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Research Article

Phage Therapy: A Different Approach to Fight Bacterial Infections

Zigale Hibstu ^*

Habtamu Belew

Yibeltal Akelew

Hylemariam Mihiretie Mengist

*Corresponding Author: Zigale Hibstu, Department of Medical Laboratory Science, College of Health Sciences, Debre Markos University, Debre Markos, Ethiopia, P.O. Box 269.

Citation: Zigale Hibstu, Habtamu Belew, Yibeltal Akelew, Hylemariam Mihiretie Mengist. (2023), Phage therapy: A different approach to fight Bacterial Infections. J, Clinical Case Reports and Studies, 4(4); DOI:10.31579/2690-8808/168

Copyright: © 2023 Zigale Hibstu, This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Received: 26 November 2022 | Accepted: 27 February 2023 | Published: 05 April 2023

Keywords: lysogenization; lytic phage, modified phages; resistance to phages; crispr; immunity; conquering crispr; phage encoded products

Abstract

Phage therapy is one of the alternatives to treat infections caused by both antibiotic sensitive and resistant bacteria with no or low toxicity to patients. It was started a century back although rapidly growing bacterial antimicrobial resistance impacting large morbidity, mortality, and financial cost initiated the revival of it. It involves the use of live lytic, bio-engineered, phage-encoded biological products and in combination with chemical antibiotics to treat bacterial infections. Importantly, phages will be removed from the body after seven days of clearing infection. They target specific bacterial strains and cause minimal disruption of microbial balance in humans. Phages for medication must be screened for the absence of resistant genes, virulent genes, cytotoxicity, and their interaction with the host tissue and organs. Since they are immunogenic, applying high phage titer for therapy exposes them and activates the host immune system. Up to date, no serious side effects are reported with phage human therapy. In this review, we narrated phage - phagocyte interaction, bacterial resistance to phages, how phages conquer bacterial resistance, the role of genetic engineering and other technologies in phage therapy, therapeutic application of modified phages and phage-encoded products. We also highlighted the comparison of antibiotics and lytic phage therapy, pros and cons of phage therapy, determinants of human phage therapy trials, phage quality and safety requirements, phage storage and handling and current challenges in phage therapy.

Introduction

Phages are obligate intracellular viruses that infect and kill bacteria. They live everywhere bacteria live and there are about 10 [30,31] phages in the biosphere. They have high durability in natural systems and inherent potential to reproduce rapidly in their appropriate host [1,2]. They are made up of proteins or proteolipid capsids containing fragments of deoxy nucleic acid (DNA) or ribonucleic acid (RNA) (figure 1). Their genome size ranges from thousands to 498kbs [3]. They have no machinery to generate energy and ribosomes to make proteins even if they carry the genetic information needed to replicate in the right host cell.
Figure 1. Classification of phage

Phages are generally more specific and their specificity is determined by phage-host receptor surface, genetic and host physical defense mechanisms, the nature of phage(s), and their co-evolution. Phage lytic enzymes (endolysin) have broader specificity at the genus and/or species level. However, their specificity varies from infecting many bacteria to a single strain. Limitations in sensitivity to a single phage therapy during poly-microbial infection are solved by applying phage cocktails [4].
Phage therapy is a way of delivering virulent phages to a clinically ill patient to rapidly kill pathogenic bacteria [5]. It involves the use of lytic phages, bioengineered phages, and purified lytic proteins of phages to infect and lyse bacteria at the site of infection. Phages and their lytic proteins can be used specifically to treat multidrug resistant (MDR) bacteria lonely or in supplement with antibiotics. Phage therapeutic approach is rapidly increasing although there is still no adequate knowledge on phage-phage, phage-bacteria, phage-human interactions mainly due to safety and efficacy concerns [6]. Novel concepts of phage therapy involve direct treatment of bacterial infections, phage-mediated prevention of bacterial infection, and exploration of phage diversity in environmental and human ecological niches (Figure 2) [7]. Currently, the human phage therapy trial is largely used, although its therapeutic use is limited to Georgia, Poland, and Russia [8]. Phage therapy is a promising approach to fight bacterial infections as phages have unique bacteria killing mechanism and life cycles; either lytic or lysogenic growth cycles (figure 3). Only lytic phages are used for therapy purposes.They inhibit the emergence of resistant bacteria by killing the bacteria they infect and are preferable to antibiotics due to less damage to the general micro-biome [9]. Lytic phage therapy involves replication of phages in phages infected bacteria that disrupt bacterial metabolism and kill them.
Figure 2. Novel concepts of phage therapy.
Figure3: Phage lytic life cycle

