phage applications, such as on host virulence reduction, biofilm degradation, genetic engineering and phage display. The versatile use of temperate phages coupled with their inherent properties, such as economy, ready accessibility, wide variety and host specificity, make temperate phages a solid candidate in tackling bacterial infections.  As the most prevalent and widely distributed group of viruses on earth, phages are estimated to be around 10iosphere . Moreovehia coli . In recehia coli , researchia coli ,7,8,9,10hia coli . Due to hia coli ,12,13,14hia coli . For inshia coli . Cell lyhia coli . Throughhia coli . Meanwhihia coli ,20. ThusTaxonomically, phages are categorized primarily according to their morphology and genome, and more refined and comprehensive classification is under way . Based oVibrionaceae family [Flavobacterium psychrophilum, which currently causes considerable economic losses in salmonid aquaculture [Temperate phages are detected in a large proportion of bacteria . When ine family , membersaculture . Around aculture , whereasaculture . E. coli, is perhaps the most thoroughly studied and widely applied temperate phage [To date, phage lambda, which infects te phage . The lamte phage . Geneticte phage . Althougte phage .Phage lambda could reproduce many generations in a lysogenic cycle. The lytic-driving genes persist in prophage, yet repressed, which is the key lysogenic maintaining force . Under cBordetella bronchiseptica and decreased the virulence of parental strain B. bronchiseptica Bb01 in mice [Siphoviridae temperate phage infecting S. aureus and shows stronger host inhibition activity than antibiotic ceftazidime [Of note, the interactions between temperate phages and the host genome are complicated. Prophage-induced lysis can impose beneficial effects on the bacterial population and, thus, is preferred in the interest of the overall situation . Most of in mice . vB_SauStazidime .Using lambda phage as a role model, we can obtain basic insights of the life cycle of temperate phages and the host–phage interrelationship. Temperate phage development that may occur through the whole life cycle could be categorized into five major phases. (1) Diffusion: the phage gets through the biofilm to approach the host bacteria. (2) Absorption and injection: the phage binds with receptor proteins on the surface of the bacteria and injects the phage genome. (3) Integration and replication: the phage genome inserts into the bacteria genome and becomes a prophage, or persists independently as plasmid. (4) Induction and packaging: the prophage becomes activated and enters the lytic cycle. (5) Lysis of host bacterium: the progeny phage is released from the bacteria ,44,45,46There is expansive diversity in each step to help the phage adapt to multiple conditions . Upon eaHere, we introduce the mechanism and interaction in the order of the temperate phage life cycle. Understanding the close relationship between the phage and host in a life-cycle order will help us to build a more holistic and intuitive perception towards phage antibacterial activity .In this initial phase, phages diffuse and penetrate through the biofilm to locate and target bacteria. Composed of polymeric substances and bacteria-secreted enzymes and proteins, the biofilm matrix wraps around the microbial community and serves as a powerful physical barrier against not only phages but also immune system and antimicrobial agents ,49. BactThere is growing evidence supporting the theory that phages can promote biofilm formation ,54. HoweWhen initiating infection, phages first adsorb to specific receptors on the surface of bacteria . BacteriHowever, most of the temperate phages are limited in their receptor binding proteins (RBPs) and are obligated to specific hosts ,62. EvolListeria monocytogenes is key in escaping cell phagosomes [Many phages encode an integrase that integrates phage DNA into the host chromosome . Phages agosomes . Lysogenic conversion is the process of prophage gene expressing as a part of host genome and is reciprocal for the prophage and host . ProphagTo make sure of their chance of survival, phages develop a number of defense mechanisms to eliminate unwanted host-sharing. The known mechanisms are divided into three groups: blocking genome injection, expressing repressor protein, and binding inhibition . In addiL. monocytogenes strain 10403S, the two coexisting prophages regulate simultaneous induction and lytic activity under SOS conditions. Moreover, the host can also benefit from the cooperation of its habitants. To maintain harmonious coexistence of two prophages, AriS is discovered as a conserved phage protein and is demonstrated to be capable of avoiding SOS response and phage induction by inhibiting RecA [With regard to phage–phage interaction within one same host, intricate coordination can be achieved upon triggering SOS responses by two phages sharing one host. In ing RecA .Pseudomonas aeruginosa exhibits a selective induction to phage phiMBL3 [Prophages can be induced into the lytic cycle under a series of stressors, such as antibiotics and UV rays . Some of phiMBL3 . In addi phiMBL3 . Separat phiMBL3 .Normally, the progeny phage is packaged, released, and keeps infecting the next host . HoweverSingle-stranded DNA phages cause hydrolysis of the host bacterial cell wall by synthesizing enzymes that interfere with host bacterial