Pharmacological and Immunological Aspects of Phage Therapy

Review Article OPEN Pharmacological and Immunological Aspects of Phage Therapy Prasanth Manohar1, Ashok. J. Tamhankar2,3, Sebastian Leptihn4,5, Nachimuthu Ramesh1,Y Abstract Downloaded from https://journals.lww.com/imd by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCywCX1AWnYQp/IlQrHD3KirptPALrnDByZCQs6J7rHr0d191xjlhnNibrT7KDa9mGB14LP3U9g== on 01/09/2020 Bacteriophages, or viruses of microbes, when used as a medical strategy, might be able to solve the current crisis mankind faces with the increasing number of pathogens being antibiotic-resistant, where chemical drugs seized to show any therapeutic effect. The socalled phage therapy may be one of the most promising alternatives to treat infections caused by antibiotic-resistant bacteria, which are killed after infection by a phage. While phages that destroy the host by lysis are chosen for therapy, many pharmacological and immunological aspects of phages as medicines have not been established so far. The immune system plays an important role in a process called phage acceptance where both, innate and adaptive immune responses of the host are involved. However, not only medical aspects but also social ones such as lacking public awareness or acceptance, and lack of structured regulatory guidelines are challenges that have to be addressed in the near future to establish phage therapy as a reliable and safe alternative for the treatment of infections. This review focuses on the unique pharmacological and immunological aspects of phages used in therapy. Keywords: phage therapy; phage immune response; phage stability; bacteriophages; phage divide. During the lytic cycle, the viral genome is replicated immediately after infection without prior integration into the host DNA.2 Lysogenic phages are able to enter a lytic phase, were replication and assembly is followed by cell lysis of the bacterium. Both lytic and lysogenic cycles produce a large amount of progeny. As the aim of phage therapy (PT) is the clearance of pathogens by killing the bacteria through lysis, phages in therapy have to be lytic, as the integration of the viral DNA is undesired. PT is not a new concept, described in the next paragraph; due to multidrug-resistant bacteria it is re-emerging after a century to kill pathogens as chemical drugs become ineffective.1 Hundred years into the discovery of phages; the first report of antibacterial activity of phages was made by British bacteriologist Ernest Hankin in 1896, who observed antibacterial activity in water samples of the Ganges and the Yamuna rivers in India against Vibrio cholerea, limiting the spread of cholera.1 A similar phenomenon was observed 2 years later for Bacillus subtilis by Russian bacteriologist, Gamaleya.3 It was Frederick Twort, a medically trained bacteriologist from England, who suggested in 1914 that it may have been due to a virus.4 Felix d’Herelle, a French-Canadian microbiologist who was the first to observe clear zones without growth on bacterial lawns on agar plates, which he called “plaques.” After a few years from his initial finding, he proposed the name “bacteriophage,” literally the “bacteria-eater.”5 Medical tests on human patients by d’Herelle began in 1917, under the supervision of Prof. Victor-Henri Hutinel at the Hospital des Enfants-Malades in Paris. He demonstrated the efficacy of phages by administrating a solution to a 12-year-old boy with severe dysentery. With the single treatment, the patient made a complete recovery without any side effects of the treatment.4 The same anti-dysentery phage was later administrated to three more patients, all of which recovered within 24 hours after the treatment.6 In 1923, the first tests were performed in the United States, at the Baylor University of Medicine in Texas. Here, successful medical trial results were conducted and the physicians concluded the enormous possibilities to fight against bacterial infections by using phages.7 The rapid rise of phages as therapeutics was rather quickly dampened by the discovery of antibiotics and their introduction in the Introduction Bacteriophages (phages) are viral particles that infect and replicate inside a bacterial cell with a high selectivity for a particular host, that is, a bacterium the virus is able to infect.1 The lytic (virulent) and the lysogenic (temperate) cycles are two different viral reproduction mechanisms by which phages replicate inside the host bacterium.2 The major difference between the two cycles is the integration of the viral nucleic acid into the bacterial genome during the lysogenic cycle, where the phage genome is multiplied as the host continues to grow and Editor: Stijn van der Veen 1 Antibiotic Resistance and Phage Therapy Laboratory, Department of Biomedical Sciences, School of Biosciences and Technology, Vellore Institute of Technology (VIT), Vellore 632014, Tamil Nadu, India, 2 Global Health, Health Systems and Policy: Medicines-focusing Antibiotics, Department of Public Health Sciences, Karolinska Institutet, Stockholm, Sweden, 3 Indian Initiative for Management of Antibiotic resistance, Department of Environmental Medicine, Ruxmaniben Deepchand Gardi Medical College, Ujjain, India, 4 Zhejiang University School of Medicine, Zhejiang University-Edinburgh University (ZJU-UoE) Institute, International Campus, Zhejiang University, Haining, Zhejiang 314400, China, 5 Department