The ability of most lytic phages to encode enzymes, holins and endolysins, that degrade bacterial structures (cell membrane and cell wall) make them a new hope to fight bacterial infections. This property makes them efficacious against both antibiotic sensitive and resistant bacteria. However, some lytic phages use only endolysins. Indeed, holins degrade bacteria cytoplasm allowing endolysins to access bacteria glycoproteins. Holins control the exact point of time for endolysins to accesses bacterial murein and synchronize the holing-endolysin system to the late stage of viral replication. The synergetic holing-endolysin system causes cell lysis and release of matured lytic phage progeny [10]. Thereby, 50-200 matured phage progenies will be released from a bacterium lysis [12, 49].
Temperate phages are not used for therapeutic purpose because they integrate their genome into the host chromosome or sometimes maintain it as a plasmid to be transmitted to daughter cells during cell division or horizontally across bacterial community. Of course, they may undergo a typical lytic cycle or lysogenization. Temperate phages enter lytic life cycle when host conditions are weakened, maybe due to scarcity of nutrients; then, prophages become active. At this stage they promote the reproductive cycle, resulting in lysis of the bacterial cell. In lysogenic life cycle, the virus continues to replicate as bacteria continues to reproduce and found in all bacterial offspring. Example, phage lambda of E. coli is a common phage that has both lysogenic cycle and the lytic cycle [11]. Surprisingly, temperate phages may increase the pathogenicity of the host bacteria because bacterial virulent genes are identified from their genome [12].
Phage cocktails are used for phage therapy due to rapidly emerging bacterial resistance because many types of phages infect the same species or strain of bacterium. Thus, allowing phage cocktails to target different structural sites and metabolic activities of a bacterium. Of course, it is argued to use only a single specific phage against a pathogen to prevent the emergence of resistant bacteria since extensive use of phages may promote resistance to phage cocktails [13]. Some of the challenges to apply phage cocktail therapy are the inability to predict the effect of mass use of phages, very high phage treatment price, the issue of efficacy and being very specific [14]. Highly increased emergence and spread of resistant pathogens and lack of new drug production directed many institutions and commercial companies to be engaged in phage therapy [15]. Antibiotic resistant opportunistic pathogens are threats especially for immune-compromised and immune-incompetent patients in health care settings which are serious problems in medicine to date where phage therapy is a solution [16].
Even if phage therapy against bacterial infections is very promising with plenty of advantages, many advancements are essential to implement phage therapy at a large scale for therapeutic purposes due to emerging issues on its safety, quality, stability, and lack of enough evidences to use them for human medication [17].
2. Phage-phagocyte interaction
Phage selected for phage therapy should be resistant to phagosomal degradation to hamper or delay phage specific adaptive immunity response induction and extend the survival of phage in immunocompetent individuals [18,19]. Therapeutic phages are naturally immunogenic so that they stimulate intertwined interactions between innate and adaptive immune cells that may affect phage therapy. Bacterial elimination occurs due to stimulation of local immune responses as a result of phage and bacterial derived pathogen associated molecular patterns (PAMPs). Since phages are immunogenic they induce phage-specific humoral memory, which can hamper phage therapeutic success due to neutralization [20].
The role of phagocytic cells is to recognize and eliminate foreign antigens and to activate the adaptive immune system response whenever necessary. Leukocytes bind to phage in a time, concentration, temperature dependent manner and endocytose (phagocytosis for particles > 500 nM) to remove them. Polymorph nuclear leukocytes and macrophages can degrade phage and phage degradation is the first step to stimulate antigen presentation and development of an adaptive immune response [20,21]. When phages express proteins that mediate bacteria-phage interaction, they bind together and macrophages become activated. Macrophages phagocytize extracellular bacteria and endocytose phages along with it. Phagocytosis will be stimulated via bacteria and phage derived pathogen associated molecular patterns (PAMPs) and there will be continual phagocytosis of phage infected bacteria. Bacteria and phage derived PAMPs will again co-stimulate macrophage activity [22,23]. Antibodies produced against the bacteria will opsonize the bacteria itself and facilitate phagocytosis by macrophages, which promote bacterial clearance. Phage-antibody complexes bind to Fc receptors on macrophages, which triggers endocytosis and subsequent phage clearance.