peptidoglycan synthesis, whereas double-stranded DNA phages, such as phage lambda, hydrolyze the host bacterial peptidoglycan structure by lysin or endolysin, which are synthesized late in replication .In a nutshell, the above steps illustrate the life cycle of temperate phages, using the lambda phage as an example. The interactions between temperate phages and their bacterial hosts are complex and intimate. While new mechanisms are being discovered, we should also look at how we can better exploit the intimate relationship between phage and bacteria to help in the fight against pathogenic bacteria.Pseudomonas, and may affect the entire lung ecosystem [Pseudomonas strains that are deleted of Pf4 prophage survive significantly longer, which indicates that the presence of prophage Pf4 is a virulence contributor [As we discussed in the temperate phage life cycle, the transduction and lysogenic conversion may cause undesirable outcome such as virulence promotion . For inscosystem . Mice intributor . Furthertributor . A changtributor . In the tributor .Nevertheless, temperate phages also have their advantages that cannot be ignored. A wide variety of temperate phages are found in nature and can be easily induced in the laboratory . TemperaTemperate phages have been widely demonstrated to promote bacterial virulence, which is the most important factor in causing infections . HoweverHosts prevent invasion by reducing virulence. The presence of prophage in the form of plasmid can provide host bacteria with resistance to other foreign DNA at the cost of host virulence. p2 is proposed to be an intact plasmid prophage in Klebsiella pneumoniae. Mutant Kp1604Δp2 exhibits an increase in host virulence which is determined by mouse infection models. The mutant p2 minus strain leads to 100% mortality compared to the 70% mortality of the p2 carrying strain, indicating that the presence of p2 decreases the virulence of its host [its host .L. monocytogenes, there is a classic trade-off situation. The cell wall of Listeria and its associated proteins are responsible for most of the interactions with the mammalian host [Listeria serotype 4b host range. In order to prevent phage adsorption, L. monocytogenes 4b underwent a mutation associated with phosphoribonate glycosylation, resulting in the loss of galactose from the phosphoribonate molecule. This loss of galactose not only prevented phage adsorption, but also led to a reduction in bacterial virulence [In ian host . The temirulence .Virulence shrinks under massive phage predation: A phage mixture targeting different extracellular structures such as receptors causes a huge impact on the bacterial fitness, virulence, and pathogenicity of P. aeruginosa. Secretory virulence factors, such as elastase, pyocyanin, and pyoverdine, significantly facilitate the P. aeruginosa colonization of new niches but are not directly related to the cell response to phage infection. The change in P. aeruginosa PAO1 biology is related to the number of phages that cause selection pressure on the population. The more phages appear in the environment, the deeper and more noticeable are the phenotypic changes involving a reduction of various virulence factors’ production levels [n levels .B. bronchiseptica Bb01 has been elucidated in detail. Isolated from sewage water, temperate phage PHB09 is reported to attenuate host virulence by lysogenization. Not only does the temperate phage reduce the virulence of its host both in vivo and in vitro, most likely by inserting and thus disrupting the pilin protein gene, but the vaccine made of lysogenic B. bronchiseptica strain Bb01+ also showed effective protection of mice challenged with virulent B. bronchiseptica. Moreover, in the sight of possible risks rising from prophage induction and phage releasing, neither antibiotic resistance genes nor reversion of bacteria virulence are observed. All induced bacteria are lysed eventually. Thus, the successful virulence attenuation of prophage PHB09 proposes a promising frontier of temperate phages being developed as vaccines [To further investigate temperate phages’ potential to benefit clinical treatment, temperate phage PHB09′s interaction with its host vaccines .Conducted by Bao’s team, an alleviating effect of temperate phage pre-treatment is observed on intestinal dysbiosis and inflammation in challenged mice. As opposed to the streptomycin treatment, the pre-treatment of mice with temperate phages safeguarded a stable and more diverse gut ecosystem and protected the intestinal system of mice against the pathogen challenge .Biofilms can inhibit drug penetration, and thereby significantly reduce the killing efficiency of antimicrobials . BiofilmTemperate phage cocktails enhanced with ions. Biofilm can provide a sanctuary for bacteria being hunted by antibiotics. The thick matrix of biofilm formed by bacteria is a special shield against antibiotics because of the reduced drug penetration and the accessibility [Siphovirdae family, administered with metal ions Ca2+ and Zn2+, shows the enhanced bactericidal impact both in vitro and in vivo compared to the phage cocktail alone. In this experiment, the reason ions such as Ca2+ and Zn2+ can offer an advantage to the phage cocktail is likely because of the promoted fluidity and stability of cocktail phages in the biofilm. The possible gene transfer through these temperate phages is avoided