of Infectious Diseases, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China. Y Corresponding author: Nachimuthu Ramesh, SMV-203A, Antibiotic Resistance and Phage Therapy Laboratory, Department of Biomedical Sciences, School of Bio Sciences and Technology, Vellore Institute of Technology (VIT), Vellore 632014, Tamil Nadu, India. E-mail: drpnramesh@gmail.com Author contributions: All authors PM, AJT, SL, and NR share equal contribution in conception, design, analysis, and writing this review. Funding: The author(s) received no specific funding for this work. Conflicts of interest: The authors reported no conflicts of interest. Copyright © 2019 the Author(s). Published by Wolters Kluwer Health, Inc. This is an open access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NCND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. Infectious Microbes & Diseases (2019) 1:2 Received: 6 October 2019 / Received in final form: 25 October 2019 / Accepted: 28 October 2019 http://dx.doi.org/10.1097/IM9.0000000000000013 34 Manohar et al., Infectious Microbes & Diseases (2019) 1:2 https://journals.lww.com/imd subsequent years.7 Almost a century after the discovery of phages and the first medical trials, the need for an alternative therapy against infectious diseases is larger than ever, as antibiotic resistance in bacteria has become a very serious human health problem. Again, PT is gaining major attention all over the world.4 Studies had demonstrated the effectiveness of PT clinically. A study by Mie˛ dzybrodzki et al. showed that the patients affected by Staphylococcus aureus infections showed positive results (either bacterial eradication or improvement in patient health) for more than 36% of the patients and similar study also showed recovery of patients with orthopaedic infections when phages are administered orally or topically.16 Pharmacologically, bacteriophages may be considered to be selectively toxic antibacterial agents.17 In that aspect, one of the advantages of using phages in the clinic is that they are more targeted “drugs” than antibiotics, which show a lesser degree of specificity towards bacteria, thus also affecting the microbiome of the patient.17 In addition, the discovery and the manipulation of the phage genetic elements are easier with the technological tools available today. The reconsideration of phages in therapy is due to the pharmacological advantages it has over the antibiotics. Pharmacokinetics of phages, that is, the movement of a pharmacological element in the body, demonstrates the benefit of using phages compared to antibiotics, as they are specific to the target bacteria, meaning that the phages would travel in the system only to adsorb onto the specific host where they exhibit their action. The increase in phage numbers observed in the presence of the specific host bacteria cause no or minimal impact on non-targeted bacterial strains and tissues.18 The specific nature of the bacteriophage with a very narrow host range (being specific to one or a very few strains of a bacterial species) also reduces the chances of a possible secondary infection because it will not affect the other non-target bacteria.18 Phages that have been administered via the oral route have been observed in the bloodstream after about 2–4 hours postadministration.18 Prolonged presence of phages is monitored in several cases but is found to be harmless.19 The number of phages in the system correlates with the concentration of the host bacteria, while full biological activity is observed only at the site of infection. Phages are cleared by the normal activity of the kidneys.20 The response of the body’s humoral and innate immune system towards the phage varies depending on the type of phage and the infection.21,22 In many cases, antibodies are produced against the phage antigens, while in other cases, the human body seems to be non-responsive with no antibody production. This points towards a possible continuous presence, and normal existence of such phages in the human system.22,23 If phages are a natural component of the human microflora, phages that enter a lytic stage but display lysogenic life cycle, might increase the chance of the genomic integration of virulent genes.13 Pharmacokinetics in PT mainly deals with the dosage of phage or phage cocktail required to reach the target site, infect the pathogens and eradicate the bacterial infection. In general, pharmacokinetics of any drug is important to understand the systematic application of the compound. In case of topical administration pharmacokinetics are ideal as the phages are applied directly to the site of infections, for example, an open wound.24,25 Here, the benefit is that the phages do not have to be efficiently distributed throughout the body to reach the site of infection, but are in addition able to penetrate biofilms, which does not reduce absorption rates of phages to the surface of target bacteria.4,26 This stands in stark contrast to the effectiveness of chemical antibiotics which sometimes are unable to diffuse into the chemically complex biofilms.26 Yet another clinical aspect of PT questions the possibility of occurrence of phage resistant bacterial mutants by natural selection (bacterial strains that are capable of becoming resistant to bacteriophages that would infect them previously). Such an Pharmacology of PT Many reviews discuss the advantages and disadvantages of using