Generally, for phage therapy to be effective there must be a strong interaction between hosts derived ligands and host pattern recognition receptor. Unfortunately, weak pattern recognition receptor activation in immune deficient individuals affects the individual innate immune responses. Thus, different clinical studies need to be done by enrolling individuals with different immune deficiencies to apply phage therapy.
3. Phage- adaptive immune system interaction
Phages strongly influence adaptive immunity via its effects on humeral immunity and effector polarization. They modulate immune response and bring a profound effect on the outcome of bacterial infection [24]. Individuals exposed to phage therapy or natural existing phages will clearly develop antibody because phages are composed of densely packed immunogenic DNA or RNA and a protein coat [23,25]
Phages alone are not sufficient to fight bacterial infection. Combined effect of the immune system along with phage therapy is essential to fight bacterial infections. Phages are themselves immunogenic microbes which can activate the human adaptive immune system. Phage mediated bacterial lysis stimulates the human adaptive immune response that enhances phage therapy efficacy. However; adverse phage treatment may cause toxicity due to the release of endotoxin as a result of bacterial lysis [20,26].
Phage-immune interactions depend on immune recognition through pattern recognition receptor (PRR), the immunogenic nature of phage, and multiplication rate of phage. Pattern recognition receptor recruits phagocytes to site of infection to resolve the infection. It recognizes phage derived DNA and RNA resulting in phage-mediated activation of innate immune cells. Pattern recognition receptor commitment and level of immune activation depends on phage type, phage dose, and nucleic acid synthesis activity [27,28].
Immunogenic nature of phage enhances repeated phage administration. Therefore, immunogenicity should be considered before phages are used for therapy [29]. Actually, phages were widely administered intravenously decades ago to diagnose and monitor primary and secondary immunodeficiency without reported complication even in patients with prolonged phage survival in their bloodstream. This implies their inherently low toxic effect [30]. Fifty healthy volunteers who were not involved in phage therapy or in phage work were evaluated for anti-phage antibody production against phage T4 and they were positive for the naturally occurring phage anti-body [23]. Remarkable decline in phage activity was observed in 81% of sero-positive participants for phage antibody. In these positive sera, natural IgG antibodies specific to the phage proteins gp23*, gp24*, Hoc, and Soc were identified. These findings show that anti-T4 phage antibodies are frequent in the human population [31]. Multiplication rate of phage can be hampered by IgG or IgA. Phage can be removed from the human body by high antibody levels and Fc receptor-mediated uptake of phage/antibody complexes by macrophages. One of the drawbacks of phage therapy is that phages are immunogenic so that antibodies are produced against them that neutralizes phages and hinder infection of a bacterium by phages [20]. Up to date, there is knowledge gap on whether this regulatory function of anti-phage antibodies can prevent the appearance of resistance to phage and pre-existing immunity to natural phage affecting phage therapy. More importantly, it is not known which phage specific factors are responsible for the mechanism of phage clearance [32]. To evade adaptive humeral immunity, further work on phage modification to loss their immunogenicity and retain their lytic effect is required.
4. Bacterial resistance to phages
Bacteria can develop resistance to phage therapy due to spontaneous mutations, acquisition of restriction modification systems (RM), adaptive immunity via the clustered regular interspaced short palindromic repeat (CRISPR-Cas) system, plasmids, temperate genes and mobile genetic islands (that can carry genes coding for resistance to antibiotics) [33,34]. These mechanisms can be used by a bacterium to target different steps of the phage life cycle, including phage attachment, penetration, replication, and host cell lysis [35]. Prominent resistance phenotypes are noticed as a result of distinct resistance mechanisms. There are different prominent resistance phenotypes depending on whether the resistance is partial or complete, the fitness cost associated with resistance, and whether the mutation can be countered by a mutation in the infecting phage [36].
Spontaneous bacterial mutation results in emergence of phage resistance and phage-bacterium co-evolution [37] which may grant phage resistance by modifying phage-associated receptors on the bacterial surface. Importantly, such alterations may associate with reduced fitness relative to non-resistant strains [38]. When mutation occurs in bacterial lipopolysaccharide or when it undergoes impaired growth due to mutation in genes involved in essential cell function, phage-resistant bacteria may become less virulent [39].