thanks to the lack of virulence gene in the studied four phages. In addition, by decreasing host virulence, prophage can also be a good helper in outcompeting other bacteria. The antibacterial effect of this recipe is determined by the biofilm removal efficiency, where added ions proved a higher bacterial CFU reduction ability. Moreover, using Galleria mellonella larvae as animal model against MRSA S-18 infection, the survival rate resulting from ions–phages therapy is 10% higher than the phage cocktail alone [sibility . A phageil alone .Temperate phage encoded enzyme to eradicate biofilm. A temperate phage of Pseudomonas has been proved to be able to produce a lyase, LKA1gp49, to degrade LPS. LKA1gp49 is a lyase that degrades the O5-serotype specific polysaccharide. This enzyme degrades LPS molecules embedded in the cell envelope and disperses the biofilm matrix, resulting in an increased diffusion rate for small molecules. LKA1gp49 lyase efficiently reduces P. aeruginosa virulence in the in vivo G. mellonella infection model and sensitizes bacterial cells to the lytic activity of the serum complement. LKA1gp49 could also be a potential additive for antimicrobials, as it does not interrupt the efficacy of ciprofloxacin and gentamicin [ntamicin .Combined with antibiotics: Temperate phage can provide enhancement efficacy in killing bacteria with antibiotics [Burkholderia cenocepacia is one of the most important opportunistic pathogens in causing high mortality rates in cystic fibrosis (hereafter CF) patients [ibiotics ,123. Theibiotics . For inspatients . CF is apatients ,127. Thepatients . With unBurkholderia phage AP3 combined with antibiotics demonstrates increased bactericidal effects in in vivo experiments with moth larvae. This finding could be considered as a potent lead against bacterial strains belonging to B. cenocepacia IIIA lineage, which are commonly isolated from CF patients [P. aeruginosa is the cause of a typically challenging infection endocarditis. Increased resistance to antibiotics and a broadened host range of P. aeruginosa is observed, along with disease progression [Temperate patients . Attentigression .E. coli temperate phage HK97 and antibiotic ciprofloxacin. This synergy works in line with the depletion of lysogens which ciprofloxacin specially targets [For temperate phages, the prophage induction through the SOS response and resensitization to antibiotics are the two main synergy mechanisms . In the  targets . The res targets .In addition to functioning as a killing machine, a temperate phage can be a powerful and stable vector, for instance, carrying vaccines into bacteria and acting as a medium for protein expression. The engineering of a phage integration site or the inhibition of the toxins’ gene expression can also take a toll on host virulence. Temperate phages are a robust platform for genetic engineering and modification .Phage vaccine: Temperate phage lambda’s potential for delivering a DNA vaccine has been exploited and substantiated [antiated . It is eantiated . With alantiated . A vacciantiated . Using tantiated . Vaccineantiated .Lambda PLP: Phage-like particles (PLPs) derived from phage lambda have physicochemical properties compatible with drug standards, and in vitro particle tracking and cellular targeting is achieved by displaying fluorescein-5-carboximide (F5M) and trastuzumab (Trz), respectively. Phage-derived nanodrugs are modular systems that can be easily adapted to combined approaches, including imaging, biomarker targeting and the intracellular delivery of therapeutics. A ‘designer nanoparticle’ system that can be rapidly engineered in a tunable and unambiguous manner, trz-PLP binds to oncogenically active human epidermal growth factor receptor 2 (HER2) and is internalized by HER2 overexpressing subtypes of breast cancer cells, but not by breast cancers lacking HER2 amplified breast cancers. The robust internalization of Trz PLPs resulted in increased intracellular Trz concentrations, prolonged cell growth inhibition and the regulation of cellular programs associated with HER2 signaling, proliferation, metabolism and protein synthesis compared to Trz treatment. The robustness and flexibility of lambda PLP provides a platform that adapts to a wide range of utility and customized features [features .Engineered endolysins: Bacteriophage-derived endolysins are cell wall hydrolases which could hydrolyze the peptidoglycan layer from inside and outside bacterial pathogens [Gardnerella phage exhibits the ability to completely disrupt bacterial biofilms of Gardnerella vaginalis. G. vaginalis is a common vaginal bacterium, but can cause bacterial vaginosis under abnormal growth. This engineered endolysin PM-477 has a strong specificity and efficiency against Gardnerella strains, and has no effect on beneficial Lactobacillus or other species of vaginal bacteria [athogens . A genetbacteria .Temperate phage display: The temperate phage M13 phage has a wide range of applications in biomedical materials, and is used for different therapeutic applications [Chlamydia trachomatis, a globally prevalent human pathogen for which there is no effective approved vaccine [C. trachomatis infection alone, engineered phages stably express RGD motifs and C. trachomatis peptides and significantly reduce C. trachomatis infection in HeLa and primary cervical cells [ications . This is vaccine . Compareal