bacteriophages in therapy compared to antibiotics.8–10 Pharmacological aspects have to be mentioned even when complex biological entities, such as bacterial viruses, are being used as drugs. Pharmacodynamics, that is, the impact of the drug on human body and pharmacokinetics, that is, the body’s reaction towards drug, have to be discussed. Pharmacodynamic studies of a drug refer to the toxicity as well as the side effects and possible impact of the compound on non-target tissues. To define the pharmacodynamic parameters of phages, it is necessary to study the specificity of administrated phages during therapy. While PT is being used as treatment in countries like Russia and Georgia, no defined studies on the pharmacodynamic aspects of PT have been performed to date.11 For its implementation especially in the western world where antibiotics are still the main basis for treatment of bacterial infections, it is necessary to overcome possible legislative hurdles as well as to create public awareness for PT.12 Phage’s used in therapy should not contain any genes that encode virulence factors or protein toxins. A phage which is found to be lysogenic, that is, has the ability to integrate into the host’s genome, or contains toxic genes should not be used, whereas lytic/virulent phages which cause lysis of the infected cell can be considered for in vitro characterization and possible subsequent therapeutic applications.13 A temperate phage undergoing lysogeny (ie, the viral genome is integrated into the bacterial host DNA where it gets multiplied by cell division of the bacteria), might make the pathogen even more virulent as the phage might carry genes delivering additional virulence factors, or manipulating the host behavior, which does not eliminate the pathogen from the infected individual but now requires yet another specific and lytic phage to clear the infection.14,15 Thus, it is absolutely necessary to find the most suitable phage for treatment of an infection. Both in vitro and in vivo, preclinical studies should find the phage fulfilling all criteria in order to be considered for clinical trials and for the development into a pharmaceutical product (Figure 1).14 Pharmacodynamic approaches from safe application of the phage solution to killing titer and the so-called multiplicity of infection (the number of phages per cell) are factors that need to be carefully determined before using a phage as a treatment strategy. Killing titre and multiplicity of infection are related to the number of phages or phage cocktail that is required to infect and kill all infection-causing bacterial cells completely. The safety of a phage solution does not only concern the phage itself, such as the absence of lysogeny or virulence genes, but also the liquid in which the virus is contained. Here, no antigenic or pyrogenic substances should be found that might be causing a direct or indirect immune response, for example, bacterial components, such as lipopolysaccharide. Another factor that is necessary to be considered is that fact that during PT a large amount of bacterial toxins (due to bacterial killing) may be released, which not only affects the immune system but potentially impacts on the balance of gut microbiota, a particular aspect which requires more attention to improve PT. 35 Manohar et al., Infectious Microbes & Diseases (2019) 1:2 Infectious Microbes & Diseases Figure 1. Future challenges for phage therapy. could imply that the human immune system might not perceive bacteriophages as a threat. Clear evidence has yet to be established regarding an altered immune response towards phages, including the clearance of microbial viruses by the immune system.34 By simply comparing the size, bacteriophages are larger than some eukaryotic viruses yet it is currently not clear whether phages induce immune responses and how the interaction with the immune system is.34 Simple immunization of humans or animals, that is, the injection of a solution containing phages produced phage antisera (antibodies against phages) with low levels of antibodies present in previously nonimmunized humans/animals.36–38 It was also predicted that “natural antibodies” may be present in the human body for Tlike bacteriophages due to the fact that they are commonly found in the normal flora.38 In an experiment where mutant lambda phages were introduced in the circulatory system, the phages were removed by innate immune system and their circulating time in blood was reduced, indicating that the immune response may be triggered against the viral particles.39–40 There are not many studies that could demonstrate the mechanisms of an anti-phage cellular response, and also a controversy about how phages are presented to the T-cells by Antigen-presenting cells (to initiate antibody production and memory responses in future), passing through epithelial barrier has not been solved, though several hypotheses have been developed.41 Microorganisms such as occurrence is expected in the long run when phages are constantly in therapeutic use.27 This might be a resultant phenomenon (occurrence of bacteriophage insensitive mutants) due to phage inactivation or modification of phage receptor.28 However, while the phage resistant mutants may arise in similar frequency in which antibiotic-resistant superbugs occur, unlike antibiotics (that are chemical entities not capable of self-modification) phages are biological entities