Bacterial restriction-modification systems are often called primitive immune systems in bacteria which are ubiquitous [40]. They are important defense mechanisms against invading phage genomes. It consists of two contrary enzymatic activities: a restriction endonuclease (REase) and a methyltransferase (MTase). Mechanism of bacterial RM systems as defense is due to its RM systems that recognize the methylation status of invading phage genomes. Methylated sequences are recognized as self, while sequences on the invading phage genome lacking methylation are recognized as foreign and are cleaved by the restriction endonuclease (REase). The role of REase is to recognize and cleave non-self-nucleic acid sequences at specific sites, while the role of MTase activity is to ensure identification between self and foreign nucleic acid, by transferring methyl groups to the same specific nucleic acid sequence within the bacterial genome [41].
Clustered regular interspaced short palindromic repeats regulate bacterial adaptive immunity. Bacteria can develop adaptive immunity against phages by acquiring a unique bit of phages’ DNA CRISPR-Cas machinery called spacers from prior exposure or infection. Bacterial adaptive immunity against phages is unique from other defense mechanisms because they are able to recognize prior infections by storing pieces of phage DNA (spacers) in its own DNA to neutralize future infections. Surprisingly, bacteria do not only recognize prior infections by using CRISPR-Cas but also transfer this experience for the coming generations 42.
Bacterial mobile genetic elements may promote bacterial resistance to phage therapy. They are responsible for horizontal transfer of phage resistant bacterial genes among bacteria. For instance, phage resistance conferring conjugative plasmids can disseminate quickly both within and between bacterial species by expressing mating pair complexes that are physically in close proximity [43]. Regardless to antibiotic selection, antibiotic resistance plasmids survive at large abundance in bacterial population because plasmids cause minimal bacterial fitness cost. In case, if it causes fitness cost, it will be balanced rapidly by mutation in the phage, bacteria or in both [44,45]. Phages that specifically bind to the mating-pair complex encoded by conjugative, drug-resistance-conferring plasmids have the potential to limit the spread of antibiotic resistance-conferring plasmids.
5. Conquering CRISPR
Clustered regularly interspaced short palindromic repeats-Cas is a genome editing system found in bacteria that helps a bacterium to defend against phages by inhibiting the integration of phage DNA to a bacterium via CRISPR and endonuclease activity (Cas). Phages undergo point mutation or deletions to escape bacterial adaptive immunity [46]. Therefore, CRISPR-Cas (the effector molecule) fails to recognize and cut phages’ specific genomic sequences that had point mutations and/or deletions. This implies in CRISPR–Cas systems, a single mutation in the protospacer-adjuscent motif is enough to avoid targeting [47-49]. Unfortunately, some phage mutations may promote CRISPR immunity by enhancing gaining of many new spacers [50,51]. Phages can evade by deleting its part or the entire protospacer target. Despite of conquering CRISPR, this strategy can have a fitness cost to phage depending on the region deleted [52].
Phages produce different proteins that inhibit CRISPR-Cas defense, of which anti-CRISPR (Acr) proteins being the most prominent [53]. Inhibition mechanisms of CRISPR-Cas defense include blocking target binding by DNA mimicry or steric blocking and prevention of DNA cleavage by nucleases [54]. An emerging concept to inhibit CRISPR–Cas activity is utilizing phages’ subversion of cellular regulatory pathways that bypass CRISPR–Cas activity [55]. Phages may possess regulatory protein homologs to bacterial proteins that suppress bacterial defense known as bacterial CRISPR–Cas repressor [56]. Otherwise, phages can use proteins that bind and inhibit bacterial regulators. Multiple bacteria modify their CRISPR–Cas activity via quorum sensing, but this behavior may be manipulated by phage-encoded proteins. Genomic modifications are other ways for phage to escape from CRISPR-Cas attack [57,58]. For example, five distinct anti-CRISPR genes are present in P. aeruginosa temperate phages. These genes encode a small protein that can immediately neutralize the immune system of the host by interfering with the formation or action of CRISPR–Cas ribonucleic protein [59].
Two general models were implemented to manage the risk of bacterial resistance to phage therapy. These were applying phage cocktails and adapting single phage to each patient [60]. Combining many phages in cocktails provides them a wide host range and improves their effectiveness. Synergizing different phages and targeting different receptors on the bacterial surface reduces bacterial resistance to phages. Such an approach has a major benefit to empirical treatment [61]. Personalized phage therapy approach utilizes single phages or targeted phage cocktails directly based on the etiologic agent isolated [62]. It is more flexible with respect to phage spectrum and minimizes the emergence of bacterial resistance effectively, but it needs higher cost for treatment [63].