cells .Gene insertion led to attenuated phenotype: The Rickettsia parkeri mutant strain is genetically modified by inserting a transposon into the gene encoding the phage integrase in the bacterial genome. Such a mutant exhibits significantly reduced virulence, significantly smaller phage plaques and improved histopathological alterations in intravenously infected mice compared to the parental wild type. Furthermore, single-dose intradermal immunization of this mutant strain provided mice with complete protection against the lethal R. parkeri rickettsioses in mice. Such a live attenuated rickettsial mutant strain could be used as a novel potential vaccine candidate for the treatment of spotted fever rickettsial disease [ disease .Modification of phage genes to inhibit toxin production: Produced by some E. coli, Shiga toxin (Stx) is causative of gastrointestinal diseases and hemolytic uremic syndrome with high incidence and lethality [E. coli is one of the four pathogens among the mostly benign intestinal commensal E. coli strains [ethality . Shiga-p strains . NotablyE. coli, and the genetic mosaicism of the λ phage was exploited to create a hybrid phage capable of overcoming the phage resistance mechanism. The phage demonstrated superior toxin inhibition in both in vivo and in vitro infections. In the foodborne pathogen EHEC, the λ prophage 933W both produces Stx2 and inhibits phage overlap infection of other λ phages [To curb toxin release, the temperate phage λ was genetically engineered to express a deterrent that neutralizes Stx production in λ phages .Gene-modified phage with CRISPR-Cas3 system: CRISPR -Cas (CRISPR associated) system is a defense system in bacteria, possessed by around 40% of bacteria [bacteria . To exclbacteria . Temperabacteria . In typebacteria .E. coli infection and validates the superior performance over wild-type phages through in vitro and in vivo experiments. In addition, there is no evidence in this study showing that EHEC developed resistance to an engineered lambda phage [A genetically engineered lambda phage exhibits enhanced killing ability and host specificity when incorporated with the CRISPR-Cas3 system and knockdown of the lytic gene cro. This engineered phage specifically and effectively eliminates enterohemorrhagic da phage .Encode proteins that block the QS system: The Quorum Sensing (QS) system is a communication system amongst bacteria to modulate community behaviors, a regulatory system that controls the expression of virulence factors and secreted public goods [vibriophage VP882, through spying on the host-produced anti inducers during QS process, VP882 is capable of manipulating its own cycle switch [ic goods . These cic goods . Moreovee switch .P. aeruginosa phage DMS3 can protect bacteria from the attack of other phages by inhibiting bacterial quorum sensing. DMS3 encodes a QS anti-activator protein aqs1 that is expressed immediately after phage infection. aqs1 inhibits the activity of LasR, a major QS regulator, and restrains twitching motility and superinfection. Although there is a 100-fold increase in the number of cells killed by DMS3aqs1 infection compared to wild-type DMS3 infection, no more phages were produced. This suggests a role for anti-phage mechanisms. Aqs 1 offers a counterstrategy through which phages might simultaneously silence multiple antiphage defenses [defenses .Converted into the stable lytic phage: A virulent mutant SA13m obtained through the random deletion of temperate phage SA13 exhibits active lytic activity and no sign of lysogenicity. The application of SA13m in sterilized milk showed that S. aureus was reduced a non-detectable levels, suggesting that SA13m can efficiently control the growth of S. aureus in food [Enterococcus faecalis temperate phage is converted to a lytic phage for therapeutical purposes. By the deletion of the putative lysogeny gene module and replacement of the putative cro promoter from the recombinant phage genome with a 50 nisin-inducible promoter, the temperate phage is rendered virulent and with expanded host range [ in food . Two tem in food . An Entest range .Temperate phages, because of their natural biological properties, play an integral and indispensable role in the war between phage and bacterium. Temperate phages have contributed a variety of new genetic resources to the bacterial gene pool. Found in half of the bacteria, temperate phages have more accessibility than virulent phages . With prHowever, there is also room for improvement, for instance in the underutilization of the induction of hidden lysogenic phages. The actual employment of induced temperate phages can be more technically demanding, considering the need to remove integrase and integrate related genes. Targeted embedding of phages to disrupt virulence genes is a promising direction for research, of course making sure that the phages do not carry any virulence genes or integrases.In summary, this paper focuses on the life cycle of temperate phages and the interactions they have with their host bacteria. Most importantly, this paper illustrates an array of temperate phage applications we could employ in order to combat bacterial infection and benefit clinical treatment. Many experiments have demonstrated the great efficacy and usefulness of temperate phages in the treatment of bacterial diseases, but more in-depth studies are yet to be discovered.