that have the capability to undergo natural evolution and hence result in self-modification thereby co-evolving with their specific host strain.29 The phage resistant bacterial mutants arise after a lag, the length of which will define “how common a bacterial species can exist before being a target for the phages which co-evolves with the bacterial strain.”30 Immunologics of PT Immunological responses of the human body as a result of exposure to phages by ingestion or presence of phages in the bloodstream are important aspects of PT. The immune system is a complex network that reacts to the presence of foreign particles, including microbial viruses. There is growing evidence demonstrating that bacteriophages are part of the healthy human microbiota/virobiota and many studies are being conducted to evaluate their role on the microbiome.31–35 Bacteriophages are present in the human body in large numbers, which possibly 36 Manohar et al., Infectious Microbes & Diseases (2019) 1:2 https://journals.lww.com/imd Figure 2. Pharmacological and immunological aspects of phage therapy. in this case a drastic decrease in the C reactive protein, was observed after phage administration although the infection was not eliminated entirely.50 In another study, the activation of NFkappa B was found after allogeneic skin transplantation in mice that diminished cellular infiltration.51 Surprisingly, even just the presence of the coat protein gp12 from phage (T4-like), not the entire phage, resulted in reduced levels of interleukin-1 and interleukin-6 in serum of murine models.52 As phage particles were also found not to produce inflammatory mediators nor reactive oxygen species during therapy potentially make them anti-inflammatory drug candidates.20,53,54 As phages are part of the human microbiota, it is not surprising that they can act as immunomodulatory and probiotics-like.20 Due to the effects observed upon administration of phages as therapeutics, it becomes clear that the success of phages in therapy depends not only on its anti-bacterial but also on anti-inflammatory characteristics as phages are good immunomodulators.55 Studies also showed that phagocytosis of hosts’ cells are in some instances influenced by phages while there is no evidence for phages to activate phagocytosis. Interestingly, phages have been reported to still be able to multiply inside the bacterial cells which have been phagocytosed.55–57 Studies had also proven that intracellular killing of pathogen/bacteria by phagocytes (granulocytes and monocytes) is not influenced by the presence of phages.58 bacteriophages may interfere by a so far unknown mechanism with the immune system of their hosts as studies suggest that bacteriophages are beneficial and play a significant role in maintenance of bacterial diversity in humans.42 While human PT studies are being conducted for several years now, our knowledge of the complex interaction of therapeutic phages with the immune system has only marginally increased.43 A study by ŁusiakSzelachowska et al. provided information on the neutralization of phages in patient sera receiving PT. The study concluded that the mode of administration of phages is critical as one of the aims is to reduce the anti-phage response of the patient during PT.44 In more recent research, the ability of phages to interact with the immune cells/mammalian cells is being extensively studied, also encompassing phage immunogenicity and phage immunomodulation.45 In the vaccine development field, phages have been used as vaccine vehicles. Here, it has been shown that phages can stimulate innate and adaptive immune response; D’Apice et al. demonstrated that bacteriophage fd virion particles are sensed by dendritic cells as pathogens and both innate as well as the adaptive immune system are activated.46 Another study by Smith et al. found that bacteriophages are safe candidates for immunization of patients with antibody-mediated immunodeficiency.47 Though the immunological applications of bacteriophages are enormous, such as the above-mentioned vaccine development strategies,48 aspects of immunity need to be considered for PT as well (Figure 2). For PT to be successful, the administrated phages should reach the infection site and multiply rapidly for eradication of infection.49 The effectiveness of a medical therapy also depends on anti-inflammatory properties. An anti-inflammatory response, Immunocompetent and immunocompromised patients Over the recent years, the number of publications and research projects in the field of PT and possible clinical or technological 37 Manohar et al., Infectious Microbes & Diseases (2019) 1:2 Infectious Microbes & Diseases applications of phages has been constantly increasing. When studying the application of phages and their interaction with their hosts, a logical question to ask aims towards the understanding of PT in immunocompromised patients.59 An immunocompromised individual displays a reduced ability to react towards microbes as the immune system is inefficient in inducing an immunogenic response against a foreign antigen.59 In contrast, immunocompetent persons are individuals with a normal and active immune response and such individuals are widely preferred when understanding PT studies. Information regarding the immune status of a person on recorded details in PT trials is typically rare.59 However, therapeutic efficiency and safety concerning usage