In summary, although bacteria have potential to develop resistance to phage therapy via different mechanisms, phages have several mechanisms to escape bacterial resistance against them. Therefore, upgrading current practices and knowledge on phage interaction with phages, bacteria and human is promising to treat bacterial infections in the era of increasing incidence and transmission of multidrug resistant bacterial species and strain where production of new antibiotics is limited.
6. Role of engineering and other genetic technologies for phage therapy
Role of phage genetic engineering for phage therapy is to produce phages of broader host range and recombination of two distinct phages, while the role of synthetic biology is to construct all genomes of phages so that artificial phages that can infect bacteria can be developed. Whole genome sequencing allowed the production of new phage variants with an expanded host range and few phage strains to cover diversified bacteria [6]. Genetic engineering helps to incorporate bacteriocins, enzybiotics, quorum sensing inhibitors, and biofilm degrading enzymes into phages. These molecules inhibit bacterial metabolism and other vital bacterial activities. Therefore, when a bacterium is infected by phage carrying these molecules, they will die. For example, an engineered T7 phage was engineered to encode lactonase (Lactonase has a broad activity and inhibits quorum sensing molecules in bacteria required for biofilm formation) [64]. Engineered phages improved conventional methods used to kill bacteria. This implies that phages can be engineered with entirely novel mechanisms to kill bacteria and alter mode of gene expression of targeted bacteria [65]. Recently, cystic fibrosis patient with a disseminated Mycobacterium abscessus infection was treated by applying engineered bacteriophages for the first time. There are over 1800 mycobacterial phages in a bank but only one of them effectively killed the clinical isolate of M. abscessus [66].
Advancement of sequencing technology and synthetic biology provided new opportunities to modify and use temperate genes to fight the ever increasing antibiotic resistance [67]. The gene editing technology, clustered regularly inter-spaced palindromic repeats), is used to target a specific genome sequence for site-specific cleavage. For example, it has been applied to carbapenem-resistant Enterobacteriaceae and Enterohemorrhagic E. coli [10].
7. Modified phages and their therapeutic applications
Bioengineered phages highly minimized the drawbacks of conventional phage therapies due to their ability to reach and kill the targeted pathogens or reverse the drug resistance of bacteria. Use of phage derived biochemical endolysin (phage lysin), is efficient against Gram positive bacteria and it can be used for bacterial treatment instead of viable phages [7,15].
Modified phages are phages whose specificity is altered into non-native forms deliberately. Their host recognition specificity is conferred by receptor binding domains found on phages [68]. They are used to target non-native hosts and designed to serve as a vehicle by which antimicrobials are incorporated or attached to its surface and to suppress host SOS DNA repair system [69,70]. Modifications enable the phage to overcome a narrow host range of phages, lessen bacteria potential to develop resistance, avoid challenges in phage manufacturing, exclude systemic side effects (especially endotoxin release), and to prevent attack of phages by the immune system. To avoid the release of endotoxins by gram-negative bacteria mainly due to lyitc phages or antibiotic treatment, phages can be made lysine deficient. For example, MRSA infected mice were successfully treated by lysine deficient phages because the bacterium was killed without lysis. Another means of using modified phages is a targeted gene delivery system to the site of infection by using engineered filamentous phages [71]. Interestingly, modified phages bypass the host immune system, persist within the body, and deliver lethal genes to the bacterial host. Many experiments in animal models revealed that engineered phages are efficient in treating infections. Filamentous phages that do not lyse the host are used as a vehicle to provide lethal genes or substances like holins, lethal transcription regulators, and addiction toxins to induce apoptosis specifically to the site of infection [72].
8. Phage encoded products
Phages encode proteins that recognize and adhere to sites on the bacterial surface like peptidoglycan, pili, flagella, or efflux pumps and to specific sugar moieties in lipopolysaccharide [73]. They encode two types of lysosomes, porins endolysins and phage tail-associated murein lytic enzymes. These enzymes degrade the cell wall of the host. Endolysin with the help of holin lyse the bacterial cell wall from the inside and allow phage progenies to be released [74]. Amazingly, endolysin ability to bind firmly to substrates on the host cell wall minimizes the turnover of endolysins and the requirement of many endolysin molecules to degrade bonds on the cell of the host [75]. Phage tail-associated murein lytic enzyme hydrolyzes the cell wall after phages’ adsorption to the host cell wall from the outside [76] and its activity is limited to Gram positive bacteria because Gram negative bacteria have an outer membrane that blocks direct enzyme contact to the peptidoglycan of the host cell wall. Recently, structurally engineered phage lysosomal molecules that contain specific binding and fusing ability with other modified lysosomes showed an encouraging results against gram negative bacteria [77].