of phages as antimicrobial agents in immunocompromised patients is highly important for the screening and diagnosis of the efficacy of the treatment.60 Treating immunodeficient or -compromised patients would allow scientists and physicians to gain exact data on the activity and the efficacy of therapeutic phages, ultimately allowing to standardize the dosage and the route of administration.59 The clinical efficacy of PT in vivo is based on two parameters: The first one is the lytic life cycle of the phage itself which results in the killing of the host pathogenic bacteria by lysis. The second parameter is a potential ability of the bacteriophage used in therapy to induce a direct or an indirect immunogenic response by the organism against the bacterial pathogen, or by being immunoregulatory. Often, the lysed bacterial cell components trigger an immune response even if intact bacterial cells are unable to elicit such an immunogenic action.59 If prepared inadequately, phage preparations can contain compounds derived from bacterial cells such as lipopolysaccharide,61 which results in the observation that some phage lysates may possess immunostimulatory properties. However, this might be beneficial in some cases as an immunostimulatory effect could play in favor for clearing the infectious agent, as well as when we consider the extensive suppression of immune response in immunocompromised patients.59 Most of the in vivo testing of PT has been performed in immunocompetent individuals, where the action of phages is potentially acting synergistically with the immune system of the individual. The therapeutic efficiency of phages may be different when phages are administered to immunocompromised individuals. Studies conducted on mouse models that are immunocompetent revealed that lysis of bacteria by the phages are the main reason for the therapeutic effect of the phage, while induction of the immune response seemed to have no effect on the eradication of microbes.62 When phage lysates were used to treat infections in immunocompromised mice, lysis was again the reason for the clearance of the pathogenic bacteria, while the potential capability of the lysate to induce an immunogenic response had no effect on the eradication of pathogen from the host.62 Considering the data obtained from immunodeficient patients with antibiotic-resistant infections, we can conclude that PT is highly valuable.63 Bacteriophages were successfully used for therapy to cure bacterial infections in cancer patients and renal allograft recipients.59,63 PT was also tested successfully in immunodeficient mice models to cure S. aureus infections.64 These studies demonstrated that PT may be effective in patients with autoimmune diseases as well. Together with their hosts, phages can be found in the oral, respiratory, gastrointestinal, and urinary tract, but also in the blood serum, where no microbes are found in a healthy individual.67 Large numbers of phages are present in the infant intestines which decrease when the person grows up. Still, the amount of bacteriophages in the human body is enormous which might be providing a basis as biological stabilizers to the gut microbiota.67 The most predominant phages in the human gut belong to the family of Caudovirales (Myoviridae, Siphoviridae, and Podoviridae), which is also the same family of phages used for PT. Studies on healthy individuals proved the existence of more than 1000 viral genotypes in the human gut. Most of these are microbial viruses, including the family of Caudovirales and prophages within bacterial genomes.65 An important aspect of phages in human body is their ability to translocate within different tissues and organs.65,68 Studies have shown that phages can move between the mucosal barriers and blood, which could influence the immune response of the host.65 The influence on the immune system is an observation which is an often overseen or disregarded aspect of bacteriophages, which have been long thought to exist only to control the bacterial population in the human gut.65 There is a considerable difference between the bacteriophages observed in the human gut of a healthy individual compared to that of an unhealthy person, with potential strong immunological implications. Studies have suggested that the translocation of phages in patients with gastrointestinal tract diseases may be higher than that of healthy individuals because the permeability is higher in the affected tissues during the disease.69 There is a disparity in reports regarding the presence of phages in blood samples collected during PT; one study reported the presence of phages in patient blood during oral administration of T4 phages for the duration of an entire month while a second study reported the absence of phages in serum after oral administration of phages in healthy volunteers.65,70 If phage destroys gut bacteria, they may inhibit the translocation of these target bacteria into the patient tissue influencing inflammatory factors.55 Phages can exhibit a strong regulatory effect on gut immune cells; here, they possibly result in down-regulation of the immune cell activities to reduce proinflammatory responses, but more detailed studies are required.55 Bacteriophages have been reported to be present in 45% of clinical samples when bacterial infections were analyzed towards the presence or the absence of microbial viruses. Considering this fact, bacteriophages can have a strong interference during the clinical practices. The role of bacteriophages in diagnosis is not very clear; the interference of phage DNA with bacterial DNA during molecular analysis might contaminate the patient samples. Patients with intestinal diseases are found to have an increased number of phage particles, possibly due to the induction of prophages.65,71 This is possibly due to the effect that some antibiotics which target the bacterial DNA replication (eg, quinolone drugs), increase the lysogenic phage particle numbers, while on the other hand, drugs that are acting on the bacterial cell membrane will result in a lower phage count due to lack of host bacteria.71 In the case of the inflammatory bowel disease (IBD), a decrease in microbial diversity is considered as one of the more important factors for the severity of the disease. Bacteriophages, which might have a strong influence on bacterial populations in patients with IBD should be investigated.72,41 A study suggested that the abundance of bacteriophages can cause a reduced bacterial diversity in the human gut that may lead to Crohn disease.73 In contrast to this, another bowel disease, ulcerative colitis, was found to have no correlation with virobiota itself, while the lysis Bacteriophages in human – an important component of the virobiome Bacteriophages are the most abundant virobiota in the human body especially in the gut where show the largest diversity.65,66 38 Manohar et al., Infectious Microbes & Diseases (2019) 1:2 https://journals.lww.com/imd of bacterial cells due to bacteriophage infection is discussed as a cause of the inflammation, which might be caused by the release of bacterial toxins.65 A recent study suggested that the use of bacteriophage cocktail could reduce the abundance of gut microbiota which in turn might lead to an increased permeability of intestinal membrane, which was discussed as a potential reason for bowel disease.74 Again, as the underlying reason, the release of bacterial toxins due to the lysis of the pathogen was discussed, which can have a strong influence on microbial diversity that potentially can lead to other symptomatic causes. Contradictory to this observation, another study suggested the important role of bacteriophages in maintaining human health during IBD by influencing the microbial diversity,75 as they observed a correlation of the diversity of phages with the diversity of bacteria. Most of the studies were performed using murine models which are of course not translatable to the clinical setting. In general, phages have been found to outnumber the bacteria in mucosal layer and the presence of phages in mucosa is believed to be involved in the selection of commensals.76 Bacteriophages can act as immunomodulatory elements by directly acting on immune responses but the immunosuppressive role of phages in controlling inflammatory and autoimmune response has not been investigated conclusively.77 The future of PT looks promising as there are many important studies being performed at different parts of the world.78–86 The importance of phages in anti-inflammatory and immunomodulatory activities will prove to be advantageous for PT. Much data was gained from investigating phages and their properties, while the importance of bacteriophages in the human microbiome and immune system remains to be fully elucidated. Therefore, further studies on the role of bacteriophages in human health are required to fully understand the complex and ancient relationship of the human body with bacteria and their phages. Clinical trials are currently being undertaken with Phase 1 trials shown no adverse reactions associated with the use of phage cocktails, thus paving the way for Phase 2 clinical trials (eg, APS Biocontrol Limited, Dundee, UK). The success of this work will set a positive model for western medicine if Phase 3 trials can be completed with a positive outcome. Wright et al. outlined that success of PT greatly depends on the susceptibility of the target bacteria present at the site of infection and accentuates the importance of correctly identifying the pathogen before the phage treatment.88,89 One concept which has received little attention in PT is their use as “bacteriophage-based probiotics,” a novel, safe and effective application.90 “Probiotic phages” eliminate potentially pathogenic bacteria in the gut without interfering with the healthy gut microflora, therefore preventing an illness. Because of their specificity, phages can be used to specifically manipulate the gut microflora, thereby providing protection against, for example, diarrhea-causing microorganisms. Phages can be used as powerful vehicles in the development of vaccines; however, this application requires further research and development. Phage-based vaccines may not only serve as a preventive platform for the infectious microbial diseases but also to combat against non-infectious diseases.91 Effect of phagebased vaccines is achieved through immunization which in turn harvests the innate ability of the natural immune defense to fight diseases. This could direct phage-based vaccine towards the treatment of cancers, neurodegenerative disorders, drug addiction, and so on. Genetic engineering of phages can furthermore improve the efficiency to deliver therapeutic cargoes to eukaryotic hosts. Phage display permits the expression of wide variety of antigens on the surface of