Phage encoded products are used to kill pathogens directly. Its merits over viable phage therapy are its enhanced ability to penetrate and diffuse to the site of action by bypassing sequestration by the spleen, lymph node, and other organs. Lysine is one of the examples of phage products assumed as antibacterial weapons. It is safe and efficient against bacteria and resistance to lysine is less frequent compared to antibiotics. They are successful in animal models against Gram positive bacteria including S.pneumonia, S. pyogenes, B. anthracis, E. faecium, and S. aureus but not to Gram negative bacteria up to date [78].
It is unlikely for use a bacterium to evolve resistance to lysins since lysins target sites on the peptidoglycan which is very vital for bacterial cell viability [79,80]. Additionally, preparing engineered recombinant lytic proteins in mass and administering is much easier than preparing a mass of actual phages and administering. Modified products of phages are more interesting than natural phages because viable phages have limitations due to short shelf life, sequestration by the reticuloendothelial system, and the potential to induce neutralizing antibodies [81]. Applying phage lysin therapy in combination with antibiotics is more effective than a single use of either lysins or antibiotics [82,83].
9. Phage Therapy in Humans
During phage therapy lytic phages are mainly applied to kill their respective bacterial hosts with no effect on human cells and no or minimal disturbance on human microbiota relative to conventional antibiotics. Phage therapy is rapidly reviving with encouraging effects in life-saving therapeutic use and multiple clinical trials. However, it is hosting obstacles with respect to regulations and policy issues for clinical use and implementation [84].
For phage therapy clinical trial, phages should be sufficiently characterized and there must be selection of right phage, human, bacteria and consideration of right disease target for phage therapy. Additionally, information on phage formulation, dosage and efficacy are vital for effective therapy. For example, very specific phages are desired for monobacterial disease. However, it may be a limitation during polybacterial infections unless it is provided in combination with antibiotics. This approach is relevant for patient safety because removal of a single pathogen and growth of other bacteria may threaten patient life [85]. In fact, broad host range phages may be more abundant than currently identified phages although further investigations are required [86]. Principally, all cautions, policies and regulations for phage therapy needs standardization as antibiotics for human use.
Lacks of validated and sufficiently controlled clinical trials are current challenges for phage therapy for clinical use. Planning and designing, pharmacological aspects and their dosage are major activities to consider for clinical use [87]. Dissemination of phages in the body may reduce their efficacious because phages require direct contact with the bacteria with optimum concentration to effectively act on bacteria. Topical applications and many other methods are applied to administer phages. Phage therapy can be given as monotherapy, combination therapy or phage cocktails but the later provides broad spectrum activity and low risk for resistance development. More importantly, combination therapy largely elevates the challenge of diagnosing inflammatory effects, potential for gene transfer and phage resistance development for all phages in cocktail [88]. Another vital consideration to be addressed before clinical trial is onset of toxic shock as phages are bactericidal [89].
9.1. Clinical trials involving phages
Phage therapy clinical trial practices in Georgia and Poland were discussed by Kutter et al. that are prominently mentioned [90] in many literatures confirming safety of phages to treat venous leg ulcers [91] and safety and efficacy in chronic otitis [92]. Rhoads and colleagues reported no adverse side effects from a patient with venous leg ulcers in a small phase I phage therapy clinical trial [93]. Efficacy and safety of anti-pseudomonal phages to late stage recurrent otitis which was mainly controlled by MDR-P.aeroginosa was shown by Wright and et al. Although phage therapy is incorporated in the health policy of Eastern Europe countries, above mentioned controlled clinical trials are among the first trials made in humans in the western world. Now a day, many clinical trials are registered [91,92].
Scientifically sounded clinical trials are essential to phage therapy to be accepted by western clinical world. Although, many observational phage therapy studies are conducted and were effective, they have limitations due to small sample size and poor control. Additionally, there are promising case studies despite strong clinical trial data is expected by regulators to prepare guide lines for phage therapy [84] to be approved by United States Food and Drug Administration Agency.
9.2. Determinants of human phage therapy trials
a) Virulent genes
Pathogenicity and severity of the disease may increases when virulent genes are acquired from other virulent species which may cuase treatment failure [93]. Phages can carry virulent genes that may increase the virulence and pathogenicity of bacteria during lysogenization. Phage virulent genes are detected in many human pathogen s like E.coli, P.aeroginosa, S. aureus, and S. pyogenes. Currently, all phage encoded virulent genes are not identified. Therefore, the metagenome and each genome sequence are vital to databases of bacterial virulent genes and antibiotic resistance genes to assure safety [94, 95].
b) Transduction
Bacteria can acquire virulent and antibiotic resistant genes through phage mediated transduction. In fact it can be mitigated by avoiding known transducing phages [96].
c) Disturbance of commensal micro-biota
It is crucial to know and inquire interactions of phages with human niche microbiota while using phages as therapeutic agent and any possible perturbation in the human microbiota due to strong selective pressure of lytic phages and specifically. Additional mechanistic investigations that clearly show the nature of host-phage dynamics to niche microbiota helps in fastening the process to obtain regulatory approval for phage therapy in Western medicine [97]. It is sometimes mentioned as a concern. However, phages are generally species specific so that disturbance of the commensal micro-biota is minimal [98].
d) Quality and safety requirements
Safety and quality of phage preparation determines the success of phage therapy. Production of phages for therapy must reside on strict regulations to assure their quality for their intended use even if there are no yet clear guidelines to manufacture phages [99]. Presence of impurities like endotoxins must be avoided or below the threshold in phage preparations but none has reached optimal so far. Quality of phage therapy is regulated by assessing and checking their stability, sterility, cytotoxicity, and performing ph measurements regularly [100].
e) Phage storage and handling
Phages for therapy are mainly in the form of water suspension and freshly made. There is no enough understanding how to process phages with well-defined pharmaceutics and stability. Phages lack stability which is one of the ideal characteristics of drugs like specificity, high affinity, solubility, safety and stability [101,102]. They are partially stable in solution due to their proteineious structure. Structural instability of them makes long-term storage difficult and cooling is a must to store phage preparations. Phages in aqueous solutions can be stabilized by the addition of stability enhancers or by processing them into another formulation like lyophilization, spray drying, incorporating phages into ointments, biodegradable polymer matrices, or micro particles [103, 104].
10. Conclusion and perspectives
Current contests encountering phage therapy are compatibility to the current quality and safety requirements, warranting the stability of phage preparations for long periods of time, designing an effective assays for phage screening, overwhelming the limited activity of phages in biofilms, controlling and disabling the appearance of bacterial resistance to phages, production of antibodies neutralizing phages, sequestration by spleen and liver and launching a regulatory framework more adequate to phage products [34,106]. Comparison of different antibacterial treatment mechanisms by administering antibiotics and phages are shown in Table 1 and the Pros and cons of phage therapy are shown in Table 2.
Table 1: Comparison of antibiotic and lytic phage therapy
Table 2: Pros and cons of lytic and temperate phage therapy
Phage therapy is a promising approach to combat bacterial infections including multidrug resistant bacteria. For efficacious phage therapy, phages need to be present in high concentration, stable, able to encounter bacteria with no restriction, and replicate. It can be used either alternatively or in a supplement to antibiotics. The increment of antibiotic resistant bacteria can be reduced by using phage cocktails, phage-derived lytic proteins, bioengineered phage, and/or antibiotics. Phage therapy is highly specific, effective in lysing the targeted bacteria, safe as seen in Eastern Europe, quickly modifiable to fight newly emerging bacterial threats. Although it is effective in some clinical trials, many of the trials do not meet the existing difficult standards for clinical trials and many questions are remaining regarding to phage therapeutic use. Better understanding of phage-host, phage-human interactions, phage diversity, phage dynamics, and genome function are essential to develop a new strategy to fight against bacterial infections and combat challenges with phage therapy. Nevertheless, of the numerous studies done about phage therapy in the last decades in Western world and before that in Eastern Europe, there are no approved phage therapies for humans by European Union and/or Food and Drug Administration Agency of United States of America (FDA). Finally, further all-inclusive intensive studies are necessary to warrant phage therapy for large-scale clinical use.