phage particles which is a major advantage in the field of vaccine development. Removal of phages by the immune system is a major barrier for bacteriophage delivery. Kim et al. constructed phages that were conjugated with polyethylene glycol, which prolonged the blood circulation time and decreased the level of T-Helper cells compared to non-modified phages.92 In animal models, both unmodified phages and polyethylene glycol-conjugated phages resulted in rapid neutralization.92 When encapsulated into liposomes, phages were able to efficiently reduce the number of Klebsiella pneumoniae in mice models,93 with increased retention time as compared to the free phage.93 A promising avenue is the encapsulation of phages including the construction of phages as micro- and nanoparticles in vesicles, emulsions, foams, nanogels, micelles, capsules, membrane emulsification, and core-shell particles.94 Currently, PT focuses on comparably fast-dividing bacterial species while for others PT has neither been explored nor developed. Two examples are Mycobacterium africanum and Mycobacterium leprae which exhibits very slow division times; however, PT might be a promising solution for these microbes.25,35,66,95 Several challenges lie ahead for the therapeutic application of phages such as the availability of phages against specific host bacteria, difficulties in phage identification for rapid and immediate therapy, elimination of bacterial toxins in the phage products, response of body’s immune system to phages such as neutralization, choice of delivery system to the host (mode of administration and dosage), awareness in the public, and the lack of regulatory guidelines. In conclusion, though several therapeutic applications of bacteriophages are currently being exploited, uncertainties in regulatory guidelines for phage products along with the difficulties in patenting laws might reduce the financial commitment of pharmaceutical companies. A tremendous effort from governments, companies and applied as Future Perspectives PT is a field that will provide innumerable benefits to science in general, but also to applied fields such as veterinary science, and of course medicine in particular by offering a possible solution to overcome the increasing problem of antibioticresistant pathogens. Combining antibiotic therapy and PT, the use of phage cocktails, or the use of phage protein products may be the most promising strategies for the treatment of bacterial infections involving phage.83,85 Therefore, the focus of PT should not lie on the discovery of phages alone, but on investigating such strategies involving combinational therapies or phage products. Phages have a tremendous potential in reducing or eliminating the amount of infectious and resistant bacteria in environments such as hospital wards (intensive care unit) to reduce the risk of nosocomial infections. In addition, the use of phages in reducing the number of multi- and extreme drug-resistant bacteria in wastewater treatment plants could help us to fight the global threat of the so-called superbugs. For the production of phages on an industrial scale, major issues have not been adequately addressed such as the removal of endotoxins and pyrogens, which are being released from the disrupted cell during phage lysis. Merabishvilli et al. recently described a protocol that employs an endotoxin removal kit which provides sufficient purity for the phages for clinical trials. It will be necessary to be able to scale up and optimize such processes if more commercial entities move into PT in near future.87 39 Manohar et al., Infectious Microbes & Diseases (2019) 1:2 Infectious Microbes & Diseases well as academic research is a necessity translate PT from bench to bedside. [12] Conclusions [13] Bacteriophage therapy is a promising but challenging field with regulatory and technical hurdles that need to be overcome to ensure its successful application in medicine. Technical challenges connected with PT will require new strategies for addressing phage manufacturing, phage delivery, and systemic side effects. Phage treatment may be able to establish itself in western medicine, predominantly in areas where it can also reduce large medical costs. Antibacterial resistance has been recognized as a global threat, with the World Health Organization calling it one of the largest challenges of the 21st century and has resulted in a Global Action Plan to address the problem. The whole world is searching of solutions for keeping antibiotics useful for the foreseeable future. PT has the potential to provide a solution posed by multidrug-resistant bacterial pathogens. However, PT may offer solutions in at least some areas but will not be able to replace antibiotics completely. Considering that renewed research efforts are being made to exploit the use of PT, it may be anticipated that over next 5–10 years PT may find its way in clinical practices in life-threatening chronic bacterial infections that show resistance towards currently available antibiotics. Although bacteria acquire resistance against phages, this resistance is not as problematic as resistance towards antibiotics, as phages are biological entities that can adapt to resistant hosts. Phage researchers are making relentless efforts to elucidate every aspect of the science behind PT, ultimately contributing in saving human lives. 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