Authors' contributions

All authors made a significant contribution to the work reported, whetherthat is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journalto which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors declare that there are no conflicts of interest.

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I would like to express my gratitude towards you process of article review and submission. I found this to be very fair and expedient. Your follow up has been excellent. I have many publications in national and international journal and your process has been one of the best so far. Keep up the great work.

Douglas Miyazaki

We are grateful for this opportunity to provide a glowing recommendation to the Journal of Psychiatry and Psychotherapy. We found that the editorial team were very supportive, helpful, kept us abreast of timelines and over all very professional in nature. The peer review process was rigorous, efficient and constructive that really enhanced our article submission. The experience with this journal remains one of our best ever and we look forward to providing future submissions in the near future.

Dr Griffith

I am very pleased to serve as EBM of the journal, I hope many years of my experience in stem cells can help the journal from one way or another. As we know, stem cells hold great potential for regenerative medicine, which are mostly used to promote the repair response of diseased, dysfunctional or injured tissue using stem cells or their derivatives. I think Stem Cell Research and Therapeutics International is a great platform to publish and share the understanding towards the biology and translational or clinical application of stem cells.

Dr Tong Ming Liu

I would like to give my testimony in the support I have got by the peer review process and to support the editorial office where they were of asset to support young author like me to be encouraged to publish their work in your respected journal and globalize and share knowledge across the globe. I really give my great gratitude to your journal and the peer review including the editorial office.

Husain Taha Radhi

I am delighted to publish our manuscript entitled "A Perspective on Cocaine Induced Stroke - Its Mechanisms and Management" in the Journal of Neuroscience and Neurological Surgery. The peer review process, support from the editorial office, and quality of the journal are excellent. The manuscripts published are of high quality and of excellent scientific value. I recommend this journal very much to colleagues.

S Munshi

Dr.Tania Muñoz, My experience as researcher and author of a review article in The Journal Clinical Cardiology and Interventions has been very enriching and stimulating. The editorial team is excellent, performs its work with absolute responsibility and delivery. They are proactive, dynamic and receptive to all proposals. Supporting at all times the vast universe of authors who choose them as an option for publication. The team of review specialists, members of the editorial board, are brilliant professionals, with remarkable performance in medical research and scientific methodology. Together they form a frontline team that consolidates the JCCI as a magnificent option for the publication and review of high-level medical articles and broad collective interest. I am honored to be able to share my review article and open to receive all your comments.

Tania Munoz

“The peer review process of JPMHC is quick and effective. Authors are benefited by good and professional reviewers with huge experience in the field of psychology and mental health. The support from the editorial office is very professional. People to contact to are friendly and happy to help and assist any query authors might have. Quality of the Journal is scientific and publishes ground-breaking research on mental health that is useful for other professionals in the field”.

George Varvatsoulias

Dear editorial department: On behalf of our team, I hereby certify the reliability and superiority of the International Journal of Clinical Case Reports and Reviews in the peer review process, editorial support, and journal quality. Firstly, the peer review process of the International Journal of Clinical Case Reports and Reviews is rigorous, fair, transparent, fast, and of high quality. The editorial department invites experts from relevant fields as anonymous reviewers to review all submitted manuscripts. These experts have rich academic backgrounds and experience, and can accurately evaluate the academic quality, originality, and suitability of manuscripts. The editorial department is committed to ensuring the rigor of the peer review process, while also making every effort to ensure a fast review cycle to meet the needs of authors and the academic community. Secondly, the editorial team of the International Journal of Clinical Case Reports and Reviews is composed of a group of senior scholars and professionals with rich experience and professional knowledge in related fields. The editorial department is committed to assisting authors in improving their manuscripts, ensuring their academic accuracy, clarity, and completeness. Editors actively collaborate with authors, providing useful suggestions and feedback to promote the improvement and development of the manuscript. We believe that the support of the editorial department is one of the key factors in ensuring the quality of the journal. Finally, the International Journal of Clinical Case Reports and Reviews is renowned for its high- quality articles and strict academic standards. The editorial department is committed to publishing innovative and academically valuable research results to promote the development and progress of related fields. The International Journal of Clinical Case Reports and Reviews is reasonably priced and ensures excellent service and quality ratio, allowing authors to obtain high-level academic publishing opportunities in an affordable manner. I hereby solemnly declare that the International Journal of Clinical Case Reports and Reviews has a high level of credibility and superiority in terms of peer review process, editorial support, reasonable fees, and journal quality. Sincerely, Rui Tao.

Rui Tao

Clinical Cardiology and Cardiovascular Interventions I testity the covering of the peer review process, support from the editorial office, and quality of the journal.

Khurram Arshad

Clinical Cardiology and Cardiovascular Interventions, we deeply appreciate the interest shown in our work and its publication. It has been a true pleasure to collaborate with you. The peer review process, as well as the support provided by the editorial office, have been exceptional, and the quality of the journal is very high, which was a determining factor in our decision to publish with you.

Gomez Barriga Maria Dolores

The peer reviewers process is quick and effective, the supports from editorial office is excellent, the quality of journal is high. I would like to collabroate with Internatioanl journal of Clinical Case Reports and Reviews journal clinically in the future time.

Lin Shaw Chin

Clinical Cardiology and Cardiovascular Interventions, I would like to express my sincerest gratitude for the trust placed in our team for the publication in your journal. It has been a true pleasure to collaborate with you on this project. I am pleased to inform you that both the peer review process and the attention from the editorial coordination have been excellent. Your team has worked with dedication and professionalism to ensure that your publication meets the highest standards of quality. We are confident that this collaboration will result in mutual success, and we are eager to see the fruits of this shared effort.

Maria Dolores Gomez Barriga

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